NEUROMUSCULAR TRANSMISSION IN A NATURALLY OCCURRING MOUSE MUTANT OF THE β SUBUNIT OF THE NEURONAL CALCIUM CHANNEL BY ELIZABETH MOLINA CAMPOS A DISSERTATION Submitted to Michigan State University In partial fulfillment of the requirements For the degree of DOCTOR OF PHILOSOPHY GENETICS 2011 ABSTRACT NEUROMUSCULAR TRANSMISSION IN A NATURALLY OCCURRING MOUSE MUTANT OF THE β SUBUNIT OF THE NEURONAL CALCIUM CHANNEL By ELIZABETH MOLINA CAMPOS Entry of Ca 2+ through voltage gated calcium channels (VGCCs) into nerve terminals is a necessary step coupling the action potential to release of acetylcholine (ACh). VGCCs are heteromultimeric complexes of α1, α2δ, and β subunits, and sometimes γ subunits. The specific α1-β combination assembled determines the channel properties. The mouse mutant lethargic (lh) has severe neurological defects due to a mutation that deletes α1 subunit interaction domain of the β4 subunit. β4 normally associates with the α1A subunit of the P/Q-type VGCCs, and has a major role in stabilizing the final α1A subunit conformation and targeting it to the cell membrane. Loss of the β4 subunit could alter the channel characteristics and localization of α1A. The overall goal of this dissertation was to test the hypothesis that disruption of the β4 subunit affects the function of the α1A subunit of the P/Q-type VGCCs. Electrophysiological recordings were performed at neuromuscular junctions (NMJs) of adult lh and wild type (wt) mice. The quantal content and phrenic nerve evoked release showed a significant decrease in lh with respect to wt. The frequency of spontaneous release of ACh also decreased significantly, although the reduction was only evident when Ca 2+ was replaced by Sr 2+ 2+ or Ba as charge carriers. The amplitude of spontaneous release was not affected by this mutation, implying that each vesicle contains approximately the same amount of ACh in wt and lh mice. These results are due to a significantly slower process of neurotransmitter vesicles release, as confirmed by FM1-43 staining method. There are specific VGCCs antagonists that can be used to determine the contribution of the different types of VGCCs in nerve-stimulated ACh release from motor nerve terminals. ω-agatoxin IVA and SNX-482, specific antagonists for P/Q- and R-type VGCCs respectively, significantly reduced the quantal content in adult lh mice. Immunolabeling of VGCC subunits revealed an increase in α1E, β1 and β3, but no apparent change in the levels of α1A at adult lh neuromuscular junctions. Therefore, lh animals control ACh release by P/Q- and R-type VGCCs. The studies of this dissertation provide evidence for: 1) decreased nerve-evoked ACh release in lh mice, 2) slowed vesicle release process in lh mice, 3) increased level of β1 and β3, compensating for the lack of β4 subunit, and 4) P/Q- and R-type VGCC involvement in release of ACh from motor nerve terminals. To Gastón, a great brother, who always had faith in me. iv ACKNOWLEDGEMENTS First and foremost I would like to thank my advisor, Dr. William Atchison for his invaluable guidance during my graduate career. His support, advice and guidance have helped me become an independent researcher. I could not have chosen a better mentor to provide the right amount of oversight and independence necessary for my success as a student and researcher. I would also like to thank my committee members, Dr. Ke Dong, Dr. John Fyfe, and Dr. Colleen Hegg. Their expertise, ideas, technical assistance and thoughtful critiques were instrumental in the development and progression of this research project. I also want to acknowledge Ms. Jeannine Lee and Dr. Barbara Sears, from the genetics program, for all their help, guidance and assistance. Dr. Stephen Schneider helped in correcting and editing my dissertation, and Dr. Nicole Pardo a previous member of Dr Atchison‟s lab, who guided me through electrophysiology and her assistance in various aspects of my research, Dr. Ravindra Hajela for his help in genotyping animals, and Dr. Yukun Yuan for all his valuable advice regarding my research, as well as Ms. Dawn Autio for her incredible support and guidance with immunohistochemistry. I must acknowledge my lab mates, Brenda Marrero-Rosado, Sara Fox, Aaron Bradford, Alexandra Colon, Heidi Hannon, Dr. Wen-hsin Ku, Dr. Frank Johnson, Dr. Erin Wakeling, and Dr. Sameera Dasari, and the girls from Puerto Rico (Desiree, Maryvi, Angie, Chesky, Katiria and Rosa) for making the lab a nice enjoyable place and keeping me sane. To all my friends here and elsewhere, sincere thanks for your v love and support. I am eternally thankful to Gabriel, for his love, incredible patience, help and for always being by my side in the hardest times; graduate school would not have been the same without you. Finally, I am forever thankful to my family, Mom, Dad, Florencio, Martina and Gastón for always being there by my side, thank you for all your love and support despite being so far away from home. I could not have done this without you. vi TABLE OF CONTENTS LIST OF TABLES ............................................................................................................. ix LIST OF FIGURES ............................................................................................................ x LIST OF ABBREVIATIONS ........................................................................................... xii CHAPTER 1 ....................................................................................................................... 1 INTRODUCTION A. General Introduction ............................................................................................ 2 B. Neuromuscular Junction and Vesicle Release ..................................................... 5 C. Voltage – Gated Calcium Channels ................................................................... 20 1. General Description…………………………………………………….. 20 2. The α1 Subunit………………………………………………………….. 23 D. E. F. 3. The α2δ Subunit...………………………………………………………. 35 4. The γ Subunit…………………………………………………………… 37 5. The β Subunit…………………………………………………………… 38 5.1. Structure of the β Subunit………………………………………………. 42 The Lethargic (lh) Mutation ............................................................................... 46 Regulation of Calcium Channels By Small G Proteins...................................... 47 Specific Aims ..................................................................................................... 50 CHAPTER 2 ..................................................................................................................... 55 CHARACTERIZATION OF NEUROMUSCULAR TRANSMISSION IN LETHARGIC (lh) MICE A. Abstract .............................................................................................................. 56 B. Introduction ........................................................................................................ 58 C. Materials and Methods ....................................................................................... 62 D. Results ................................................................................................................ 67 E. Discussion .......................................................................................................... 72 CHAPTER 3 ..................................................................................................................... 91 ACETYLCHOLINE RELEASE IS CONTROLLED BY P/Q- AND R-TYPE CALCIUM CHANNELS IN ADULT LETHARGIC (lh) MICE A. Abstract .............................................................................................................. 92 B. Introduction ........................................................................................................ 94 C. Materials and Methods ....................................................................................... 97 D. Results .............................................................................................................. 102 E. Summary and Conclusion ................................................................................ 106 CHAPTER 4 ................................................................................................................... 129 SUMMARY AND CONCLUSIONS A. Summary and Conclusions ............................................................................... 130 vii REFERENCES ............................................................................................................... 151 viii LIST OF TABLES Table 1.1: Pharmacological and biophysical properties of VGCC subtypes. .................. 26 Table 1.2: Human and mouse disorders associated with VGCCs subunits. .................... 28 ix LIST OF FIGURES Figure 1.1: Neuromuscular transmission. .......................................................................... 9 Figure 1.2: Configuration of the interactions of synaptic vesicle and plasma membrane proteins before (a) and after (b) the formation of the SNARE complex. ......................... 13 Figure 1.3: Structure of voltage-gated calcium channels (VGCCs). ............................... 16 Figure 1.4: Structure of the nicotinic acetylcholine (nACh) receptor.............................. 19 Figure 1.5: Molecular organization of VGCCs. ............................................................... 22 Figure 1.6: Structure of the α1 subunit............................................................................. 24 Figure 1.7: Structure of the β subunit. ............................................................................. 43 Figure 1.8: Interaction of VGCCs α1 subunit and β subunit............................................ 48 Figure 2.1: Spontaneous release of ACh measured in solutions containing 2, 4 or 8 mM CaCl2................................................................................................................................. 76 Figure 2.2: Spontaneous release of ACh measured in solutions containing SrCl2 (2 or 4 mM) or BaCl2 (0.5 or 1 mM)............................................................................................ 78 Figure 2.3: Nerve evoked release of ACh measured in solutions containing 2, 4 or 8 mM CaCl2................................................................................................................................. 80 Figure 2.4: Quantal content measured in solutions containing 2, 4 or 8 mM CaCl2. ...... 82 Figure 2.5: Nerve evoked release of ACh measured in solutions containing 2 or 4 mM SrCl2. ................................................................................................................................ 83 Figure 2.6: Quantal content measured in solutions containing 2 or 4 mM SrCl2. ........... 84 Figure 2.7: Time course of FM1-43 destaining triggered by perfusion application of 40 mM KCl in wt and lh animals. .......................................................................................... 85 Figure 2.8: FM1-43 destaining in wt and lh motor nerve terminals. ............................... 86 Figure 2.9: Time course of FM1-43 destaining triggered by hypertonic solution (500 mM) in wt and lh animals. ................................................................................................ 89 x Figure 2.10: Time course of FM1-43 destaining triggered by α-LTx in wt and lh animals. ........................................................................................................................................... 90 Figure 3.1: Effect of lh mutation on cerebellar protein levels. ...................................... 109 Figure 3.2: ACh release is controlled by P/Q- and R-type VGCCs in adult lh mice. .... 113 Figure 3.3: Effect of Aga IVA and SNX-482 on quantal content of lh and wt mice. .... 116 Figure 3.4: Immunostaining of wt and lh neuromuscular junctions (NMJs) with α1A/α1E and β4. ............................................................................................................................. 118 Figure 3.5: Immunostaining of wt and lh NMJs with α1A/α1E and β1. ......................... 121 Figure 3.6: Immunostaining of wt and lh NMJs with α1A/α1E and β3. ......................... 124 Figure 3.7: Relative fluorescence of α1 and β subunits at wt and lh NMJs.................... 127 Figure 3.8: Percent juxtaposition of α1 and β subunits of wt and lh NMJ with ACh receptors. ......................................................................................................................... 128 Figure 4.1: Structure of the α1A subunit showing the tg mutation. ............................... 134 Figure 4.2: Proposed model of the NMJ of adult lh mice. ............................................. 148 xi LIST OF ABBREVIATIONS ABP AID-binding pocket ACh Acetylcholine AChE Acetylcholinesterase Aga-IVA ω -agatoxin IVA AID α-interaction domain α-LTx α-latrotoxin AMPA α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid α-SNAP α-soluble NSF attachment protein BCA Bicinchoninic acid BID -interacting domain ChAT Choline-O-acetyl-transferase CNS Central nervous system Ctx GVIA -conotoxin GVIA DAP 3,4-diaminopyridine FITC Fluorescein isothiocyanate EDL Extensor digitorum longus EDTA Ethylenediaminetetraacetic acid EGTA Ethylene glycol tetraacetic acid ER Endoplasmic reticulum EPP End-plate potential GABA γ-aminobutyric acid xii GK Guanylate kinase Glu Glutamate Gly Glycine HEPES 2-hydroxyethyl-1-piperazineethanesulfonic acid HVA High voltage activated IHC Immunohistochemistry 2+ [Ca ]i Intracellular calcium concentration LEMS Lambert-Eaton myasthenic syndrome lh Lethargic LTP Long term potentiation LVA Low voltage activated m Quantal content MEPP Miniature end-plate potential nACh Nicotinic acetylcholine NMDAR N-methyl-D-aspartate receptor NMJ Neuromuscular junction NSF N-ethylmaleimide-sensitive factor PBS Phosphate-buffered saline SDS PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SEM Standard error of the mean SH3 Src homology 3 SNAP-25 Synaptosomal protein of 25 kD xiii SNARE Soluble N-ethylmaleimide-sensitive factor attachment protein receptors stg Stargazer Synprint Synaptic protein interaction TARPs Transmembrane AMPA receptor regulatory proteins tg Tottering 4J tg 5J tg la tg rol Tottering 4 Jackson Tottering 5 Jackson Tottering leaner tg Tottering rolling Nagoya TS Triangularis sterni VGCCs Voltage gated calcium channels wt Wild type xiv CHAPTER 1 INTRODUCTION 1 A. General Introduction Voltage gated calcium channels (VGCCs) contribute to the entry of Ca 2+ into nerve terminals. This is a necessary step coupling the action potential to release of acetylcholine (ACh) (Augustine et al., 1987; Llinas et al., 1976; Katz and Miledi, 1970). Although multiple VGCCs subtypes are known to coexist in the same cell, the specific channel subtype involved in release of ACh from motor nerve terminals is both speciesand age-dependent (Catterall, 1998; Rosato Siri and Uchitel, 1999). During early stages of development, motor nerve terminals have multiple subtypes of VGCCs (Rosato Siri and Uchitel, 1999; Santafe et al., 2001). Mature motor nerve terminals, however, contain primarily one subtype of VGCCs involved in the release of ACh. Mature mammalian motor nerve terminals utilize P/Q-type (Cav2.1) (Katz et al., 1995), whereas amphibians (Sano et al., 1987) and birds (De Luca et al., 1991) rely mainly on N-type (Cav2.2) VGCCs to control the release of ACh. However the complement of VGCCs does not necessarily seem to be fixed. Under specific conditions, subtypes of VGCCs that are not normally associated with ACh release at motor nerve terminals can mediate it (Flink and Atchison, 2002; Pardo et al., 2006). VGCCs are categorized into two main classes depending on the extent of depolarization from rest needed to activate them. These are: low voltage-activated and high voltage-activated VGCCs. The former are activated by small depolarizations from rest, and hence open at highly negative membrane potentials, while the latter require strong depolarizations and activate at more depolarized membrane potentials. 2 The different VGCCs can be distinguished by the genes that encode them and their pharmacological and biophysical characteristics (Catterall et al., 2005; Zhang et al., 1993). VGCCs are formed by α1, β, and α2δ subunits (Tsien et al., 1991). Some also contain a γ subunit. The α1 subunits make up the selective pore for Ca 2+ and determine most of the subtype-specific attributes of VGCCs. They contain binding sites for various pharmacological agents as well as the gating regions of the channel (Catterall, 1995; Zhang et al., 1993). There are at least five α1 subunits for neuronal VGCCs which fall under the high voltage activated (HVA) subclass. They include: α1A, α1B, and α1E subunits which represent the P/Q- (Cav2.1), N- (Cav2.2), and R-type (Cav2.3) VGCCs, respectively; while the α1C, α1D, α1F and α1S represent the L-type channels (Cav1.2-1.3) (Catterall, 1995; Tsien et al., 1991). In VGCCs the β subunit is a cytoplasmic protein that regulates the assembly and membrane localization of the α1 subunits (Dolphin, 2003). The β subunit also strongly influences the current amplitude, rate and voltage-dependence of activation and inactivation, and ligand-binding sites on the channel (Brice and Dolphin, 1999; Catterall, 1995; Dolphin, 2003; Walker and De Waard, 1998). There are four different types of β subunits (β1-4) which each are encoded by different genes (Chien et al., 1995). The β4 subunit is a common constituent of neuronal P/Q- VGCCs (Wittemann et al., 2000). The correct β subunit is essential for its interaction with its corresponding α1 subunit for proper targeting, membrane insertion, channel density, kinetic parameters such as 3 activation and inactivation, as well as interactions with vesicular release site proteins (Murakami et al., 2003; Wittemann et al., 2000). However, in the absence of the normally associating β subunit, alternate β subunits may interact with α1 subunits to restore most of the VGCCs‟ functions, although in an altered manner (Burgess et al., 1999). The β4 subunit, which typically associates with the α1A subunit, is normally widely expressed in the brain. It has been reported that spontaneous mutations in this subunit cause several neurological syndromes in mice (Burgess et al., 1997; Catterall, 1995); it produces various effects in VGCC expression and function, such as dramatically reducing the VGCCs‟ targeting, assembly, membrane insertion, and channel density. It also alters the characteristic kinetic parameters, vesicular release and synaptic transmission (Burgess et al., 1999; Catterall, 1995; Catterall et al., 2005; Walker et al., 1998). Also, the loss of a functional β4 subunit can impact the function of α1A – containing VGCCs (P/Q-type) (Helton and Horne, 2002). A four base pair insertion into a splice donor site within the β4 gene in mouse chromosome 2 leads to the lethargic (lh) mutation. This insertion leads to exon skipping, translational frameshift, and protein truncation, with omission of 60% of the C-terminal of the β4 subunit relative to wild type (wt), including loss of the α1-binding site. This suggests that a defect in VGCC assembly could be one cause for the pathogenesis in lh phenotype (Burgess et al., 1997; Burgess et al., 1999). The lh mice suffer from ataxia, lethargic behavior, spike-wave epilepsy, and paroxysomal dyskinesia. The onset of ataxia is two weeks after birth (Khan and Jinnah, 2002). Electrophysiologically and pharmacologically, these seizures are similar to the absence seizures present in the 4 human petit mal epilepsy and to those present in tottering (tg) mice (Burgess et al., 1999; Hosford et al., 1992). In addition to the neurological signs, lh mice show reduced body weight and immunological problems when compared with unaffected litter mates (Sidman et al., 1965). B. Neuromuscular Junction and Vesicle Release The neuromuscular junction (NMJ) is a chemical synapse that occurs between the axons of lower motor neurons of the spinal cord or brainstem neurons and skeletal muscle. It is comprised of a specialized cleft or synapse (which is 20 – 30 nm wide) between motor nerve terminals and the muscle‟s end plate, where nicotinic ACh (nACh) receptors are clustered in junctional folds. Neuromuscular synaptic transmission is fast and reliable. An action potential in the motor axon, leads to the release of chemical neurotransmitters from the motor nerve terminals causing an action potential in the muscle cell leading to muscle contraction. This reliability is accounted for, in part, by structural specializations of the NMJ. The presynaptic terminal contains a large number of highly specialized regions known as active zones, which are the sites of neurotransmitter release in the presynaptic membrane (Ceccarelli et al., 1979; Dreyer et al., 1973; Heuser et al., 1974; Heuser et al., 1979; Heuser and Reese, 1981; Rash et al., 1974). In addition, the post-synaptic membrane at the motor end-plate contains a series of shallow folds packed with neurotransmitter receptors. The NMJ contains several proteins which are involved in mediating the connections between the motor nerve endings in the presynaptic region with the muscle‟s end plate in the postsynaptic region. 5 Fast synaptic transmission at most central nervous system (CNS) synapses is mediated by glutamate (Glu), γ-aminobutyric acid (GABA), and glycine (Gly). The motor nerve terminal contains the chemical neurotransmitter ACh, which also mediates fast synaptic transmission. Chemical synaptic transmission requires that neurotransmitters be synthesized and ready for release. Neurons contain specific enzymes that synthesize neurotransmitters from various metabolic precursors. The synthesizing enzymes for both amino acid and amine neurotransmitters are transported to the axon terminal, where they locally and rapidly direct transmitter synthesis. Once synthesized in the cytoplasm of the axon terminal, amino acid and amine neurotransmitters must be taken up by synaptic vesicles through transporters. ACh is synthesized in the terminals from acetyl-CoA and choline. This reaction is catalyzed by choline-O-acetyl-transferase (ChAT) (Browning and Schulman, 1968; Hebb, 1972; Tucek, 1982). Once ACh is synthesized it is transported to synaptic vesicles for storage and release via an ATPasedependent transport system (Anderson et al., 1982; Breer et al., 1977; Parsons and Koenigsberger, 1980). There are two populations of synaptic vesicles. Those vesicles that are immediately available for synaptic transmission, the “readily releasable pool”, are found close to the nerve terminals in the active zone (the regions involved with neurotransmitter release). The other population includes those vesicles that serve to replenish the readily releasable pool once it is empty and are known as the “reserve pool”. This movement of vesicles between both populations of vesicles is known as “mobilization” (Delgado et al., 2000; Kuromi and Kidokoro, 2002; Richards et al., 2000). It is believed that synapsin, which is a synaptic vesicle protein, is involved in this process 6 by a series of phosphorylation / dephosphorylation – dependent interactions with cytoskeletal proteins such as F-actin (Humeau et al., 2001; Llinas et al., 1991; Petrucci and Morrow, 1987). In the case of cholinergic synapses, the refilling of the readily releasable pool occurs by synthesis of new ACh (Collier, 1986). However, it is not an allor-nothing process, there is a process known as “kiss and run” in which there is incomplete release of vesicles. This process has been measured at the NMJs, hippocampal synapses and chromaffin cells. (Ceccarelli et al., 1973; Fesce and Meldolesi, 1999; Klingauf et al., 1998; Kraszewski et al., 1996; Palfrey and Artalejo, 1998; Verstreken et al., 2008; Zefirov et al., 2004). ACh is released in defined packets known as quanta (Boyd and Martin, 1956; del Castillo and Katz, 1954; Katz and Miledi, 1967b), in which each synaptic vesicle is believed to represent a single quantum of ACh (Heuser and Reese, 1973; Heuser et al., 1979). The ACh release can either be spontaneous or induced by an action potential. The spontaneous release of ACh represents single packets of quanta that are released asynchronously. These single packets produce small depolarizations at the end-plate region of the muscle, known as miniature end-plate potentials (MEPP) (Katz and Miledi, 1963, 1967b). MEPPs have an amplitude distribution around mean amplitude equal to that of a small end-plate potential (EPP). These small depolarizations are not enough to reach threshold, and so an action potential in the postsynaptic membrane does not occur (Sellin et al., 1996). Action potentials evoke ACh release of several quanta from the motor nerve terminal in a synchronous way; this results in a large depolarization of the muscle membrane, known as an EPP (Boyd and Martin, 1956; del Castillo and Katz, 1954; Fatt and Katz, 1951). 7 + An action potential in the nerve terminal, produced by the movement of Na and + K down their respective electrochemical gradients, invades the terminal membrane (Fig. 1.1). This action potential eventually ceases near the end of the axon, but the currents spread electrotonically and depolarize the membrane (Mallart and Brigant, 1982; Mallart, 1985a). As a result of membrane depolarization, VGCCs open, allowing Ca 2+ to move down its electrochemical gradient from the extracellular environment into the nerve terminal (Augustine et al., 1987; Katz and Miledi, 1967a, 1970; Llinas et al., 1981a). 2+ This rapid increase in intracellular [Ca ] occurs in distinct domains at active zones (Llinas et al., 1992) and induces synaptic vesicle fusion with the surface membrane, releasing the neurotransmitter molecules into the synaptic cleft (Ceccarelli et al., 1979; Ellisman et al., 1976; Harris and Sultan, 1995; Heuser et al., 1974; Heuser et al., 1979). Even though neurotransmitter release is triggered by Ca 2+ entry via VGCCs in nerve endings, it is not the only factor required for synaptic vesicle exocytosis. The delay 2+ observed between presynaptic Ca influx and the changes produced by the ACh in the end-plate is very short, as little as 200 µs (Llinas et al., 1981b; Sabatini and Regehr, 1996). The rapidity of this response led to the supposition that fusion of synaptic vesicles with the plasma membrane was already preformed prior to Ca 2+ entry into the nerve terminal (Jahn and Südhof 1999; Lonart and Südhof, 2000). Moreover, there can be 2+ release of ACh in the absence of Ca . The release can be induced by high osmolarity conditions or in the presence of α-latrotoxin (α-LTx) (Hubbard et al., 1968; Rosenmund 8 Figure 1.1: Neuromuscular transmission. (1) Stimulation of the nerve leads to initiation and propagation of an action potential. (2) Depolarization of presynaptic terminal leads to opening of VGCCs, which allow rapid influx of Ca 2+ to the nerve terminal. (3) Ca 2+ causes synaptic vesicles to fuse with the synaptic membrane. The neurotransmitter (ACh) is released to the synaptic cleft via exocytosis. (4) Two ACh molecules bind to receptor molecules in the postsynaptic membrane (nicotinic ACh receptors), leading to the opening of the ACh receptor + associated channel located on the end-plate region of the muscle. Diffusion of Na and + K across the channel causes depolarization which opens voltage gated sodium channels in the adjacent muscle membrane which allows for muscle action potential and muscular contraction. (5) The effects of ACh are rapidly terminated by the action of AChE, which hydrolyzes ACh into acetate and choline. Choline is taken back into the cytoplasm of the nerve terminal by a high affinity transporter. Choline is then combined with acetylCoA by the enzyme choline-O-acetyltransferase (ChAT) to form new molecules of ACh, which are then stored in the synaptic vesicles via an ATP-dependent transport system. (6) Recycling of vesicular membrane from plasma membrane occurs through a clathrinmediated cycle. (Adapted from Purves et al., 2008) 9 Figure 1.1 (cont’d) 1 6 3 5 2 4 For interpretation of the reference to color of this and all other figures, the reader is referred to the electronic version of this dissertation. 10 and Stevens, 1996). α-LTx is a component of the black widow spider venom, and produces massive exocytosis of synaptic vesicles. Additionally Ba 2+ and Sr 2+ readily enter the cell through VGCCs and can support the generation of MEPPs in the absence of Ca Ca Sr 2+ 2+ 2+ 2+ 2+ (Silinsky, 1977; 1978; 1981; 1985). However, Sr , but not Ba can also replace 2+ to support an EPP in the process of synchronous-evoked release. Using Ba and for neurotransmitter release at the squid synapse has shown that the release is much lower than for an equivalent amount of Ca 2+ influx. The amplitude of post-synaptic current elicited by presynaptic transmitter release and the amplitude of postsynaptic potential changes was reduced when Ca Sr 2+ or Ba 2+ 2+ was replaced with equivalent concentrations of (Augustine and Eckert, 1984). This suggests that the ion-sensitive step in the release process prefers Ca Ba 2+ 2+ strongly over other ions in the order of Ca 2+ > Sr 2+ (Dodge et al., 1969; Meiri and Rahamimoff, 1971; Miledi, 1966). However, Ba and Sr 2+ cause a much higher MEPP frequency than Ca 1978; 1981). Therefore, it seems that Ca 2+ 2+ > 2+ (Mellow et al., 1982; Silinsky, most likely acts as a key regulator, but not an absolute requirement for exocytosis. There are four molecules that are central to the exocytosis of synaptic vesicles. They are synaptotagmin, synaptobrevin (this is the only synaptic vesicle protein), syntaxin and SNAP-25 (synaptosomal associated protein of 25 kDa). Fusion of synaptic vesicles with the plasma membrane involves the formation of stable core complexes of proteins known as a SNARE [Soluble N-ethylmaleimide-sensitive factor (NSF) 11 Attachment Protein Receptors] complex. SNAREs consist of two groups. The nerve terminal or t-SNAREs are composed by SNAP-25 and syntaxin, and are localized on the plasma membrane. The synaptic vesicle or v-SNARE, is composed of synaptobrevin, and found on vesicle membranes (Bennett et al., 1992a; Bennett et al., 1992b; Oyler et al., 1989; Trimble and Scheller, 1988). SNAREs have been shown to mediate membrane trafficking and secretion in both mammalian and yeast cells (Fig 1.2) (Bennett et al., 1992a; Bennett et al., 1992b). The formation of the core complex is a highly regulated event in which a protein known as munc18 is involved (Hata et al., 1993; Pevsner et al., 1994). This is a highly soluble protein found in the nerve endings. It is not part of the fusion complex, however it is able to bind syntaxin, and this binding reaction prevents syntaxin from joining the SNARE complex (Misura et al., 2000; Pevsner et al., 1994; Weber et al., 1998). Once munc18 dissociates, a highly stable core complex is formed in such a manner that promotes a condition amenable to synaptic vesicle and plasma membrane fusion. The interaction between synaptobrevin, syntaxin and SNAP-25 is a very tight one. For transmitter release to proceed normally, the SNARE complex must become disassembled. This process is carried out by the binding of NSF along with the αsoluble NSF accessory protein (α-SNAP) (Sollner et al., 1993). The SNARE complex (v- and t- SNARE) mediates the fusion of synaptic vesicles to the plasma membrane resulting in exocytosis. Syntaxin (member of membrane integrated Q-SNARE protein complex participating in exocytosis) and synaptotagmin (Ca 2+ sensor synaptic vesicle protein) have been shown to co-localize with N-type VGCCs (Martin-Moutot et al., 1993) indicating that VGCCs interact with proteins involved in the release apparatus. Direct interaction of N- and P/Q-type VGCCs is known 12 Figure 1.2: Configuration of the interactions of synaptic vesicle and plasma membrane proteins before (a) and after (b) the formation of the SNARE complex. (Adapted from Levitan and Kaczmarek, 1997) Synaptotagmin Synaptobrevin Syntaxin Munc-18 SNAP-25 13 Figure 1.2 (cont’d) (a) (b) Plasma membrane VGCC 14 to occur at the synprint (synaptic protein interaction) site of the α1 subunit, which is 2+ regulated by Ca . This site has been shown to be necessary for the docking of vesicles via SNAREs to the plasma membrane and subsequent vesicular fusion and transmitter release (Sheng et al., 1991), as well as the interaction site with synaptotagmin (Fig. 1.2). Different types of VGCCs show differential subcellular distributions in neurons, with N-type and P/Q-type channels located on presynaptic axon terminals, and L-type channels primarily on the cell body and dendrites (Cao et al., 2004; Gomez-Ospina et al., 2006; Li et al., 2004). There is evidence that assembly with different VGCC β isoforms is a factor in determining the subcellular distribution of VGCC (Wittemann et al., 2000). It is, however, now clear that the VGCC α1 subunit itself is a major determinant of subcellular targeting. Both N- and P/Q-type VGCCs contain a synprint site within the domain II-III linker region that assembles with synaptic proteins such as syntaxin, SNAP25 and synaptotagmin (Jarvis and Zamponi, 2005). This association is seen to couple synaptic vesicles and the source of extracellular calcium, and serves as a regulatory element by which proteins may control VGCCs function (Jarvis and Zamponi, 2005). This region also appears to play a major role in VGCC targeting. In addition, the Cterminal region of the N-type VGCC contains interaction sites for the adaptor proteins Mint-1 and CASK, both of which are expressed at the nerve terminal (Fig. 1.3) (Zamponi, 2003). This suggests that there must be other factors involved in synaptic targeting. Mochida‟s group created chimeric VGCCs and showed that the synprint site is involved in synaptic targeting of P/Q-type VGCCs (Mochida et al., 2003). Additionally, Szabo‟s group, by using naturally occurring domain II-III linker splice variants of human 15 Figure 1.3: Structure of VGCCs. The α1 subunit defines the channel subtype, and is composed of four homologous transmembrane domains, which are connected by large cytoplasmic loops. The domain III linker interacts with the β subunit of the VGCCs through the β-interaction domain (BID). In N- and P/Q-type VGCCS, the II-III linker region contains a synaptic protein interaction site (Synprint), which binds syntaxin, SNAP-25 and synaptotagmin. The Cterminus region of the N-type VGCCs contains scaffolding proteins CASK and Mint-1 (Adapted from Spafford and Zamponi, 2003). BID Synprint Mint-1 16 CASK N-type VGCCs reported that the presence of an intact synprint site was a crucial determinant of synaptic targeting of N-type VGCCs (Szabo et al., 2006). Furthermore, these authors showed that even N-type channels lacking a synprint region still targeted to axonal compartments rather than remaining in the soma. It appears then, that there are at least two important structural elements in N-type VGCC that are involved in synaptic targeting: the synprint site in the domain II-III linker and an adapter protein interaction site in the C-terminus. These two interaction motifs may be involved at different stages during synaptic vesicle targeting, and the presence of both motifs appears necessary for the channel to reach its presynaptic locus. In the case of P/Q-type VGCCs, the synprint site appears to have a more prominent role, but it is possible that there may be other channel regions, or perhaps preferential association with the β4 subunit, that may contribute to synaptic targeting. Along these lines, it is worth noting that R-type channels are also located in presynaptic nerve terminals and participate in neurotransmitter release (Kamp et al., 2005) even though they do not have a synprint region, nor Mint-1 and CASK interaction sites in the C-terminus. The rab3A protein is also involved in the process of membrane trafficking and release. This is a GTP-binding protein that associates with synaptic vesicles (Fischer von Mollard et al., 1990). In the GTP-bound form, rab3A associates with synaptic vesicle and other proteins (Cao et al., 1998; Shirataki et al., 1993). The association of these proteins may act to fuse and target synaptic vesicles to the membrane (Orci et al., 1998). During or after exocytosis, GTP bound to rab3A is hydrolyzed to GDP leading to the removal of rab3A by guanine nucleotide dissociation inhibitor (Ullrich et al., 1993), thus limiting the fusion of synaptic vesicles with the plasma membrane. Regulation of synaptic vesicle 17 exocytosis by Ca as the Ca 2+ 2+ is thought to involve the vesicle protein, synaptotagmin, which acts sensor (Geppert et al., 1994; Li et al., 1995a; Li et al., 1995b). Synaptotagmin 2+ has two Ca binding regions on its cytoplasmic side that are homologous to a region within protein kinase C (Davletov and Südhof, 1993; Perin et al., 1990). It is thought that binding of Ca 2+ induces electrostatic changes of synaptotagmin to facilitate association with syntaxin and phospholipids leading to rapid exocytosis (Shao et al., 1998; Ubach et al., 1998). Once synaptic vesicles fuse with the nerve terminal membrane, ACh is released and diffuses across the synaptic cleft where it binds specifically to post-synaptic ACh receptors localized in the end-plate region of the muscle (Landau, 1978). The nACh receptor is a large pentamer complex arranged in a ring around a central ion pore (Fig. 1.4). There are two α subunits, and a β, a γ, and a δ subunit (Ashcroft, 2000). The ACh binding sites are on the α subunits, and full activation of the channel requires that both sites be occupied. When two ACh molecules bind to an ACh receptor, a conformational change in the receptor opens the central pore and allows cations to flow into or out of the + + muscle cell (Takeuchi, 1963). Since generally more Na flows in than K leaves (due to electrochemical gradients), the muscle cell is depolarized (Boyd and Martin, 1956; Fatt and Katz, 1951). If enough ACh is released, then sufficient ACh receptor channels are activated and the muscle cell membrane is depolarized to threshold for opening voltagegated Na + channels nearby and initiating a muscle action potential. Generally, stimulation of a motoneuron automatically releases enough ACh into the cleft to 18 Figure 1.4: Structure of the nicotinic acetylcholine (nACh) receptor. The nACh receptor contains a β, γ and δ subunits as well as two α subunits which contain the ACh binding site. (Adapted from Levitan and Kaczmarek, 1997). EXTRACELLULAR α ACh γ β δ INTRACELLULAR 19 stimulate the adjacent muscle cell to threshold. Thus, an action potential (with ion fluxes similar to the neuronal action potential) is initiated at the postsynaptic site on the muscle cell membrane. The muscle action potential then activates a cascade of events that leads to muscle contraction. The activation of the ACh receptors comes to an end when the enzyme acetylcholinesterase (AChE) hydrolyzes ACh to acetate and choline (Fig. 1.1) (Gaspersic et al., 1999; Rotundo et al., 1998). The AChE is found in high concentrations in the 4 5 synaptic cleft. Due to its high concentration and fast catalytic rate (10 to 10 substrate molecules hydrolyzed per second), the concentration of ACh in the synaptic cleft drops very quickly following its release. Choline is then taken up into the nerve terminal via a high-affinity sodium-dependent choline up-take system and reused to synthesize new ACh molecules which are then packaged into synaptic vesicles for posterior use (Marchbanks, 1982). C. Voltage – Gated Calcium Channels 1. General Description VGCCs are expressed in the plasma membrane of virtually all excitable cells that transduce electrical activity into intracellular biochemical signals. At resting membrane potential, these channels are normally closed, but they become activated (open) at depolarized membrane potentials. When channels are activated, Ca 2+ enters the cell, acting as a second messenger that plays vital roles in cellular metabolism, excitability, contraction, gene regulation, hormonal and neurotransmitter release, depending on the cell type (Augustine et al., 1987; Miller, 1987). Since Ca 20 2+ plays so many important roles in various physiological functions, a slight alteration of the intracellular Ca 2+ homeostasis will produce a wide array of phenotypes and disorders. The importance of Ca 2+ in cellular functions requires precise spatial and temporal control, and as such, various mechanisms exist to control Ca 2+ levels within the cell. One of these is the gating of VGCCs. The existence of multiple types of VGCCs, each with distinct biophysical properties, distribution, and densities on cell membranes, allows precise control of Ca 2+ entry into the cell. Another important difference between VGCCs is their sensitivity to depolarization. Some channels activate with small depolarizations, and therefore belong to the class of low voltage-activated (LVA) channels. These types of channels usually have rapid, voltage-dependent inactivation. The other class is the high voltage-activated (HVA) channels, which require large depolarizations to activate. These kinds of channels often lack rapid inactivation, and therefore their activity can be recorded in isolation from LVA currents starting from depolarized holding potentials. Later studies, further classified Ca 2+ currents into L, N, P, Q, R, and T based upon their molecular, biochemical, pharmacological, and electrophysiological characteristics (Table 1.1) (Catterall, 2000; Randall and Tsien, 1995; Snutch et al., 1990; Tsien et al., 1988; Zhang et al., 1993). Modern nomenclature refers to them as Cav 1.1-1.4 (L-type), Cav 2.1-2.3 (P/Q-, N- and R-type), and Cav 3.1-3.3 (T-type). The Cav 2 is specifically found in neurons. 21 Figure 1.5: Molecular organization of VGCCs. Subunit composition of HVA VGCCs. The α1 subunit is a transmembrane protein composed of four domains. The α2δ subunit showing the glycosylphosphatidylinositol (GPI) anchor consists of ethanolamine (orange), three mannose rings (blue), glucosamine (pink), and inositol (yellow). The β subunit is located in the cytoplasm, and interacts with the α1 subunit. Some VGCCs may have an additional transmembrane protein, the γ subunit. (Davies et al., 2010). 22 The VGCCs are composed of five subunits: α1, α2, β, δ, and γ. α2 and δ are linked posttranslationally by disulfide bonds into a single subunit referred to as α2δ (Takahashi et al., 1987). L-, N-, P/Q- and R-type channels are made up of α1, α2δ, β, and in some tissues γ subunits (Fig. 1.5). T-type channels, on the other hand, appear to require only an α1 subunit (Perez-Reyes, 2003; 2006). The α1 subunit is responsible for their unique biophysical and pharmacological properties. However, proper trafficking and functioning of L-, N-, P/Q- and R-type channels require the presence of the auxiliary subunits. In particular, the β subunit plays a very important role in trafficking the channels to the plasma membrane, fine-tuning channel gating and regulating channel modulation by other proteins and signaling molecules. 2. The α1 Subunit The α1 subunit is the principal subunit of VGCCs. It is a 190-250 kDa protein containing four homologous repeats (I-IV) connected through cytoplasmic loops (Fig. 1.6). Each repeat has six predicted transmembrane segments (S1-S6) and a reentrant pore forming loop (P-loop) between S5 and S6. The four P-loops form the ion-selectivity filter, where four highly conserved negatively charged amino acids (glutamate or aspartate), one from each P-loop, form a signature locus that is essential for selecting and conducting Ca 2+ (Kim et al., 1993; Kuo and Hess, 1993; Sather and McCleskey, 2003; Yang et al., 1993). The S6 segments form the inner pore (Zhen et al., 2005), and the S4 23 Figure 1.6: Stucture of the α1 subunit. Schematic representation of the predicted transmembrane topology of the α1 subunit, with the localization of the β-interaction domain (BID) marked in red (Adapted from Burgess et al., 1997). EXTRACELLULAR I 1 2 345 II III IV 6 BID NH2 COOH INTRACELLULAR 24 segments‟ positively charged amino acids form part of the voltage sensor. The voltagedependent movement of this sensor results in channel opening and closing. Furthermore, the majority of drug and toxin binding sites are located on the α1 subunit (Catterall, 2000). Thus, the α1 subunit possesses all the key features that define a VGCC, including pharmacological and biophysical properties such as gating, ion selectivity, and permeation. Mammalian α1 subunits are encoded by 10 distinct genes (Table 1.2). Based on amino acid sequence similarity, the α1 subunit is divided into three subfamilies: Cav1, Cav2, and Cav3 (Arikkath and Campbell, 2003; Catterall, 2000; Ertel et al. 2000; Yang and Berggren, 2006). The Cav1 subfamily includes channels that conduct L-type Ca 2+ currents; the Cav2 subfamily includes channels that conduct N-, P/Q-, and R-type Ca currents; and the Cav3 subfamily includes channels that conduct T-type Ca 2+ 2+ currents. The Cav1 subfamily, or L-type, is composed of α1C, α1D, α1F or α1S. They belong to the HVA subfamily of VGCCs. They are located mainly in skeletal, smooth (Almers et al., 1981; Rosenberg et. al., 1986; Sanchez and Stefani, 1978), and cardiac muscle (Reuter, 1983; Tsien, 1983), endocrine cells where they initiate hormone release (Milani et al., 1990), and in neurons where they are important in regulation of gene expression and in integration of synaptic inputs (Bean, 1989; Llinas et al., 1992; Tsien et 2+ al., 1988; Zhang et al., 1993). L-type VGCCs replenish cellular Ca during periods of rapid activity and increase intracellular calcium in response to tetanic stimulation, which 25 Table 1.1: Pharmacological and biophysical properties of VGCC subtypes (Adapted from Hille, 2000; Urbano et al., 2008). HVA HVA LVA L P/Q, N, R T Cav 1.1, 1.2, 1.3, 1.4 Cav 2.1, 2.2, 2.3 Cav 3.1, 3.2, 3.3 α1 subunits α1C, α1D, α1F, α1S α1A, α1B, α1E α1G, α1H, α1I Activation range Positive to -10 mV Positive to -20 mV Positive to -70 mV Inactivation range -60 to -10 mV -120 to -30 mV -100 to -60 mV Very slow ( > 500 ms) Partial ( ~ 50 – 80 ms) Complete ( ~ 20 – 50 ms) Deactivation rate Rapid Slow Rapid Single-channel conductance 25 pS 13 pS 8 pS Single-channel openings Continual reopening Long burst Brief burst, Inactivation Ca 2+ current type Structural nomenclature Inactivation Relative conductance Ba 2+ > Ca 2+ Ba 2+ > Ca 2+ Ba 2+ = Ca 2+ ω-conotoxin GVIA Resistant Cav2.2 sensitive Resistant Dihydropyridines Sensitive Resistant Resistant ω-agatoxin IVA Resistant Calciseptine SNX-482 Divalent block Cav2.1 sensitive Sensitive Resistant Cav2.3 sensitive Resistant Cd 2+ > Ni 2+ 2+ Cd 26 > Ni 2+ Resistant Resistant Resistant Ni 2+ 2+ > Cd leads to increased contractile force. The main physiological role for skeletal muscle VGCCs is to serve as a voltage sensor in excitation-contraction coupling in cardiac, skeletal, and smooth muscles (Bean, 1989). VGCCs in the transverse tubule membranes are thought to interact physically with the calcium release channels located in the sarcoplasmic reticulum membrane. Voltage-driven conformational changes in VGCCs then activate calcium release from the sarcoplasmic reticulum via ryanodine receptormediated stores (Adams and Beam, 1990; Catterall, 1991; Rios and Brum, 1987). L-type VGCCs mediate “long-lasting” currents when Ba 2+ is the current carrier (this characteristic gave rise to the channel‟s name) (Nowycky et al., 1985). With slow voltage-dependent inactivation, they have a large single-channel conductance (~ 25 pS) and a high voltage of activation, and they are specifically inhibited by the organic dihydropyridine compounds (Catterall and Striessnig, 1992; Hofmann et al., 1994). Ltype VGCCs are also antagonized by the peptide toxin, calciseptine, isolated from the black mamba snake, Dendroaspis polylepsis polylepsis, as well as the antihypertensive drugs diltiazem and verapamil. There are several disorders associated with mutations in L-type channels (Table 1.2). The disorder type varies according to which α1 subunit is mutated. Mutations in the human α1C subunit (CACNA1C) result in Timothy‟s Syndrome, a multi-system disorder including syndactyly (a condition in which two or more digits are fused together), immune deficiency and long QT interval (the QT interval represents electrical depolarization and repolarization of the left and right ventricles. A prolonged QT interval is a biomarker for ventricular tachyarrhythmias and a risk factor for sudden death) and 27 Table 1.2: Human and mouse disorders associated with VGCCs subunits (Adapted from Burgess et al., 1999; Mckeown et al., 2006) Subunit Channel type Human gene Associated disorder (mouse and human) α1A P/Q CACNA1A Humans: Episodic ataxia type-2; familial hemiplegic migraine; spinal cerebellar ataxia type-6, sporadic hemiplegic migraine with ataxia and nystagmus. la rol 4J 5J Mice: tg, tg , tg , tg , and tg α1B N CACNA1B α1C L CACNA1C Humans: Timothy syndrome. Mice: knockout is lethal due to cardiac dysfunction. α1D L CACNA1D Humans: Cav 1.3 has been postulated to be related to Parkinson‟s disease, though not necessarily due to a mutation. Mice: congenital deafness. α1E R CACNA1E Humans: no mutations have been associated with this gene. Mice: mutations in this gene affect glucosestimulated insulin release from pancreatic β cells by facilitating the entry of calcium needed for granule replenishment. α1F L CACNA1F Humans: X-linked blindness type 2. α1G T CACNA1G Humans: no mutations have been associated with this gene. Mice: reduced sleep patterns, bradycardia and delayed atriventricular conduction. Humans: Mutations in this gene have not been reported. -/α1B mice: problems in nociception, decrease in sympathetic nervous system function and alterations in response to ethanol and anesthetics. 28 congenital stationary Table 1.2 (cont’d) Subunit Channel type Human gene α1H T CACNA1H Associated disorder (mouse and human) Humans: childhood absence epilepsy, idiopathic generalized epilepsy type 6, autism spectrum disorders. α1I T CACNA1I Humans: No mutations have been associated with this gene. α1S L CACNA1S Humans: hypokalemic periodic paralysis, malignant hyperthermia susceptibility. Mice: Muscular dysgenesis (lethal). n.a. CACNB1 β2 n.a CACNB2 β3 n.a CACNB3 β4 P/Q CACNB4 α2δ1 n.a. CACNA2D1 Humans: malignant hyperthermia α2δ2 n.a. CACNA2D2 Mice: ducky, they seizures and ataxia. α2δ3 n.a. CACNA2D3 α2δ4 n.a. CACNA2D4 γ1 n.a. CACNG1 β1 Humans: episodic ataxia, juvenile myoclonic epilepsy, generalized epilepsy. Mice: lethargic present Humans: retinal cone dystrophy 4 29 spike-wave Table 1.2 (cont’d) Subunit Channel type Human gene Associated disorder (mouse and human) γ2 n.a. CACNG2 Mice: stargazer, they present ataxic gait, paroxysmal dyskinesia, frequent spike–wave discharges, characteristic of absence seizures in humans. γ3 n.a. CACNG3 γ4 n.a. CACNG4 γ5 n.a. CACNG5 γ6 n.a. CACNG6 γ7 n.a. CACNG7 γ8 n.a. CACNG8 30 ventricular arrhythmias during infancy (Splawski et al., 2004; Splawski et al., 2005). Another subunit present in L-type VGCCs is the α1F (CACNA1F) subunit in which several mutations have been identified in patients with incomplete X-linked congenital stationary night blindness (Miyake et al., 1986; Tremblay et al., 1995), as well as in families with X-linked cone dystrophy (Jalkanen et al., 2006). On the other hand, missense mutations in the CACNA1S gene have been associated with human cases of hypokalemic periodic paralysis and malignant hyperthermia susceptibility (Elbaz et al., 1995; Jurkat-Rott et al., 1994; Wang et al., 2005). Using the skeletal muscle α1 subunit as a probe, five additional genes encoding α1 subunits of VGCC have been identified by cDNA cloning and sequencing (Snutch and Reiner, 1992; Soong et al., 1993; Zhang et al., 1993). The α1 subunits fall into two groups based on amino acid sequence similarity, the L-type and the non-L type. The class C and D genes encode the L-type in which sequences are greater than 75% identical to skeletal muscle L-type α1 subunits. The class C gene is mainly present in calcium channels localized in the heart and is widely expressed in other tissues. The class D gene is expressed in neuroendocrine cells and neurons. Class A, B, and E genes encode non-L type channel, expressed primarily in neurons, in which the amino acid sequences are only 25 to 40% identical to the skeletal muscle α subunits. In general, the level of amino acid sequence identity among the α1 subunits is greatest in the transmembrane regions and least in the large intracellular loops connecting domains I, II, and III and in the 31 intracellular amino-terminal and carboxy terminal domains. Most of the α1 subunit genes also encode alternatively spliced segments that increase their molecular diversity. The non-L-type calcium channels (Cav 2.x) contain α1A (which forms part of the P/Q-type), α1B (N-type) or α1E (R-type), all of which are distinct from the L-type VGCC and belong to the Cav2 subfamily. All these channels are present in neurons, and have 2+ intermediate single channel conductances (~ 15 pS) and can mediate Ca currents with varying rates of voltage-dependent inactivation, depending on their subunit composition and other factors. They are best distinguished by their pharmacological properties. P-type Ca 2+ current was initially identified in Purkinje cells, and is blocked at low concentrations by the spider venom ω-agatoxin IVA (Aga-IVA) (Katz et al., 1995; Mintz et al., 1992). An Aga-IVA sensitive current also was found in cerebellar granule cells. However, this Ca 2+ current exhibits properties resembling those described using cloned P-like channel proteins and were named Q-type channels (Randall and Tsien, 1995; Wheeler et al., 1994; Zhang et al., 1993). They are found throughout the nervous system controlling fast neurotransmitter release, mainly at excitatory synapses. Recessive mutations in the α1A subunit in mice lead to the tottering phenotype (Table 1.2) which is characterized by ataxia, polyspike discharges, and intermittent dystonic episodes. Mutations in the tottering locus disrupt the Cacna1a gene, which encodes the α1A subunit of P/Q-type (Doyle et al., 1997; Fletcher et al., 1996). There are la five alleles at the tottering locus, which include: totttering (tg), tottering leaner (tg ), 32 rol tottering rolling Nagoya (tg 4J 5J ), tottering 4 Jackson (tg ), and tottering 5 Jackson (tg ) (Green and Sidman, 1962; Lorenzon et al., 1998; Oda, 1981; Tsuji and Meier, 1971). Mice homozygous for the original allele, tg, exhibit three distinct, recessively inherited neurological phenotypes: epilepsy, ataxia, and episodic (or paroxysomal) dyskinesia. These phenotypes become apparent at 3 weeks of age and continue throughout life. However, it was recently shown that the N- and R- type VGCCs control ACh release at the adult NMJ (Pardo et al., 2006). R-type VGCCs also control ACh release at some enteric synapses (Naidoo et al., 2010). In humans, mutations in the CACNA1A gene give rise to three distinct neurological disorders: episodic ataxia type-2, familial hemiplegic migraine type 1, spinal cerebellar ataxia type 6, and certain cases of absence epilepsy (Bidaud et al., 2006; Jeng et al., 2006; Ophoff et al., 1996; Pietrobon, 2005; Zhuchenko, et al., 1997). Moreover, the α1A subunit is the main target for the attack of autoantibodies of Lambert Eaton myasthenic syndrome (LEMS), an autoimmune disorder. LEMS is a neuromuscular disorder characterized by reduced ACh release, due to autoantibodies directed against VGCCs, mainly the P/Q-type (Flink and Atchison, 2003; Pinto et al., 2002). These disorders illustrate the extensive diversity of phenotype that can arise from alterations in a single VGCC subunit isoform. The R-type (CaV2.3) was observed to be resistant to the action of all the VGCC antagonists, however it was sensitive to SNX-482, a synthetic peptide whose structure was based on a peptide derived from the venom of the tarantula Hysterocarates gigas. This channel is mainly localized in the somata and dendrites of central neurons, as well as nerve terminals of central synapses (Breustedt et al., 2003; Kamp et al., 2005). No human 33 mutations have been identified with Cav2.3 (Table 1.2). However, studies done in knockout mice (Jing et al., 2005) show that it affects glucose-stimulated insulin release from pancreatic β cells by facilitating the global entry of calcium needed for granule replenishment. The N-type VGCCs are specifically inhibited by ω-conotoxin GVIA (Ctx GVIA), which is derived from the Conus geographus snail (Grantham et al., 1994; Hillyard et al., 1992; McCleskey et al., 1987; Tsien et al., 1988).They are also non-selectively inhibited by ω-conotoxin MVIIC (Cntx MVIIC) which is derived from the Conus magus snail. They have intermediate voltage-dependence and rate of inactivation: more negative and faster than L-type, but more positive and slower than T-type (Fox et al., 1987a,b; Nowycky et al., 1985). This gave rise to its name, since its current was neither L- nor Ttype. There are no reports of mutations in the CACN1B gene in the human population (Table 1.2). Studies done in β1B -/- knockout mice show nociception problems (Saegusa et al., 2002), decreased sympathetic nervous system function (Ino et al., 2001) and alterations in response to ethanol (Newton et al., 2004) and anesthetics (Takei et al., 2003). N-type VGCCs have been shown to play a prominent role in neuropathic pain in humans and are the target of a drug derived from the ω-conotoxins such as Ziconotide® (Berecki et al., 2010; Lee et al., 2010; Schmidtko et al., 2010). The T-type VGCCs, belong to the Cav3 subfamily, and are a subtype of LVA class VGCCs. They were designated T-type because of their transient kinetics and tiny conductance. In comparison to L-type, these Ca 2+ currents activate at much more negative membrane potentials, inactivate and deactivate rapidly, have small single 34 channel conductance, and they are insensitive to VGCC antagonists (Carbone and Lux, 1984; Nowycky et al., 1985). People with mutations in the CACNA1H gene can suffer from idiopathic generalized epilepsy, childhood absence epilepsy (Chen et al., 2003; Robinson and Gardiner, 2000) or autism spectrum disorders (Splawski et al., 2006) (Table 1.2). 3. The α2δ Subunit The Cav1 and Cav2 subfamilies contain an auxiliary α2δ subunit (Davies et al., 2007). To date there are four known α2δ subunits (α2δ1 - α2δ4), each encoded by a unique gene and all possessing splice variants. Each α2δ protein is encoded by a single messenger RNA (mRNA) and is cleaved posttranslationally into α2 (extracellular) and δ (transmembrane) subunits. These are then linked by disulfide bonds (Klugbauer et al., 1999; Klugbauer et al., 2003; Qin et al., 2002). The δ peptide, originally presumed to be transmembrane but recently shown to be attached to the membrane through a glycosylphosphatidylinositol linker (Davies et al., 2010), anchors the larger extracellular α2 peptide in place (Fig. 1.5). α2δ subunits can modify channel biophysical properties (Canti et al., 2003; Singer et al., 1991; Wakamori et al., 1994), but their main role is to 2+ increase Ca current (Canti et al., 2003; Davies et al., 2006; Gao et al., 2000; Klugbauer et al., 1999; Klugbauer et al., 2003; Singer et al., 1991; Wakamori et al., 1994) by promoting trafficking of the α1 subunit to the plasma membrane and/or by increasing its retention there (Bernstein and Jones, 2007; Canti et al., 2005; Gurnett et al., 1997; 35 Sandoval et al., 2004). More recently, it was reported that α2δ functioned as a thrombospondin receptor to regulate excitatory synaptogenesis, independently from its regulation of VGCC activity (Eroglu et al., 2009; Kurshan et al., 2009). The ducky phenotype results from a naturally occurring mutation caused by the loss of the α2δ2 protein (Table 1.2). It is characterized by shortened life span, absence epilepsy, spike wave seizures, cerebellar ataxia, decreased whole cell P/Q-type current densities, and decreased Purkinje cell dendritic arborization and firing rates (Barclay et al., 2001; Davies et al., 2007; Donato et al., 2006; Klugbauer et al., 2003). α2δ2 knockouts also have abnormalities in the cardiovascular, immune, respiratory, and nervous systems. Irregularities in the cardiovascular system are also found in α2δ1 knockouts (Fuller-Bicer et al., 2009). α2δ3-null Drosophila are not viable, and the mutants have significantly impaired synaptic transmission (Dickman et al., 2008; Kurshan et al., 2009). Upregulation of α2δ1, on the other hand, is associated with neuropathic pain (Li et al., 2004; Li et al., 2006). In humans, linkage analysis has indicated that mutations in the α2δ1 subunit may play a role in malignant hyperthermia susceptibility, although no mutations have yet been found in the CACNL2A gene that encodes for the α2δ1 subunit (Illes et al., 1994). The α2δ1 subunit is the target of a pharmacological agent (Gabapentin) used to treat intractable pain; the mechanism by which this reduces nociception is unknown (Field et al., 2007; Hendrich et al., 2008; Kusunose et al., 2010; Van Elstraete et al., 2008). 36 4. The γ Subunit There are eight different γ subunit genes (γ1-8), all yielding proteins with four transmembrane segments flanked by cytoplasmic N- and C- termini. This subunit increases the current densities (Yasuda et al., 2004), and alters pharmacological and functional properties of the channels. γ1 was the first cloned γ subunit (Glossmann et al., 1987; Jay et al., 1990; Takahashi et al., 1987) and was copurified with muscle VGCCs, consistent with its primary role as a VGCC subunit. γ1 knockout mice are viable, 2+ morphologically indistinguishable from wt mice, but have larger Ca currents with altered inactivation kinetics (Freise et al., 2000). γ2, γ3, and γ4 also associate with VGCCs (Kang et al., 2001; Sharp et al., 2001). γ1-4 subunits have been shown to produce varying effects on VGCC activity, depending on the partnered α1 and β subunit (Eberst et al., 1997; Freise et al., 2000; Held et al., 2002; Kang et al., 2001; Klugbauer et al., 2000; Letts et al., 1998; Rousset et al., 2001; Singer et al., 1991; Wei et al., 1991). The most consistent effect is a small reduction of current, caused mainly by a hyperpolarizing shift of inactivation voltage and/or a positive shift of activation voltage. The γ2, γ3, γ4, and γ8 regulate the trafficking, localization, and biophysical properties of α-amino-3-hydroxy-5methyl-4-isoxazole propionic acid (AMPA) receptors (Black, 2003; Chen et al., 2007; Kang and Campbell, 2003; Osten and Stern-Bach, 2006; Tomita et al., 2004). They are, therefore, referred to as transmembrane AMPA receptor regulatory proteins (TARPs). Indeed, acting as TARPs seems to be the primary role of γ2, γ3, γ4, γ8, and probably of γ7 37 (Kato et al., 2007). While the function of γ5 remains unknown, γ6 is suggested to inhibit Cav3.1 channels (Lin et al., 2008), and γ7 is involved in the turnover of the mRNA of Cav2.2 and other proteins (Ferron et al., 2008; Moss et al., 2002). Mutations in γ2 underlie the stargazer mouse phenotype (Letts et al., 1998), which includes early onset ataxia, spike-wave seizures, absence epilepsy (Noebels et al., 1990), and defects in the cerebellum and inner ear. 5. The β Subunit Purified Cav1 and Cav2 channels contain a tightly bound cytosolic β protein (Fig. 1.5). There are four subfamiles of β (β1-4), each with splice variants, encoded by four distinct genes, Cacnb 1-4. They all have 14 exons except Cacnb3, which has 13; each β subunit has 2 or more splice variants. The β subunits are abundantly expressed in excitable tissues such as brain, heart, and muscles. The expression of some variants is developmentally regulated. For example, β1b and β4 expression increases with development (McEnery et al., 1998; Pichler et al., 1997; Witcher et al., 1995), whereas β2c, β2d and β2e expression is decreased in adults (Chu et al., 2004; Herzig et al., 2007; Hullin et al., 1992). All four β subunits can dramatically enhance Ca 2+ channel currents when they are coexpressed in heterologous expression systems along with a Cav1 or Cav2 α1 subunit (Lacerda et al., 1991; Mikami et al., 1989; Mori et al., 1991; Pragnell et al., 1994; Shistik 38 et al., 1995; Varadi et al., 1991; Wei et al., 1991; Williams et al., 1992). Since the association between the β subunit and α1 subunit is promiscuous (i.e., any full length β subunit can associate with any Cav1 or Cav2 α1 subunit), alternative splicing greatly increases the molecular diversity and functionality of HVA Ca 2+ channels. β subunits change the voltage-dependence and kinetics of activation and inactivation (Josephson and Varadi, 1996; Lacerda et al., 1991; Mori et al., 1991; Singer et al., 1991; Stea et al., 1993; Stephens et al., 2000; Varadi et al., 1991). However, they do not affect ion permeability (Gollasch et al., 1996; Shistik et al., 1995; Wakamori et al., 1993). The β subunits antagonize endoplasmic reticulum (ER) retention of the α1 subunit. For P/Q-type channels it has been proposed that the α1 subunit may contain multiple ER retention motifs that are masked upon β subunit binding (Geib et al., 2002), thus allowing for cotrafficking of the α1-β subunit protein complex to the plasma membrane. The β subunits have multiple consensus sites for phosphorylation. Co-expression of different β subunits may induce different regulation by protein phosphorylation/dephosphorylation mechanisms. Furthermore, the β subunit either regulates or is indispensable for the modulation of Cav1 and Cav2 channels by protein kinases, G proteins, and small RGK proteins. The β subunit can lower the energy barrier for opening the channel pore and thereby increase peak Ca 2+ currents without increasing the number of VGCCs expressed. In the case of β1 and β2, knockout to these subunits results in the animals being nonviable (Chien et al., 1995; Gregg et al., 1996; Strube et al., 1996; Strube et al., 1998); 39 in the case of β3 and β4 it results in severe phenotype (Burgess el at., 1999; McEnery et al., 1998; Murakami et al., 2003a,b; Namkung et al., 1998). In 1989, Ruth‟s group successfully cloned the first β subunit using a classical approach based on peptide sequences derived from a purified skeletal muscle β subunit (Ruth et al., 1989). This β subunit is now referred to as β1a. Later, using a labeled skeletal muscle β1a cDNA, another group screened a rat brain cDNA library (Pragnell et al., 1991) and cloned a new β subunit, which was a splice variant of β1, and was named β1b (Powers et al., 1992). Using a rat brain cDNA library with β1a, and low-stringency hybridization, β2a was discovered (Perez-Reyes et al., 1992). While using a cardiac cDNA library, β2a, two other splice variants (β2b and β2c) were found, and cDNA for β3 was isolated (Hullin et al., 1992). β3 and β4 subunits were cloned from a rat brain cDNA library, using degenerate primers corresponding to the conserved domains of β1 and β2 (Castellano et al., 1993). All β subunits are expressed in the brain. However, β1 is the only one that is also expressed in skeletal muscle. The importance of this subunit is demonstrated by mutations in the β1 subunit (Table 1.2), which result in the loss of excitation contraction coupling in skeletal muscle, and is lethal (Gregg et al., 1996; Strube et al., 1998). 40 Mutations in the β3 subunit lead to altered kinetics in dorsal root ganglia and sympathetic cervical ganglion neurons (Murakami et al., 2003; Namkung et al., 1998). The β4 subunit, which normally associates with the α1A subunit of the P/Q-type VGCCs, is widely expressed in the brain. It has been reported that spontaneous mutations in this subunit cause several neurological syndromes in mice (Burgess et al, 1997), producing effects on VGCC expression and function. These involve reduction in VGCC targeting, assembly, membrane insertion, channel density and alteration in kinetic parameters, vesicular release and synaptic transmission. Also, the loss of a functional β4 subunit can impact the function of α1A – containing calcium channels. The β4 subunit has the ability to interact with any one of at least four neuronal calcium channel α1 subunits in addition to its preferred partner, α1A. In wt mouse brain, α1A preferentially pairs with β4 subunit. While preferential α1/β pairings (α1 + β4 > β3 >> β1b  β2) are inferred from binding studies of in vitro translated subunits (Liu et al., 1996); coimmunoprecipitation experiments demonstrate that each of the four known β subunits can associate with native L (α1C, α1D), N (α1B) and P/Q-type (α1A), channels in brain (Liu et al., 1996; Pichler et al., 1997; Scott et al., 1996). They are thus likely to regulate more than a single α1 subtype. Other experiments show that heterogeneous α1-β pairings can occur within a presumably homogeneous population of cultures of PC12 cells (Liu et al., 1996). Thus, while mutations in α1 subunit genes affect the function of a single specific channel type, mutation of a β subunit can simultaneously modify multiple 41 channel types, and independently alter Ca 2+ current properties at various sites and developmental stages in the CNS. It has been shown that truncation and missense mutations in CACNB4 gene are associated with families having juvenile myoclonic epilepsy, generalized epilepsy and episodic ataxia (Escayg et al., 2000) (Table 1.2). 5.1 Structure of the β subunit The β subunit has a modular structure consisting of five distinct regions (Birnbaumer et al., 1998; Colecraft et al., 2002; De Waard et al., 1994; Hanlon et al., 1999; Opatowsky et al., 2003; Pragnell et al., 1994). The first, third, and fifth regions are variable in length and amino acid sequence, whereas the second and fourth regions are highly conserved and are more homologous to the Src homology 3 (SH3) and guanylate kinase (GK) domains, respectively. The SH3 domain is a common protein interaction module present in diverse groups of proteins. The GK domain is also engaged in proteinprotein interactions (Elias and Nicoll, 2007; Funke et al., 2005; Takahashi et al., 2004). The middle three regions of the β subunit constitute the β subunit core, which is able to reconstitute many key functions of the β subunit (Chen et al., 2004; Chen et al., 2009; De Waard et al., 1994; Gao et al., 1999; McGee et al., 2004; Opatowsky et al., 2003). Early studies determined that the β subunit binds with high affinity to α1. This high affinity site is located in the cytoplasmic loop connecting the first two homologous repeats (i.e., the III loop) of the α1 subunit and was named the β-interaction domain or BID (Pragnell et al., 1994) (Fig. 1.7). The BID is comprised of 18 residues with a conserved consensus motif 42 Figure 1.7: Structure of the β subunit. This structure reveals the following regions: SH3 domain (Src homology 3), GK domain (guanylate kinase) and the AID (VGCCs α1 subunit interaction domain) and the linker sequence connecting SH3 and GK domains (Vendel et al., 2006). Linker AID SH3 GK COOH 43 in all Cav1 and Cav2 α1 subunits. The BID binds to all four β subunits (De Waard et al., 1995). The affinity of the BID-β interaction ranges from 2 to 54 nM, depending on the BID/β or α1/β pair (Bell et al., 2001; Butcher et al., 2006; Canti et al., 2001; De Waard et al., 1995; De Waard et al., 1996; Geib et al., 2002; Opatowsky et al., 2003; Richards et al., 2004; Scott et al., 1996; Van Petegem et al., 2008). Single mutations of several conserved residues in the BID greatly weaken the BID-β interaction, as indicated by in vitro binding experiments and by the reduction or abolishment of β-induced stimulation of Ca 2+ channel current in heterologous expression systems (Berrou et al., 2002; Berrou et al., 2005; Butcher et al., 2006; De Waard et al., 1996; Gerster et al., 1999; GonzalezGutierrez et al., 2008; Hidalgo et al., 2006; Leroy et al., 2005; Pragnell et al., 1994; Van Petegem et al., 2008). The core of the β subunit contains an SH3 and a GK domain, which are connected by a linker region (Chen et al., 2004; Opatowsky et al., 2004; Van Petegem et al., 2004). The β subunit structures show that β-GK binds tightly to the BID in α1 (Chen et al., 2004; Opatowsky et al., 2004; Van Petegem et al., 2004). The β-SH3 domain has a similar fold as SH3 domains, but its last two β sheets are noncontinuous, separated by the linker region (Chen et al., 2004; Opatowsky et al., 2004; Van Petegem et al., 2004). The β-SH3 domain contains a well preserved proline-rich motif binding site and therefore has the potential to bind proteins that contain the same motif. However in the crystal structures, this binding site is partly shielded by the linker region and a long loop connecting two of the four continuous β sheets. Thus, access to this site requires movement of these two regions. Such conformational changes are 44 conceivable when the β subunit is bound to the α1 subunit. Variation among the different β subunits comes from the fact that the linker region, as well as the NH2 and COOH termini of the β subunit are highly variable in length and amino acid composition among the β subfamilies. In the β subunit there is a 31 amino acid segment that is referred to as the αinteraction domain (AID), which is the main binding site for the BID of the α1 subunit (De Waard et al., 1994). The AID was able to enhance slightly Ca 2+ current amplitude and modulate gating. Several AID point mutations were able to weaken the β/α1 interaction and reduce AID-stimulated Ca 2+ currents (De Waard et al., 1994; De Waard et al., 1996). However it was later found that the BID does not bind to the AID as was originally thought (Chen et al., 2004; Opatowsky et al., 2004; Van Petegem et al., 2004). Instead, the BID binds to a hydrophobic groove in the GK domain termed the AIDbinding pocket (ABP) (Chen et al., 2004; Van Petegem et al., 2004, Van Petegem et al., 2008). The AID occupies only a tiny fraction of the β subunit‟s surface area, raising the possibility that other domains of the β subunit are involved in interactions with other regions of the α1 subunit or with other proteins. The BID-GK domain interactions are extensive and predominantly hydrophobic. These interactions account for the affinity measured in the BID-β binding. Functional studies show that mutating two or more key residues in the ABP severely weakens or completely abolishes the BID-β interaction (He et al., 2007; Zhang et al., 2008). 45 The binding of the BID with β does not significantly alter the β subunit‟s structure, except for some small and localized changes near the ABP. Importantly, however, the BID undergoes a dramatic change in secondary structure when it is engulfed by the ABP. When alone, the BID exists as a random coil in solution, as determined by circular dichroism spectrum measurements (Opatowsky et al., 2004). When bound to the β subunit, the BID forms a continuous α-helix. Together with the observation that the 22amino acid linker between the BID and the first S6 segment of α1 (i.e. IS6) also forms an α-helix (Arias et al., 2005), a picture emerges that the entire region encompassing IS6 and the BID adopts a continuous α-helical structure in the presence of the β subunit. This structural hallmark is crucial for the regulation of calcium channel gating by the β subunit. D. The Lethargic (lh) Mutation The lethargic locus encodes the VGCCs β4 subunit, and is the first example of a mammalian neurological disease caused by an inherited defect in a non-pore forming subunit of a VGCCs (Burgess et al., 1997). The lh mutation is caused by a four-base pair insertion into a splice donor site within the Cacnb4 gene on mouse chromosome 2 that produces two abnormally spliced mutant mRNA isoforms. Both of these result in translational frameshift and a premature stop codon. The predicted β4 protein is truncated - missing 60% of the C-terminal of the β4 protein, including the AID (Fig. 1.8) (Burgess et al., 1997). The predicted β4 protein is likely to be completely non-functional, and the 2J lh mutation can thus be considered a β4 null mutant. A second allele, lh , mutated in the 46 promoter region, appears to eliminate β4 mRNA expression and results in a mutant phenotype identical to that seen in tottering, including spike-wave epilepsy, ataxia, and paroxysmal dyskinesia. The onset of ataxia is 2 weeks after birth. In addition to the neurological disorders, lethargic homozygotes show a slightly reduced body weight and transient defects in nonneuronal tissues including the immune system, splenic and thymic involution, and renal cysts (Dung, 1977; Dung and Swigart 1971, 1972). Electrophysiologically and pharmacologically, these seizures are similar to the absence seizures present in the human petit mal epilepsy and to those present in tg mice. Neuropathological observations in the lethargic mouse brain show reduction in the size of the cerebellar molecular layer. Regional increases in the levels of GABAB receptor binding have been reported (Lin et al., 1993), providing an example of the downstream responses that may occur in a brain attempting to compensate for a persistent physiological abnormality. Additionally, T-type VGCC upregulation (by ~50%) in thalamic neurons of lh mice has been reported, and hypothesized that they might contribute to the seizures (Zhang et al., 2002). These mice also present lower N-type channel expression in the forebrain and cerebellum (McEnery et al., 1998), reduced excitatory neurotransmission in the thalamus (Caddick et al., 1999). E. Regulation of Calcium Channels By Small G Proteins RGK is a family of small GTP-binding proteins, which can regulate VGCCs‟ function and surface expression. RGK has four members – Rad, Gem (and its mouse homolog Kir), Rem, and Rem2, all of which have a conserved Ras GTPase-like core (Kelly, 2005). RGK proteins present the following characteristics: (1) they have low 47 Figure 1.8: Interaction of VGCCs α1 subunit and β subunit. The binding site for the α1 I-II cytoplasmic loop is indicated by the orange segment of the β subunit. The red arrow indicates the relative position of the 4 base-pair insertion in the intron, resulting in exon skipping, translational frameshift and posterior protein truncation of the lh Cchb4 proteins (Adapted from Burgess et al., 1997). EXTRACELLULAR α1 1 2 3 4 5 6 1 234 5 6 1 2 34 5 6 1234 5 6 COOH β INTRACELLULAR NH2 COOH 48 intrinsic GTPase activity, secondary at least in part to modifications of key amino acids involved in GTP binding and hydrolysis in Ras GTPases (Chen et al., 2005; Kelly, 2005), (2) they have an extended N-terminal with a 14-3-3 binding domain and an extended C2+ terminal domain that contains binding sites for 14-3-3 and Ca /calmodulin (Béguin et al., 2005; Béguin et al., 2006; Kelly, 2005). All members of the RGK family have the ability to interact with the β subunit. Rem2, however, has a unique characteristic in that it is the only member of the RGK family expressed in high levels in the CNS (Finlin et al., 2006). Another member of this family, Gem, was shown to inhibit potently the trafficking of Cav1.2 channels to the cell surface in HEK293 cells (Béguin et al., 2001), a finding subsequently demonstrated for the regulation of calcium channels by Rem2 (Béguin et al., 2005) as well as Rad and Rem (Béguin et al., 2006). It was proposed that the subcellular distribution of all the members of the RGK family is regulated by 14-3-3 proteins and calmodulin (Béguin et al., 2001; Béguin et al., 2005; Béguin et al., 2006). Hence, the abilities of RGK proteins to sequester the β subunit within the ER are under dynamic control, thus allowing 14-3-3 proteins and calmodulin to regulate indirectly the trafficking of the α1 subunits, and thus their membrane expression. In addition to their role in trafficking, it has been shown that RGKs have the ability to potently inhibit high-voltage Ca 2+ currents. Exogenous overexpression of RGK proteins in heterologous expression systems and neurons consistently demonstrates almost complete inhibition of VGCC currents (Béguin et al., 2005; Chen et al., 2005; Finlin et al., 2003), with further experiments in Xenopus oocytes demonstrating that this may be a concentration-dependent effect (Seu and Pitt, 2006). It was recently shown that 49 the VGCC subunit β2a, within its highly conserved GK domain, contains separate sites for interacting with the α1 subunit (via the AID domain) and Rem (and even maybe with other members of the RGK family) (Finlin et al., 2006). Furthermore, the β subunit can simultaneously bind both the BID motif of the α1 subunit and Rem. Moreover, it was recently shown that a β subunit is required for Gem and Rem2 inhibition of Cav1.2 (Seu and Pitt, 2006). This suggests that the RGK regulation of VGCCs function is not due to a physical uncoupling between the α1 and β subunits, but may instead be due to an alteration of the functional properties of α1/β subunit complexes. In support of such a mechanism, electrophysiological studies (Chen et al., 2005) suggest that Rem2 interaction with the N-type calcium channel results in a non-conducting β subunit containing complex. Taken together, these data suggest that in some cases, RGKs have two roles in regulating VGCCs activity: they alter membrane trafficking (independently, and via calmodulin and 14-3-3), and they directly inhibit membrane-associated channel complexes. G. Specific aims In wt mice ACh release is primarily controlled by P/Q-type VGCCs in which the α1A subunit normally coassociates with the β4 subunit. The interaction of the correct β subunit with its corresponding α1 subunit is essential for proper targeting, membrane insertion, channel density, channel kinetics and interaction with vesicular release proteins (Murakami et al., 2003; Wittemann et al., 2000). 50 lh mice have a naturally occurring mutation in which there is a four base pair insertion into a splice donor site within the Cacnb4 gene on mouse chromosome 2. This mutation results in exon skipping, translational frameshift, and protein truncation missing 60% of the C-terminal relative to wt, including the loss of the AID site, suggesting that this defect in the VGCC assembly might be the cause of the pathogenesis in the lh phenotype (Burgess et al., 1997; Burgess and Noebels, 1999). This phenotype is characterized by ataxia, lethargic behavior, spike-wave epilepsy, and paraxysomal dyskinesia. The onset of ataxia is two weeks after birth. Additionally, lh mice show reduced body weight and immunological problems when compared with unaffected litter mates (Sidman et al., 1965); only 20% of homozygous lh mice survive after the weaning period. β4 mainly coassociates with α1A subunit of the neuronal P/Q-type VGCC (McEnery et al., 1998), therefore it is not a surprise that lh mice have a similar phenotype to tg mice which carry a mutation in the α1A subunit. Hence, it is normal to speculate that the β4 mutation present in lh mice is having an effect on α1A subunit of the neuronal P/Qtype VGCC. The overall goal of this dissertation is to characterize the lh mutation. How does the absence of the β4 subunit affect the ACh release, and which compensatory mechanisms (if any) are present in lh mice to compensate for this mutation in these animals? To answer these questions, I propose to examine the following specific aims: 51 Specific Aim 1: VGCCs are involved, among other functions, in controlling ACh release from motor nerve terminals. The lh mutation disrupts the normal conformation of the neuronal P/Q-type VGCC, in which the β4 subunit is absent in animals with this mutation. Chapter 2 deals with determining how ACh release at motor nerve terminals is affected by the alteration of the normal complement of calcium channel β subunit. For this purpose intracellular recordings at the neuromuscular junction of adult lh and wt mice were 2+ obtained. Sr since the Ca 2+ and Ba 2+ 2+ 2+ and Ba in asynchronous spontaneous release selectivity filter of α1 of VGCCs allows for the binding of these 3 divalent cations. Moreover, the Ca Sr 2+ can substitute for Ca 2+ permeability pore is more permeable to Sr 2+ 2+ and Ba , and 2+ have a higher frequency of spontaneous release as compared to Ca . Therefore if this mutation was affecting in a subtle way certain characteristic of the channel, using cations that are more sensitive than Ca 2+ would allow me to determine possible changes in the channel‟s characteristics. Therefore, I measured spontaneous 2+ 2+ release (MEPPs) of ACh in the presence of different divalent cations (Ca , Sr or 2+ Ba ) and evaluated whether this mutation alters in any way the frequency or amplitude of spontaneous release. I also measured nerve evoked release (EPP) of ACh (with the buffer containing either Ca 2+ or Sr 2+ as the charge carrier) and examined whether this mutation altered the EPP amplitude or the quantal content. To have a further understanding on how this mutation might affect ACh release I measured the kinetics of 52 the synaptic vesicle release by means of FM1-43 fluorescence. Destaining, as a measure of synaptic vesicle release, was induced by high KCl (40 mM KCl), and the destaining process was measured. Neurotransmitter release can be induced by high KCl or by nerve stimulation. A high KCl solution induces massive asynchronous exocytosis, while synchronous exocytosis can be induced by stimulation of the motor nerve. This allowed me to evaluate whether this mutation affected the synaptic vesicle kinetics. Additionally, ACh release can be induced in the absence of Ca 2+ by either high osmolarity conditions (Palma et al., 2011; Sons and Plomp, 2006) or in the presence of α-latrotoxin (α-LTx) (Hubbard et al., 1968; Sons and Plomp, 2006; Rosenmund and Stevens, 1996; Rossetto et al., 2004; Tedesco et al., 2009; Xu et al., 2002). These sets of experiments allowed me to 2+ evaluate the kinetics of release in the absence of Ca . Specific Aim 2: Chapter 3 determined whether the P/Q-type calcium channel is expressed at motor nerve terminals of lh mice. As a first step, I tried to assay for protein level in diaphragm, however, due to the scarcity of VGCCs proteins present in this tissue and the poor sensitivity of western blots I was unable to probe against any subunit (no bands were revealed in these blots). Therefore, to answer this question I performed western blots on proteins isolated from the cerebellum of lh and wt mice and probed for the different β subunits as well as α1A. Since β4 is normally coassociated with α1A, then it is plausible that a mutation affecting β4 might alter the levels of α1A. Additionally this allowed me to evaluate whether the protein level of any other β subunit was altered in response to this mutation. I also performed intracellular recordings in wt and lh adult mice in the presence 53 of ω-agatoxin IVA, a specific antagonist of the P/Q-type calcium channel, to determine whether the P/Q-type calcium channel controls ACh release in lh mice, and if so, whether its involvement in ACh release is to the same extent as it is in wt mice. All these results were corroborated with immunohistochemistry assays to determine the localization of α1A and the possible determination of which β subunit(s) is present at the NMJ when α1A is present. Specific Aim 3: Chapter 3 deals with determining if there are other types of VGCCs contributing to the control of the release of ACh at motor nerve terminals of adult lh mice. To answer this question I performed intracellular recordings in wt and lh adult mice in the presence of specific antagonist for the different types of calcium channels (ω-conotoxin GVIA which is a specific antagonists of N-type, nimodipine to antagonize L-type and SNX-482 to block R-type calcium channels). This allowed me to evaluate the extent of the involvement of the different calcium channels in the control of ACh release. Moreover, I performed immunohistochemistry assays to determine the localization of the different α1 subunits and the possible determination of which β subunit is present at the NMJ. 54 CHAPTER 2 CHARACTERIZATION OF NEUROMUSCULAR TRANSMISSION IN LETHARGIC (lh) MICE 55 Abstract: Voltage gated calcium channels (VGCCs) are heteromultimeric complexes composed of α1, β, and α2δ subunits. The β subunits have a role in directing the VGCCs complexes to the plasma membrane as well as regulating its channel properties. The α1 subunits make up the selective pore for Ca 2+ and determine most of the subtype-specific attributes of VGCCs. A mutation in the β4 subunit of the P/Q-type (Cav2.1) calcium channel present in lethargic (lh) homozygous mice causes ataxia and lethargic behavior at 15 days of age. To determine how acetylcholine (ACh) released at motor nerve terminals is affected by this mutation, electrophysiological recordings at neuromuscular junctions (NMJs) were compared from adult lh mice with wild type (wt). Different physiological saline solutions were used for the recordings and the quantal content of the lh mice was compared to that of controls using CaCl2 (2, 4, 8 mM), and SrCl2 (2, 4 mM). lh responses were reduced by: 72%, 59%, 32%, 41% and 50%, respectively. The spontaneous release of ACh, measured as the frequency of occurrence of miniature end plate potentials (MEPPs) remained unchanged for the various Ca 2+ concentrations, but a 54% and 52% reduction was observed when using 2 mM and 4 mM SrCl2, respectively. A 56% and 57% reduction in the spontaneous release of ACh was observed when using BaCl2 (0.5, 1 mM) respectively, in the lh mice as compared to control animals. The reduction in the ACh level could be due to a slower process of release of the neurotransmitter vesicles. To determine the vesicle dynamic process, we used the FM1- 56 43 staining method. We determined that the vesicle dynamic process is severely affected by the lh mutation. 3.5 minutes of external stimulation by high KCl (40 mM) in wt mice caused an almost complete destain of the FM1-43 fluorescence. In lh mice under the same conditions after the same amount of time 38% of stain remained. This shows that the vesicle release process is significantly slower in lh mice as compared to wt. Inasmuch as the β4 subunit is normally associated with the α1A at mammalian NMJs, its disruption in lh mice might be anticipated to result in aberrant neuromuscular transmission. 57 Introduction: Voltage gated calcium channels (VGCCs) are formed by α1, β, and α2δ subunits, and some times γ (Tsien et al., 1991), where the α1 subunits make up the selective pore for Ca 2+ and determine most of the subtype-specific attributes of the channel. The α1 subunit contains binding sites for various pharmacological agents as well as the gating regions of the channel (Catterall et al., 2005; Zhang et al., 1993). The cytoplasmic auxiliary β subunit regulates the assembly and membrane localization of the α1 subunits, enhances Ca 2+ currents and modifies the voltage-dependence and kinetics of activation and inactivation (Lacerda et al., 1991; Stea et al., 1993). It also has antagonistic effects on Ca 2+ currents by regulating different aspects of channel function. The β-subunit contains a guanylate kinase (β-GK) and a Src homology 3 (β-SH3) domains. While β-GK binds to a conserved site within the α1-pore-forming subunit and facilitates channel opening, βSH3 also promotes endocytosis by binding to dynamin, a GTPase responsible for endocytosis. Channel activation and internalization are two mutually exclusive functions of the β subunit (Miranda-Laferte et al., 2011). There are four different types of β subunit (β1-4), in which the β4 subunit normally coassociates with the α1A subunit of the P/Q-type VGCC. However, there is a naturally occurring mutation of the β4 subunit in the P/Q-type (Cav2.1) VGCCs present in lethargic (lh) mice, which causes a series of neurological signs that mimic certain 58 human neurological diseases. Mice homozygous for lh mutation exhibit ataxia, wobbly gait, smaller body size, and lethargic behavior beginning at 15 days of age (Burgess et al., 1997; Burgess and Noebels, 1999). In neurons, the primary role of VGCCs is to mediate transmitter release from nerve terminals both in the central and peripheral nervous system by conducting presynaptic Ca 2+ et al., 1992). Ca influx required for neurotransmitter release (Mintz et al., 1995; Uchitel 2+ is vital for many processes, such as acting as second messengers that play crucial roles in cellular metabolism, excitability, contraction, gene regulation, vesicle cycle, hormone and neurotransmitter release (Augustine et al., 1987; Catterall, 1995; Katz and Miledi, 1970; Llinas et al., 1976; Miller, 1987). The synaptic vesicle cycle allows these vesicles to be used repeatedly in the processes releasing neurotransmitters from chemical synapses at nerve endings. The cycle includes several stages: exocytosis (fusion of vesicles with the presynaptic membrane and secretion of a quantum of neurotransmitter into the synaptic cleft), endocytosis (formation of a new vesicle), and intracellular transport of synaptic vesicles (Zefirov, 2007; Zefirov et al., 2006). The rate of the vesicle cycle provides for efficient secretion of neurotransmitter during prolonged high-frequency synapse activity. Ca 2+ plays an important role in the mechanisms of the vesicle cycle. In natural conditions, the influx of Ca 2+ through VGCCs in the active zones of motor nerve terminals triggers exocytosis (Chapman, 2008; Südhof, 2004). Ca 2+ has both inducing and inhibiting roles in the processes of vesicle recycling (Zefirov, 2007). 59 2+ The process of vesicle fusion involves different Ca -binding sites, whose activation triggers different types of neurotransmitter secretion: synchronous, phasic (lasting a few milliseconds after the presynaptic action potential), and spontaneous asynchronous (Van Der Kloot and Molgó, 1994), which facilitates neurotransmitter secretion in conditions of high-frequency activity (Zefirov and Mukhamed‟yarov, 2004). 2+ These sites differ in terms of their locations, affinities for Ca , and sensitivities to other divalent cations, such as Sr 2+ Mukhamed‟yarov, 2004). The Ca Sr 2+ 2+ and Ba 2+ (Van Der Kloot and Molgó, 1994; Zefirov and binding site for asynchronous release is sensitive to 2+ and Ba , while the synchronous exocytosis is sensitive to Sr 2+ only. The β4 subunit normally co-associates with the α1A subunit of the neuronal P/Qtype VGCC (McEnery et al., 1998). Therefore, it is not a surprise that lh mice have a similar phenotype to tottering (tg) mice which carry a mutation in the α1A subunit. The main role of P/Q-type VGCC is to mediate neurotransmitter release from nerve terminals. Hence, it is normal to speculate that the β4 mutation present in lh mice is having an effect on α1A subunit of the neuronal P/Q-type VGCC, which might be reflected in an alteration in ACh release from motor nerve terminals. In lh mice the normal complement of the α1A subunit is missing, therefore, we wanted to determine how this alteration of the normal complement of the P/Q-type VGCCs affected neurotransmitter release at motor nerve terminals. To test this, we recorded miniature end plate potentials (MEPPs) and end plate potentials (EPPs) from control and lh NMJ to calculate the quantal content of 60 acetylcholine (ACh). Additionally, we tested whether this mutation affected the kinetics of release of synaptic vesicles. Vesicular release is regulated by the Ca 2+ influx through VGCCs. Therefore, any alteration in the normal complement of VGCCs could alter the characteristics of vesicular release. 61 Materials and Methods: Mice. Breeding pairs of heterozygous Cacnb4lh4J mice were obtained from Jackson Laboratory (Bar Harbor, ME) and subsequently maintained in a breeding colony at Michigan State University Laboratory Animal Resources. Litters were genotyped at weaning, 3 weeks after birth. lh (homozygous) mice were also identified by their characteristic phenotype consisting of a mild ataxia, wobbly gait behavior, and smaller body size. Adult mice between 3 to 9 months of age were used for all the experiments. All experiments were performed in accordance with local university (Michigan State University Laboratory Animal Resources) and national guidelines (National Institutes of Health of the USA - NIH) and were approved by the University Animal Use and Care Committee. Electrophysiology. Animals were sacrificed by decapitation following anesthesia (80% O2 + 20% CO2). The diaphragm with its attached phrenic nerve was removed from each animal and pinned in a Sylgard-coated chamber. The tissue was superfused continuously with 100% oxygenated physiological saline solution (137.5 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 11 mM d-glucose, and 4 mM HEPES, adjusted to pH 7.4). MEPPs and EPPs were recorded from hemidiaphragms of lh and wt mice using intracellular microelectrodes (made from 1.0 mm o.d. glass capillaries; WPI, Sarasota, FL) with a resistance of 5 – 15 M when filled with 3 mM KCl. Electrodes were localized at the end plate. EPPs were elicited by phrenic nerve stimulation at 0.5 Hz by means of a suction electrode attached to a stimulus isolation unit (Grass SIU5; Grass Instruments, Quincy, 62 MA) and stimulator (Grass S88). Signals were amplified using an Axoclamp-2 amplifier (Molecular Devices, Sunnyvale, CA), digitized using a PC-type computer and Axoscope 9.0 software (Molecular Devices, Sunnyvale, CA), and analyzed using MiniAnalysis 6.0 Software (Synaptosoft, Decatur, GA). Recordings were obtained from 5-10 end-plates for a period of 5 mins each for each mouse. This is important since there can be burst in MEPP frequency, giving rise to “clusters” of MEPPs (Fatt and Katz, 1952; Kriebel and Stolper, 1975; Vautrin and Kriebel, 1991). If the recordings are done over short periods of time these bursts of MEPPs could lead to erroneous interpretation of MEPP frequency. Sampling over a larger period of time, gives a better representation of what could naturally be occurring. Muscle action potentials were inhibited by addition of 2.5 μM of μ-conotoxin GIIIB (Alomone Labs, Jerusalem, Israel). The amplitude of MEPPs and EPPs was normalized to a membrane potential of -75 mV using the following formula: Vc = [Vo x (-75)]/E, where Vc is the corrected amplitude of MEPPs and EPPs, Vo is the actual recorded amplitude of MEPPs and EPPs and E is the membrane potential at which the recording was made (Magleby and Stevens, 1972; McLachlan and Martin, 1981). A recording was rejected if the 10-90% rise time exceeded 1.5 ms, and/or, if the membrane potential was more depolarized than -55 mV. The quantal content (m) was calculated using the corrected mean amplitude values of EPPs and MEPPs, where m = EPP/MEPP (Hubbard et al., 1969). We also tested ACh release in response to different divalent cations, and different concentrations of each of them. In some experiments Ca 8 mM) was substituted for Sr 2+ (2 or 4 mM) or Ba 2+ (2, 4 or (0.5 or 1 mM). Separate preparations were used for each cation and concentration tested. 63 2+ FM1-43 fluorescence. The thin, almost translucent trangularis sterni (TS) muscle preparation was chosen for these experiments because FM1-43 loads more efficiently and it can be observed more clearly. For double labeling of ACh receptors and terminals, preparations were sequentially exposed to 4 μg/ml rhodamine-conjugated α-bungarotoxin (Sigma, St. Louis, MO). The muscle was exposed to α-bungarotoxin (1:200 in physiological saline) for 20 mins, washed for 20 mins, then exposed to 8 μM FM 1-43 (Invitrogen, Carlsbad, CA) (Betz and Bewick, 1992; Betz et al., 1992; Ribchester et al., + 1994) in 30 mM K physiological saline solution for 3 mins, and then washed with 5 mM + K physiological saline for 20 – 40 mins. At least 2 terminals were examined per animal used. Destaining of the terminal was induced by perfusion of the preparation with a solution containing 40 mM KCl and 2.5 μM μ-conotoxin GIIIB. Images were obtained every 10 secs. The tissue was examined using a rhodamine filter module (excitation at 530 nm and emission of 590 nm) on a Nikon Optiphot-2 (Nikon Instruments, Tokyo, Japan) microscope equipped with a 40X water immersion objective. For observation of FM1-43, the FITC excitation filter module was used (excitation at 460 nm, emission at 520 nm). To reduce the possibility of photobleaching and phototoxicity, illumination was kept at a minimum. Images were acquired using a 40 ms exposure time. Images were captured using an Andor Ixon+ camera (Andor Technology, South Windsor, CT), connected to a PC type computer with Till Photonics software (TILL Photonics GmbH, Munich, Germany). To calculate the destaining process, the average brightness of the entire terminal was measured relative to the background. Averages of the mean values of fluorescence obtained over the different time points from all the nerve terminals sampled 64 were calculated and compared between the lh and wt preparations using Metamorph software (Molecular Devices, Sunnyvale, CA). ACh release can be induced in the absence of Ca 2+ by either hypertonic conditions (Palma et al., 2011; Sons and Plomp, 2006) or in the presence of α-latrotoxin (α-LTx) (Alomone), which induces a massive release of neurotransmitter (Hubbard et al., 1968; Rosenmund and Stevens, 1996; Rossetto et al., 2004; Sons and Plomp, 2006; Tedesco et al., 2009; Xu et al., 2002). Therefore to evaluate the kinetics of transmitter 2+ release in the absence of Ca , we induced exocytosis by high osmolarity solution (500 mM) and in the presence of α-LTx. α-LTX in its tetrameric form interacts with receptors (neurexins and latrophilins) on the neuronal membrane, which leads to insertion of α-LTX into the membrane. The toxin forms pores in the lipid membrane which are permeable to Ca2+. This influx stimulates synaptic vesicle exocytosis. The pore is also permeable to neurotransmitters which causes massive depletion of the neurotransmitter pool in the cytoplasm (Ushkaryov et al., 2004). At nM concentrations of α-LTx, bursts of neurotransmitter release occur, followed by prolonged periods of steady-state release (Henkel and Sankaranarayanan, 1999; Ushkaryov, et al., 2008). Hypertonic solutions induce spontaneous release of ACh from the motor nerve terminal, as reflected by an increase in MEPP frequency. The effects of osmolarity changes are not produced by changes in nerve terminal polarization (Hubbard et al., 1968). The high osmotic pressure induces dehydration of the nerve terminals, which may decrease a hydration barrier preventing synaptic vesicles from making contact with the 65 nerve terminal membrane and so discharging their contents (Bass and Moore, 1966).The increase in MEPP frequency maybe due to the outward flow of water through the nerve terminal (Hubbard et al., 1968) Statistical Analysis. Differences between genotypes were analyzed using a one way analysis of variance followed by Tukey‟s test. Differences between concentration response were analyzed using a two way analysis of variance followed by Tukey‟s test. P values were set to < 0.05 for all tests. Measurements are expressed as means ± S.E.M for n ≥ 7. 66 Results: Spontaneous ACh release To test whether the lh mutation affected spontaneous (asynchronous) release of ACh, we performed intracellular recordings from hemidiaphragms of wt and lh mice in saline solution containing Ca 2+ (2, 4 or 8 mM) in. A change in the Ca 2+ concentration when performing spontaneous release of ACh, translates in an increase on the frequency of occurrence. Additionally an increase in Ca 2+ concentration, leads to an increase in vesicle release, which in turn increases recycling. Therefore if this mutation affected the frequency of release, it would be easier to visualize using a higher extracellular Ca 2+ concentration, which would induce a faster frequency of release. We measured the amplitude and frequency of MEPPs (Fig. 2.1a and b). There was no difference in the amplitude or frequency of the spontaneous release of ACh between lh and wt mice using Ca 2+ as a charge carrier. Sr 2+ 2+ and Ba can substitute for Ca 2+ in the generation of MEPPs (Anwyl et al., 1982; Elmqvist and Feldman, 1965; Mellow et al, 1982; Silinsky, 1978). Spontaneous exocytosis involves the activation of a low-threshold exocytosis with a calcium microdomain (Zefirov and Grigor‟ev, 2008). This site is not selective and can be 2+ 2+ activated by Ca , Sr and Ba 2+ (Zefirov and Grigor‟ev, 2010). The most likely molecular candidate for this site is synaptagmin III (Chapman, 2008; Sudhof, 2004) which is a high-affinity isoform which has been shown to be able to bind to these divalent 67 cations (Fukuda et al., 1997; Sudhof, 2004). Using different divalent cations we could determine if characteristics of the channel were altered by this mutation, since they all 2+ 2+ 2+ have different mobility through the channel (relative conductance: Ba > Sr > Ca ) 2+ (Hagiwara and Ohmori, 1982). As such, we substituted Ca for Ba 2+ or Sr 2+ in lh animals to determine if the release process responded differentially to these ions (Fig. 2.2a and b). The amplitude of MEPPs was not altered for either lh or wt mice by substituting Ba 2+ 2+ or Sr 2+ for Ca . This implies that there is no difference in the amount of ACh each synaptic vesicle carries. However, MEPP frequency was significantly decreased in lh mice when used Ba 2+ or Sr 2+ as charge carriers. Nerve evoked ACh release We wanted to evaluate whether this mutation also affected the nerve-evoked release of ACh. For this purpose we performed intracellular recordings in saline solution containing Ca 2+ (2, 4 or 8 mM) in hemidiaphragms of wt and lh mice, and measured the amplitude of EPPs evoked by nerve stimulation (Fig. 2.3). When inducing evoked release 2+ of ACh, the amplitude of the EPPs depends on the extracellular concentration of Ca , meaning the higher the concentration of Ca 2+ the greater the EPP amplitudes, since the change in concentration is increasing release. We observed that regardless of the concentration of Ca 2+ we were using in the physiological saline solution, we still had a significant decrease in the amplitude of EPPs in lh mice as compared to wt. Using the amplitude of EPPs and the amplitude of MEPPs, we calculated the quantal content of the 68 mice (Fig. 2.4). We observed that the quantal content in lh mice was significantly decreased as compared to wt. Strontium is capable of supporting synaptic transmission. The mechanism of phasic synchronous exocytosis includes activation of a high-threshold site mediated by the calcium microdomain close to an open VGCC (Neher, 1998; Stanley, 1993). The site for phasic exocytosis is only sensitive to Ca 2+ Sr , and it is insensitive to Ba 2+ 2+ 2+ and Sr , although it has lesser affinity for (Zefirov and Grigor‟ev, 2010). This site consists of the low-affinity isoform synaptotagmin I (Sudhof, 2004). When Sr 2+ is used for synaptic 2+ transmission replacing Ca , peak release is reduced and the duration of release is prolonged. When we performed the recordings in the presence of Sr 2+ as a charge carrier, we saw that the amplitude of EPPs (Fig. 2.5) as well as the quantal content (Fig. 2.6) were also significantly decreased in lh mice. These results show that the lh mutation severely affects ACh release from adult motor nerve terminals. FM1-43 Based on these results, we wanted to examine whether the vesicular dynamics were affected by the absence of the β4 subunit in lh mice, which could explain the decrease in ACh release. These experiments are designed to investigate how the lh mutation affects the availability of quanta release, the exocytosis of vesicles and their recycling. Synaptic vesicle exocytosis, and internal vesicular processing were examined 69 using the motor nerve terminals in the triangularis sterni (TS) muscle of wt and lh mice. ACh depletion was achieved by massive asynchronous exocytosis induced by high KCl (40 mM KCl) solution. Then, the tissue was incubated with the fluorescent dye FM1-43, which was taken up into synaptic vesicles during the endocytic re-uptake of released vesicles. This technique can be used to estimate synaptic vesicle pool size and monitor synaptic vesicle dynamics (Betz and Bewick, 1993; Reid et al., 1999). We visualized the FM1-43 labeling dynamics observing the amount of decrease in FM1-43 fluorescence in the presynaptic terminals after KCl-induced depolarization of the nerve terminals. This translates to transmitter progressively released in real time. After 3.5 mins, most of the fluorescence was lost from wt NMJs, however a large amount of fluorescence remained in nerve terminals of lh mice. KCl evoked-released induced unloading of FM1-43 up to ~ 46.4 ± 1.6% and 8.7 ± 0.8% in lh and wt respectively (Fig. 2.7). It took ~ 15 mins for lh preparations to achieve a level of destaining similar to that obtained at 3.5 mins in wt mice (Fig. 2.8). Additionally, to test whether this difference in the kinetics of release was due to a pool size problem or Ca 2+ current vesicle release was induced by α-latrotoxin (α-LTx) or by hypertonic solution (500 mM). The observed reduction in ACh release may be caused by a reduction of the number of transmitter vesicles that is ready for release. In order to probe for this vesicle pool we added hypertonic medium (500 mM sucrose-Ringer) (Stevens and Tsujimoto, 1995) and measured the FM1-43 destaining process. Exposure to hypertonic solution causes exocytosis of ACh from the readily releasable pool (Stevens and Tsujimoto, 1995) and acts in a Ca 2+ independent manner (Rosenmund and Stevens, 1996). Hypertonic solution increases the frequency (Van Der Kloot, 1991), amplitude and 70 duration of spontaneous release (Yu and Van Der Kloot, 1991). The rate of destaining induced by hypertonic solution was the same in lh and wt mice (Fig. 2.9). This result suggests unaltered size of the readily releasable pool at NMJs of lh mice. As an alternative means to evoke transmitter release, we applied 2 μg/ml of α-LTx 2+ to wt and lh NMJs. This toxin binds to presynaptic receptors and evokes Ca -dependent and -independent spontaneous neurotransmitter release by insertion of ion pores and stimulation of secondary messengers, emptying the readily releasable and the reserve pool together (Grishin, 1998; Rosenthal and Meldolesi, 1989; Ushkaryov et al., 2004; 2008). There was no difference between genotypes in the rate of destaining induced by αLTx (Fig. 2.10). This result suggests that the size of both the readily releasable and the reserve pool is unaltered in lh mice. Therefore, destaining of FM1-43 appeared to be slower and less complete in lh mice. The lh mutation might affect the Ca could result in the slow process of release. 71 2+ current which Discussion: Mice harboring the lh mutation exhibit obvious neuromuscular impairment. It has been shown that the N-terminal region of the β4a isoform interacts with synaptotagmin I, and hence is involved in the vesicle release process (Vendel et al., 2006). Therefore, lh mice which are null for β4 could have affected ACh release at motor nerve terminals. We studied how ACh release responded to the use of different charge carriers and different 2+ 2+ concentration of the charge carriers. In the absence of Ca , Ba the generation of MEPPs. Sr 2+ and Ba 2+ and Sr 2+ can support act on different “sensors” in the release process (Van Der Kloot and Molgó, 1994; Zefirov and Grigor‟ev, 2010; Zefirov and Mukhamed‟yarov, 2004). We also wanted to understand how this mutation affected the kinetics of the neurotransmitter release, by studying the vesicle dynamics by means of the fluorescent dye FM1-43. Results of the present study are consistent with the following conclusions: (1) ACh release following nerve stimulation (EPP) is significantly decreased in lh animals. (2) The quantal content is significantly reduced in lh mice as compared to wt. (3) There is a decrease in MEPP frequency in lh animals as compared to wt when using SrCl2 and BaCl2, but not CaCl2. (4) The rate of FM1-43 destaining induced by high [KCl] is slower in lh mice as compared to wt. (5) The rate of FM1-43 destaining in lh animals is the same as wt when the release is induced either by high osmolarity or α-LTx. Evoked ACh release from motor nerve terminals is significantly reduced in lh mice as compared to wt. The mutation had no effect on MEPP amplitude, but it does 72 affect MEPP frequency. Moreover, the EPP amplitude is significantly reduced in lh mice; leading to an overall decrease in the quantal content in these mice. These results suggest the possibility that there is a slower process of release in lh mice. To address this question, we used the FM1-43 staining method, which would help us in determining the vesicle dynamic process. KCl (40 mM) depolarization of wt mice caused almost complete destaining, reflecting a depletion of fluorescently labeled vesicles. However, under the same conditions, in lh mice there is still 38% of stain remaining. Thus the vesicle release process is significantly slower in lh mice. This result could imply that there is a problem with the vesicle pool size in lh mice. To determine if this was the case we induced vesicle depletion by two mechanisms: hypertonic solution and α-LTx. In both cases the rate of release was the same for both genotypes. This result implies that in lh mice there might be a problem in the Ca 2+ current affecting ACh release, but that this mutation does not affect the vesicle‟s pool size (neither the readily releasable nor the reserve pool). It seems that the effect on Ca 2+ current is translated in a slower vesicle dynamics which is then perceived as a decreased quantal content. Therefore in the lh mouse, the severe neurological phenotype might be due in part to altered vesicle dynamics. The lh NMJ has not been studied extensively. Work done by Kaja et al. (2007) showed that the lh mutation did not affect quantal content, EPP amplitude or MEPP frequency or amplitude. Our study corroborates some of these findings, but differs in others. The finding that lh mice have no difference in ACh release as their wt littermates proposed by Kaja (2007) differs from our results. They saw that this mutation had no effect on MEPP amplitude or frequency, or in EPP amplitude or quantal content. 73 However, the study of Kaja et al. (2007) did not include different divalent cations to substitute Ca 2+ as charge carriers, which might explain why they did not see any difference in MEPP frequency, since there is no obvious difference in the frequency of spontaneous release when using Ca become apparent when using Ba 2+ 2+ as a charge carrier. However, but these differences 2+ or Sr . Differences between our two studies are most likely due to age-related factors. Kaja‟s group use 6 wk old mice, when we carry out all our studies in mice that are between 3 – 9 mos of age. It is possible that at 6 wks of age it is too early to have full expression of VGCCs to see difference between genotypes in ACh release. Moreover, there is evidence that during the first 6 wks of life of mice there is a significant and rapid change in parameters (quantal content, resting membrane potential, MEPP frequency) (Kelly, 1978). Hence, it is important that mice are properly matched according to age. However, this would need to be examined further to substantiate the differences between our results. The influx of extracellular Ca 2+ through VGCCs stimulates neurotransmitter release by exocytosis. Exocytosis of synaptic vesicles occurs in the active zones enriched 2+ 2+ with VGCCs due to activation of high-affinity Ca -site in Ca -macrodomain (Zefirov and Grigor‟ev, 2008). The β subunit regulates the assembly, membrane localization of the 2+ α1 subunits, enhances Ca currents and modifies the voltage-dependence and kinetics of activation and inactivation (Lacerda et al., 1991; Stea et al., 1993). Lethargic mice lack the β4 subunit which is the normal complement of P/Q-type VGCCs. This alteration in the normal composition of these channels could alter the relative abundance of P/Q-type 74 2+ VGCCs at motor nerve terminals, as well as Ca currents. Both effects could explain a slower vesicle process, leading to a decrease in ACh release. Exposure to hypertonic solution and α-LTx causes exocytosis of ACh in a Ca independent manner. When we induce vesicle release independently of Ca 2+ 2+ - the rate of FM1-43 destaining is similar between genotypes. This also supports the hypothesis that Ca 2+ influx might be altered, affecting neurotransmitter release at motor nerve terminals of lh mice, which could account for their lethargic behavior. 75 Figure 2.1: Spontaneous release of ACh measured in solutions containing 2, 4 or 8 mM CaCl2. Intracellular recordings were performed in hemidiaphrams of lh and wt mice. The amplitude and frequency of MEPPs were measured for both genotypes. (a) Amplitude of MEPPs measured in hemidiaphragms of wt and lh mice. There is no difference in amplitude between genotypes. Each value represents the mean ± S.E.M of 7 animals. (b) Frequency of spontaneous release measured in wt and lh mice. There is no difference in frequency between genotypes. Each value represents the mean ± S.E.M of 7 animals. (a) MEPP Am plitude (m V) 2 wt lh 1 0 2 4 2+ [Ca ] (mM) 76 8 Figure 2.1 (cont’d) (b) MEPP Frequency (1/s) 2 wt lh 1 0 2 4 2+ [Ca ] (mM) 77 8 Figure 2.2: Spontaneous release of ACh measured in solution containing SrCl2 (2 or 4 mM) or BaCl2 (0.5 or 1 mM). Intracellular recordings were made in hemidiaphrams of lh and wt mice. The amplitude and frequency of MEPPs were measured for both genotypes. (a) Amplitude of MEPPs measured in hemidiaphragms of wt and lh mice. There is no difference in amplitude between genotypes. Each value represents the mean ± S.E.M of 7 animals. (b) Frequency of spontaneous release measured in wt and lh mice. There is significant difference in frequency between genotypes. Each value represents the mean ± S.E.M of 7 animals. The asterisk (*) indicates a significant difference between the two genotypes. (a) MEPP Am plitude (m V) 2 wt lh 1 0 Sr 2 Sr 4 Ba 0.5 2+ [Me ] (mM) 78 Ba 1 Figure 2.2 (cont’d) (b) MEPP Frequency (1/s) 4 wt lh 3 2 * 1 0 Sr 2 * * Ba 0.5 Sr 4 2+ * Ba 1 [Me ] (mM) 79 Figure 2.3: Nerve evoked release of ACh measured in solution containing 2, 4 or 8 mM CaCl2. Intracellular recordings were performed in hemidiaphrams of lh and wt mice. EPPs were elicited by phrenic nerve stimulation at 0.5 Hz by means of a suction electrode attached to a stimulus isolation unit. (a) Shows EPPs from neuromuscular junction preparations isolated from homozygote lethargic (lh) (orange trace) and wt mice (black trace). (b) The EPP amplitude was measured for both genotypes. There is a significant difference in amplitude between genotypes. Each value represents the mean ± S.E.M of 7 animals. The asterisk (*) indicates a significant difference between the two genotypes. (a) wt lh 80 Figure 2.3 (cont’d) (b) EPP Am plitude (m V) 25 wt lh 20 15 * * * 10 5 0 2 4 [Ca 2+] (mM) 81 8 Figure 2.4: Quantal content measured in solution containing 2, 4 or 8 mM CaCl2. The m value (quantal content) was calculated for each neuromuscular junction preparation using the ratio of the average EPPs amplitude to the average MEPPs amplitude (data from Fig. 2.1a and Fig. 2.3). The quantal content was significantly decreased in lh mice compared to wt at all Ca 2+ concentrations used. Each value represents the mean ± S.E.M of 7 animals. The asterisk (*) indicates a significant difference between the two genotypes. The number sign (#) indicates a significant difference between Ca 2+ concentrations. Quantal Content ( m) 25 wt lh 20 * 15 10 *# *# 5 0 2 4 2+ [Ca ] (mM) 82 8 Figure 2.5: Nerve evoked release of ACh measured in solution containing 2 or 4 mM SrCl2. Intracellular recordings were performed in hemidiaphrams of lh and wt mice. EPPs were elicited by phrenic nerve stimulation at 0.5 Hz by means of a suction electrode attached to a stimulus isolation unit. The amplitude EPPs were measured for both genotypes. There is significant difference in amplitude between genotypes. Each value represents the mean ± S.E.M of 7 animals. The asterisk (*) indicates a significant difference between the two genotypes. EPP Am plitude (m V) 20 wt lh 10 * * 0 2 4 2+ [Sr ] (mM) 83 Figure 2.6: Quantal content measured in solution containing 2 or 4 mM SrCl2. The m value (quantal content) was calculated from each neuromuscular junction preparation using the ration of the average EPP amplitude to the average MEPP amplitude (data from Fig. 2.2a and Fig. 2.5). There is significant difference in amplitude between genotypes. Each value represents the mean ± S.E.M of 7 animals. The asterisk (*) indicates a significant difference between the two genotypes. Quantal Content ( m) 15 wt lh 12 9 * * 6 3 0 2 4 2+ [Sr ] (mM) 84 Figure 2.7: Time course of FM1-43 destaining triggered by perfusion application of 40 mM KCl in wt and lh animals. KCl evoked FM1-43 release appears to occur faster in wt nerve terminals as compared to lh. The data are presented as the amount of fluorescence normalized to the average fluorescence values before KCl-evoked release. Each value represents the mean ± S.E.M of 9 animals. The asterisk (*) indicates a significant difference between the two genotypes. 125 wt Intensity (% of T0) 100 lh * 75 50 25 0 0 1 2 Time (min) 85 3 Figure 2.8: FM1-43 destaining in wt and lh motor nerve terminals. (a) Representative images of lh and wt nerve terminals stained with FM1-43 dye, showing the distaining process evoked by 40 mM KCl over time. (b) Quantification of the destaining process in wt and lh mice. The KCl evoked FM1-43 release appears to occur faster in wt nerve terminals as compared to lh. It takes more than 15 minutes for lh mice to achieve a level of distaining similar to that of wt under the same conditions. Each value represents the mean ± S.E.M of 9 animals. The asterisk (*) indicates a significant difference between the two genotypes. 86 Figure 2.8 (cont’d) (a) Representative images of a distaining process induced by 40 mM KCl, of lh (upper panel) and wt (lower panel) nerve terminal stained with FM1-43 0 min 1.5 min 3.5 min 7 min lh wt 87 10 min 15 min Figure 2.8 (cont’d) (b) 125 wt lh Intensity (% of T0) 100 75 * 50 25 0 0 1.5 3.5 7 Time (min) 88 10 15 Figure 2.9: Time course of FM1-43 destaining triggered by hypertonic solution (500 mM) in wt and lh animals. FM1-43 release evoked by high osmolarity appears to occur at the same rate in wt and lh nerve terminals. The data are presented as the amount of fluorescence normalized to the average fluorescence values before hypertonic solution-evoked release. Each value represents the mean ± S.E.M of 9 animals. 125 wt lh Intensity (% of T0) 100 75 50 25 0 0 2 4 6 8 Time (min) 89 10 12 14 Figure 2.10: Time course of FM1-43 destaining triggered by α-LTx in wt and lh animals. FM1-43 release evoked by α-LTx appears to occur at the same rate in wt and lh nerve terminals. The data are presented as the amount of fluorescence normalized to the average fluorescence values before α-LTx -evoked release. Each value represents the mean ± S.E.M of 9 animals. 125 wt lh Intensity (% of T0) 100 75 50 25 0 0 1 2 3 4 5 Time (min) 90 6 7 8 9 CHAPTER 3 ACETYLCHOLINE RELEASE IS CONTROLLED BY P/Q- AND R-TYPE CALCIUM CHANNELS IN ADULT LETHARGIC (lh) MICE 91 Abstract: Voltage gated calcium channels (VGCCs) are formed by α1, β and α2δ and sometimes γ subunits. The α1 subunit makes up the selective pore for Ca 2+ and determines most of the subtype-specific attributes of calcium channels. This subunit contains binding sites for various pharmacological agents as well as the gating regions of the channel. The β subunit regulates the assembly and membrane localization of the α1 subunits, and strongly influences the physiological features of the protein. The β4 subunit normally coassociates with the α1A subunit of the P/Q-type calcium channels at adult mammalian neuromuscular junctions. A natural occurring mutation present in lethargic (lh) mice due to a 4 base pair insertion into a splice donor site within the β4 gene leads to loss of 60% of the C-terminal of the β4 subunit relative to wild type (wt), including loss of the α1-binding site. Mice with this mutation suffer from ataxia, lethargic behavior, spikewave epilepsy, and paroxysomal dyskinesia. We wanted to identify which calcium channel subtypes control nerve-stimulated acetylcholine (ACh) release from motor nerve terminals of adult lh mice. ω-Agatoxin IVA and SNX 482 significantly reduced the quantal content in adult lh mice (60% and 46%, respectively), however neither toxin affected the miniature end plate potential (MEPP) frequency or amplitude. Neither ωconotoxin GVIA nor nimodipine had any effect on ACh release. Immunolabeling of calcium channel subunits revealed an increase in α1E, β1 and β3, but no apparent change in the levels of α1A at adult lh neuromuscular junctions. This presumably compensates 92 for the absence of β4. Therefore, it seems that in these animals the ACh release is controlled by P/Q- and R-type calcium channels, which could account for the reduction in ACh release. 93 Introduction: Voltage gated calcium channels (VGCCs) contribute to entry of Ca 2+ into neurons and muscle cells (Catterall, 1995; Katz and Miledi, 1970; Llinas et al., 1976). Although multiple VGCC types coexist in the same cell, the specific channel subtype involved in release of acetylcholine (ACh) from motor nerve terminals is both speciesand age-dependent (Catterall, 1998; Rosato Siri and Uchitel, 1999). Mature mammalian motor nerve terminals contain primarily P/Q-type VGCCs (Katz et al., 1995). However, under specific conditions, subtypes of VGCCs that are not normally associated with ACh release at motor nerve terminals can mediate it (Flink and Atchison, 2002; Pardo et al., 2006; Smith et al., 1995). An example of this is seen in the tottering (tg) mutation, in which ACh release in adult tg mice is controlled by N- and R-type VGCCs (Pardo et al., 2006). The different types of VGCCs can be distinguished by the genes that encode them, as well as their pharmacological and biophysical characteristics (Catterall et al., 2005; Zhang et al., 1993). VGCCs are formed by α1, β, and α2δ subunits (Tsien et al., 1991). The α1 subunits make up the selective pore for Ca 2+ and determine most of the subtype-specific attributes of the VGCC. This subunit contains binding sites for various pharmacological agents as well as the gating regions of the channel (Catterall, 1995; Zhang et al., 1993). There are at least five α1 subunits for neuronal VGCC which fall under the high voltage activated (HVA) subclass (Catterall, 1995; Tsien et al., 1991). They include: α1A, α1B, and α1E subunits which comprise the P/Q- (Cav2.1), N(Cav2.2), and R-type (Cav2.3) Ca 2+ channels, respectively; while the α1C or α1D subunits 94 comprise the L-type channels (Cav1.2-1.3) (Catterall, 1995; Tsien et al., 1991). The β subunit regulates the assembly and membrane localization of the α1 subunits. The β subunit also strongly influences the current amplitude, rate and voltage-dependence of activation and inactivation, and ligand-binding sites on the neuromuscular junction (NMJ) (Brice and Dolphin, 1999; Catterall, 1995; Walker and De Waard, 1998). There are four different types of β subunits (β1-4) each encoded by a separate gene (Chien et al., 1995). The β4 subunit is typically associated with P/Q-type VGCC (Wittemann et al., 2000). The interaction of the correct β subunit with its corresponding α1 subunit is essential for proper targeting, membrane insertion, channel density, kinetic parameters such as activation and inactivation as well as interactions with vesicular release site proteins (Murakami et al., 2003; Wittemann et al., 2000). In the absence of the normally associating β subunit, alternate β subunits may interact with α1 subunits to restore most of the VGCC‟s functions, although in an altered manner (Burgess et al., 1999). The β4 subunit is normally widely expressed in the brain. Spontaneous mutations in this subunit cause several neurological syndromes in mice (Burgess et al., 1997; Catterall, 1995). These mutations produce various effects in VGCCs‟ expression and function, such as dramatically reducing the VGCCs targeting, assembly, membrane insertion, channel density and altering characteristic kinetic parameters, vesicular release and synaptic transmission (Burgess and Noebels, 1999; Catterall, 1995; Catterall et al., 2005; Walker et al., 1998). Also, the loss of a functional β4 subunit can impact the function of α1A – containing calcium channels (P/Q-type). The lh mutation is caused by a 95 four base pair insertion into a splice donor site within the β4 gene on mouse chromosome 2. This insertion leads to exon skipping, translational frameshift, and protein truncation, missing 60% of the C-terminal of the β4 subunit relative to wt, this includes loss of the α1-binding site, suggesting that this defect in VGCC assembly could be one cause for the pathogenesis in lh phenotype (Burgess et al., 1997; Burgess and Noebels, 1999). Lethargic mice suffer from ataxia, lethargic behavior, spike-wave epilepsy, and paroxysomal dyskinesia. Electrophysiologically and pharmacologically, the seizures are similar to the absence seizures present in the human petit mal epilepsy and to those present in tottering (tg) mice (Burgess and Noebels, 1999; Hosford et al., 1992). The onset of ataxia is two weeks after birth (Khan and Jinnah, 2002). In addition to the neurological symptoms, lh mice show reduced body weight and immunological problems when compared with unaffected litter mates (Sidman et al., 1965). Since lh animals live to become adults, some type of VGCC must assume control of release of ACh at motor nerve terminals. Therefore, the objectives of the present study were to determine which subunits of the VGCCs are present at the motor nerve terminals of adult lh mice and control the release of Ach, and in these mice which β subunit(s) substitute for the β4 subunit which normally co-associates with α1A. Electrophysiological recordings of spontaneous and evoked release of ACh were made in wt and lh animals at intact NMJs in the presence of various VGCC antagonists. Additionally, the protein levels of different β subunits were compared in lh mice. To confirm our electrophysiological and protein assay results, immunostaining of cryosections of the extensor digitorum longus (EDL) muscle of lh and wt mice was done. 96 Materials and Methods: Mice. Breeding pairs of heterozygoous Cacnb4lh4J mice were obtained from Jackson Laboratory (Bar Harbor, ME) and subsequently maintained in a breeding colony at Michigan State University Laboratory Animal Resources. Litters were genotyped at weaning, 3 wks after birth. lh (homozygous) mice were also identified by their characteristic phenotype consisting of a mild ataxia, wobbly gait behavior and smaller body size. Adult mice between 3 to 9 mos of age were used for all the experiments. All experiments were performed in accordance with local university (Michigan State University Laboratory Animal Resources) and national guidelines (National Institutes of Health of the USA - NIH) and were approved by the University Animal Use and Care Committee. Drugs and chemicals. The presence of the various VGCCs was tested by the use of specific antagonists. To test for the presence of the L-type VGCC, the tissue was treated with 10 μM nimodipine (Nim. -Sigma-Aldrich, St. Louis, MO). The involvement of P/Qtype VGCC was determined with the use of 100 nM of ω-agatoxin IVA (Aga IVA Alomone Labs, Jerusalem, Israel). The presence of the N-type VGCC was assessed with the use of 3 μM of ω-conotoxin GVIA (Ctx GVIA – Bachem California, Torrance, CA). The R-type VGCC was determined using 1 μM SNX 482 (Ascent Scientific, Princeton, NJ). Antibodies against the various α1 subunits were obtained from Alomone Labs (Jerusalem, Israel). To probe for the presence of the various β subunits we used different antibodies, for β1,2 and 4 (Neuromab, UC Davis, University of California, CA) and β3 97 (Santa Cruz Biotechnology Inc., Santa Cruz, CA) subunits. Fluorescein (FITC)conjugated goat anti-rabbit IgG (heavy + light chains) was purchased from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA). Pacific Blue goat anti-mouse IgG (heavy + light chains) was obtained from Invitrogen (Carlsbad, CA) and tetramethylrhodamine α-bungarotoxin was purchased from Sigma (St. Louis, MO). All antibodies were used in a dilution of 1:200. Electrophysiology. Animals were sacrificed by decapitation following anesthesia with 80% O2 + 20% CO2. The diaphragm with its attached phrenic nerves was removed from each animal and pinned in a Sylgard-coated chamber. The tissue was superfused continuously with 100% oxygenated physiological saline solution (137.5 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 11 mM d-glucose, and 4 mM HEPES, adjusted to pH 7.4). MEPPs and EPPs were recorded from lh and wt mice using conventional intracellular recording techniques. EPPs were elicited by phrenic nerve stimulation at 0.5 Hz using a suction electrode attached to a stimulus isolation unit (Grass SIU5; Grass Instruments, Quincy, MA) and stimulator (Grass S88). Signals were amplified using an Axoclamp-2 amplifier (Molecular Devices, Sunnyvale, CA), digitized using a PC-type computer and Axoscope 9.0 software (Molecular Devices), and analyzed using MiniAnalysis 6.0 Software (Synaptosoft, Decatur, GA). Recordings were performed from 5-10 end-plates for a period of 5 mins each for each mouse. Muscle action potentials were inhibited by addition of 2.5 μM μ-conotoxin GIIIB (Alomone Labs, Jerusalem, Israel). MEPPs and EPPs were recorded using intracellular glass microelectrodes (1.0 mm- o.d.; WPI, Sarasota, FL) which had a resistance between 5 and 15 MΩ, when filled 98 with 3M KCl. MEPP and EPP amplitudes were normalized to a membrane potential of 75 mV using the following formula: Vc = [Vo x (-75)]/E, where Vc is the corrected amplitude of MEPP and EPP, Vo is the actual recorded amplitude of MEPP and EPP and E is the membrane potential at which the recording is made (Magleby and Stevens, 1972; McLachlan and Martin, 1981). A recording was rejected if the 10-90% rise time was greater than 1.5 ms, also, if the membrane potential was more depolarized than -55 mV. The m value (quantal content) was calculated using the corrected mean amplitude values of EPP and MEPP, where m = EPP/MEPP (Hubbard et al., 1969). Protein Isolation and Western Blot Analysis. Western blots were not sufficiently sensitive to detect the scarcity of VGCCs subunits in the presynaptic area of the diaphragm muscle. Therefore, I performed western blots to determine the protein levels in cerebellum of lh and wt mice. The protein level of α1A, β1 - β4 subunits at lh and wt mice was compared by western blots of protein isolated from cerebellum which expresses of high concentrations of Cav2.1. The tissue sample was placed in a mortar containing 1 ml of 2X lysis buffer with 50 µl of each protease (20X stock solution of pepstatin, leupeptin, ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA) and protease inhibitors (Roche)). The tissue was ground, the lysate transferred to a microfuge tube, and centrifuged for 10 mins at 13000 RPM. The supernatant was stored at -80ºC. The protein concentration was determined using the bicinchoninic acid assay (BCA), and quantified with the Beckman Du 640 spectrophotometer (Beckman, Brea, CA). The proteins were loaded to a 10% SDS PAGE gel and migrated at a constant current of 40 mA. They were then transferred to a nitrocellulose membrane, this was 99 done at 4C and constant voltage of 90 mV. The membrane was probed against β-actin (dilution 1:20,000) as a loading control, and against α1A (dilution 1:200) and β1-4 subunits (dilution 1:200). Immunohistochemistry. Extensor digitorum longus (EDL) from wt and lh mice was used for immunohistochemistry. The muscle was dissected and fixed for 30 min in 4% (w/v) paraformaldehyde in 0.1M phosphate-buffered saline (PBS; composition 137 mM NaCl, 2.7 mM KCl, 1.4 mM NaH2PO4, and 4.3 mM Na2HPO4, pH 7.4). Then the tissue was washed in PBS for 1 min and treated with 0.1% (w/v) Triton X-100 in PBS for 30 min; after which the tissue was washed for 15 min with PBS and cryoprotected in 20% and 30% (w/v) sucrose each for 24 hs. The tissue was then placed in optimal cutting temperature compound (Tissue Tek, Tokyo, Japan) in a plastic mold and stored at -20ºC until used. Longitudinal sections (20 µm thick) were cut in a cryostat (Cryostat model Microm HM 525, Thermo Shandon Inc., Pittsburgh, PA) and mounted onto gelatincoated slides. After this the tissue was stained with specific antibodies. α-Bungarotoxin was used as a marker for the ACh receptor in the muscle. The nerve was stained with antibodies against α1 and β1-4 subunits. The preparations were viewed on a Nikon Eclipse TE 2000-U Diaphot-TMD microscope (Nikon, Melville, NY) with a Hamamatsu Orca 285 charge-coupled device camera (Bridgewater, NJ), and images were acquired using Metamorph software (Molecular Devices, Sunnyvale, CA). 100 Statistical Analysis. Differences between genotypes were analyzed using a one way analysis of variance followed by Tukey‟s test. Differences between treatments were analyzed using a two way analysis of variance followed by Tukey‟s test. P values were set to < 0.05 for all tests. Measurements are expressed as mean ± S.E.M for n ≥ 5. 101 Results: Effect of the lh mutation in cerebellar protein levels Since lh animals lack the β4 subunit, we first wanted to determine whether this mutation affected the protein levels of the remaining different β subunits of adult lh mice. We first tried to assay for protein level in diaphragm and phrenic nerve. However, due to the scarcity of VGCC proteins present in this tissue and the poor sensitivity of western blots we were unable to probe against any subunit (no bands were revealed in these blots, Fig. 3.1a). Next we probed for the different VGCC subunits in cerebellum. We chose this tissue since the β4 subunit is normally extensively expressed in cerebellum and this region of the brain is responsible for motor coordination. Lh mice exhibit poor motor coordination and balance, indicating that cerebellar dysfunction likely occurs. Because the β4 subunit normally co-associates with α1A, we also wanted to assay for α1A levels to determine if the absence of β4 affects the levels of α1A. Protein level analysis, through western blot assays showed that even though lh animals lack the β4 subunit, the protein levels of the α1A subunit are the same for lh and wt animals. This result may be due to the observed significant increased levels of β1 and β3, which might compensate for the lack of β4 (Fig.3.1 b and c). ACh release is controlled by P/Q- and R-type VGCCs in adult lh mice According to the western blot results, the lh mutation appeared not to affect the α1A protein levels as compared to their wt littermates. However, these mice are 102 apparently compensating for the absence of β4 with increased levels β1 and β3. Based on these results, we wanted to determine if this atypical association affected the types of VGCC being expressed in NMJs of adult lh mice. For this purpose we performed intracellular recordings in the diaphragms of adult lh and wt mice, in the presence of different VGCC antagonists. ACh release following phrenic nerve stimulation is significantly decreased both in lh as well as wt animals (60% and 76%, respectively) after treating the tissue with ωagatoxin IVA (ω-Aga IVA; 100 nM), a specific antagonist of the P/Q-type VGCC. The difference between both genotypes was not statistically significant. We therefore wanted to test whether other types of VGCC are present and contribute to the release of ACh in lh mice. The antagonist of the R-type VGCC, SNX 482 (1 μM), significantly decreased the quantal content of lh (46%), but not wt mice (8%). This implied that R-type VGCCs contribute to the control of the ACh release in the NMJ of adult lh but not in wt mice. In contrast, the contribution of L- and N-type VGCCs to the control of ACh release both in wt and lh mice was negligible. ω-Conotoxin GVIA (ω-ctx GVIA) (3 μM) a specific antagonist of the N-type VGCC, did not significantly change the quantal content of either lh or wt mice. The same result occurred when nimodipine (Nim) (10 μM) was applied to test for the presence of L-type VGCC. Nimodipine did not induce any change in the ACh release in either lh or wt mice. These results imply that L- and Ntype VGCCs do not contribute significantly to control ACh release at the NMJ of adult lh or wt mice. When we co-applied ω-Aga IVA and SNX 482 they significantly reduced ACh release in lh to a level similar to that observed after the application of ω-Aga IVA alone 103 in wt mice. Taken together, these results implicate that in lh animals ACh release is controlled by P/Q- and R- type VGCCs, and that both types contribute to a similar extent (Fig.3.2). However this is not the case in wt mice, since when we combined the two antagonists we saw no significant change from the results obtained following application of ω-Aga IVA alone (Fig.3.3). Additionally as a control, we applied Cd 2+ (10 µM), a non-specific blocker of VGCCs, to some preparations. This concentration of Cd 2+ effectively blocked ACh release in both wt and lh mice (data not shown). Immunohistochemistry data Western blot results showed an increased level of β1 and β3 subunits, while electrophysiological results indicated that P/Q- and R-type VGCCs control ACh release at NMJs. Consequently, we wanted to determine the presence of the different α1 and β subunits at NMJs of adult mice. The localization of the different α1 and β subunits at lh and wt mice NMJ was examined using fluorescence immunohistochemistry in sections of extensor digitorum longus (EDL) muscle. The sections were stained with specific antibodies against the various α1 and β subunits as well as the muscle type nACh receptor (as a postsynaptic marker). As demonstrated by the representative immunostaining images (Fig.3.4) wt animals have staining of α1A (green), β4 (blue), and α-bungarotoxin (red); adult lh mice 104 have no immunostaining of β4, but there is immunostaining of β3, β1 (blue), α1A, α1E (green), and α-bungarotoxin (red) (Figs.3.5 and 3.6). Figure 3.6 compares the relative fluorescence for each subunit in both genotypes. There is a significant increase in α1E, β3, and β1 in lh mice as compared to their wt littermates. However the levels of α1A in lh mice are slightly decreased as compared to wt, yet this change is not significantly different (p>0.05). This is consistent with the results of both western blot and electrophysiology. We also quantified the extent of juxtaposition of the VGCC subunits against the nACh receptor present in both genotypes (Fig. 3.7). In lh mice there is a 58% and 44% juxtaposition of α1A and α1E respectively, while in wt there was 73% juxtaposition of α1A with α-bungarotoxin. With regards to the β subunits, in lh mice β1 and β3 juxtaposed with the nACh receptor at 88% and 65%, respectively. The immunohistochemistry data confirmed the electrophysiology data; both α1A and α1E staining overlaps that of nACh receptors at the NMJ. We were also able to determine that there is an increase in β3 and β1 subunit in lh mice, which might compensate for the absence of β4 subunit. 105 Discussion: In normal adult mammals, ACh release is mainly controlled by P/Q-type VGCCs (Katz et al., 1995). We know that α1A knockout mice do not survive more than 15-21 days after birth (Jun et al., 1999). We also know that under specific conditions, different subtypes of VGCCs can mediate ACh release when they would not do so normally, such as in tg mice (Flink and Atchison, 2002; Pardo et al., 2006). The β subunit regulates the assembly and membrane localization of the α1-pore forming subunit of the VGCCs (Walker and De Waard, 1998). β4 subunits normally coassociate with α1A pore forming subunit of the neuronal P/Q-type VGCCs (Burgess et al., 1997; Catterall, 1995). Based on this information we hypothesized that lh mice might present compensation by other types of VGCC due to the absence of the β4 subunit. These mice live to adulthood, and are fertile. Moreover, they show no obvious neuromuscular impairment. Our results show the following: (1) Even though lh animals lack the β4 subunit, levels of the α1A subunit are similar for lh and wt animals. This may be due to the observed increased level of β1 and β3, to compensate for the lack of the β4 subunit. (2) ACh release following nerve stimulation is significantly decreased both in lh as well as wt animals after treating the tissue with ω-Aga IVA, a specific antagonist of P/Q-type VGCCs. (3) ACh release following nerve stimulation is significantly decreased in lh animals after treating the tissue with SNX 482, a specific antagonist of R-type VGCCs. (4) In lh mice β1 and β3 subunits seem to associate with α1A and α1E. 106 Unlike the tg mouse for which a direct mutation occurs in α1A (Pardo et al., 2006), the lh mouse maintains a functional complement of P/Q-type VGCCs and association of β4 with α1A is not obligatory for functional expression of the protein, albeit at apparently reduced abundance. The effect of ω-Aga IVA on wt and lh NMJ was not significantly different, however it did show an interesting trend in which wt mice showed inhibition of 76% after application of the P/Q-type antagonist, while in lh the same protocol produced 60% inhibition. It was this difference which led us to think that other VGCCs might be present at the NMJ of adult lh mice. The significantly differential effect of SNX 482 on wt and lh mice indicates that ACh release in adult lh NMJ is controlled in part by R-type VGCC, while this subtype is not involved in ACh release at NMJ of adult wt mice. Increased immunofluorescence of α1E at NMJs of EDLs in lh animals, confirmed the electrophysiological results showing the involvement of the R-type VGCC. We also saw similar levels of fluorescence in wt and lh mice in the α1A subunit, which also implies that there are P/Q-type VGCCs present at the NMJ. With regards to the auxiliary cytoplasmic β subunit, we saw an increase in fluorescence of the β1 and β3 in lh mice, which may compensate for the absence of the β4 subunit. Although this work shows some results that are similar to those presented by Kaja (2007), most of the results presented in this work differ from his. Kaja et al. (2007) determine that P/Q-type VGCCs are the only type of channel controlling ACh release at 107 adult lh NMJ. The difference between the results of the two studies could reside in the age difference between the animals used. While he performed his entire study in 6 wk old mice, we used 3-9 mo old mice for our studies. At the comparatively young age of mice at which Kaja (2007) examined ACh release, there might have been a greater dependence on P/Q-type and a lesser contribution of R-type channels than in 3 to 9 mo old animals. Additionally there are slight differences in the methodology used between us. These differences could possibly lead to differences in our results. This would need to be examined further, by repeating the experiments using his protocol. In conclusion, the type of channel that can control ACh release at mammalian NMJ is not fixed. It has been shown that recruitment of alternative types of VGCCs to compensate for a deficit is possible. For example in tg mice, which carry a point mutation in the α1A subunit of the P/Q-type VGCCs compromising the channel‟s function, ACh release is controlled by N- and R- type VGCCs (Pardo et al., 2006). Therefore, it is not unlikely that in lh mice, there is a form of neuronal plasticity in ACh release to compensate for their mutation. 108 Figure 3.1: Effect of lh mutation on protein levels. Western blots were performed to probe for protein levels of the different VGCCs subunits. β-Actin was used as a loading control. a) Representative western blot of VGCCs subunits present in diaphragm and phrenic nerve from wt mice. The tissue was loaded in the following order: (1) diaphragm (40 µg), (2) diaphragm (60 µg), (3) phrenic nerve (40 µg), (4) phrenic nerve (60 µg). Protein levels from wt mice preparations were probed with antibodies for α1A and β1-4. The western blots did not reveal any VGCCs subunit in these tissues, but was sensitive to β-Actin, which was used as a loading control. (b) Representative western blot of VGCCs subunits present in cerebellar proteins (40 µg). Protein levels from lh and wt cerebellar preparations were probed with antibodies for α1A and β1-4. (c) Protein levels were quantified using the program Image J®. Each value represents the mean ± S.E.M of 7 animals. The asterisk (*) indicates a significant difference between the two genotypes. 109 Figure 3.1 (cont’d) (a) 1 2 3 4 α1A 268 88 β1 55 78 β2 58 β3 50 β4 42 β-Actin 110 Figure 3.1 (cont’d) (b) lh wt α1Α 268 88 β1 55 78 β2 58 β3 50 β4 42 β-Actin 111 Figure 3.1 (cont’d) (c) lh  Protein level (o.d.) (% of wt) *  *      *     Subunits 112   Figure 3.2: ACh release is controlled by P/Q- and R-type VGCCs in adult lh mice. Effect of VGCC antagonists on nerve-evoked ACh release from motor nerve terminals of adult lh and wt mice. The tissue of wt or lh mice was treated with VGCC antagonists. (a c) Shows EPPs from neuromuscular junction preparations isolated from homozygote lethargic (lh) and wt mice with no pharmacological treatment (black control trace) or treated by incubation with ω-Aga IVA (orange trace), ω-Ctx GVIA (light blue trace), Nimodipine (purple trace) or SNX 482 (green trace). (d) 100 nM of ω-Aga IVA reduced the quantal content in lh animals to 40%, while the same concentration reduced the quantal content in wt mice to 24%. This difference in the reduction level suggests the possible presence of other VGCC controlling the ACh release in lh mice. The application of ω-Cntx GVIA and nimodipine did not significantly changed the quantal content of either wt or lh mice. The use of SNX 482 significantly reduced the quantal content in lh animals, but not in wt mice. The asterisk (*) indicates significant difference from the control pretreated preparation. The number sign (#) indicates a significant difference between genotypes. Data represent the mean ± S.E.M. (n ≥ 8). 113 Figure 3.2 (cont’d) 114 Figure 3.2 (cont’d) (d) Quantal Content (% of pre-treatm ent) 125 wt lh 100 75 *# * 50 25 0 * AgaIVA CtxGVIA Nim Antagonist 115 SNX 482 Figure 3.3: Effect of Aga IVA and SNX-482 on quantal content of lh and wt mice. EPPs were recorded from NMJ preparations of adult lh and wt mice. SNX 482 and ω-Aga IVA were applied simultaneously to measure the combined effect of both antagonists. The application of both toxins in lh animals had a similar effect to that observed in wt with ω-Aga IVA alone. There was no significant change in preparations from wt mice when SNX 482 and ω-Aga IVA were applied together or when ω-Aga IVA was applied alone. However when both antagonists were applied together there was a further decrease in the quantal content in lh preparations as compared to that obtained when ω-Aga IVA or SNX 482 were applied alone. The asterisk (*) indicates a significant difference from the control pretreated preparations. The number sign (#) indicates a significant difference between genotypes. Data represent the mean ± S.E.M. (n ≥ 5). 116 Figure 3.3 (cont’d) Quantal Content (% of pre-treatm ent) 125 SNX 482 Aga IVA Aga IVA + SNX 482 100 75 *# * 50 25 0 * * * wt lh Genotype 117 Figure 3.4: Immunostaining of wt and lh neuromuscular junction with α1A/α1E and β4. EDL muscles from wt and lh mice were stained with specific antibodies against VGCCs α1 (green) and β4 (blue) subunits as well as α-bungarotoxin (red). The tissue was then observed under the Nikon Eclipse TE 2000-U fluorescent microscope. The composite shows the superimposition of the various subunits. The bar scale is 10 µm. 118 Figure 3.4 (cont’d) (a) AChR α1A β4 Composite wt lh 119 Figure 3.4 (cont’d) (b) AChR α1E β4 Composite wt lh 120 Figure 3.5: Immunostaining of wt and lh neuromuscular junction with α1A/α1E and β1. EDL muscles from wt and lh mice were stained with specific antibodies against VGCCs α1 (green) and β1 (blue) subunits as well as α-bungarotoxin (red). The tissue was then observed under the Nikon Eclipse TE 2000-U fluorescent microscope. The composite shows the superimposition of the various subunits. The bar scale is 10 µm. 121 Figure 3.5 (cont’d) (a) AChR α1A β1 Composite wt lh 122 Figure 3.5 (cont’d) (b) AChR α1E β1 Composite wt lh 123 Figure 3.6: Immunostaining of wt and lh neuromuscular junction with α1A/α1E and β3. EDL muscles from wt and lh mice were stained with specific antibodies against VGCCs α1 (green) and β3 (blue) subunits as well as α-bungarotoxin (red). The tissue was then observed under the Nikon Eclipse TE 2000-U fluorescent microscope. The composite shows the superimposition of the various subunits. The bar scale is 10 µm. 124 Figure 3.6 (cont’d) (a) AChR α1A β3 Composite wt lh 125 Figure 3.6 (cont’d) (b) AChR α1E β3 Composite wt lh 126 Figure 3.7: Relative fluorescence of α1 and β subunits at wt and lh NMJs. Relative pixel count of fluorescence corresponding to α1 and β subunits at wt and lh NMJ. EDL preparations were probed with antibodies against the specific VGCCs subunits. The fluorescence levels were quantified using MetaMorph®. The asterisk (*) indicates a significant difference from wt preparations. (n = 8). % of fluorescence relative to wt lh  * * *    *           C   VGCCs Subunits 127   Figure 3.8: Percent juxtaposition of α1 and β subunits of wt and lh NMJ with ACh receptors. Percent juxtaposition of α-bungarotoxin and various subunits of VGCC in lh and wt NMJ. The values are taken from the data for the NMJ samples depicted in Fig. 3.7. The % of juxtaposition (V GC C subunit/A C hR ) asterisk (*) indicates a significant difference from wt preparations. (n = 8). wt 125 100 lh * 75 * * 50 25 0 *      C    VGCCs Subunits 128    CHAPTER 4 SUMMARY AND CONCLUSIONS 129 Summary and Conclusions The overall aim of this dissertation was to examine acetylcholine (ACh) release in lethargic (lh) mice, and determine what effect disruption of the β4 subunit has on the function of the α1A subunit in lh mice. We know that the α1A subunit is vital to the survival of adult mammals; α1A knockout mice do not live past 2 weeks of age (Jun et al., 1999). Normally α1A combines with β4 to form functional P/Q-type channels. Lethargic mice live to adulthood, therefore these mice must compensate for the absence of β4 either (1) by having a different voltage gated calcium channel (VGCC) controlling the ACh release (as is the case in the tottering (tg) mouse mutation), and/or (2) by substituting a different β subunit for β4. My dissertation‟s work has shown that even though the lh mutation does not affect miniature end-plate potential (MEPP) amplitude, it does affect the frequency of spontaneous release of ACh when induced by Sr 2+ 2+ 2+ or Ba , but not by Ca . Additionally, it alters the amplitude of the nerve evoked ACh release, which translates to a decrease in quantal content. Moreover, the vesicle release process is slower in lh mice, as shown by the FM1-43 staining experiments. However when release is induced by hypertonic solution or α-latrotoxin (α-LTx), FM1-43 destaining is the same in both lh and wild-type (wt) mice. Even though lh mice lack the β4 subunit, the protein levels of the α1A subunit in cerebellum are similar to wt animals. This may be due to the observed increased levels of β1 and β3, which might compensate for the lack of β4 subunit. To test 130 which VGCCs are controlling ACh release, intracellular recordings were performed in the presence of various VGCC antagonists. In adult lh mice, ACh release is controled by P/Q- as well as R-type VGCCs at the motor nerve terminal. This was corroborated by the immunohistochemistry results showing that there is an increased level of fluorescence of α1E, but the levels of α1A remained unaltered by this mutation, and there is an increase in β1 and β3 in the motor nerve terminal of extensor digitorum longus (EDL) muscle to compensate for the absence of β4 subunit (Fig.3.7). In lh animals neurologic signs occur despite the normal level of α1A. This might be due to the abnormal association in the lh animals of α1A- β1 or α1A-β3, and the presence of α1E- β1 or α1E-β3, which might affect the Ca 2+ current. VGCCs regulate diverse neuronal functions by mediating the entry of Ca 2+ into nerve terminals. VGCCs are multiple subunit structures which contain the α1 subunit the major pore-forming subunit, and several auxiliary subunits - α2δ, β and γ (Catterall, 1995, 2000; Hoffmann et al., 1999; Zhang et al., 1993). The β subunits are cytoplasmic proteins. They regulate the assembly and membrane localization of the α1 subunits, influence the current amplitude, rate, activation/inactivation kinetics, and ligand-binding sites on motor nerve VGCCs (Brice and Dolphin, 1999; Catterall, 1995; Walker and De Waard, 1998). There are four different types of β subunits (β1-4) each encoded by different genes (Chien et al., 1995). There are different types of VGCCs which can be classified according to their pharmacological and physical properties. In mature 131 mammalian motor nerve terminals, it is the P/Q-type calcium channels that control ACh release (Katz et al., 1995). However the complement of VGCCs is not necessarily fixed. Under specific conditions, subtypes of VGCC that are not normally associated with the ACh release at motor nerve terminals can mediate it. Lambert-Eaton Myasthenic syndrome (LEMS) is a paraneoplastic disorder, in which autoantibodies target the α1A subunit of the P/Q-type VGCC. When wt mice are induced to show LEMS like signs by repeated administration of plasma from LEMS patients, these mice showed that L-type VGCCs were contributing to ACh release from the motor nerve terminal (Flink and Atchison, 2002). Moreover, tg mice have a point mutation in the α1A subunit of the P/Qtype VGCC. This causes tg mice to show an ataxic phenotype. In this case, tg mice compensate for the mutation in the P/Q-type VGCC, by controlling ACh release by Nand R-type VGCCs. Recruitment of alternate types of VGCCs at motor nerve terminals to control neurotransmitter release is a frequent method of neuronal plasticity (Pardo et al., 2006). The β4 subunit, which is widely expressed in the brain (Burgess et al., 1997; Catterall, 1995), is normally associated with the α1A subunit of the neuronal P/Q- VGCC (Witterman et al., 2000). There is a naturally-occurring mutation that leads to the lh mutant mouse. There is a four bp insertion into a splice donor site, in mouse chromosome 2 (Fig.1.8), which results in exon skipping, translational frameshift, and protein truncation. Reduced levels of transcripts are produced from this allele and are present at approximately 20% of the normal levels in homozygous mice (Burgess et al., 1997). The β4 subunit of these animals is missing 60% of the C-terminal region which includes the 132 α1-binding site, suggesting that this defect in calcium channel assembly could be one cause for the pathogenesis in lh phenotype (Burgess et al., 1997; Burgess and Noebels, 1999). Mice with this mutation have a very distinct phenotype that becomes apparent at 15 days of age (Khan and Jinnah, 2002). They suffer from ataxia, lethargic behavior, spike-wave epilepsy and paroxysomal dyskinesia, reduced body weight and immunological problems, when compared with unaffected litter mates (Sidman et al., 1965). Loss of a functional β4 subunit can impact the function of α1A – containing VGCCs (P/Q-type). The tg mouse mutation is an example of neuronal plasticity. These mice have a point mutation which causes an amino acid substitution of proline-to-leucine in the S5-S4 linker region of repeat domain II of the α1A subunit (Fig. 4.1) (Fletcher et al., 1996). This leads to a non-functional α1A subunit. These mice, therefore, control ACh release at the motor nerve terminal by N- and R-type VGCCs (Pardo et al., 2006). Despite showing an obvious phenotype, lh mice live to become adults. Consequently, I hypothesized that the lh mice might also be presenting some form of neuronal plasticity. First I wanted to determine if and how ACh release at motor nerve terminals was affected by the alteration of the normal complement of VGCCs β subunit. To answer this question, intracellular recordings at the neuromuscular junction (NMJ) of adult lh and wt mice were obtained. The recordings were done in the presence of different concentrations of Ca 2+ in physiological saline to determine if varying the Ca 133 2+ concentration affected Figure 4.1: Stucture of the α1A subunit showing the tg mutation. Schematic representation of the predicted transmembrane topology of the α1 subunit, with the localization of the β-interaction domain (BID) marked in red. The cylinders indicate transmamebrane repeats (I-IV) of 6 transmembrane segments (1-6). The position of the tg mutation is indicated by an arrow (Adapted from Fletcher et al., 1996). tg 12345 I 6 II III IV NH2 COOH 134 2+ the frequency of spontaneous release. Also, an increase in Ca concentration, leads to an increase in vesicle release, which in turn increases recycling. Therefore if this mutation affected the frequency of release, it would be easier to visualize by using a higher extracellular Ca 2+ concentration, which would induce a faster frequency of release. There was no difference in amplitude or frequency of spontaneous release (MEPPs) in lh mice as compared to wt (Fig. 2.1). This led to the question of what would happen if the charge carrier was changed to either Sr 2+ 2+ or Ba . These cations can substitute for Ca 2+ in the generation of MEPPs (Anwyl et al., 1982; Elmqvist and Feldman, 1965; Mellow et al, 1982; Silinsky, 1978). 2+ Neurotransmitter release occurs through vesicle fusion which involves different Ca 2+ binding sites. These sites involved in asynchronous release are sensitive to Sr 2+ and Ba , while those involved in synchronous exocytosis are only sensitive to Sr 2+ (Van Der Kloot and Molgó, 1994; Zefirov and Grigor‟ev, 2010; Zefirov and Mukhamed‟yarov, 2004). Therefore, we substituted Ca 2+ 2+ for Ba 2+ or Sr to determine if the asynchronous release process responded differentially to these ions (Fig. 2.2). There was no change in MEPP amplitude in lh as compared to wt (Fig. 2.2a). This implies that there is no difference in the amount of ACh each synaptic vesicle carries. However, when the frequency of spontaneous release was measured in the presence of Ba 2+ 2+ or Sr , wt mice showed an increase in frequency with the use of these cations. In lh mice, however, these 135 cations did not alter the frequency of MEPPs (Fig 2.2b).This could imply that this mutation has a presynaptic effect. Nerve-evoked release of ACh (EPPs) and quantal content in lh mice in the presence of Ca 2+ as a charge carrier, were significantly altered (Figs. 2.3 and 2.4). As 2+ 2+ stated before, Sr , but not Ba , can replace Ca 2+ in supporting synaptic transmission, since the calcium-binding site for synchronous exocytosis is sensitive to Sr 2+ ions (Dodge et al., 1969; Meiri and Rahamimoff, 1971; Miledi, 1966; Silinsky, 1977, 1981, 1985; Zefirov and Grigor‟ev, 2010). When utilizing Sr 2+ as a charge carrier, I also observed that EPP amplitude and quantal content in lh mice were significantly decreased as compared to wt mice (Figs. 2.5 and 2.6). This may be due to the lower amount of current carried by Sr 2+ in the VGCC. Burgess et al. (1999) found that despite the loss of β4 subunit, whole cell P-type current was present at normal amplitude in lh cerebellar Purkinje neurons. McEnery et al. (1998) showed that in forebrain and cerebellum of lh mice they express an immature form of N-type VGCCs, which in turn alters Ca 2+ currents. The results presented so far in my dissertation opened the possibility that the electrophysiological changes I was measuring in lh mice could be due to alterations in the Ca 2+ current flowing through VGCCs. To test whether this hypothesis is true, it would be interesting to measure Ca 2+ current flowing through motor nerve terminals of lh mice and compared them to wt mice. These measurements could be performed in triangularis sterni (TS) muscle with its 136 intercostal nerves in adult lh and wt mice. Changes in the voltage potential arise due to the summation of currents flowing longitudinally outside of the axon and within the perineural sheath of motor neurons isolated from the intercostal nerve present in TS muscle. K + channel blockers {such as Tetraethylammonium (TEA) and 3,4- diaminopyridine (DAP)} should be used to unmask the Ca 2+ currents present (that are + normally obscured by the presence of K currents) (Brigant and Mallart, 1982; Mallart, 1985; McArdle et al., 1981; Smith et al., 1995; Xu and Atchison, 1996). Since the β subunit is involved in modifying the kinetics of the VGCCs. I suspect that lh mice will have a decreased peak Ca 2+ current flowing through them. Based on these results, I also wanted to examine whether the vesicular release dynamics were affected by the absence of the β4 subunit in lh mice, which could explain the decrease in ACh release. In order to do this, I performed FM1-43 staining of the nerve terminal. These experiments are designed to investigate how the lh mutation affects the availability of quanta release, the exocytosis of vesicles and their recycling (Betz and Bewick, 1993; Reid et al., 1999). The entire process of destaining took approximately 3.5 min to be completed in wt mice; however at that time there was still a large amount of fluorescence remaining in nerve terminals of lh mice (Fig. 2.7). It took ~ 15 min for lh preparations to achieve a level of destaining similar to that measured in wt mice (Fig. 2.8). Thus the vesicle release process is significantly slower in lh mice. However this finding opens additional questions. The slower process could be due to a releasable pool size problem or a reduced Ca 2+ current. To test this, vesicle release was induced by two 137 different mechanisms: α-LTx (Hubbard et al., 1968; Rosenmund and Stevens, 1996; Rossetto et al., 2004; Sons and Plomp, 2006; Tedesco et al., 2009; Xu et al., 2002) or hypertonic solution (Palma et al., 2011; Sons and Plomp, 2006). In each case, the rate of the destaining process was the same in lh and wt mice; suggesting that a decreased Ca 2+ current might contribute to the slow process in the release, and not the vesicle pool size (readily releasable or reserve pool) in lh mice. Therefore, destaining of FM1-43 appeared to be slower and less complete in lh mice. However, it would be of interest to determine if the vesicle release process is altered between lh and wt mice, when Ca for Sr 2+ 2+ or Ba 2+ is substituted when performing FM1-43 experiments. In conclusion, ACh release at somatic motor nerve terminals is significantly altered in the lh mice. This might be due to an effect in the Ca 2+ current which could then translate to a slower vesicle release or recycling process, leading to a decrease in ACh release. This could explain the abnormal phenotype seen in lh mice. In the future it would be interesting to study what would happen if these animals + were treated with DAP. DAP blocks K channel efflux in nerve terminals. This way the duration of action potentials is increased, allowing VGCCs to remain open for longer periods of time. Hence, a greater amount of ACh is released from the motor nerve terminal. According to the results of this dissertation, the lh mutation affects ACh release, which could account for their phenotypic “lethargic” behavior, but it would be interesting to know what would happen if we increase the availability of ACh by + inhibiting motor nerve terminals K channels. Would this change the phenotype of these 138 mice? Would it alter their behavior since they would have more ACh available to stimulate the muscle? This experiment could be done in two ways. In the first part diaphragm dissections could be treated with an inhibitor of DAP and then intracellular recordings from the NMJ could be performed; this would enable one to see if there was a change in the postsynaptic response. If there is an increase in postsynaptic response as measured by an increase in EPP amplitude which translates to an increase in quantal content, then I could potentially go to animal trial. Mice would be treated with an DAP and the progress of the mice would be assessed by behavioral tests such as foot print test to quantify motor function and the stride length, rearing-climbing to assess motor movement and coordination, and grip strength test to measure forelimb grip strength as an indicator of neuromuscular function. Additionally weight changes and eating habits should be monitored, since lh mice have reduced body weight as compared to wt mice. Since DAP has been tried in LEMS patients and proven to have negligible side effects (perioral and digital paresthesia), and laboratory studies showed no evidence of toxicity affecting liver, renal, hematologic, endocrinologic, encephalographic, or electrocardiologic function (Sanders et al., 2000), therefore, it could be safe to consider this treatment for lh mice, although constant monitoring of the animals is advised. Based on the fact that lh mice have a decrease in ACh release and a slower process of release, I wondered if there was an alteration in the Ca 2+ current, then, what was responsible for this alteration. Therefore I wanted to determine if the P/Q-type VGCCs are involved in controlling ACh release at adult lh motor nerve terminals. Additionally, I also wanted to assess the possible involvement of other types of VGCCs in controlling ACh release. Finally, I wanted to determine which β subunits were 139 substituting for the absence of β4. The diaphragm muscle presents a scarcity of VGCC protein levels. When performing western blots in this tissue, the western blot was not sufficiently sensitive to detect VGCCs subunits in the presynaptic area. Therefore, I performed western blots to determine the protein levels in cerebellum of lh and wt mice. Since lh animals lack the β4 subunit, I first wanted to determine whether this mutation affected the protein levels of the remaining different β subunits in cerebellum of adult lh mice. Additionally I wanted to assay for α1A levels since the β4 subunit normally coassociates with α1A; therefore I wanted to test if the absence of β4 affected the levels of α1A. Protein level analysis showed that even though lh animals lack the β4 subunit, the protein levels of the α1A subunit are the same for lh and wt animals. This could be due to the significantly higher levels of β1 and β3, which might compensate for the lack of β4 (Fig. 3.1). This could imply that ACh release was controlled by P/Q-type VGCCs, in which the α1A subunit was abnormally associating with either β1 and/or β3 subunit. To determine if this was correct, I performed intracellular recordings using ω-agatoxin IVA (ω-Aga IVA) (a specific antagonist of P/Q-type VGCCs). ω-Aga IVA significantly decreased evoked ACh release in lh and wt animals (60% and 76%, respectively) (Fig. 3.2). Since the effect of ω-Aga IVA was lower in lh mice, this could be explained by the fact that maybe other types of VGCCs are also controlling ACh release in adult lh mice. To determine if this was the case, I performed additional intracellular recordings in the 140 presence of other VGCCs antagonists. The use of specific antagonists for L- and N-type VGCC (nimodipine and ω-Ctx GVIA respectively) did not affect quantal content, implying that neither L- nor N-type VGCCs control ACh release in either wt or lh mice. However, SNX 482 an R-type VGCC antagonist significantly decreased quantal content in lh (46%), but not wt mice (8%) (Fig. 3.2), thus implying that R-type VGCC contribute to the control of the ACh release at motor nerve terminals of adult lh but not wt mice. Co-application of ω-Aga IVA and SNX 482 significantly reduced ACh release in lh mice to a level similar to that observed after the application of ω-Aga IVA alone in wt mice. This suggests that ACh release in lh animals is controlled by P/Q- and R- type VGCCs, and that both types seem to contribute to a similar extent (Fig. 3.3). The western blots showed increased level of β1 and β3 subunits, which might be compensating for the absence of β4 subunit. Additionally, electrophysiology had shown that ACh release is controlled by P/Q- and R-type VGCCs in lh mice. I, therefore, wanted to verify the presence of the different α1 and β subunits at the NMJ of adult mice by performing immunohistochemistry in sections of EDL muscle. I chose this muscle to permit me use the diaphragm for electrophysiological studies and the EDL for immunohistochemical studies from the same animals. Some subtle differences exist between diaphragm and EDL. The diaphragm is a mix fiber type of muscle, and the EDL is a fast twitching type of muscle. However, there is enough similarity between them that it will not present a problem. EDL sections were stained with specific antibodies against the various α1 and β subunits as well as the nicotinic ACh receptor (nACh - as a postsynaptic marker). 141 When the relative amount of fluorescence was quantified for each subunit in both genotypes (Fig 3.5), I found a significant increase in α1E, β3 and β1 in lh mice as compared to their wt littermates. However, the levels of α1A in lh mice were not significantly different from wt mice. These results are consistent with my western blot and electrophysiology findings. When the juxtaposition of the VGCCs subunits against the somatic nACh receptor was quantified, it showed that in lh mice there is a 58% and 44% juxtaposition of α1A and α1E respectively, while in wt the α1A subunit had a 73% juxtaposition. With regards to the β subunits, in lh mice β1 and β3 juxtaposed with the nACh at 88% and 65%, respectively (Fig. 3.6). The immunohistochemistry data confirmed the electrophysiology data; both α1A and α1E staining overlap that of nicotinic receptors at the NMJ. Determining additionally that there is an increase in β3 and β1 subunit in lh mice, which might compensate for the absence of β4 subunit. Taken together, these results suggest that ACh release is mediated by P/Q- as well as R-type VGCCs in lh mice. However, it would be interesting to confirm these results by determining whether there is a change in the mRNA level of the different β subunits. RT-PCR assays could be performed, in which mRNA is isolated from spinal cords of wt and lh mice. We know that the β3 subunit is extensively expressed in the brain, especially in the hippocampus (Ludwig et al., 1997; Namkung et al., 1998). It has been shown that the β3 -/- mouse exhibits enhanced long term memory and N-methyl-D-aspartate receptor 142 (NMDAR)-dependent long term potentiation (LTP) as well as NMDAR-mediated synaptic responses and increase NR2B level in the hippocampus (Jeon et al., 2008). It is therefore possible that β3 subunits normally suppress NMDA receptors, although whether this effect is a result of a direct interaction remains to be determined (Jeon et al., 2008). Based on these results, and considering that the β3 subunit is significantly increased in lh mice, it would be interesting to determine whether these animals have an impairment in memory formation. To study memory formation I propose two separate sets of tests. First a set of behavioral tests, given the low motor coordination these animals present, some commonly used tests to evaluate memory formation are not going to be able to be used (such as the case of water maze or contextual fear conditioning). Therefore, I propose we perform social transmission of food preference and novel object recognition memory task. Second, I propose we evaluate LTP by whole-cell patch clamp recording on hippocampal slices and compare the results to those obtained in the wt littermates. Additionally, Lambert-Eaton Myasthenic Syndrome (LEMS) is an autoimmune disease, characterized by muscle weakness, in which autoantibodies primarily target the α1A subunit of the P/Q-type calcium channels (Flink and Atchison, 2003; Hewett and Atchison, 1992; Nagel et al., 1988; Suzuki, 2010; Takamori, 2008). One goal of future studies would be to determine how lh mice respond to LEMS antibodies. Since lh mice have both P/Q- and R-type VGCCs we could test whether R-type VGCCs are also targeted by LEMS autoantibodies as are P/Q-type. Additionally we could test whether there is a compensation by different VGCCs localized at the motor nerve terminals, since it has been shown that after treating wt mice with LEMS plasma they compensate with Ltype calcium channels for the attack on the P/Q-type performed by LEMS autoantibodies 143 (Flink and Atchison, 2002). In order to perform this study, lh mice would be exposed to LEMS plasma by passive transfer of 1.5 ml of plasma from LEMS patients over a period of 30 days (Hewett and Atchison, 1992; Smith et al., 1995), after which time the mice would be sacrificed and evaluated to determine whether LEMS antibodies had any effect on them. The involvement of the different VGCCs would be assessed by intracellular recordings of the NMJ in the presence of different VGCC antagonists. The phrenic nerve will be stimulated at 0.5 and 50 Hz to determine whether there is facilitation, a characteristic of LEMS, which would show whether the passive transfer was successful and if the mice acquired LEMS-like characteristics. Additionally, immunohistochemistry could be performed to determine the localization of the different subunits of VGCC present in animals treated with LEMS plasma. Although some of my results are similar to those presented by Kaja (2007), there are several areas of disagreement. When evaluating which VGCCs control ACh release, he used only one antagonist, ω-Aga IVA which is the specific antagonist for the P/Qtype VGCCs, and did not investigate the possible involvement of other VGCCs. Moreover he did not evaluate protein levels or subunits localizations by the use of western blots or immunohistochemistry studies to confirm the results he obtained. Additionally, he determines that P/Q-type VGCCs are the only ones responsible for the control ACh release in lh mice. These differences between our results could be explained by the age difference between the animals we used. While he worked with 6 wks old mice, I performed all the experiments present in this dissertation using mice between the ages of 3 to 9 mos. Several studies have demonstrated developmental changes in the expression of the α1 subunits of VGCCs (Gray et al., 1992; Rosato Siri and Uchitel, 144 1999). It has been determined that β subunits regulate the channel properties and targeting of α1. Studies done in rat brain showed that the immature N-type VGCC is comprised mainly of β1b, while the mature N-type is comprised of β3> β1b> β4 (Ludwig et al., 1997; McEnery et al., 1998; Tanaka et al., 1995; Vance et al, 1998). It is not clear what is the developmental pattern of expression for the β subunits in nerve terminals, and if the pattern of expression is finalized by 6 wks, especially in lh mice. Full expression of α1E and the different β subunits may not occur until later than 6 wks postnatal in lh mice. Thus at the comparatively young age of mice in which Kaja (2007) examined ACh release, there might have been a greater dependence on P/Q-type, as seen by a larger percentage of sensitivity of his animals to ω-Aga IVA, and a lesser contribution of R-type channels than in 3 to 9 mo old animals that I used. These differences could possibly lead to differences in our results. Kaja‟s work showed that the lh mutation did not affect MEPP amplitude or frequency, or in EPP amplitude or quantal content. Something that over the length of this dissertation I have shown using different techniques to assess for ACh release (both spontaneous and nerve evoked release). The study performed by Kaja et al. (2007) did not include the use of different divalent cations to substitute Ca 2+ as charge carriers, which might explain why he did not see any difference in MEPP frequency, since there is 2+ no difference in the frequency of spontaneous release when using Ca carrier; but these differences become apparent when substituting Ca 2+ as a charge 2+ by Ba or Sr 2+ (chapter 2). Another possible explanation for our differences might be methodological. 145 While I performed recordings from 5 to 10 end-plates of up to 5 minutes each; Kaja‟s work was done by recording at least 30 EPPs and 30 MEPPs at each NMJ. At their reported frequencies in their article (0.81 MEPPs/s in lh and 0.93 MEPPs/s in wt) this implies recordings no longer than 40 s. I sampled over a much longer interval. At a MEPP frequency of 1Hz for 5 minutes, I sampled ~300 MEPPs. Given the fact that we both stimulated EPPs at 0.5 Hz, this implies his recordings were about 90 s long. At a stimulation of 0.5 Hz, for 5 minutes I sampled 150 EPPs. Short time intervals could reflect inaccurate results, which could not be representative of what is going on over longer intervals. This would need to be examined more rigorously, by repeating the experiments using his protocol. This is not the first time that neuronal plasticity at the NMJ has been reported (Flink and Atchison, 2002; Pardo et al., 2006; Xu et al., 1998). The lh mutation seems to present a compensatory mechanism in which VGCCs present at motor nerve terminals are formed by α1A-β1/-β3 and α1E-β1/-β3 (Fig. 4.2). This is consistent with the fact that the AID consensus sequence is highly conserved among all the subunits (de Waard et al., 1994). Additionally, the work done in rabbit brain membranes by Liu et al. (1996), has shown that there is a preferential α1- β pairing (β4 > β3 >> β1b  β2). Although my findings do not show a preference between β3 and β1 pairing with either α1A or α1E. This difference might suggest that the interaction of the α1A and β subunits in vivo may be dependent upon the species, spatial and temporal expression of β subunits and the affinity of α1A-β subunit interaction. However, this abnormal association between α1A- β1/-β3 146 and the presence of α1E might be a possible cause for the slower vesicle release process observed in lh mice. 147 Figure 4.2: Proposed model of the NMJ of adult lh mice. (a) In motor nerve terminals of adult wt mice we find α1A which normally co-associates with β4 since in wt ACh release is primarily controlled by P/Q-type VGCCs. Additionally wt mice have a faster vesicle release process. (b) In motor nerve terminals of adult lh mice there is localization of α1A and α1E which seem to associate with either β1 or β3 indiscriminately. Thus in lh mice ACh release is controlled by P/Q- and R-type calcium channels, which are also characterized by a slower release process. α1A α1E β4 β1 β3 ACh nACh receptors 148 Figure 4.2 (cont’d) (a) wt NMJ 149 Figure 4.2 (cont’d) (b) lh NMJ 150 REFERENCES 151 REFERENCES Adams, B A, and K G Beam. “Muscular dysgenesis in mice: a model system for studying excitation-contraction coupling.” FASEB 4, no. 10 (1990): 2809-2816. Almers, W, R Fink, and P T Palade. “Calcium depletion in frog muscle tubules: the decline of calcium current under maintained depolarization.” J Physiol 312 (1981): 177207. Anderson, D C, S C King, and S M Parsons. “Proton gradient linkage to active uptake of [3H]acetylcholine by Torpedo electric organ synaptic vesicles.” Biochem 21, no. 13 (1982): 3037-3043. Arias, Juan Manuel, Janet Murbartián, Iuliia Vitko, Jung-Ha Lee, and Edward Perez2+ Reyes. “Transfer of beta subunit regulation from high to low voltage-gated Ca channels.” FEBS Letters 579, no. 18 (2005): 3907-3912. Arikkath, Jyothi, and Kevin P Campbell. “Auxiliary subunits: essential components of the voltage-gated calcium channel complex.” Curr Opin Neurobiol13, no. 3 (2003): 298307. Ashcroft, F M. “Ion Channels and Disease: Channelopathies.” Nat Cell Biol. Vol. 2. Academic Press, 2000. Augustine GJ, Eckert R. “Divalent cations differentially support transmitter release at the squid giant synapse.” J Physiol 346 (1984): 257-271. Augustine, G J, M P Charlton, and S J Smith. “Calcium action in synaptic transmitter release.” Annl Rev Neurosc 10 (1987): 633-693. Barclay, J, N Balaguero, M Mione, S L Ackerman, V A Letts, J Brodbeck, C Canti, et al. “Ducky mouse phenotype of epilepsy and ataxia is associated with mutations in the Cacna2d2 gene and decreased calcium channel current in cerebellar Purkinje cells.” J Neurosc 21, no. 16 (2001): 6095-6104. Bass, L. and Moore, W. J. “Electrokinetic mechanism of miniature postsynaptic potentials.” Proc Natn Acad Sci U.S.A. 55 (1966): 1214-1217. Bean, B P. “Neurotransmitter inhibition of neuronal calcium currents by changes in channel voltage dependence.” Nature 340, no. 6229 (1989): 153-156. Bell, D C, A J Butcher, N S Berrow, K M Page, P F Brust, A Nesterova, K A Stauderman, G R Seabrook, B Nürnberg, and A C Dolphin. “Biophysical properties, 152 pharmacology, and modulation of human, neuronal L-type (alpha(1D), Ca(V)1.3) voltage-dependent calcium currents.” J Neurophys 85, no. 2 (2001): 816-827. Bennett, M K, N Calakos, and R H Scheller. “Syntaxin: a synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones.” Science 257, no. 5067 (1992a): 255-259. Bennett, M K, N Calakos, T Kreiner, and R H Scheller. “Synaptic vesicle membrane proteins interact to form a multimeric complex.” J Cell Biol 116, no. 3 (1992b): 761-775. Berecki, G, L Motin, A Haythornthwaite, S Vink, P Bansal, R Drinkwater, C I Wang, et al. “Analgesic (omega)-conotoxins CVIE and CVIF selectively and voltage-dependently block recombinant and native N-type calcium channels.” Mol Pharm 77, no. 2 (2010): 139-148. Bernstein, Geula M, and Owen T Jones. “Kinetics of internalization and degradation of N-type voltage-gated calcium channels: role of the alpha2/delta subunit.” Cell Calcium 41, no. 1 (2007): 27-40. Berrou, L, H Klein, G Bernatchez, and L Parent. “A specific tryptophan in the I-II linker is a key determinant of beta-subunit binding and modulation in Ca(V)2.3 calcium channels.” Biophys J 83, no. 3 (2002): 1429-1442. Berrou, Laurent, Yolaine Dodier, Alexandra Raybaud, Audrey Tousignant, Omar Dafi, Joelle N Pelletier, and Lucie Parent. “The C-terminal residues in the alpha-interacting domain (AID) helix anchor CaV beta subunit interaction and modulation of CaV2.3 channels.” J Biol Chem 280, no. 1 (2005): 494-505. Betz, W J, and G S Bewick. “Optical monitoring of transmitter release and synaptic vesicle recycling at the frog neuromuscular junction.” J Physiol 460 (1993): 287-309. Bidaud, Isabelle, Alexandre Mezghrani, Leigh Anne Swayne, Arnaud Monteil, and Philippe Lory. “Voltage-gated calcium channels in genetic diseases.” Biochimica et Biophysica Acta 1763, no. 11 (2006): 1169-1174. Birnbaumer, L, N Qin, R Olcese, E Tareilus, D Platano, J Costantin, and E Stefani. “Structures and functions of calcium channel beta subunits.” J Bioenergetics and Biomembranes 30, no. 4 (1998): 357-375. Black, John Logan. “The voltage-gated calcium channel gamma subunits: a review of the literature.” J Bioenergetics and Biomembranes 35, no. 6 (2003): 649-660. Bourinet, E, G W Zamponi, A Stea, T W Soong, B A Lewis, L P Jones, D T Yue, and T P Snutch. “The alpha 1E calcium channel exhibits permeation properties similar to lowvoltage-activated calcium channels.” J Neurosc 16, no. 16 (1996): 4983-4993. 153 Boyd, I A, and A R Martin. “The end-plate potential in mammalian muscle.” J Physiol 132, no. 1 (1956): 74-91. Breer, H, S J Morris, and V P Whittaker. “Adenosine triphosphatase activity associated with purified cholinergic synaptic vesicles of Torpedo marmorata.” FEBS 80, no. 1 (1977): 313-318. Breustedt, J, K E Vogt, R J Miller, R A Nicoll, and D Schmitz. “Alpha1E-containing Ca2+ channels are involved in synaptic plasticity.” PNAS 100, no. 21 (2003): 1245012455. Brice, Nicola L, and Annette C Dolphin. “Differential plasma membrane targeting of voltage-dependent calcium channel subunits expressed in a polarized epithelial cell line.” J Physiol 515, no. 3 (1999): 685-694. Brigant JL, and Mallart A. “Presynaptic currents in mouse motor endings.” J Physiol 333 (1982): 619-636. Browning, E T, and M P Schulman. “(14C) acetylcholine synthesis by cortex slices of rat brain.” J Neurochem 15, no. 12 (1968): 1391-1405. Burgess, D L, G H Biddlecome, S I McDonough, M E Diaz, C A Zilinski, B P Bean, K P Campbell, and J L Noebels. “beta subunit reshuffling modifies N- and P/Q-type Ca2+ channel subunit compositions in lethargic mouse brain.” Mol Cell Neurosc 13, no. 4 (1999): 293-311. Burgess, D L, J M Jones, M H Meisler, and J L Noebels. “Mutation of the Ca2+ channel beta subunit gene Cchb4 is associated with ataxia and seizures in the lethargic (lh) mouse.” Cell 88, no. 3 (1997): 385-392. Burgess, D L, and J L Noebels. “Single gene defects in mice: the role of voltagedependent calcium channels in absence models.” Epilepsy Research 36, no. 2-3 (1999): 111-122. Butcher, Adrian J, Jérôme Leroy, Mark W Richards, Wendy S Pratt, and Annette C Dolphin. “The importance of occupancy rather than affinity of CaV(beta) subunits for the calcium channel I-II linker in relation to calcium channel function.” J Physiol 574, no. Pt 2 (2006): 387-398. Béguin, P, R N Mahalakshmi, K Nagashima, Damian Hwee Kiat Cher, N Kuwamura, Y Yamada, Y Seino, and W Hunziker. “Roles of 14-3-3 and calmodulin binding in subcellular localization and function of the small G-protein Rem2.” Biochem J 390, no. Pt 1 (2005): 67-75. 154 Béguin, P, K Nagashima, T Gonoi, T Shibasaki, K Takahashi, Y Kashima, N Ozaki, K Geering, T Iwanaga, and S Seino. “Regulation of Ca2+ channel expression at the cell surface by the small G-protein kir/Gem.” Nature 411, no. 6838 (2001): 701-706. Béguin, Pascal, Ramasubbu Narayanan Mahalakshmi, Kazuaki Nagashima, Damian Hwee Kiat Cher, Hiroki Ikeda, Yuichiro Yamada, Yutaka Seino, and Walter Hunziker. “Nuclear sequestration of beta-subunits by Rad and Rem is controlled by 14-3-3 and calmodulin and reveals a novel mechanism for Ca2+ channel regulation.” J Mol Biol 355, no. 1 (2006): 34-46. Caddick SJ, Wang C, Fletcher CF, Jenkins NA, Copeland NG, Hosford DA. “Excitatory but not inhibitory synaptic transmission is reduced in lethargic [Cacnb4(lh)] and tottering (Cacna1atg) mouse thalami.” J Neurophysiol 81 (1999): 2066-2074. Canti, C, A Davies, N S Berrow, A J Butcher, K M Page, and A C Dolphin. “Evidence for two concentration-dependent processes for beta-subunit effects on alpha1B calcium channels.” Biophys J 81, no. 3 (2001): 1439-1451. Canti, C, A Davies, and A C Dolphin. “Calcium Channel alpha2delta Subunits: Structure, Functions and Target Site for Drugs.” Curr Neuropharmacol 1 (2003): 209–218. Cantí, C, M Nieto-Rostro, I Foucault, F Heblich, J Wratten, M W Richards, J Hendrich, et al. “The metal-ion-dependent adhesion site in the Von Willebrand factor-A domain of α2δ subunits is key to trafficking voltage-gated Ca2+ channels.” PNAS 102, no. 32 (2005): 11230-11235. Cao, X, N Ballew, and C Barlowe. “Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins.” EMBO 17, no. 8 (1998): 21562165. Cao, Yu-Qing, Erika S Piedras-Rentería, Geoffrey B Smith, Gong Chen, Nobutoshi C Harata, and Richard W Tsien. “Presynaptic Ca2+ channels compete for channel typepreferring slots in altered neurotransmission arising from Ca2+ channelopathy.” Neuron 43, no. 3 (2004): 387-400. Carbone, E, and H D Lux. “A low voltage activated fully inactivating calcium conductance in embryonic chick sensory neurones.” Nature 310 (1984): 501-502. Castellano, A, X Wei, L Birnbaumer, and E Perez-Reyes. “Cloning and expression of a third calcium channel beta subunit.” J Biol Chem 268, no. 5 (1993): 3450-3455. Catterall, W A. “Functional subunit structure of voltage-gated calcium channels.” Science 253, no. 5027 (1991): 1499-1500. Catterall, W A. “Structure and function of neuronal Ca2+ channels and their role in neurotransmitter release.” Cell Calcium 24, no. 5-6 (1998): 307-323. 155 Catterall, W A. “Structure and function of voltage-gated ion channels.” Ann Rev Biochem 64, no. 12 (1995): 493-531. Catterall, W A. “Structure and regulation of voltage-gated Ca2+ channels.” Ann Rev Cell Dev Biol16, no. 13 (2000): 521-555. Catterall W A, and J Striessnig. “Receptor-sites for Ca2+ channel antagonists.” TIPS 13, no. 6 (1992): 256-262. Catterall WA, Perez-Reyes E, Snutch TP, and Striessnig J. “International Union of Pharmacology. XLVIII. Nomenclature and structure-function relationships of voltagegated calcium channels.” Pharmacol Revs 57, no. 4 (2005): 411-425. Ceccarelli B, Grohovaz F, and Hurlbut WP. “Freeze-fracture studies of frog neuromuscular junctions during intense release of neurotransmitter. II. Effects of electrical stimulation and high potassium.” J Cell Biol81, no. 1 (1979): 178-192. Ceccarelli, B, Hurlbut WP, and Mauro A. “Turnover of transmitter and synaptic vesicles at the frog neuromuscular junction.” J Cell Biol 57, no. 2 (1973): 499-524. Chapman ER. “How does synaptotagmin trigger neurotransmitter release?” Ann Rev Biochem 77 (2008): 615-641. Chen YH, Li MH, Zhang Y, He LL, Yamada Y, Fitzmaurice A, Shen Y, Zhang H, Tong L, and Yang J. “Structural basis of the alpha1-beta subunit interaction of voltage-gated Ca2+ channels.” Nature 429, no. 6992 (2004): 675-680. Chen YH, He LL, Buchanan DR, Zhang Y, Fitzmaurice A, Yang J. “Functional dissection of the intramolecular Src homology 3-guanylate kinase domain coupling in voltage-gated Ca2+ channel beta-subunits.” FEBS Letters 583, no. 12 (2009): 1969-1975. Chen Y, Lu J, Pan H, Zhang Y, Wu H, Xu K, Liu X, et al. “Association between genetic variation of CACNA1H and childhood absence epilepsy.” Ann Neurol 54, no. 2 (2003): 239-243. Chen Z, Kujawa SG, and Sewell WF. “Auditory sensitivity regulation via rapid changes in expression of surface AMPA receptors.” Nat Neurosc 10, no. 10 (2007): 1238-1240. Chien AJ, Zhao X, Shirokov RE, Puri TS, Chang CF, Sun D, Rios E, Hosey MM. “Roles of a membrane-localized beta subunit in the formation and targeting of functional L-type Ca2+ channels.” J Biol Chem 270, no. 50 (1995): 30036-30044. Chu PJ, Larsen JK, Chen CC, and Best PM. “Distribution and relative expression levels of calcium channel beta subunits within the chambers of the rat heart.” JMCC 36, no. 3 (2004): 423-434. 156 Colecraft HM, Alseikhan B, Takahashi SJ, Chaudhuri D, Mittman S, Yegnasubramanian V, Alvania RS, Johns DC, Marbán E, and Yue DT. “Novel functional properties of Ca(2+) channel beta subunits revealed by their expression in adult rat heart cells.” J Physiol 541, no. 2 (2002): 435-452. Collier, B. “Choline analogues: their use in studies of acetylcholine synthesis, storage, and release.” CJPP 64, no. 3 (1986): 341-346. Davies A, Douglas L, Hendrich J, Wratten J, Tran Van Minh A, Foucault I, Koch D, Pratt WS, Saibil HR, and Dolphin AC. “The calcium channel alpha2delta-2 subunit partitions with CaV2.1 into lipid rafts in cerebellum: implications for localization and function.” J Neurosc 26, no. 34 (2006): 8748-8757. Davies A, Hendrich J, Tran Van Minh A, Wratten J, Douglas L, and Dolphin AC. “Functional biology of the alpha(2)delta subunits of voltage-gated calcium channels.” TIPS 28, no. 5 (2007): 220-228. Davies A, Kadurin I, Alvarez-Laviada A, Douglas L, Nieto-Rostro M, Bauer CS, Pratt WS, and Dolphin AC. “The α2δ subunits of voltage-gated calcium channels form GPIanchored proteins, a posttranslational modification essential for function.” PNAS 107, no. 4 (2010): 1654-1659. Davletov BA, and Sudhof TC. “A single C2 domain from synaptotagmin I is sufficient for high affinity Ca2+/phospholipid binding.” J Biol Chem 268, no. 35 (1993): 2638626390. De Luca A, Rand MJ, Reid JJ, Story DF. “Differential sensitivities of avian and mammalian neuromuscular junctions to inhibition of cholinergic transmission by omegaconotoxin GVIA.” Toxicon. 29, no. 3 (1991):311-320. De Waard M, Pragnell M, and Campbell KP. “Ca2+ channel regulation by a conserved beta subunit domain.” Neuron 13, no. 2 (1994): 495-503. De Waard M, Scott VE, Pragnell M, and Campbell KP. “Identification of critical amino acids involved in alpha1-beta interaction in voltage-dependent Ca2+ channels.” FEBS Letters 380, no. 3 (1996): 272-276. De Waard M, Witcher DR, Pragnell M, Liu H, and Campbell KP. “Properties of the alpha 1-beta anchoring site in voltage-dependent Ca2+ channels.” J Biol Chem 270, no. 20 (1995): 12056-12064. Del Castillo J. “Quantal Components of the end plate potential.” J Physiol 124 (1954): 560-573. Del Castillo J and Katz B. “Quantal components of the end-plate potential.” J Physiol 124, no. 3 (1954): 560-573. 157 Delgado R, Maureira C, Oliva C, Kidokoro Y, and Labarca P. “Size of vesicle pools, rates of mobilization, and recycling at neuromuscular synapses of a Drosophila mutant, shibire.” Neuron 28, no. 3 (2000): 941-953. Dickman DK, Kurshan PT, and Schwarz TL. “Mutations in a Drosophila alpha2delta voltage-gated calcium channel subunit reveal a crucial synaptic function.” J Neurosc 28, no. 1 (2008): 31-38. Dodge FA, Miledi R, and Rahamimoff R. “Strontium and quantal release of transmitter at the neuromuscular junction.” J Physiol 200, no. 1 (1969): 267-283. Dolphin, AC. “Beta subunits of voltage-gated calcium channels.” JOBB 35, no. 6 (2003): 599-620. Donato R, Page KM, Koch D, Nieto-Rostro M, Foucault I, Davies A, Wilkinson T, Rees M, Edwards FA, and Dolphin AC. “The ducky2J mutation in Cacna2d2 results in reduced spontaneous Purkinje cell activity and altered gene expression.” J Neurosc 26, no. 48 (2006): 12576-12586. Doyle J, Ren X, Lennon G, and Stubbs L. “Mutations in the Cacnl1a4 calcium channel gene are associated with seizures, cerebellar degeneration, and ataxia in tottering and leaner mutant mice.” J Intern Mamm Gen Soc 8, no. 2 (1997): 113-120. Dreyer F, Peper K, Akert K, Sandri C, and Moor H. “Ultrastructure of the "active zone" in the frog neuromuscular junction.” Brain Res 62, no. 2 (1973): 373-380. Dung HC. “Deficiency in the thymus-dependent immunity in "lethargic" mutant mice.” Transpl 23, no. 1 (1977): 39-43. Dung HC and Swigart RH. “Experimental studies of “lethargic” mutant mice.” Texas Rep Biol Med 29 (1971): 273-288. Dung HC and Swigart RH. “Histo-pathologic observations of the nervous and lymphoid tissues of “lethargic” mutant mice.” Texas Rep Biol Med 30 (1972): 23-39. Eberst R, Dai S, Klugbauer N, and Hofmann F. “Identification and functional characterization of a calcium channel gamma subunit.” Pflugers Archiv European J Physiol 433, no. 5 (1997): 633-637. El Far O, Martin-Moutot N, Leveque C, David P, Marqueze B, Lang B, Newsom-Davis J, Hoshino T, Takahashi M, and Seagar MJ. “Synaptotagmin associates with presynaptic calcium channels and is a Lambert-Eaton myasthenic syndrome antigen.” Ann NYAS 707, no. 1 (1993): 382-385. Elbaz A, Vale-Santos J, Jurkat-Rott K, Lapie P, Ophoff RA, Bady B, Links TP, et al. “Hypokalemic Periodic Paralysis and the Dihydropyridine Receptor (CACNLIA3): 158 Genotype/Phenotype Correlations for two Predominant Mutations and Evidence for the Absence of a Founder Effect in 16 Caucasian Families.” AJHG 56, no. 2 (1995): 374380. Elias GM, and Nicoll RA. “Synaptic trafficking of glutamate receptors by MAGUK scaffolding proteins.” Tends in Cell Biol 17, no. 7 (2007): 343-352. Ellisman MH, Rash JE, Staehelin LA, and Porter KR. “Studies of excitable membranes. II. A comparison of specializations at neuromuscular junctions and nonjunctional sarcolemmas of mammalian fast and slow twitch muscle fibers.” J Cell Biol 68, no. 3 (1976): 752-774. Eroglu C, Allen NJ, Susman MW, OʼRourke NA, Young Park C, Ozkan E, Chakraborty C, et al. “Gabapentin receptor alpha2delta-1 is a neuronal thrombospondin receptor responsible for excitatory CNS synaptogenesis.” Cell 139, no. 2 (2009): 380-392. Ertel EA, Campbell KP, Harpold MM, Hofmann F, Mori Y, Perez-Reyes E, Schwartz A, et al. “Nomenclature of voltage-gated calcium channels.” Neuron, 2000. Escayg A, De Waard M, Lee DD, Bichet D, Wolf P, Mayer T, Johnston J, Baloh R, Sander T, and Meisler MH. “Coding and noncoding variation of the human calciumchannel beta4-subunit gene CACNB4 in patients with idiopathic generalized epilepsy and episodic ataxia.” AJHG 66, no. 5 (2000): 1531-1539. Fatt P, and Katz B. “An analysis of the end-plate potential recorded with an intracellular electrode.” J Physiol 115, no. 3 (1951): 320-370. Fatt P, and Katz B. “Spontaneous subthreshold activity at motor nerve endings.” J Physiol (Lond) 117 (1952): 109-128. Ferron L, Davies A, Page KM, Cox DJ, Leroy J, Waithe D, Butcher AJ, et al. “The stargazin-related protein gamma 7 interacts with the mRNA-binding protein heterogeneous nuclear ribonucleoprotein A2 and regulates the stability of specific mRNAs, including CaV2.2.” J Neurosc 28, no. 42 (2008): 10604-10617. Fesce R, and Meldolesi J. “Peeping at the vesicle kiss.” Nat Cell Biol 1 (1999): 3-4. Field MJ, Li Z, and Schwarz JB. “Ca2+ channel alpha2-delta ligands for the treatment of neuropathic pain.” J Med Chem 50, no. 11 (2007): 2569-2575. Finlin BS, Correll RN, Pang C, Crump SM, Satin J, and Andres DA. “Analysis of the complex between Ca2+ channel beta-subunit and the Rem GTPase.” J Biol Chem 281, no. 33 (2006): 23557-23566. Finlin BS, Crump SM, Satin J, and Andres DA. “Regulation of voltage-gated calcium channel activity by the Rem and Rad GTPases.” PNAS 100, no. 24 (2003): 14469-14474. 159 Fischer Von Mollard G, Mignery GA, Baumert M, Perin MS, Hanson TJ, Burger PM, Jahn R, and Südhof TC. “rab3 is a small GTP-binding protein exclusively localized to synaptic vesicles.” PNAS 87, no. 5 (1990): 1988-1992. Fletcher CF, Lutz CM, O‟Sullivan TN, Shaughnessy JD, Hawkes R, Frankel WN, Copeland NG, and Jenkins NA. “Absence epilepsy in tottering mutant mice is associated with calcium channel defects.” Cell 87, no. 4 (1996): 607-617. Flink, MT, and Atchison WD. “Passive transfer of Lambert-Eaton syndrome to mice induces dihydropyridine sensitivity of neuromuscular transmission.” J Physiol 543, no. 2 (2002): 567-576. Flink, MT, and Atchison WD. “Ca2+ channels as targets of neurological disease: Lambert-Eaton Syndrome and other Ca2+ channelopathies.” JOBB 35, no. 6 (2003): 697718. Fox AP, Nowycky MC, and Tsien RW. “Kinetic and pharmacological properties distinguishing three types of calcium currents in chick sensory neurones.” J Physiol 394, no. 1 (1987): 149-172. Fox AP, Nowycky MC, and Tsien RW. “Single-channel recordings of three types of calcium channels in chick sensory neurones.” J Physiol 394, no. 1 (1987): 173-200. Freise D, Held B, Wissenbach U, Pfeifer A, Trost C, Himmerkus N, Schweig U, et al. “Absence of the gamma subunit of the skeletal muscle dihydropyridine receptor increases L-type Ca2+ currents and alters channel inactivation properties.” J Biol Chem 275, no. 19 (2000): 14476-14481. Fuller-Bicer GA, Varadi G, Koch SE, Ishii M, Bodi I, Kadeer N, Muth JN, et al. “Targeted disruption of the voltage-dependent calcium channel alpha2/delta-1-subunit.” Am J Physiol Heart Circ Physiol 297, no. 1 (2009): H117-H124. Funke L, Dakoji S, and Bredt DS. “Membrane-associated guanylate kinases regulate adhesion and plasticity at cell junctions.” Ann Rev Biochem 74 (2005): 219-245. Fukuda M, Kojima T and Mikoshida K. “Regulation by bivalent cations of phospholipid binding to the C2A domain of synaptotagmin.” Biochem J 323 (1997): 421-425. Gao B, Sekido Y, Maximov A, Saad M, Forgacs E, Latif F, Wei MH, et al. “Functional properties of a new voltage-dependent calcium channel alpha(2)delta auxiliary subunit gene (CACNA2D2).” J Biol Chem 275, no. 16 (2000): 12237-12242. Gao T, Chien AJ, and Hosey MM. “Complexes of the alpha1C and beta subunits generate the necessary signal for membrane targeting of class C L-type calcium channels.” J Biol Chem 274, no. 4 (1999): 2137-2144. 160 Gaspersic R, Koritnik B, Crne-Finderle N, and Sketelj J. “Acetylcholinesterase in the neuromuscular junction.” Chem Bio Int 119-120 (1999): 301-308. Geib S, Sandoz G, Cornet V, Mabrouk K, Fund-Saunier O, Bichet D, Villaz M, Hoshi T, Sabatier JM, and De Waard M. “The interaction between the I-II loop and the III-IV loop of Cav2.1 contributes to voltage-dependent inactivation in a beta -dependent manner.” J Biol Chem 277, no. 12 (2002): 10003-10013. Geppert M, Goda Y, Hammer RE, Li C, Rosahl TW, Stevens CF, and Südhof TC. “Synaptotagmin I: a major Ca2+ sensor for transmitter release at a central synapse.” Cell 79, no. 4 (1994): 717-727. Gerster U, Neuhuber B, Groschner K, Striessnig J, and Flucher BE. “Current modulation and membrane targeting of the calcium channel α1C subunit are independent functions of the β subunit.” J Physiol 517, no. 2 (1999): 353-368. Glossmann H, Striessnig J, Ferry DR, Goll A, Moosburger K, and Schirmer M. “Interaction between calcium channel ligands and calcium channels.” Circ Res 61, no. 4 (1987): I30-I36. Gollasch M, Ried C, Liebold M, Haller H, Hofmann F, and Luft FC. “High permeation of L-type Ca2+ channels at physiological [Ca2+]: homogeneity and dependence on the alpha 1-subunit.” American J Physiol 271, no. 3 (1996): C842-C850. Gomez-Ospina N, Tsuruta F, Barreto-Chang O, Hu L, and Dolmetsch R. “The C terminus of the L-type voltage-gated calcium channel Ca(V)1.2 encodes a transcription factor.” Cell 127, no. 3 (2006): 591-606. Gonzalez-Gutierrez G, Miranda-Laferte E, Nothmann D, Schmidt S, Neely A, and Hidalgo P. “The guanylate kinase domain of the β-subunit of voltage-gated calcium channels suffices to modulate gating.” PNAS 105, no. 37 (2008): 14198-14203. Grantham CJ, Bowman D, Bath CP, Bell DC, and Bleakman D. “Omega-conotoxin MVIIC reversibly inhibits a human N-type calcium channel and calcium influx into chick synaptosomes.” Neuropharm 33, no. 2 (1994): 255-258. Gray DB, Brusés JL, and Pilar GR. “Developmental switch in the pharmacology of Ca2+ channels coupled to acetylcholine release.” Neuron 8, no. 4 (1992): 715-724. Green MC, and Sidman RL. “Tottering--a neuromusclar mutation in the mouse. And its linkage with oligosyndacylism.” J Heredity53 (1962): 233-237. Gregg RG, Messing A, Strube C, Beurg M, Moss R, Behan M, Sukhareva M, et al. “Absence of the β subunit (cchb1) of the skeletal muscle dihydropyridine receptor alters expression of the α1 subunit and eliminates excitation-contraction coupling.” PNAS 93, no. 24 (1996): 13961-13966. 161 Grishin EV. “Black widow spider toxins: The present and the future.” Toxicon 36, no. 11 (1998): 1693-1701. Gurnett CA, Felix R, and Campbell KP. “Extracellular interaction of the voltagedependent Ca2+ channel alpha2delta and alpha1 subunits.” J Biol Chem 272, no. 29 (1997): 18508-18512. Hagiwara S, and Ohmori H. “Studies of calcium channels in rat clonal pituitary cells with patch electrode voltage clamp.” J Physiol 331 (1982): 231-252. Hanlon MR, Berrow NS, Dolphin AC, and Wallace BA. “Modelling of a voltagedependent Ca2+ channel beta subunit as a basis for understanding its functional properties.” FEBS Letters 445, no. 2-3 (1999): 366-370. Harris KM, and Sultan P. “Variation in the number, location and size of synaptic vesicles provides an anatomical basis for the nonuniform probability of release at hippocampal CA1 synapses.” Neuropharm 34, no. 11 (1995): 1387-1395. Hata Y, Slaughter CA, and Südhof TC. “Synaptic vesicle fusion complex contains unc-18 homologue bound to syntaxin.” Nature 366, no. 6453 (1993): 347-351. He LL, Zhang Y, Chen YH, Yamada Y, and Yang J. “Functional Modularity of the βSubunit of Voltage-Gated Ca2+ Channels.” Biophys J 93, no. 3 (2007): 834-845. Hebb C. “Biosynthesis of acetylcholine in nervous tissue.” Physiol Revs 52, no. 4 (1972): 918-957. Held B, Freise D, Freichel M, Hoth M, and Flockerzi V. “Skeletal muscle L-type Ca2+ current modulation in γ1-deficient and wildtype murine myotubes by the γ1 subunit and cAMP.” J Physiol 539, no. 2 (2002): 459-468. Helton TD, and Horne WA. “Alternative splicing of the beta 4 subunit has alpha1 subunit subtype-specific effects on Ca2+ channel gating.” J Neurosc 22, no. 5 (2002): 1573-1582. Hendrich J, Tran Van Minh A, Heblich F, Nieto-Rostro M, Watschinger K, Striessnig J, Wratten J, Davies A, and Dolphin AC. “Pharmacological disruption of calcium channel trafficking by the α2δ ligand gabapentin.” PNAS 105, no. 9 (2008): 3628-3633. Henkel AW, and Sankaranarayanan S. "Mechanisms of alpha-latrotoxin action." Cell Tiss Res 296, no.2 (1999): 229-233. Herzig S, Khan IFY, Gründemann D, Matthes J, Ludwig A, Michels G, Hoppe UC, et al. “Mechanism of Ca(v)1.2 channel modulation by the amino terminus of cardiac beta2subunits.” FASEB 21, no. 7 (2007): 1527-1538. 162 Heuser JE, and Reese TS. “Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction.” J Cell Biol 57, no. 2 (1973): 315344. Heuser JE, Reese TS, Dennis MJ, Jan Y, Jan L, and Evans L. “Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release.” J Cell Biol 81, no. 2 (1979): 275-300. Heuser JE, Reese TS, and Landis DM. “Functional changes in frog neuromuscular junctions studied with freeze-fracture.” J Neurocyt 3, no. 1 (1974): 109-131. Heuser JE, and Reese TS. “Structural changes after transmitter release at the frog neuromuscular junction.” J Cell Biol. 88, no. 3 (1981): 564-80. Hewett SJ and Atchison WD. “Specificity of Lambert-Eaton myasthenic syndrome immunoglobulin for nerve terminal calcium channels.” Brain Res 599, no. 2 (1992): 324332. Hidalgo P, Gonzalez-Gutierrez G, Garcia-Olivares J, and Neely A. “The alpha1-betasubunit interaction that modulates calcium channel activity is reversible and requires a competent alpha-interaction domain.” J Biol Chem 281, no. 34 (2006): 24104-24110. Hille B. “Ionic channels of excitable membranes.” Sinauer Associates Inc 2nd Edition. Vol. 3, 2001. Hillyard DR, Monje VD, Mintz IM, Bean BP, Nadasdi L, Ramachandran J, Miljanich G, Azimi-Zoonooz A, McIntosh JM, and Cruz LJ. “A new Conus peptide ligand for mammalian presynaptic Ca2+ channels.” Neuron 9, no. 1 (1992): 69-77. Hofmann F, Biel M, and Flockerzi V. “Molecular basis for Ca2+ channel diversity.” Ann Rev Neurosc 17, no. 1 (1994): 399-418. Hosford DA, Clark S, Cao Z, Wilson WA, Lin FH, Morrisett RA, and Huin A. “The role of GABAB receptor activation in absence seizures of lethargic (lh/lh) mice.” Science 257, no. 5068 (1992): 398-401. Huanmian C, Puhl HL, Niu SL, Mitchell DC, and Ikeda SR. “Expression of Rem2, an RGK family small GTPase, reduces N-type calcium current without affecting channel surface density.” J Neurosc 25, no. 42 (2005): 9762-9772. Hubbard JI, Jones SF, and Landau EM. “An examination of the effects of osmotic pressure changes upon transmitter release from mammalian motor nerve terminals.” J Physiol 197, no. 3 (1968): 639-657. Hubbard JI, Llinas R, and Quastel DMJ. “Electrical properties of nerve and muscle. In Electrophysiological Analysis of Synaptic Transmission” Williams & Wilkins (1969). 163 Hullin R, Singer-Lahat D, Freichel M, Biel M, Dascal N, Hofmann F, and Flockerzi V. “Calcium channel beta subunit heterogeneity: functional expression of cloned cDNA from heart, aorta and brain.” the EMBO 11, no. 3 (1992): 885-890. Humeau Y, Doussau F, Vitiello F, Greengard P, Benfenati F, and Poulain B. “Synapsin controls both reserve and releasable synaptic vesicle pools during neuronal activity and short-term plasticity in Aplysia.” J Neurosc 21, no. 12 (2001): 4195-4206. Iles DE, Lehmann-Horn F, Scherer SW, Tsui LC, Olde Weghuis D, Suijkerbuijk RF, Heytens L, et al. “Localization of the gene encoding the α 2 /δ-subunits of the L-type voltage-dependent calcium channel to chromosome 7q and analysis of the segregation of flanking markers in malignant hyperthermia susceptible families.” Hum Mol Gen 3, no. 6 (1994): 969-975. Ino M, Yoshinaga T, Wakamori M, Miyamoto N, Takahashi E, Sonoda J, Kagaya T, et al. “Functional disorders of the sympathetic nervous system in mice lacking the alpha 1B subunit (Cav 2.2) of N-type calcium channels.” PNAS 98, no. 9 (2001): 5323-5328. Jalkanen R, Mäntyjärvi M, Tobias T, Isosomppi J, Sankila EM, Alitalo T, and BechHansen NT. “X linked cone-rod dystrophy, CORDX3, is caused by a mutation in the CACNA1F gene.” J Med Gen 2006. Jarvis SE, and Zamponi GW. “Masters or slaves? Vesicle release machinery and the regulation of presynaptic calcium channels.” Cell Calcium 37, no. 5 (2005): 483–488. Jay SD, Ellis SB, McCue AF, Williams ME, Vedvick TS, Harpold MM, and Campbell KP. “Primary structure of the gamma subunit of the DHP-sensitive calcium channel from skeletal muscle.” Science 248, no. 4954 (1990): 490-492. Jeng CJ, Chen YT, Chen YW, and Tang CY. “Dominant-negative effects of human P/Qtype Ca2+ channel mutations associated with episodic ataxia type 2.” Am J Physiol Cell Physiol 290, no. 4 (2006): C1209-C1220. Jing X, Li DQ, Olofsson CS, Salehi A, Surve VV, Caballero J, Ivarsson R, et al. “CaV2.3 calcium channels control second-phase insulin release.” J Clin Inves115, no. 1 (2005): 146-154. Josephson IR, and Varadi G. “The beta subunit increases Ca2+ currents and gating charge movements of human cardiac L-type Ca2+ channels.” Biophys J 70, no. 3 (1996): 1285-1293. Jun K, Piedras-Rentería ES, Smith SM, Wheeler DB, Beom Lee S, Lee TG, Chin H, et al. “Ablation of P/Q-type Ca2+ channel currents, altered synaptic transmission, and progressive ataxia in mice lacking the α1A-subunit.” PNAS 96, no. 26 (1999): 1524515250. 164 Jurkat-Rott K, Lehmann-Horn F, Elbaz A, Heine R, Gregg RG, Hogan K, Powers PA, Lapie P, Vale-Santos JE, and Weissenbach J. “A calcium channel mutation causing hypokalemic periodic paralysis.” Hum Mol Gen 3, no. 8 (1994): 1415-1419. Kaja S, Todorov B, Van De Ven RCG, Ferrari MD, Frants RR, Van Den Maagdenberg AMJM, and Plomp JJ. “Redundancy of Cav2.1 channel accessory subunits in transmitter release at the mouse neuromuscular junction.” Brain Res 1143 (2007): 92-101. Kamp MA, Krieger A, Henry M, Hescheler J, Weiergräber M, and Schneider T. “Presynaptic „Ca2.3-containing‟ E-type Ca channels share dual roles during neurotransmitter release.” European J Neurosc 21, no. 6 (2005): 1617-1625. Kang MG, Chen CC, Felix R, Letts VA, Frankel WN, Mori Y, and Campbell KP. “Biochemical and biophysical evidence for gamma 2 subunit association with neuronal voltage-activated Ca2+ channels.” J Biol Chem 276, no. 35 (2001): 32917-24. Kang MG, and Campbell KP. “Gamma subunit of voltage-activated calcium channels.” J Biol Chem 278, no. 24 (2003): 21315-8. Kato AS, Zhou W, Milstein AD, Knierman MD, Siuda ER, Dotzlaf JE, Yu H, et al. “New transmembrane AMPA receptor regulatory protein isoform, gamma-7, differentially regulates AMPA receptors.” J Neurosc 27, no. 18 (2007): 4969-4977. Katz B, and Miledi R. “A study of spontaneous miniature potentials in spinal motorneurones.” J Physiol 168, no. 2 (1963): 389-422. Katz B, and Miledi R. “The timing of calcium action during neuromuscular transmission.” J Physiol 189, no. 3 (1967a): 535-544. Katz B, and Miledi R. “A study of synaptic transmission in the absence of nerve impulses.” J Physiol 192, no. 2 (1967b): 407-436. Katz B, and Miledi R. “Further study of the role of calcium in synaptic transmission” J Physiol 207 (1970): 789-801. Katz E, Ferro PA, Cherksey BD, Sugimori M, Llinás R, and Uchitel OD. “Effects of Ca2+ channel blockers on transmitter release and presynaptic currents at the frog neuromuscular junction.” J Physiol 486, no. 3 (1995): 695-706. Kelly K. “The RGK family: a regulatory tail of small GTP-binding proteins.” Tends Cell Biol 15, no. 12 (2005): 640-643. Kelly, SS. “The effect of age on neuromuscular transmission.” J Physiol 274 (1978): 5162. 165 Khan Z, and Jinnah HA. “Paroxysmal dyskinesias in the lethargic mouse mutant.” J Neurosc 22, no. 18 (2002): 8193-8200. Kim MS, Morii T, Sun LX, Imoto K, and Mori Y. “Structural determinants of ion selectivity in brain calcium channel.” FEBS Letters 318, no. 2 (1993): 145-148. Klingauf J, Kavalali ET, and Tsien RW. “Kinetics and regulation of fast endocytosis at hippocampal synapses.” Nature 394, no. 6693 (1998): 581-585. Klugbauer N, Dai S, Specht V, Lacinová L, Marais E, Bohn G, and Hofmann F. “A family of gamma-like calcium channel subunits.” FEBS Letters 470, no. 2 (2000): 189197. Klugbauer N, Lacinová L, Marais E, Hobom M, and Hofmann F. “Molecular diversity of the calcium channel alpha2delta subunit.” J Neurosc 19, no. 2 (1999): 684-691. Klugbauer N, Marais E, and Hofmann F. “Calcium channel alpha2delta subunits: differential expression, function, and drug binding.” JOBB 35, no. 6 (2003): 639-647. Kraszewski K, Daniell L, Mundigl O, and De Camilli P. “Mobility of synaptic vesicles in nerve endings monitored by recovery from photobleaching of synaptic vesicle-associated fluorescence.” J Neurosc 16, no. 19 (1996): 5905-5913. Kriebel ME and Stolper DR. “Non-Poisson distribution in time of small- and large-mode miniature end-plate potentials.” Am J Physiol 229 (1975): 1321-1329. Kuo CC, and Hess P. “Ion permeation through the L-type Ca2+ channel in rat phaeochromocytoma cells: two sets of ion binding sites in the pore.” J Physiol 466 (1993): 629-655. Kuromi H, and Kidokoro Y. “Selective replenishment of two vesicle pools depends on the source of Ca2+ at the Drosophila synapse.” Neuron 35, no. 2 (2002): 333-343. Kurshan PT, Oztan A, and Schwarz TL. “Presynaptic alpha2delta-3 is required for synaptic morphogenesis independent of its Ca2+-channel functions.” Nat Neurosc 12, no. 11 (2009): 1415-1423. Kusunose N, Koyanagi S, Hamamura K, Matsunaga N, Yoshida M, Uchida T, Tsuda M, Inoue K, and Ohdo S. “Molecular basis for the dosing time-dependency of anti-allodynic effects of gabapentin in a mouse model of neuropathic pain.” Mol Pain 6, no. 1 (2010): 83. Lacerda AE, Kim HS, Ruth P, Perez-Reyes E, Flockerzi V, Hofmann F, Birnbaumer L, and Brown AM. “Normalization of current kinetics by interaction between the alpha 1 and beta subunits of the skeletal muscle dihydropyridine-sensitive Ca2+ channel.” Nature 352, no. 6335 (1991): 527-530. 166 Landau EM. “Function and structure of the ACh receptor at the muscle end-plate.” Prog Neurobiol 10, no. 4 (1978): 253-288. Lee S, Kim Y, Back SK, Choi HW, Lee JY, Jung HH, Ryu JH, Suh HW, Na HS, Kim HJ, Rhim H, Kim JI. ”Analgesic effect of highly reversible ω-conotoxin FVIA on N type Ca2+ channels.” Mol Pain. 6, no. 97 (2010): 1-12. Leroy J, Richards MW, Butcher AJ, Nieto-Rostro M, Pratt WS, Davies A, and Dolphin AC. “Interaction via a key tryptophan in the I-II linker of N-type calcium channels is required for beta1 but not for palmitoylated beta2, implicating an additional binding site in the regulation of channel voltage-dependent properties.” J Neurosc 25, no. 30 (2005): 6984-6996. Letts VA, Felix R, Biddlecome GH, Arikkath J, Mahaffey CL, Valenzuela A, Bartlett FS, Mori Y, Campbell KP, and Frankel WN. “The mouse stargazer gene encodes a neuronal Ca2+-channel gamma subunit.” Nat Genet 19, no. 4 (1998): 340-347. Levitan IB, and Kaczmarek LK. “The neuron: cell and molecular biology”. Oxford University Press (1997). Li C, Davletov BA, and Südhof TC. “Distinct Ca2+ and Sr2+ binding properties of synaptotagmins. Definition of candidate Ca2+ sensors for the fast and slow components of neurotransmitter release.” J Biol Chem 270, no. 42 (1995a): 24898-24902. Li C, Ullrich B, Zhang JZ, Anderson RG, Brose N, and Südhof TC. “Ca(2+)-dependent and -independent activities of neural and non-neural synaptotagmins” Nature 375, no. 6532 (1995b): 594-599. Li CY, Song YH, Higuera ES, and Luo ZD. “Spinal dorsal horn calcium channel alpha2delta-1 subunit upregulation contributes to peripheral nerve injury-induced tactile allodynia.” J Neurosc 24, no. 39 (2004): 8494-8499. Li CY, Zhang XL, Matthews EA, Li KW, Kurwa A, Boroujerdi A, Gross J, et al. “Calcium channel alpha2delta1 subunit mediates spinal hyperexcitability in pain modulation.” Pain 125, no. 1-2 (2006): 20-34. Li W, Thaler C, and Brehm P. “Calcium channels in Xenopus spinal neurons differ in somas and presynaptic terminals.” J Neurophysiol 86, no. 1 (2001): 269-279. Lin FH, Cao Z, Hosford DA. ” Increased number of GABAB receptors in the lethargic (lh/lh) mouse model of absence epilepsy.” Brain Res 608, no. 1 (1993):101-106. Lin Z, Witschas K, Garcia T, Chen RS, Hansen JP, Sellers ZM, Kuzmenkina E, Herzig S, and Best PM. “A critical GxxxA motif in the γ6 calcium channel subunit mediates its inhibitory effect on Cav3.1 calcium current.” J Physiol 586, no. 22 (2008): 5349-5366. 167 Liu H, De Waard M, Scott VE, Gurnett CA, VA Lennon, and Campbell KP. “Identification of three subunits of the high affinity omega-conotoxin MVIIC-sensitive Ca2+ channel.” J Biol Chem 271, no. 23 (1996): 13804-13810. Liu H, Felix R, Gurnett CA, De Waard M, Witcher DR, and Campbell KP. “Expression and subunit interaction of voltage-dependent Ca2+ channels in PC12 cells.” J Neurosc 16, no. 23 (1996): 7557-7565. Llinás R, Gruner JA, Sugimori M, McGuinness TL, and Greengard P. “Regulation by synapsin I and Ca(2+)-calmodulin-dependent protein kinase II of the transmitter release in squid giant synapse.” J Physiol 436, no. 21 (1991): 257-282. Llinás R, Steinberg IZ, and Walton K. “Presynaptic calcium currents in squid giant synapse.” Biophys J 33, no. 3 (1981a): 289-321. Llinás R, Steinberg IZ, and Walton K. “Relationship between presynaptic calcium current and postsynaptic potential in squid giant synapse.” Biophys J 33, no. 3 (1981b): 323-351. Llinás R, Sugimori M, Hillman DE, and Cherksey B. “Distribution and functional significance of the P-type, voltage-dependent Ca2+ channels in the mammalian central nervous system.” TINS 15, no. 9 (1992): 351-355. Llinás R, Sugimori M, and Silver RB. “Presynaptic calcium concentration microdomains and transmitter release.” J Physiol (Paris) 86, no. 1-3 (1992): 135-138. Llinás R, Steinberg IZ, and Walton K. “Presynaptic calcium currents and their relation to synaptic transmission: voltage clamp study in squid giant synapse and theoretical model for the calcium gate.” PNAS 73, no. 8 (1976): 2918-2922. Lorenzon NM, Lutz CM, Frankel WN, and Beam KG. “Altered calcium channel currents in Purkinje cells of the neurological mutant mouse leaner.” J Neurosc 18, no. 12 (1998): 4482-4489. Ludwig A, Flockerzi V, and Hofmann F. “Regional expression and cellular localization of the alpha1 and beta subunit of high voltage-activated calcium channels in rat brain.” J Neurosc 17, no. 4 (1997): 1339-1349. Magleby KL and Stevens CF. “A quantitative description of end-plate currents.” J Physiol 223, no. 1 (1972): 173-197. Mallart A, Brigant JL. “Electrical activity at motor nerve terminals of the mouse.” J Physiol (Paris) 78, no. 4 (1982): 407-411. Mallart A. “Electric current flow inside perineurial sheaths of mouse motor nerves.” J Physiol 368 (1985): 565-575. 168 Marchbanks RM. “The activation of presynaptic choline uptake by acetylcholine release.” J Physiol 78, no. 4 (1982): 373-378. Martin PT, Ettinger AJ, and Sanes JR. “A synaptic localization domain in the synaptic cleft protein laminin beta 2 (s-laminin).” Science 269, no. 5222 (1995): 413-416. McArdle JJ, Angaut-Petit D, Mallart A, Bournaud R, Faille L and Brigant JL. “Advantages of the traingularis sterni muscle of the mouse for investigations of synaptic phenomena.” J Neurosci Meth 4 (1981): 109-115. McCleskey EW, Fox AP, Feldman DH, Cruz LJ, Olivera BM, Tsien RW, and Yoshikami D. “Omega-conotoxin: direct and persistent blockade of specific types of calcium channels in neurons but not muscle.” PNAS 84, no. 12 (1987): 4327-4331. McEnery MW, Copeland TD, and Vance CL. “Altered expression and assembly of Ntype calcium channel alpha1B and beta subunits in epileptic lethargic (lh/lh) mouse.” J Biol Chem 273, no. 34 (1998): 21435-21438. McEnery MW, Vance CL, Begg CM, Lee WL, Choi Y, and Dubel SJ. “Differential expression and association of calcium channel subunits in development and disease.” JOBB 30, no. 4 (1998): 409-418. McGee AW, Nunziato DA, Maltez JM, Prehoda KE, Pitt GS, and Bredt DS. “Calcium channel function regulated by the SH3-GK module in beta subunits.” Neuron 42, no. 1 (2004): 89-99. McKeown L, Robinson P, and Jones OT. “Molecular basis of inherited calcium channelopathies: role of mutations in pore-forming subunits.” Acta Pharmacol Sin 27, no. 7 (2006): 799-812. McLachlan EM, and Martin AR. “Non-linear summation of end-plate potentials in the frog and mouse.” J Physiol 311 (1981): 307-324. Meiri U, and Rahamimoff R. “Activation of transmitter release by strontium and calcium ions at the neuromuscular junction.” J Physiol 215, no. 3 (1971): 709-726. Mellow AM, Perry BD, and Silinsky EM. “Effects of calcium and strontium in the process of acetylcholine release from motor nerve endings.” J Physiol 328 (1982): 547562. Mikami A, Imoto K, Tanabe T, Niidome T, Mori Y, Takeshima H, Narumiya S, and Numa S. “Primary structure and functional expression of the cardiac dihydropyridinesensitive calcium channel.” Nature 340, no. 6230 (1989): 230-233. 169 Milani D, Malgaroli A, Guidolin D, Fasolato C, Skaper SD, Meldolesi J, and Pozzan T. “Ca2+ channels and intracellular Ca2+ stores in neuronal and neuroendocrine cells.” Cell Calcium 11, no. 2-3 (1990): 191-199. Miledi R. “Strontium as a substitute for calcium in the process of transmitter release at the neuromuscular junction.” Nature 212, no. 5067 (1966): 1233-1234. Miller RJ. “Multiple calcium channels and neuronal function.” Science 235, no. 4784 (1987): 46-52. Mintz IM, Sabatini BL, and Regehr GW. “Calcium control of transmitter release at a cerebellar synapse.” Neuron 15, no. 3 (1995): 675-688. Mintz IM, Venema VJ, Swiderek KM, Lee TD, Bean BP, and Adams ME. “P-type calcium channels blocked by the spider toxin omega-Aga-IVA.” Nature 355, no. 6363 (1992): 827-829. Miranda-Laferte E, Gonzalez-Gutierrez G, Schmidt S, Zeug A, Ponimaskin EG, Neely A, Hidalgo P. “Homodimerization of the Src homology 3 domain of the calcium channel {beta}-subunit drives dynamin-dependent endocytosis.” J Biol Chem. In Press (2011) Misura KM, Scheller RH, and Weis WI. “Three-dimensional structure of the neuronalSec1-syntaxin 1a complex.” Nature 404, no. 6776 (2000): 355-362. Miyake Y, Yagasaki K, Horiguchi M, Kawase Y, and Kanda T. “Congenital stationary night blindness with negative electroretinogram: a new classification.” Arch Ophtalmol 104, no. 7 (1986): 1013. Mochida S, Westenbroek RE, Yokoyama CT, Zhong H, Myers SJ, Scheuer T, Itoh K, and Catterall WA. “Requirement for the synaptic protein interaction site for reconstitution of synaptic transmission by P/Q-type calcium channels.” PNAS 100, no. 5 (2003): 2819-2824. Mori Y, Friedrich T, Kim MS, Mikami A, Nakai J, Ruth P, Bosse E, Hofmann F, Flockerzi V, and Furuichi T. “Primary structure and functional expression from complementary DNA of a brain calcium channel.” Nature 350, no. 6317 (1991): 398-402. Moss FJ, Viard P, Davies A, Bertaso F, Page KM, Graham A, Cantí C, et al. “The novel product of a five-exon stargazin-related gene abolishes CaV2.2 calcium channel expression.” EMBO 21, no. 7 (2002): 1514-1523. Murakami M, Miyoshi I, Suzuki T, Sasano S, and Iijima T. “Structures of the murine genes for the beta1- and beta4-subunits of the voltage-dependent calcium channel.” J Mol Neurosc 21, no. 1 (2003): 13-21. 170 Murakami M, Yamamura H, Suzuki T, Kang MG, Ohya S, Murakami A, Miyoshi I, et al. “Modified cardiovascular L-type channels in mice lacking the voltage-dependent Ca2+ channel beta3 subunit.” J Biol Chem 278, no. 44 (2003): 43261-43267. Nagel A, Engel AG, Lang B, Newsom-Davis J, and Fukuoka T. “Lambert-Eaton myasthenic syndrome IgG depletes presynaptic membrane active zone particles by antigenic modulation.” Ann Neurol 24, no. 4 (1988): 552-558. Naidoo V, Dai X, Galligan JJ. “R-type Ca(2+) channels contribute to fast synaptic excitation and action potentials in subsets of myenteric neurons in the guinea pig intestine.” Neurogastroenterol Motil 22, no. 12 (2010): 353-363. Namkung Y, Smith SM, Beom Lee S, Skrypnyk NV, Kim HL, Chin H, Scheller RH, Tsien RW, and Shin HS. “Targeted disruption of the Ca2+ channel β3 subunit reduces Nand L-type Ca2+ channel activity and alters the voltagedependent activation of P/Q-type Ca2+ channels in neurons.” PNAS 95, no. 20 (1998): 12010-12015. Neher, E. “Vesicle pools and Ca2+ microdomains: new tools for understanding their roles in neurotransmitter release.” Neuron 20 (1998): 389-399. Newton PM, Orr CJ, Wallace MJ, Kim C, Shin HS, and Messing RO. “Deletion of Ntype calcium channels alters ethanol reward and reduces ethanol consumption in mice.” J Neurosc 24, no. 44 (2004): 9862-9869. Noebels, J L, X Qiao, R T Bronson, C Spencer, and M T Davisson. “Stargazer - A New Neurological Mutant on Chromosome-15 in the Mouse with Prolonged Cortical Seizures.” Epilepsy Research 7, no. 2 (1990): 129-135. Nowycky, M C, A P Fox, and R W Tsien. “Long-opening mode of gating of neuronal calcium channels and its promotion by the dihydropyridine calcium agonist Bay K 8644.” PNAS 82, no. 7 (1985): 2178-2182. Nowycky, M C, A P Fox, and R W Tsien. “Three types of neuronal calcium channel with different calcium agonist sensitivity.” Nature 316, no. 6027 (1985): 440-443. Oda SI, Lee KJ, Arii T, Imoto K, Hyun BH, Park IS, Kim H, Rhyu IJ. “Differential regulation of Purkinje cell dendritic spines in rolling mouse Nagoya (tg/tg), P/Q type calcium channel (α1(A)/Ca(v)2.1) mutant.” Anat Cell Biol 43, no. 3 (2010): 211-217. Opatowsky Y, Chen CC, Campbell KP, and Hirsch JA. “Structural analysis of the voltage-dependent calcium channel beta subunit functional core and its complex with the alpha 1 interaction domain.” Neuron 42, no. 3 (2004): 387-399. Opatowsky Y, Chomsky-Hecht O, Kang MG, Campbell KP, and Hirsch JA. “The voltage-dependent calcium channel beta subunit contains two stable interacting domains.” J Biol Chem 278, no. 52 (2003): 52323-52332. 171 Ophoff, R A, G M Terwindt, M N Vergouwe, R Van Eijk, P J Oefner, S M Hoffman, J E Lamerdin, et al. “Familial hemiplegic migraine and episodic ataxia type-2 are caused by mutations in the Ca2+ channel gene CACNL1A4.” Cell 87, no. 3 (1996): 543-552. Orci, L, A Perrelet, and J E Rothman. “Vesicles on strings: morphological evidence for processive transport within the Golgi stack.” PNAS 95, no. 5 (1998): 2279-2283. Osten P and Stern-Bach Y. “Learning from stargazin: the mouse, the phenotype and the unexpected.” Curr Opin Neurobiol 16, no. 3 (2006): 275-280. Oyler, G A, G A Higgins, R A Hart, E Battenberg, M Billingsley, F E Bloom, and M C Wilson. “The identification of a novel synaptosomal-associated protein, SNAP-25, differentially expressed by neuronal subpopulations.” J Cell Biol 109, no. 6 (1989): 30393052. Palfrey, H C, and C R Artalejo. “Vesicle recycling revisited: rapid endocytosis may be the first step.” Neurosc 83, no. 4 (1998): 969-989. Palma, A G, S Muchnik, and A S Losavio. “Excitatory effect of the A(2A) adenosine receptor agonist CGS-21680 on spontaneous and K(+)-evoked acetylcholine release at the mouse neuromuscular junction.” Neurosc 172 (2011): 164-176. Pardo NE, Hajela RK, and Atchison WD. “Acetylcholine release at neuromuscular junctions of adult tottering mice is controlled by N-(cav2.2) and R-type (cav2.3) but not L-type (cav1.2) Ca2+ channels.” JPET 319, no. 3 (2006): 1009-1020. Parsons, S M, and R Koenigsberger. “Specific stimulated uptake of acetylcholine by Torpedo electric organ synaptic vesicles.” PNAS 77, no. 10 (1980): 6234-6238. Perez-Reyes, E, A Castellano, H S Kim, P Bertrand, E Baggstrom, A E Lacerda, X Y Wei, and L Birnbaumer. “Cloning and expression of a cardiac/brain beta subunit of the Ltype calcium channel.” J Biol Chem 267, no. 3 (1992): 1792-1797. Perin, M S, V A Fried, G A Mignery, R Jahn, and T C Sudhof. “Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C.” Nature 345, no. 6272 (1990): 260-263. Petrucci, T C, and J S Morrow. “Synapsin I: an actin-bundling protein under phosphorylation control.” J Cell Biol105, no. 3 (1987): 1355-1363. Pevsner, J, S C Hsu, and R H Scheller. “n-Sec1: a neural-specific syntaxin-binding protein.” PNAS 91, no. 4 (1994): 1445-1449. Pichler M, Cassidy TN, Reimer D, Haase H, Kraus R, Ostler D, Striessnig J.” Beta subunit heterogeneity in neuronal L-type Ca2+ channels.” J Biol Chem 272, no.21 (1997): 13877-13882. 172 Pietrobon D. “Migraine: new molecular mechanisms.” Neuroscientist 11, no. 4 (2005): 373-386. Pinto A, Iwasa K, Newland C, Newsom-Davis J, and Lang B. “The action of LambertEaton myasthenic syndrome immunoglobulin G on cloned human voltage-gated calcium channels.” Muscle nerve 25, no. 5 (2002): 715-724. Powers, P A, S Liu, K Hogan, and R G Gregg. “Skeletal muscle and brain isoforms of a beta-subunit of human voltage-dependent calcium channels are encoded by a single gene.” J Biol Chem 267, no. 32 (1992): 22967-22972. Pragnell, M, M De Waard, Y Mori, T Tanabe, T P Snutch, and K P Campbell. “Calcium channel beta-subunit binds to a conserved motif in the I-II cytoplasmic linker of the alpha 1-subunit.” Nature 368, no. 6466 (1994): 67-70. Pragnell, M, J Sakamoto, S D Jay, and K P Campbell. “Cloning and tissue-specific expression of the brain calcium channel beta-subunit.” FEBS Letters 291, no. 2 (1991): 253-258. Purves D, Augustine GJ; FitzpatrickD, Hall WC, LaMantia AS, McNamara JO, White LE. “Neuroscience” Sinauer Associates, Inc. Fourth Edition (2008). Qin N, Yagel S, Momplaisir ML, Codd EE, and D‟Andrea MR. “Molecular cloning and characterization of the human voltage-gated calcium channel alpha(2)delta-4 subunit.” Mol Pharmacol 62, no. 3 (2002): 485-496. Randall, A, and R W Tsien. “Pharmacological dissection of multiple types of Ca2+ channel currents in rat cerebellar granule neurons.” J Neurosc 15, no. 4 (1995): 29953012. Rash, J E, L A Staehelin, and M H Ellisman. “Rectangular arrays of particles on freezecleaved plasma membranes are not gap junctions.” Exp Cell Res86, no. 1 (1974): 187190. Reid, B, C R Slater, and G S Bewick. “Synaptic vesicle dynamics in rat fast and slow motor nerve terminals.” J Neurosc 19, no. 7 (1999): 2511-2521. Reuter, H. “Calcium channel modulation by neurotransmitters, enzymes and drugs.” Nature 301, no. 5901 (1983): 569-574. Richards, D A, C Guatimosim, and W J Betz. “Two endocytic recycling routes selectively fill two vesicle pools in frog motor nerve terminals.” Neuron 27, no. 3 (2000): 551-559. Richards MW, Butcher AJ, and Dolphin AC. “Ca2+ channel beta-subunits: structural insights AID our understanding.” TIPS 25, no. 12 (2004): 626-632. 173 Rios, E, and G Brum. “Involvement of dihydropyridine receptors in excitationcontraction coupling in skeletal muscle.” Nature 325, no. 6106 (1987): 717-720. Robinson, R, and M Gardiner. “Genetics of childhood epilepsy.” Arch Dis Child 82, no. 2 (2000): 121-125. Rosato Siri MD and Uchitel OD. “Calcium channels coupled to neurotransmitter release at neonatal rat neuromuscular junctions.” J Physiol 514, no. 2 (1999): 533-540. Rosenberg, R L, P Hess, J P Reeves, H Smilowitz, and R W Tsien. “Calcium channels in planar lipid bilayers: insights into mechanisms of ion permeation and gating.” Science 231, no. 4745 (1986): 1564-1566. Rosenmund, C, and C F Stevens. “Definition of the readily releasable pool of vesicles at hippocampal synapses.” Neuron 16, no. 6 (1996): 1197-1207. Rosenthal, L, and J Meldolesi. “a-Latrotoxin and related toxins.” Pharmacol Ther 42 (1989): 115-134. Rossetto, O, M Rigoni, and C Montecucco. “Different mechanism of blockade of neuroexocytosis by presynaptic neurotoxins.” Toxicology Letters 149, no. 1-3 (2004): 91101. Rotundo, R L, S G Rossi, and H B Peng. “Targeting acetylcholinesterase molecules to the neuromuscular synapse.” J Physiol (Paris) 92, no. 3-4 (1998): 195-198. Rousset, M, T Cens, S Restituito, C Barrere, J L Black, M W McEnery, and P Charnet. “Functional roles of gamma2, gamma3 and gamma4, three new Ca2+ channel subunits, in P/Q-type Ca2+ channel expressed in Xenopus oocytes.” J Physiol 532, no. 3 (2001): 583-593. Ruth, P, A Röhrkasten, M Biel, E Bosse, S Regulla, H E Meyer, V Flockerzi, and F Hofmann. “Primary structure of the beta subunit of the DHP-sensitive calcium channel from skeletal muscle.” Science 245, no. 4922 (1989): 1115-1118. Sabatini BL, and Regehr WG. “Timing of neurotransmission at fast synapses in the mammalian brain.” Nature 384, no. 6605 (1996): 170-172. Saegusa H, Matsuda Y, and Tanabe T. “Effects of ablation of N- and R-type Ca(2+) channels on pain transmission.” Neurosc Res 43, no. 1 (2002): 1-7. Sanchez JA, Stefani E. “Inward calcium current in twitch muscle fibres of the frog.” J Physiol 283 (1978):197-209. Sanders DB, Massey JM, Sanders LL and Edwards LJ. “A randomized trial of 3,4diaminopyridine in Lambert-Eaton myasthenic syndrome” Neurobiol 54 (2000): 603-607. 174 Sandoval A, Oviedo N, Andrade A, and Felix R. “Glycosylation of asparagines 136 and 184 is necessary for the alpha2delta subunit-mediated regulation of voltage-gated Ca2+ channels.” FEBS Letters 576, no. 1-2 (2004): 21-26. Sano K, Enomoto K, Maeno T " Effects of synthetic omega-conotoxin, a new type Ca2+ antagonist, on frog and mouse neuromuscular transmission.” Eur J Pharmacol 141, no. 2 (1987):235-41. Santafé, M M, N Garcia, M A Lanuza, O D Uchitel, and J Tomás. “Calcium channels coupled to neurotransmitter release at dually innervated neuromuscular junctions in the newborn rat.” Neurosc 102, no. 3 (2001): 697-708. Sather WA and McCleskey EW. “Permeation and selectivity in calcium channels.” Ann Rev Physiol 65, no. 1 (2003): 133-159. Schmidtko A, Lötsch J, Freynhagen R, and Geisslinger G. “Ziconotide for treatment of severe chronic pain.” The Lancet 375, no. 9725 (2010): 1569-1577. Scott, V E, M De Waard, H Liu, C A Gurnett, D P Venzke, V A Lennon, and K P Campbell. “Beta subunit heterogeneity in N-type Ca2+ channels.” J Biol Chem 271, no. 6 (1996): 3207-3212. Sellin, L C, J Molgó, K Törnquist, B Hansson, and S Thesleff. “On the possible origin of giant or slow-rising miniature end-plate potentials at the neuromuscular junction.” Pflugers Archiv European J Physiol 431, no. 3 (1996): 325-334. Seu Land Pitt GS. “Dose-dependent and Isoform-specific Modulation of Ca2+ Channels by RGK GTPases.” J Gen Physiol 128, no. 5 (2006): 605-613. Shao, X, I Fernandez, T C Südhof, and J Rizo. “Solution structures of the Ca2+-free and Ca2+-bound C2A domain of synaptotagmin I: does Ca2+ induce a conformational change?” Biochem 37, no. 46 (1998): 16106-16115. Sharp, A H, J L Black, S J Dubel, S Sundarraj, J P Shen, A M Yunker, T D Copeland, and M W McEnery. “Biochemical and anatomical evidence for specialized voltagedependent calcium channel gamma isoform expression in the epileptic and ataxic mouse, stargazer.” Neurosc 105, no. 3 (2001): 599-617. Sheng, M, M A Thompson, and M E Greenberg. “CREB: a Ca(2+)-regulated transcription factor phosphorylated by calmodulin-dependent kinases.” Science 252, no. 5011 (1991): 1427-1430. Shirataki, H, K Kaibuchi, T Sakoda, S Kishida, T Yamaguchi, K Wada, M Miyazaki, and Y Takai. “Rabphilin-3A, a putative target protein for smg p25A/rab3A p25 small GTPbinding protein related to synaptotagmin.” Mol Cell Biol 13, no. 4 (1993): 2061-2068. 175 Shistik, E, T Ivanina, T Puri, M Hosey, and N Dascal. “Ca2+ current enhancement by alpha 2/delta and beta subunits in Xenopus oocytes: contribution of changes in channel gating and alpha 1 protein level.” J Physiol 489, no. 1 (1995): 55-62. Sidman, R L, S H Appel, and J F Fullier. “Neurological Mutants of the Mouse.” Science 150, no. 3695 (1965): 513-516. Silinsky, E M. “Can barium support the release of acetylcholine by nerve impulses?” Brit J Pharmacol 59, no. 1 (1977): 215-217. Silinsky, E M. “On the role of barium in supporting the asynchronous release of acetylcholine quanta by motor nerve impulses.” J Physiol 284 (1978):157-171. Silinsky, E M. “On the calcium receptor that mediates depolarization-secretion coupling at cholinergic motor nerve terminals.” Brit J Pharmacol 73, no. 2 (1981): 413-429. Silinsky, E M. “The biophysical pharmacology of calcium-dependent acetylcholine secretion.” Pharmacol Revs 37 (1985): 81-132. Singer, D, M Biel, I Lotan, V Flockerzi, F Hofmann, and N Dascal. “The roles of the subunits in the function of the calcium channel.” Science 253, no. 5027 (1991): 15531557. Smith DO, Conklin MW, Jensen PJ and Atchison WD. “Decreased calcium currents in motor nerve terminals of mice with Lambert-Eaton myasthenic syndrome.” J Physiol 487, no.1 (1995): 115-123. Snutch, T P, J P Leonard, M M Gilbert, H A Lester, and N Davidson. “Rat brain expresses a heterogeneous family of calcium channels.” PNAS 87, no. 9 (1990): 33913395. Snutch, T P, and P B Reiner. “Ca2+ channels: diversity of form and function.” Curr Opin Neurobiol 2, no. 3 (1992): 247-253. Sons MS and Plomp JJ. “Rab3A deletion selectively reduces spontaneous neurotransmitter release at the mouse neuromuscular synapse.” Brain Res 1089, no. 1 (2006): 126-134. Soong, T W, A Stea, C D Hodson, S J Dubel, S R Vincent, and T P Snutch. “Structure and functional expression of a member of the low voltage-activated calcium channel family.” Science 260, no. 5111 (1993): 1133-1136. Spafford JD and Zamponi GW. “Functional interactions between presynaptic calcium channels and the neurotransmitter release machinery.” Curr Opin Neurobiol13, no. 3 (2003): 308-314. 176 Splawski I, Timothy KW, Decher N, Kumar P, Sachse FB, Beggs AH, Sanguinetti MC, and Keating MT. “Severe arrhythmia disorder caused by cardiac L-type calcium channel mutations.” PNAS 102, no. 23 (2005): 8089-8096. Splawski I, Timothy KW, Sharpe LM, Decher N, Kumar P, Bloise R, Napolitano C, et al. “Ca(V)1.2 calcium channel dysfunction causes a multisystem disorder including arrhythmia and autism.” Cell 119 (2004): 19-31. Splawski I, Yoo DS, Stotz SC, Cherry A, Clapham DE, and Keating MT. “CACNA1H mutations in autism spectrum disorders.” J Biol Chem 281, no. 31 (2006): 22085-22091. Stanley, EF. “The calcium channel and the organization of the presynaptic transmitter release face.” Neuron, 11 (1993): 1007-1011. Stea, A, S J Dubel, M Pragnell, J P Leonard, K P Campbell, and T P Snutch. “A betasubunit normalizes the electrophysiological properties of a cloned N-type Ca2+ channel alpha 1-subunit.” Neuropharm 32, no. 11 (1993): 1103-1116. Stephens GJ, Page KM, Bogdanov Y, and Dolphin AC. “The alpha1B Ca2+ channel amino terminus contributes determinants for beta subunit-mediated voltage-dependent inactivation properties.” J Physiol 525 Pt 2, no. 2 (2000): 377-390. Stevens, C F, and T Tsujimoto. “Estimates for the pool size of releasable quanta at a single central synapse and for the time required to refill the pool.” PNAS 92, no. 3 (1995): 846-849. Strube, C, M Beurg, P A Powers, R G Gregg, and R Coronado. “Reduced Ca2+ current, charge movement, and absence of Ca2+ transients in skeletal muscle deficient in dihydropyridine receptor beta 1 subunit.” Biophys J 71, no. 5 (1996): 2531-2543. Strube, C, M Beurg, M Sukhareva, C A Ahern, J A Powell, P A Powers, R G Gregg, and R Coronado. “Molecular origin of the L-type Ca2+ current of skeletal muscle myotubes selectively deficient in dihydropyridine receptor beta1a subunit.” Biophys J 75, no. 1 (1998): 207-217. Sudhof TC. “The synaptic vesicle cycle.” Ann Rev Neurosc 27, no. 1 (2004): 509-547. Suzuki S. “Lambert-Eaton myasthenic syndrome (LEMS).” Brain Nerve 62, no. 4 (2010): 419-426. Szabo Z, Obermair GJ, Cooper CB, Zamponi GW, and Flucher BE. “Role of the synprint site in presynaptic targeting of the calcium channel CaV2.2 in hippocampal neurons.” European J Neurosc 24, no. 3 (2006): 709-718. 177 Söllner, T, M K Bennett, S W Whiteheart, R H Scheller, and J E Rothman. “A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion.” Cell 75, no. 3 (1993): 409-418. Takahashi, M, M J Seagar, J F Jones, B F Reber, and W A Catterall. “Subunit structure of dihydropyridine-sensitive calcium channels from skeletal muscle.” PNAS 84, no. 15 (1987): 5478-5482. Takahashi SX, Miriyala J, and Colecraft HM. “Membrane-associated guanylate kinaselike properties of β-subunits required for modulation of voltage-dependent Ca2+ channels.” PNAS 101, no. 18 (2004): 7193-7198. Takamori, M. “Lambert-Eaton myasthenic syndrome: search for alternative autoimmune targets and possible compensatory mechanisms based on presynaptic calcium homeostasis.” J Neuroimm 201-202 (2008): 145-152. Takei T, Saegusa H, Zong S, Murakoshi T, Makita K, and Tanabe T. “Increased sensitivity to halothane but decreased sensitivity to propofol in mice lacking the N-type Ca2+ channel.” Neuros Letters 350, no. 1 (2003): 41-45. Takeuchi, N. “Effects of calcium on the conductance change of the end-plate” J Physiol 167 (1963):141-55. Tanaka, O, H Sakagami, and H Kondo. “Localization of mRNAs of voltage-dependent Ca(2+)-channels: four subtypes of alpha 1- and beta-subunits in developing and mature rat brain.” Mol Brain Res 30, no. 1 (1995): 1-16. Tedesco E, Rigoni M, Caccin P, Grishin E, Rossetto O, Montecucco C. “Calcium overload in nerve terminals of cultured neurons intoxicated by alpha-latrotoxin and snake PLA2 neurotoxins.” Toxicon 54, no. 2 (2009):138-144. Tomita S, Fukata M, Nicoll RA, and Bredt DS. “Dynamic interaction of stargazin-like TARPs with cycling AMPA receptors at synapses.” Science 303, no. 5663 (2004): 15081511. Tremblay, F, R G Laroche, and I De Becker. “The electroretinographic diagnosis of the incomplete form of congenital stationary night blindness.” Vis Res 35, no. 16 (1995): 2383-2393. Trimble, W S, and R H Scheller. “Molecular biology of synaptic vesicle-associated proteins.” TINS, 1988. Tsien, R W. “Calcium channels in excitable cell membranes.” Annl Rev Physiol 45 (1983): 341-358. 178 Tsien, R W, D Lipscombe, D V Madison, K R Bley, and A P Fox. “Multiple types of calcium channels and their selective modulations.” TINS 11, no. 10 (1988): 431-438. Tsien RW, Ellinor PT, and Horne WA. “Molecular Diversity of Voltage-Dependent Ca2+ Channels.” TIPS 12 (1991): 349-354. Tsuji, S, and H Meier. “Evidence for allelism of leaner and tottering in the mouse.” Gen Res 17, no. 1 (1971): 83-88. Tuček, S. “The synthesis of acetylcholine in skeletal muscles of the rat.” J Physiol 129, no. 3 (1982): 81-82P. Ubach, J, X Zhang, X Shao, T C Südhof, and J Rizo. “Ca2+ binding to synaptotagmin: how many Ca2+ ions bind to the tip of a C2-domain?” EMBO 17, no. 14 (1998): 39213930. Uchitel, O D, D A Protti, V Sanchez, B D Cherksey, M Sugimori, and R Llinás. “P-type voltage-dependent calcium channel mediates presynaptic calcium influx and transmitter release in mammalian synapses.” PNAS 89, no. 8 (1992): 3330-3333. Ullrich, O, H Stenmark, K Alexandrov, L A Huber, K Kaibuchi, T Sasaki, Y Takai, and M Zerial. “Rab GDP dissociation inhibitor as a general regulator for the membrane association of rab proteins.” J Biol Chem 268, no. 24 (1993): 18143-18150. Urbano FJ, Pagani MR, and Uchitel OD. “Calcium channels, neuromuscular synaptic transmission and neurological diseases.” J Neuroimm 201-202 (2008): 136-144. Ushkaryov, Y A, K E Volynski, and A C Ashton. “The multiple actions of black widow spider toxins and their selective use in neurosecretion studies.” Toxicon 43, no. 5 (2004): 527-542. Ushkaryov YA, Rohou A, and Sugita S. “alpha-Latrotoxin and its receptors.” Exp Pharmacol, no. 184 (2008): 171-206. Van Der Kloot, W. “The regulation of quantal size.” Prog Neurobiol36, no. 2 (1991): 93130. Van Der Kloot, W, and J Molgó. “Quantal acetylcholine release at the vertebrate neuromuscular junction.” Physiol Revs 74, no. 4 (1994): 899-991. Van Elstraete AC, Sitbon P, Mazoit JX, and Benhamou D. “Gabapentin prevents delayed and long-lasting hyperalgesia induced by fentanyl in rats.” Anes 108, no. 3 (2008): 484494. 179 Van Petegem F, Clark KA, Chatelain FC, and Minor DL. “Structure of a complex between a voltage-gated calcium channel beta-subunit and an alpha-subunit domain.” Nature 429, no. 6992 (2004): 671-675. Van Petegem F, Duderstadt KE, Clark KA, Wang M, and Minor DL. “Alanine-scanning mutagenesis defines a conserved energetic hotspot in the CaValpha1 AID-CaVbeta interaction site that is critical for channel modulation.” Structure 16, no. 2 (2008): 280294. Vance, C L, C M Begg, W L Lee, H Haase, T D Copeland, and M W McEnery. “Differential expression and association of calcium channel alpha1B and beta subunits during rat brain ontogeny.” J Biol Chem 273, no. 23 (1998). Varadi, G, P Lory, D Schultz, M Varadi, and A Schwartz. “Acceleration of activation and inactivation by the beta subunit of the skeletal muscle calcium channel.” Nature 352, no. 6331 (1991): 159-162. Vautrin J and Kriebel ME. “Characteristics of slow-miniature endplate currents show a subunit composition.” Neurosc 41 (1991): 71-88. Vendel AC, Terry MD, Striegel AR, Iverson NM, Leuranguer V, Rithner Cd, Lyons BA, Pickard GE, Tobet SA, and Horne WA. “Alternative splicing of the voltage-gated Ca2+ channel beta4 subunit creates a uniquely folded N-terminal protein binding domain with cell-specific expression in the cerebellar cortex.” J Neurosc 26, no. 10 (2006): 26352644. Verstreken P, Ohyama T, Bellen HJ. ” FM 1-43 labeling of synaptic vesicle pools at the Drosophila neuromuscular junction.” Methods Mol Biol 440 (2008): 349-69. Wakamori, M, G Mikala, A Schwartz, and A Yatani. “Single-channel analysis of a cloned human heart L-type Ca2+ channel alpha 1 subunit and the effects of a cardiac beta subunit.” Biochem Biophys Res Comm 196, no. 3 (1993): 1170-1176. Wakamori, M, T Niidome, D Furutama, T Furuichi, K Mikoshiba, Y Fujita, I Tanaka, K Katayama, A Yatani, and A Schwartz. “Distinctive functional properties of the neuronal BII (class E) calcium channel.” Receptors channels 2, no. 4 (1994): 303-314. Walker, D, D Bichet, K P Campbell, and M De Waard. “A beta 4 isoform-specific interaction site in the carboxyl-terminal region of the voltage-dependent Ca2+ channel alpha 1A subunit.” J Biol Chem 273, no. 4 (1998): 2361-2367. Walker, D, and M De Waard. “Subunit interaction sites in voltage-dependent Ca2+ channels: role in channel function.” TINS 21, no. 4 (1998): 148-154. 180 Wang Q, Liu M, Xu C, Tang Z, Liao Y, Du R, Li W, et al. “Novel CACNA1S mutation causes autosomal dominant hypokalemic periodic paralysis in a Chinese family.” J Mol Med 83, no. 3 (2005): 203-208. Weber, T, B V Zemelman, J A McNew, B Westermann, M Gmachl, F Parlati, T H Söllner, and J E Rothman. “SNAREpins: minimal machinery for membrane fusion.” Cell 92, no. 6 (1998): 759-772. Wei, X Y, E Perez-Reyes, A E Lacerda, G Schuster, A M Brown, and L Birnbaumer. “Heterologous regulation of the cardiac Ca2+ channel alpha 1 subunit by skeletal muscle beta and gamma subunits. Implications for the structure of cardiac L-type Ca2+ channels.” J Biol Chem 266, no. 32 (1991): 21943-21947. Wheeler, D B, A Randall, and R W Tsien. “Roles of N-type and Q-type Ca2+ channels in supporting hippocampal synaptic transmission.” Science 264, no. 5155 (1994): 107-111. Williams, M E, D H Feldman, A F McCue, R Brenner, G Velicelebi, S B Ellis, and M M Harpold. “Structure and functional expression of alpha 1, alpha 2, and beta subunits of a novel human neuronal calcium channel subtype.” Neuron 8, no. 1 (1992): 71-84. Witcher, D R, M De Waard, H Liu, M Pragnell, and K P Campbell. “Association of native Ca2+ channel beta subunits with the alpha 1 subunit interaction domain.” J Biol Chem 270, no. 30 (1995): 18088-18093. Wittemann, S, M D Mark, J Rettig, and S Herlitze. “Synaptic localization and presynaptic function of calcium channel beta 4-subunits in cultured hippocampal neurons.” J Biol Chem 275, no. 48 (2000): 37807-37814. Xu YF and Atchison WD. “Effects of -agatoxin IVA and -conotoxin MVIIC on ++ ++ + perineural Ca and Ca -activated K currents of mouse motor nerve terminals.” JPET 279 (1996): 1229-1236. Xu YF, Autio D, Rheuben MB, Atchison WD. ”Impairment of synaptic vesicle exocytosis and recycling during neuromuscular weakness produced in mice by 2,4dithiobiuret.” J Neurophys 88, no. 6 (2002):3243-3258. Xu YF, Hewett SJ, Atchison WD. “Passive transfer of Lambert-Eaton myasthenic syndrome induces dihydropyridine sensitivity of ICa in mouse motor nerve terminals.” J Neurophys 80, no. 3 (1998):1056-1069. 181 Yang, J, P T Ellinor, W A Sather, J F Zhang, and R W Tsien. “Molecular determinants of Ca2+ selectivity and ion permeation in L-type Ca2+ channels.” Nature 366, no. 6451 (1993): 158-161. Yang SN, and Berggren PO. “The role of voltage-gated calcium channels in pancreatic beta-cell physiology and pathophysiology.” End Rev 27, no. 6 (2006): 621-676. Yasuda T, Chen L, Barr W, McRory JE, Lewis RJ, Adams DJ, and Zamponi GW. “Auxiliary subunit regulation of high-voltage activated calcium channels expressed in mammalian cells.” Eur J Neurosc 20, no. 1 (2004): 1-13. Yu, S P, and W Van Der Kloot. “Increasing quantal size at the mouse neuromuscular junction and the role of choline.” J Physiol 433 (1991): 677-704. Zamponi, G W. “Regulation of presynaptic calcium channels by synaptic proteins.” J Pharmacol Sc 92, no. 2 (2003): 79-83. Zefirov AL. “Vesicle cycle in the presynaptic nerve terminal” Ross Fiziol Zh Im I M Sechenova. 93, no. 5 (2007):544-562. Zefirov AL, Abdrakhmanov MM, Mukhamedyarov MA, Grigoryev PN. “The role of extracellular calcium in exo- and endocytosis of synaptic vesicles at the frog motor nerve terminals.” Neurosc 143, no. 4 (2006):905-910. Zefirov, A L, and P N Grigor‟ev “Topografy and affinity of the calcium sensors of exoand endocytosis in motor nerve endings.” Byull Éksperim Biol Med 150 (2008): 136-140. Zefirov, A L, and P N Grigor‟ev. “Sensitivity of intracellular calcium-binding sites for exo- and endocytosis of synaptic vesicles to Sr, Ba, and Mg ions.” Neurosc Behav Physiol 40, no. 4 (2010): 389-396. Zefirov AL, and Mukhamed'iarov MA. “The mechanisms of short-term forms of synaptic plasticity” Ross Fiziol Zh Im I M Sechenova. 90, no. 8 (2004):1041-1059 Zefirov AL, Abdrakhmanov MM, Grigor'ev PN. “Kiss-and-run quantal secretion in frog nerve-muscle synapse.” Bull Exp Biol Med 137, no. 2 (2004): 107-110. 182 Zefirov, A L, and P N Grigor‟ev . “Topography and affinity of calcium sensors of exoand endocytosis in motor nerve terminals.” Bull Exp Biol Med 146, no. 6 (2008): 667670. Zhang, J F, A D Randall, P T Ellinor, W A Horne, W A Sather, T Tanabe, T L Schwarz, and R W Tsien. “Distinctive pharmacology and kinetics of cloned neuronal Ca2+ channels and their possible counterparts in mammalian CNS neurons.” Neuropharm 32, no. 11 (1993): 1075-1088. Zhang, Y., Mori, M., Burgess, DL., Noebels JL. “Mutations in high-voltage-activated calcium channel genes stimulate low-voltage-activated currents in mouse thalamic relay neurons.” J Neurosci 22 (2002): 6362-6371. Zhang Y, Chen YH, Bangaru SD, He L, Abele K, Tanabe S, Kozasa T, and Yang J. “Origin of the voltage dependence of G-protein regulation of P/Q-type Ca2+ channels.” J Neurosc 28, no. 52 (2008): 14176-14188. Zhen XG, Xie C, Fitzmaurice A, Schoonover CE, Orenstein ET, and Yang J. “Functional architecture of the inner pore of a voltage-gated Ca2+ channel.” J Gen Physiol 126, no. 3 (2005): 193-204. Zhuchenko, O, J Bailey, P Bonnen, T Ashizawa, D W Stockton, C Amos, W B Dobyns, S H Subramony, H Y Zoghbi, and C C Lee. “Autosomal dominant cerebellar ataxia (SCA6) associated with small polyglutamine expansions in the alpha 1A-voltage-dependent calcium channel.” Nat Genet15, no. 1 (1997): 62-69. 183