um 140 Betsholtz et al. (1990) ......... T .........H. RCK3 50 Stuhmer et al. (1989) ......... T .........K. RCK4 >100 Stuhmer et al. (1989) ......... S .........V. RCK5 129 Christie et al.(1990) ......... T .........R. Ak01a >100 Pfaffinger et al.(1991) .G ...... T ......... R. 8KV1.1 >100 aConcentration of external TEA for 50% inhibition (ICm) of the current. 55 Figure 8. (A) N-terminal amino acid sequence of SKv1.1 and other Sh K channels. Basic residues are underlined. (B) Amino acid+sequences in the S4-85 loop region of SKv1.1 and other Sh K channels. Five residues involved in fast . inactivation are boxed. Dots indicate the same amino aCld residues as SKv1.1 at the corresponding positions. References; Shaker (Hoshi et al., 1990), Shah, Shal and shaw (Butler et al., 1989). (A) (B) 56 SKv1.1 MSTLSGTASTLLLPHGTLAYCNBKINQN Shaker MAAVAGLYGLGEDBQHRKKQQQQQQHQKEQL Shab Shal Shaw SKv1.1 Shaker Shab Shal Shaw MVGQLQGGQAAGQQQQQQQATQQQQHSKQQLQQQQQQQQQLQLKQHQQQ MASVAAWLPFABAAAIGWVPIATHPLPPPPMPKDRRKTDDEKLL MNLINMDSENBWLNVGGIBHETYKATLEI PATRLSRLTEA GLQI L GQ T LRA S VR EL G .... . .R . .K. . M. .. ...S . .F . ..N . YK .. ..R. . .Y . .KS C AS .. . ..K._;JI. . F.. . AK .. T 57 c. Nerthern and Southern blot analysis Northern blot analysis using adult schistosome poly(AU RNA and a probe that is specific to the 3'-untranslated region of SKv1.1 detected a single transcript of 2.8 kb, which is comparable to the size of SKv1.1 gene (Figure 9A). In order to preclude the possibility that the probe targeting the 3'-untranslated region is detecting only one form of alternatively spliced transcripts showing variation in the 3'-untranslated region, a Northern blot analysis using a probe containing the whole region of SKv1.1 (2.8 kb), rather than the 3'-untranslated region, has been tried. Again, only single band has been observed in the blot indicating the SKv1.1 mRNA may occur as a single transcript in adult S. mansoni, not as multiple alternatively spliced transcripts. Southern blot analysis also suggested that the SKv1.1 gene exists as a single copy in the whole genomic DNA of S. mansoni (Figure 9B). 58 Figure 9. Hybridization analyses of SKv1.1. (A) Northern blot analysis of SKv1.1. Three micrograms of poly(A ) RNA isolated from adult S. mansoni were fractionated on 1% formaldehyde gel, blotted and hybridized with a probe derived from the 3'-untranslated region of SKv1.1. Hybridization conditions are described in Materials and Methods. Size markers are indicated in kb. (B) Southern blot analysis of SKv1.1. Ten micrograms of genomic DNA were digested with EcoRI (E) and HindIII (H) and resolved on a. 0.8 % agarose gel. The blotted membrane was hybridized w1th the same probe that was used in Northern blot. Size markers are indicated on left in kb. 59 \\ 86.. \0‘00 Ori— 7.5- 4.4- 2.4- Ori— 9.5- 60 II. Electrophysiology A. Expression of SKv1.1 protein in Xenopus oocytes For the expression of SKv1.1 gene in Xenopus oocytes, we have initially made SKv1.1 mRNA without any modification, however, the injection of the mRNA did not result in a functional expression. Nor did the introduction of the Kozak's motif (Kozak, 1991) and a poly adenylated tail into SKv1.1 mRNA by the method described by Cestari et al. (1993). In order to maximize the functional expression of SKv1.1 gene in oocytes, two major modifications were made on SKv1.1 mRNA. Firstly, the coding region of SKv1.1 was placed between the 5' and the 3' untranslated regions (UTRs) of B-globin gene of Xenopus (Melton et al., 1984), in order to increase the stability or the translation efficiency of SKv1.1 mRNA. Secondly, in addition to introducing Kozak's motif, the fourth nucleotide (C) was changed to G in order to increase the translation efficiency of SKv1.1 mRNA. These two modifications increased the expression of SKv1.1 to a level which was high enough to be detected by oocyte voltage clamp. Injection of SKv1.1 mRNA into Xenopus oocytes produced a rapidly inactivating outward potassium current (Figure 10). Expression of a rapidly inactivating A—type current is commonly observed in oocytes by injecting Shaker channel mRNAs from invertebrates such as Drosophila, Aplysia (Pfaffinger et al., 1991). A-type currents have also 61 observed in some mammalian Shaker channels such as RCK4 (Stuhmer et al., 1989). However, most mammalian voltage- gated,KF channel genes express the slowly inactivating delayed rectifiers. The putative structural motifs that are related with the fast inactivating current of SKv1.1 will are discussed in the discussion section. 62 Figure 10. Expression of SKv1.1 protein in Xenopus oocytes. (A) Currents evoked by depolarizing voltage steps from -80 mV to test potentials of -40 to +60 mV in 20 mV increments. (B) Current-voltage relationship averaged from 9 oocytes. 63 200 - CURRENT (nA) 8 8 0| O 0 . . . I -150 -100 -50 0 50 100 TEST POTENTIAL (mV) 64 B. Voltage dependence of SKv1.1 current The current mediated by SKv1.1 was voltage-dependent, with a threshold of activation near -20 mV (Figure 10). Although the conductance was not saturated at +60 mV, test pulses to more positive potentials produced other, non-ohmic currents that confounded the isolation of the SKv1.1 component (Figure 11). Therefore, for the purpose of analysis, the conductance at +60 mV was considered to be Gmx. The Vfl,for activation was +20 mV, with a gentle slope factor, a number mV required for an e-fold change of the relative conductance value, of 15 mV. When compared with other cloned voltage-gated K+ channels, the V50 (+20 mV) of SKv1.1 current is located on the right edge of the normal range along the voltage axis. Most known Shaker channels have sz for activation ranging from -30 mV to +5 mV. The slope factor (15 mV) does not appear to be placed on the extreme edge, showing significant similarities to those measured from other Shaker currents (Papazian et al., 1991). The SKv1.1 current also displayed a voltage-dependent steady-state inactivation (Figure 11). The Vw,for inactivation was -40 mV and the slope factor was 4 mV, which is steeper than that of voltage-dependent activation. Both the V50 and the slope factor for steady-state inactivation of SKv1.1 current fall into the normal range that are commonly observed in other Shaker channels. Voltage- 65 dependent activation and steady-state inactivation showed little overlap. In the native A-type current measured from the isolated muscle fibers, a similar voltage dependence was observed (Day at al., 1993; Table 6). The V50 for activation was +5 mV with a slope factor of 12 mV, the V50 for steady—state inactivation was -50 mV with a slope factor of 14 mV. This similarity strongly supports the idea that the expression of A—type current measured in muscle fibers is directed by SKv1.1 gene. 66 Figure 11. Voltage dependence of activation (0, n=9) and steady-state inactivation (0, n=5) of the SKv1.1 current. The conductance was calculated by dividing the amplitude of the SKv1.1 current by the difference between the test potential and the hypothetical reversal potential for K, - 82 mV. The lines represent the fit of the daEDAto the Boltzmann equation in the form G/Gma x=1/(1+e ), where V = test potential, Vflfi-voltage of half activation or inactivation, k = slope factor. For steady- -state inactivation, the oocytes were held at different voltages for 5 sec before applying a depolarizing voltage step to +60 mV. Peak currents obtained by during the test pulse after a prepulse to the indicated voltage were divided by the peak currents obtained during a test pulse obtained during a test pulse after a prepulse to -80 mV. 67 O . . . . -120 -80 -40 0 40 MEMBRANE POTENTIAL (mV) 68 Table 6. Functional properties of the current mediated by SKv1.1 compared to those of a native current in muscle fibers of S. mansoni (Day et al., 1993; Day et al., 1994). SKv1.1 current muscle current Activation Threshold -20 mV Time to peak 8 ms Vso +20 mV Slope factor -15 mV Inactivation v50 -40 mV Slope factor 4 mV Time constant 17 ms Recovery from inactivation Time constant 75 Pharmacology (ICSO)a 4-AP 0.2 mM TEA >100 mM DTX >300 nM MCDP >300 nM CTX >100 nM -30 mV 8 ms +5 mV -12 mV -50 mV 14 mV 13 ms 1 mM 30 mM >300 nM 8Concentration of blockers for 50% inhibition of the current. 69 c. Kinetics of SKv1.1 current The currents mediated by SKv1.1 were rapidly activated and inactivated with a time to peak of 8 ms and a time constant of inactivation of 17 ms in response to a test pulse to +60 mV from a holding potential of -80 mV (Figure 12). Inactivation of the current fit well with a single exponential. Both activation and inactivation were voltage- dependent, becoming faster at more positive potentials. The time to peak and the time constant of inactivation of SKv1.1 were similar to those from other A-type currents as well as the native A-type currents of isolated muscle fibers (Table 6). 70 Figure 12. Time to peak (0, n=4) and the time constant of inactivation (0, n=4) of the SKv1.1 current. The holding potential was -80 mV and the time constants of inactivation were derived by fitting the data to the equation I=A(1-e M) , where t = time, T = time constant and with the single exponential n= 2. Error bars indicate standard deviations. TIME (ms) 71 40‘ N O -20 0 20 40 60 80 TEST POTENTIAL (mV) 72 D. Recovery of SKv1.1 current from inactivation Recovery from inactivation was measured using two consecutive pulses with different intervals. The ratio of the amplitude of the second peak current to that of the first peak current was plotted against the duration of intervals. (Figure 13). The time course of recovery fit well with a single exponential with a time constant of 5 sec. SKv1.1 showed a very slow recovery from inactivation. The slow recovery from inactivation has also been observed form the Aplysia Shaker channel (time constant of 2.3 sec) and the mammalian RCK4 channel with a time constant in the range of seconds (Pardo et al., 1992). 73 Figure 13. Recovery of SKv1.1 current from inactivation (n=10). (A) Current traces show a control current at a +60 mV test potential, followed by currents elicited by the same pulse following variable intervals. Shown are intervals of ls, 35, 55 and 155. (B) The amplitude of SKv1.1 current induced by a step to a test potential of +60 mV against the interval between the two pulses. I (pulse 2)" (pulse 1) P 01 74 1O 20 PULSE INTERVAL (s) 30 75 E. Pharmacology of SKv1.1 current The SKv1.1 current was extremely sensitive to block by external 4-aminopyridine (4-AP) (Figure 14A). The current was reduced 93:5% (n=5) by 1 mM 4-AP. The current was strikingly resistant to external TEAf, with 100 mM TEA+ producing no discernable blockade (n=4)(Figure 14B). Insensitivity to external TEAf‘was predicted by the amino acid sequence analysis of the SKv1.1 channel, which showed the presence of ArgB94 in the outer pore region. The current was not sensitive to 100 nM Charybdotoxin (CTX), which would also be predicted by the presence of the positively charged Arg394 in the pore region. The current was insensitive to mast cell degranulating peptide (MCDP) and dendrotoxin (DTX) at concentrations as high as 300 nM. 76 Figure 14. Pharmacological properties of the currents mediated by SKv1.1. The blockers were applied by perfusing the chamber with ND-96 containing the blockers (1-2 ml/min). A voltage step to +60mV was given to oocytes from the holding potential of -80 mV. (A) SKv1.1 current before (top) and after (bottom) the application of 1 mM external 4- aminopyridine. (B) SKv1.1 current before (top) and after (bottom) the application of 100 mM external tetraethylammonium. 77 . 14. 4‘11 mNWWW‘AP‘MWWFWPWMMWWM' “w. ”11'“ 311V] WFM'WnkVWW‘MWflM‘JWW 78 III. Immunohistochemistry A. In vitro translation and immunoprecipitation A synthetic oligopeptide based on the amino acid sequence of the cytoplasmic domain (aa 447-466) near the C- terminal end of SKv1.1 was made. Since different Sh subfamilies have unique amino acid sequences in the region around the N- and C-terminals, an antibody raised against the unique region of the Shaker protein would be expected to recognize only the Shaker channel protein and not other Sh family proteins in Schistosoma. After immunization, a SKv1.1 protein-specific antibody (ASK1), was purified using an affinity column in which the synthetic oligopeptide was immobilzed. The purified ASKl was highly specific to the oligopeptide motif, not to the carrier proteins such as BSA and KLH. In Vitro translation of the SKv1.1 gene was used to test the specificity of the ASKl antibody to its target protein by immunoprecipitation analysis. In addition, the in vitro translation was employed to test the expression of SKv1.1 gene in a heterologous system and to measure the molecular weight of the protein. The SKv1.1 mRNA for in Vitro translation was prepared by in Vitro transcription using linearlized pBTGSKl as a template. The in Vitro translation product of SKv1.1 mRNA showed a molecular weight of 55 kDa, which is close to the predicted molecular weight of 56.5 kDa based on the amino acid sequence of SKv1.1 79 (Figure 15A). Immunoprecipitation analysis showed that ASK1 can precipitate the in vitro translation product of SKv1.1 (Figure 15B, lane 2). On the other hand, the preimmune serum and the ASK1 preincubated with an excess amount of oligopeptide were unable to precipitate the SKv1.1 protein (Figure 15B, lane 1 and 3). Western blot analysis has also been tried using both the in vitro translation product of SKv1.1 and membrane proteins of S. mansoni as target proteins. No specific band was found in the Western blot membrane, indicating that the peptide motifs in the SKv1.1 protein that are recognized by ASK1 without any problem in a non-denaturating condition may be completely denatured by the presence of SDS, resulting in the loss of their native configuration. This has been observed in some peptide-specific antibodies (Sheng et al., 1992). However, since most conditions that are used in immunohistochemistry, except for Western blotting, do not eliminate the native configuration of target proteins, ASK1 has been shown to successfully recognize the target protein in immunohistochemistry on isolated muscle fibers and tissue sections. 80 Figure 15. In Vitro translation and immunoprecipitation of SKv1.1 protein. (A) Autoradiograph of in Vitro translation products of SKv1.1 protein. The mRNAs from SKv1.1 gene were added to the rabbit reticulocyte lysate in Vitro translation system containing [ S]-methionine. Lane 1, water was used instead of mRNA as a negative control; lane 2, SKv1.1 mRNA. Translation products were resolved on 10 % SDS PAGE gel. (B) Immunoprecipitation of in Vitro translation product by ASK1 antibody. Lane 1, preimmune antibody as a negative control; lane 2, immunoprecipitation with ASK1; lane 3, ASK1 was preincubated with an excess amount of free peptide before the immunoprecipitation. 45— 25- 81 205- 116- 82 B. Immunohistochemistry on isolated muscle fibers Since native A-type currents were initially measured from isolated muscle fibers of S. mansoni (Day et al., 1993), we, by immunohistochemistry, tested whether SKv1.1 protein is expressed in the isolated muscle fibers of S. mansoni. During this analysis, we have focused on finding the expression of SKv1.1 protein only in muscle fibers, not in any other cells, because the morphology of muscle fibers is relatively well characterized compared with other cell types. The expression of SKv1.1 gene in other cell types was studied using immunohistochemistry on paraffin sections. Among three types, frayed, crescent and spindle, of isolated muscle fibers of schistosome, strong immunoreactivities were observed in the most abundant frayed fibers and in the crescent fibers, where A-type currents were originally measured. ASK1 specifically recognized its target protein (Figure 16A and B) while the ASK1 preincubated with free peptide did not identify the target (Figure 16C and D). Also, the preimmune serum did not show any specific binding to the muscle preparation. This result, together with the results from the electrophysiology of SKv1.1, provides strong evidence that the expression of A-type currents measured in the frayed and the crescent muscle fibers are directed by the SKv1.1 gene. 83 Figure 16. Immunolocalization of SKv1.1 protein in the isolated muscle fibers. Light (A and C) and fluorescent (B and D) microscopy of the isolated frayed muscle fiber stained with ASK1 (A and B) and with the ASK1 preincubated with an excess amount of free peptide (C and D). 84 85 C. Immunohistochemistry on paraffin sections In order to study the expression of SKv1.1 protein in tissues other than muscle fibers, paraffin sections (2 pm) of adult female and male worms were immunostained with ASK1 antibody. Strong immunoreactivities were observed in the central and peripheral nervous system of both female and male worm (Figure 17). In the head region of the male worm, ASK1 strongly stained the anterior ganglia and the central commissures from which longitudinal nerve cords extend out both anteriorly and posteriorly (Figure 17A). On the other hand, preabsorbed ASK1 did not recognize these structures (Figure 178). Also, strong immunoreactivities were localized in the longitudinal nerve cords in the body region of both female and male worms (Figure 17C). Fine nerve fibers that emanate from main nerve cords were observed (Figure 17D). Relatively strong immunoreactivities were also observed in the nervous system innervating the oral sucker (Figure 17E), the ventral sucker (Figure 17F) and the tail region (Figure 178). In addition to the nervous system, tubercles as well as muscle fibers including longitudinal, circular and ventral muscle fibers were mildly stained (Figure 17H). 86 Figure 17. Immunolocalization of SKv1.1 protein in paraffin sections of S. mansoni (x400). Scale bar: 50 pm. (A) Head region of male showing strong immunoreactivity in the anterior ganglia (AG) and the central commissure (CC). Oral sucker (OS), ventral sucker (VS). (B) Negative control. Head region of male in an adjacent section in which ASK1 antibody was inactivated by incubating with an excess amount of free peptide. The fluorescence around the ventral sucker is an autofluorescence. Oral sucker (OS), ventral sucker (V8), anterior ganglia (AG) and central commissure (CC). (C) Longitudinal nerve cords (NC) of female (bottom) and male (top) worm located in the middle of the body. (D) Fine nerve fibers (nf) emanating from the main nerve cords (NC). (E) Oral sucker (OS) in the head region of male. Anterior ganglia (AG). (F) Ventral sucker (V8) in the head region of male. (G) Tail region of the male showing immunoreactivity in the main nerve cords (NC). (H) Tubercles (TB), longitudinal muscle fibers (LM) and circular muscle fibers (CM) in the dorsal surface of male. DISCUSSION I. Molecular cloning A. What does the existence of a Shaker channel gene in Schistosoma mean in an evolutionary sense? The first organisms that show the presence of a nervous system are coelenterates such as hydra and jelly fish. The Phylum Platyhelminthes, including Schistosoma emerged soon after the coelenterates in the evolutionary pathway about 600 million years ago, and the platyhelminths are the first that show both central and peripheral nervous system and multiple organs. Since ion channels are one of the most important constituents of the nervous system, ion channels have evolved in parallel with the evolution of the nervous system. Based upon the comparative analysis of amino acid sequences of various ion channels, it appears ancient duplications gave rise to three groups of ion channels: sodium and calcium channels, potassium channels and nucleotide-binding channels (Strong et al., 1993). K+ channels have further evolved into several groups including voltage—gated and calcium-activated K+ channels, generating much more diverse channels compared with other types of ion channels. Again, voltage-gated K+ channels have evolved into several families such as the Sh family (six membrane- spanning domains), inward rectifiers (two domains), and slow 89 90 16 channels (one domain). In the Sh family, a common ancestor has given rise to four subfamilies (Shaker, Shal, Shah and Shaw) by a gene duplication event, and each subfamily again has undergone a considerable expansion by another gene duplication during the evolution of mammalian species. For example, there are seven Shaker K+ channel genes in mammals. When did the first gene duplication event that generated the four Sh subfamilies happen? Before the cloning of the SKv1.1 gene, the most primitive Shaker channels that have been cloned were those from Drosophila and Aplysia, which are highly developed invertebrates. Also it was not clear whether the duplication happened before or after the divergence of deuterostomes (Chordata and Echinodermata) and protostomes (Annelid, Mollusc, and Arthropod) which occurred approximately 570 million years ago. Therefore, the presence of a Shaker homologue in Schistosoma indicates the gene duplication happened at least before the emergence of Schistosoma about 600 million years ago. Another interesting question arises when we consider the fact that the gene duplication event that generated the four Sh subfamilies happened before the emergence of Schistosoma. If the gene duplication event happened before the emergence of schistosome, do other Sh subfamilies such as Shal, Shah and Shaw exist in Schistosoma and contribute to the diversity of schistosomal K6 currents? They might 91 exist in modern Schistosoma unless these genes were lost in the evolutionary pathway. One possible way to prove it is to try to screen other Sh subfamilies in the schistosomal cDNA library using the SKv1.1 as a probe using a low- stringency hybridization condition. Do Sh subfamilies exist in other phyla of lower invertebrates? Theoretically, Sh subfamilies should be found in organisms of any higher phyla such as nematodes. Indeed, a Shaw homolog was found in C. elegans (Wei et al., 1991), but the full sequence of the gene has not been published. Do Sh subfamilies exist in more primitive phyla such as protozoa, sponges, coelenterates? A variety of voltage-gated.KF currents that have properties similar to mammalian channels have been detected in these phyla including coelenterates (Hagiwara, Yoshida & Yoshii, 1981) and protozoa (Eckert & Brehm, 1979; Deitmer, 1989), supporting the idea that these currents might be directed by Sh subfamilies. B. What does the analysis of SKv1.1 gene say about the diversity of RF channel currents in Schistosoma? With limited molecular and physiological data, it is not easy to discuss the diversity of K5 channel currents in Schistosoma. There are several mechanisms for the generation of diverse K5 channels in the Sh family: the presence of subfamilies (Wei et al., 1990), the formation of 92 heteromultimers (Isacoff et al., 1990; MacKinnon et al., 1991; McCormack et al., 1990; Ruppersberg et al., 1990), alternative splicing (Iverson and Rudy, 1990; Jan and Jan, 1990; Kamb et al., 1988; Schwarz et al., 1988; Stocker et al., 1990; Timpe et al., 1988; Timpe et al., 1988), gene duplication within a subfamily (Stuhmer et al., 1989) and the level of expression (Guillemare et al., 1992; Honoré et al., 1992; Moran et al., 1992). Are there multiple copies of SKv1.1 gene in Schistosoma? Southern hybridization analysis has been tried, and the result shows that there is only a single copy of SKv1.1-related gene in the schistosomal chromosome. This suggests that, in contrast to mammalian K5 channel genes, multiple copies of identical or closely related genes for a given channel do not exist in invertebrates as also reported in Drosophila and Aplysia. In general, invertebrates employ alternative splicing as a primary mechanism to generate diverse K? channels while mammals use extensive gene duplication in a given Sh subfamily even though alternative splicing has also been reported in some mammalian Shaker (Attali et al., 1993) and Shaw channels (Rettig et al., 1992; Luneau et al., 1991a; Luneau et al., 1991b). This has led us to investigate the presence of alternatively spliced transcripts of the SKv1.1 gene. However, evidence for the existence of alternatively spliced transcripts of the SKv1.1 gene could not be found. 93 The six positive clones showed the same restriction patterns. Three of the six clones contained the same nucleotide sequence in the coding region. Also, Northern blot analysis detected only a single primary transcript with a small size (2.8 kb), which may not be long enough to allow the generation of diverse alternatively spliced transcripts. What does the lack of alternative splicing and multiple copies of SKv1.1 mean? Does this mean Schistosoma might have a relatively simple profile of voltage-gated K+ channel genes? Although no conclusion can be drawn at this point due to the lack of data, Schistosoma appears to have diverse types of excitable tissues that may need diverse K+ channels. Schistosoma, as a member of the Phylum Platyhelminthes, is the first organism that shows multiple organs, tissue layers and primitive central and peripheral nervous system. In addition to several ion channels that have been measured in schistosomes (Blair et al., 1991; Day et al., 1992; Day et al., 1993), Schistosoma respond to a variety of neurotransmitters and neuropeptides that may be directly or indirectly related to K+ channels (Baker et al., 1966; Hillman 1983; Mellin et al., 1983; Fax et al., 1984; Bennett et al., 1969; Bennett and Bueding, 1971; Chou et al., 1972; Dei-Cas et al., 1971; Dei—Cas et al., 1981; Gianutsos and Bennett, 1977; Gustafsson, 1987; Machado et al., 1972; Tomosky et al., 1974; Basch and Gupta, 1988; Gupta and Bash, 1989). 94 Therefore, if any further diversity of voltage-gated K+ channels exists in the schistosome, it could results from the presence of other Sh subfamilies, alternative splicing in other Sh subfamilies, the formation of heteromultimers or the density of channels in the membrane. C. Does the SKv1.1 have a potential to be a novel target for drug development for schistosomiasis? The availability of SKv1.1 gene provides several advantages for drug development. It permits to compare the amino acid sequence of SKv1.1 to that of the host. The expression of SKv1.1 channel protein in a heterogenous system also permits a high volume screening for drug development. The nervous system and the neuromuscular system of helminths are attractive targets for drug development (Geary et al., 1992). Many anthelminthic drugs act on receptors and ion channels in the nervous system. In order for SKv1.1 to be considered as a target for drug development, the following questions need to be answered. Does SKv1.1 protein exist in the nervous or neuromuscular system of S. mansoni? Is the function of SKv1.1 protein important enough to make the chemotherapeutic intervention of SKv1.1 protein valuable? Is the amino acid sequence of the SKv1.1 protein significantly different from that of the host? Several lines of evidence in the present study show 95 that SKv1.1 proteins exist in the nervous system and the neuromuscular system of S. mansoni. The expression of SKv1.1 protein in the nervous system has been demonstrated by immunohistochemistry on paraffin sections of Schistosoma. Also, immunohistochemistry on isolated muscle fibers using the antibody raised against SKv1.1 shows SKv1.1 proteins are indeed expressed in these muscle fibers. The functions of SKv1.1 in Schistosoma are not obvious from current data. The fact that SKv1.1 shows a A-type current implies that, if this channel is present in neurons, it may regulate the frequency and shape of action potentials. On the other hand, the function of SKv1.1 protein in muscle fibers is difficult to investigate due to technical problems such as the lack of a specific blocker for the channel and the small size of the muscle fibers. However, the presence of sites for phosphorylation by protein kinase C or tyrosine kinase in SKv1.1 gene suggests that SKv1.1 channel might be modulated by many neurotransmitters and neuropeptides participating in the regulation of cellular excitability. The comparison of the amino acid sequence of SKv1.1 to other mammalian Shaker channels shows that, despite the evolutionary distance between the Schistosoma and mammals, they share high amino acid sequence identity in the middle of the channel including the tetramerization domain, the six membrane spanning domains and the pore-forming domain. 96 However, the N-terminal (aa 1-50) and C-terminal (aa 430- 512) ends show almost no sequence identity to other Shaker channels, making this region a possible novel target. Nonetheless, the presence of highly variable regions at both ends, not in the middle, of the gene holds several disadvantages for drug development. Most KP channel blockers target the pore-forming domain (H5) in the middle of the channel, where almost all Shaker channels share high amino acid sequence identities (Pongs, 1992). Also, the diversity of the.KF channel blockers is far less than that of K5 channels. Another problem arises from the fact that both N- and C-terminal regions 0f the channels are located intracellularly, so any drugs targeted against these region would have to penetrate the cell membrane. However, the N- or C-terminal ends are targeted in drug development, there are some possibilities that novel drugs might be found, for instance, a drug interfering with the inactivation of the channel because the N-terminal is involved in the rapid inactivation of channel. II. Electrophysiology A. Why does SKv1.1 current show a fast inactivation? The injection of SKv1.1 mRNA into Xenopus oocytes results in the expression of a fast inactivating current. What are the structural elements that are responsible for the fast inactivation of SKv1.1 current? 97 Fast inactivating currents have been observed in various Shaker channels from Drosophila, Aplysia and some mammals. Mechanisms underlying the fast inactivation of some Shaker channels have been very well characterized by deletion and mutation analysis. The fast inactivation of the channel appears to be the result of an interaction between the ball structure at the N-terminal end and the receptor structure in the intracellular 84-85 loop. Application of an oligopeptide mimicking the N-terminal sequence of the channel gene accelerated the inactivation of the current (Ruppersberg et al., 1991). Deletion of the N- terminal sequence also slowed down inactivation of mouse Shal current (Pak et al., 1991). Deletion of three basic amino acid residues (aa 35 - 37) in the ball structure also slowed down inactivation of rat Shal current (Baldwin et al., 1991). At the N-terminal end, in addition to the positively charged amino acids, the importance of a hydrophobic residue has been demonstrated by replacing the seventh leucine residue with a hydrophilic residue. This substitution inhibits the fast inactivation almost completely. At the receptor region in the 84-85 loop, five important amino acids including one glutamate interact with the inactivation ball (Isacoff et al., 1991). In SKv1.1, the N-terminal region contains two positively charged amino acid residues (Arg-Lys, aa 23-24) and four hydrophobic leucines (aa 4, 11-13). The receptor 98 region at the S4-85 loop contains all five amino acids that are highly conserved in other rapidly inactivating Shaker channels. Therefore, these structural features of SKv1.1 provide possible explanations for the rapid inaction of SKv1.1 current although deletion or site-directed mutagenesis analysis of the N-terminal and the receptor structure of SKv1.1 will give direct evidence for this possibility. In addition to the fast inactivation currents (N-type) described above, a slow inactivation (C-type) has been observed in some A-type currents. No slowly inactivating currents could be observed in SKv1.1 after leak-subtraction. In other Shaker channels, some channels show only the N-type inactivation, while others show both the N- and C- type inactivations. Alanine in the 86 domain is an important determinant of slow inactivation as shown by site-directed mutagenesis study (Wittka et al., 1991). Based on the sequence comparison between SKv1.1 and Shaker channels, SKv1.1 current is expected to show both the N-type and the C-type inactivation. The reason for the absence of C-type inactivation in SKv1.1 current is not clear. One possibility is that the amplitude of the slowly inactivating current is very small compared with that of the rapidly inactivating (N-type) current. Another possibility is that there are determinants other than the Ala residue in the 86 domain, for the slowly inactivating component of the 99 currents. Another interesting question that needs to be answered is whether there is a B-subunit in SKv1.1. Most of the mammalian Shaker channels show very slow inactivations when expressed in frog oocytes. However, when coexpressed with the B-subunit, the a and B complex show an altered inactivation. For instance, the RCKl, which normally shows a slow inactivation when expressed alone, exhibits a fast inactivation when expressed together with the B—subunit (Rettig et al., 1994). A more surprising result is that RCK4, which normally shows a fast inactivation even when expressed alone, exhibited a faster rate of inactivation by the coexpression. Is there a B-subunit of SKv1.1 in Schistosoma? Unfortunately, the fact that SKv1.1 alone can make a fast inactivating channel does not necessarily give any clue for the presence of a B-subunit because the inactivation of a fast inactivating channel can also be accelerated by the presence of a B-subunit as shown in mammalian Shaker channels. B. Why is the voltage dependence of activation of SKv1.1 current and native muscle current shifted to the right edge of the normal range along the voltage axis when compared with other Shaker channels? In SKv1.1 current, the V50 of the relative conductance vs. voltage relationship is +20 mV, being placed in the 100 right extreme edge of the normal range. Native A-type current from isolated muscle fibers also shows a very high V50 of +5 mV. What are the structural features that underlie this unique voltage-dependence of schistosomal A- type current? Several regions that are involved in the voltage- dependence have been suggested by extensive site-directed mutagenesis studies combined with electrophysiological studies. The first region is the 84 membrane spanning domain or the S4 segment. This domain is well known as a voltage-sensor which detects voltage changes in the membrane and initiates the conformational change of the protein for the opening of the channel. In most Shaker channels, the S4 segment contains seven positively charged amino acid residues which occur at every third position. Their movement toward the extracellular side is thought to be responsible for the gating current that is observed during the opening of the channel (McCormack et al., 1994). The importance of positively charged amino acid residues of the S4 segment in determining the voltage— dependence of the channel has been studied by substituting all the basic residues in the S4 segment with non-charged glutamines or with other conserved basic residues (Papazian et al., 1991). These mutant channels exhibit a wide range of changes in their voltage-dependences, indicating the importance of positively charged residues. This study also 101 showed that every positively charged amino acid does not contribute equally to the voltage-dependence of the channel. Detailed mechanisms for these changes have also been studied using single channel analysis of mutant channels (Shao & Papazian, 1993). The importance of hydrophobic amino acids, in addition to positively charged amino acids, in the S4 segment has also been investigated by site-directed mutagenesis analysis (Lopez et al., 1991). In the S4 segment, conservative mutations of hydrophobic residues, for instance, Leu to Ala, Leu to Val or Ala to Leu, that cause slight changes in the hydrophobicity of each residue resulted in large shifts of voltage-dependence of the current, indicating the importance of hydrophobic amino acids in determining the voltage- dependence. In addition to the S4 segment, the S4—85 loop structure has been shown to play an important role in determining the voltage-dependence (McCormack et al., 1991). A leucine zipper motif, with the occurrence of a Leu at every seventh position, is present in this loop structure. The leucine zipper is a motif commonly found in transcription factors and participates in protein-protein interaction, however, this motif in K+ channels has not been proven to be participating in the assembly of the K4' channel subunits. When each Leu is substituted by Val, these mutations cause significant changes in the voltage-dependence such as 102 voltage-dependent activation and steady-state inactivation. In order to determine if the sequence of SKv1.1 has any unique features, amino acid sequences of the S4 segment and the 84-85 loop structure containing the leucine zipper motif were compared to the corresponding regions of other Shaker channels. Surprisingly, the sequence of the S4 segment of SKv1.1 was identical to the corresponding regions of Shaker channels from Drosophila, Aplysia and most mammals which obviously show a wide range of Vw's of voltage-dependence of activation. This suggests that, although the S4 segment is an important determinant for the voltage-dependence, the S4 segment may not be the element that is responsible for the various voltage-dependences observed in many K+ channels. On the other hand, when the 84-85 loop structure of SKv1.1 containing the leucine zipper was compared to other sequences, differences were observed in three amino acid residues (Arg335, Val338 and Val345), which are non-Leu residues located within the leucine zipper motif. Unfortunately, no data are available for the possible effects of mutations of non-Leu residues in the leucine zipper motif on the voltage-dependence. An interesting experiment will be to make mutations of these three residues to highly conserved residues and observe if there is a leftward shift of the voltage-dependence of SKv1.1 along the voltage axis. Another possible explanation for the extreme voltage- 103 dependence of activation of SKv1.1 is that the voltage- dependence may be affected by regions other than the S4 segment and leucine zipper motif. Supporting evidence comes from the sequence comparison between SKv1.1 and the Aplysia 18 channel (Ak01a). Ak01a has an identical amino acid sequence both in the 84 segment and leucine zipper motif, but, it displays a different voltage-dependence of activation in a more negative position (Vfl,of +5 mV) compared with that of SKv1.1 (V50 of +30 mV). c. Why is SKv1.1 current resistant to external TEA and CTX? The current mediated by SKv1.1 is highly sensitive to 4-AP, but highly resistant to external TEA, CTX, DTX and MCDP. In general, the amino acid residues that are responsible for the sensitivity of voltage-gated K? currents to external TEA and CTX, but not for 4-AP, DTX and MCDP, are relatively well characterized. In order to understand the structural bases for the pharmacology of SKv1.1 current, the amino acid sequence of the pore region of SKv1.1 gene was compared with those from other Shaker channel genes. For external TEA, the residue at position 19 in Table 5 appears to be the most important residue in determining the sensitivity of the channel to external TEA. The channel shows a high sensitivity to external TEA when the residue is Tyr or Phe. The presence of Thr or His at this position gives the channel a medium sensitivity to external TEA. 104 However, when the residue is a positively charged residue (Arg or Lys), the channel shows a high resistance to external TEA. The importance of residue 19 has also been studied by extensive site directed mutagenesis. A Thr to Tyr mutation makes the Shaker channel current highly sensitive to TEA while mutations from Thr to positively charged residues (Arg or Lys) make the channel highly resistant to TEA (MacKinnon & Yellen, 1990). In rat brain Shaker channels, a mutation (Val to Tyr) increases the TEA sensitivity of the channel while the reverse mutation (Tyr to Val) decreases the sensitivity (Kavanaugh et al., 1991). Further evidence for the importance of residue 19 comes from an experiment using heteromultimers that are made of subunits with different pharmacology. When the channel pore is blocked by external TEA, all four subunits participate in binding one molecule of TEA (Kavanaugh et al., 1992; Heginbotham & MacKinnon, 1992). This cooperative interaction of four subunits to bind one TEA has been proven by demonstration a linear relationship between the number of Tyr-containing subunits and the sensitivity of the channel to external TEA. SKv1.1 contains an Arg (amino acid 394) at the corresponding position, which makes it possible to predict a high resistance of the SKv1.1 channel to external TEA. Indeed, the SKv1.1 current was highly resistant to external 105 TEA when expressed in Xenopus oocytes, proving the importance of the Arg residue at position 19 in determining the sensitivity of the channel to external TEA. SKv1.1 current was highly resistant to CTX, being insensitive to as much as 100 nM of CTX. CTX is a scorpion venom toxin, from Leiurus quinquestriatus, that block Cay- activated K+ channels (Miller et al., 1985) as well as voltage-gated K+ channels (Garcia-Calvo et al., 1992). The amino acids that mediate molecular recognition between CTX and Shaker K+ channel have been well characterized by site directed mutagenesis analysis of all the amino acid residues of both CTX and the receptor region in the channel (Goldstein et al., 1994). Mutations of amino acid residues in the receptor region, especially on the two extracellular loop regions of 85-H5 and H5-S6 (Figure 88), revealed five important residues for the determination of the sensitivity of the channel to CTX. When these amino acids are compared with the corresponding residues of SKv1.1, two differences in the SKv1.1 sequence were detected: L (370) rather than G in the 85-H5 loop and R (394) rather than T in the H5-S6 loop. Both differences provide reasonable explanations why SKv1.1 channel is resistant to CTX. Firstly, the presence of a bigger side chain, L rather than G, at the first position (370) gives a clue for the resistance of SKv1.1 to CTX. In the Shaker channel, when 106 the Gly residue was mutated to other residues with increasing sizes of the side chain, for instance from Gly to Ala, Val, Leu and Phe, the affinity of the toxin to the receptor was inversely related with size of the side chain (Goldstein et al., 1994). This implies that bigger side chains are sterically hindering CTX from fitting into the receptor site of the channel. For example, the Gly to Leu mutation decreased the affinity of CTX to the receptor by 2000-fold. Secondly, in position 394, the presence of a positively charged residue (Arg) probably results in the resistance of SKv1.1 current to CTX. Other Shaker channels, rat Kv1.5 and Aplysia Ak01a, that have Arg at this position are highly resistant to CTX (Pak et al., 1991; Pfaffinger et al., 1991). When the Thr residue at this position in the Drosophila Shaker channel is mutated to a positively charged Lys, the channel becomes extremely resistant to CTX, implying an electrostatic repulsion between the toxin and the receptor although an electrostatic repulsion has not been demonstrated experimentally. Taken altogether, the resistance of SKv1.1 channel to CTX appears to be due to the presence of two amino acid residues (Leu370 and Arg394) around the pore region. It is interesting to note that the residue 394, Arg in SKv1.1, is known to be an important determinant for both TEA and CTX sensitivity. These two blockers bind to the pore in 107 a mutually exclusive manner (Miller, 1988; Goldstein and Miller, 1993). This residue is also important in determining the requirement of external K+ for channel opening (Pardo et al., 1992). The rKv1.4 channel, which has a positively charged Lys at this position, shows an extreme requirement of the presence of external K+ for channel opening, but the dependence disappears when the Lys is replaced by Tyr. Since SKv1.1 has an Arg at this position, one would expect it to require the presence of external K+ for channel opening. However, this hypothesis has not been tested in this study. Only limited information is available for the molecular mechanisms for the blocking actions of 4-AP (Kirsch et al., 1993; Yao & Tseng, 1993), DTX (Hurst et al., 1991) and MCDP. Amino acid sequence comparison among SKv1.1 and other Shaker channels has not been attempted for these blockers. D. Is the expression of A-type current measured in isolated muscle fibers directed by SKv1.1 gene? The easiest way of addressing this question is to compare the electrophysiological and pharmacological properties of SKv1.1 current to those of native A-current. The electrophysiological properties of SKv1.1 current are much like those of the rapidly inactivating outward current that has been measured from the isolated muscle fibers of S. mansoni (Table 6)(Day et al., 1993; Day et al., 1994). In 108 terms of voltage-dependence, the SKv1.1 current and the native current share very similar Vw's for both activation and inactivation and a similar slope factor for activation. In terms of kinetics, SKv1.1 current and the native current display very similar time constants of activation and inactivation. The most apparent difference between the SKv1.1 current and the native muscle current is their pharmacological properties. The current mediated by SKv1.1 is almost completely blocked by 1 mM 4-AP, while the native current requires 30 mM 4-AP for complete blockade. Whereas the SKv1.1 current was not blocked by 100 mM TEA+, 30 mM TEA+ produced a 50% inhibition of the native current. There are several factors that make the direct comparison between oocyte current and native current difficult. Firstly, one-electrode patch clamping has been employed to measure native current in isolated muscle fibers while two-electrode recording has been used to measure SKv1.1 current in oocytes. Secondly, the native currents are contaminated by other currents, making it difficult to isolate a pure current. Thirdly, different cells provide different environments for the expression as well as the modulation of the channel. There are some examples in which the same channel gene, when expressed in different cells, shows different properties including conductance, kinetics and pharmacology. When rat Kv1.l channel gene is expressed in frog oocytes and 109 in a fibroblast cell line, the channel shows different conductances (Stuhmer et al., 1988; Koren et al., 1990). The time course of inactivation is much slower in oocytes compared with the expression in a mammalian cell (Grissmer et al., 1992). Also, when rat Kv1.3 channel is expressed in frog oocytes and the pharmacology of the current is compared to the native lymphocyte current, different sensitivities to external TEA, quinine and verapamil have been observed (Douglas et al., 1990). Some possible mechanisms for these differences have been suggested. Different cells have different abilities to express the channel protein resulting in a different amount of the channel protein in the cell membrane. Although the exact mechanism has not been revealed, the level of expression determines the electrophysiology and pharmacology of the channel (Guilemare et al., 1992; Honore et al., 1992). The sodium channel, when expressed in frog oocytes and in Electophorus electroplax, exhibits different sensitivity to CTX, which proves to be due to a different posttranslational modification, the glycosylation of the channel (Thornhill & Levitan, 1987). There are also many modulatory factors that affect the properties of the channel in different cells including the presence of different cytoplasmic factors (Marom et al., 1993), B-subunit of K+ channel (Rettig et al., 1994) or functionally associated protein kinases (Esguerra et al., 1994). 110 The SKv1.1 current measured from frog oocytes displayed characteristics similar to native muscle current except for the pharmacology. Further evidence comes from the immunohistochemistry on the isolated muscle fibers in which SKv1.1-specific antibody (ASK1) specifically labels two types of muscle fibers from which A-type currents were originally measured. Therefore, based on results from both electrophysiological and immunohistochemical studies, the expression of A-type currents in isolated muscle fibers appears to be directed by SKv1.1 gene. III. Immunohistochemistry A. What is the function of SKv1.1 current in the nervous system and the neuromuscular system? Strong immunoreactivity to SKv1.1 has been observed in many excitable cells including neurons and muscle fibers. A-type currents modulate the excitability of neurons. In axons, A-type currents modulate the onset, duration and frequency of action potentials (Connor and Stevens, 1971; Segal et al., 1984; Storm, 1987; Kaang et al., 1992). In nerve terminals, A-type currents also regulate neurotransmitter release and synaptic efficacy (Shimahara, 1981; Jan et al., 1977; Kang et al., 1992). A-type currents also modulate postsynaptic excitability (Cassell and McLachlan, 1986). Consistent with their diverse functions, A-type 111 currents are localized in various regions of neurons including dendrites, axon fibers and nerve terminals. Recently, subcellular segregation of various types of mammalian Shaker channels have been demonstrated in Vivo indicating differential roles of K6 channels depending on different subcellular locations (Sheng et al.,1992; Sheng et al., 1994). Obviously, determination of subcellular localization of SKv1.1 protein in the nervous system of S. mansoni could be useful for understanding the detailed functions of SKv1.1 protein in neurons. The function of SKv1.1 protein in muscle fibers is not clear. Neither oscillation of membrane potentials nor the firing of action potential have been observed in the isolated muscle fibers of S. mansoni. In addition to the distribution of SKv1.1 protein, the voltage-dependence of SKv1.1 may help to understand the function of SKv1.1 in the regulation of action potentials. A-type channels modulate the excitability of excitable cells in three ways. First, they delay the initiation of the action potential. Secondly, they decrease the duration of action potentials by facilitating the repolarization of the membrane potential. Thirdly, they, activated by an afterhyperpolarization following action potential repolarization, delay the onset of the next action potential, decreasing the frequency of action potentials. The V50 of the steady-state inactivation of SKv1.1 112 is -40 mV. The resting membrane potential of isolated or in situ (Bricker et al., 1982) muscle fibers is around -30 mV. At this membrane potential, only about 10% of SKv1.1 channels are available, not providing enough channels for the first and second functions described above. Therefore, it is more likely that SKv1.1 protein, activated by afterhyperpolarization, may modulate the frequency of action potentials rather than the onset or duration, unless there is some mechanism that cycles the membrane potential and recruits more SKv1.1 channels without hyperpolarization. Based on the sequence comparison, the SKv1.1 channel is expected to require extracellular Ki for channel opening like mammalian RCK4 channel (Pardo et al., 1992). Higher extracellular K’ concentration also increases the amplitude of the current mediated by RCK4. Therefore, it is possible that SKv1.1 may regulate the excitability of many cells in an activity-dependent manner, being more active when the surrounding concentration of K+ increases as a result the activity of neighboring cells. SKv1.1 also contains many putative sites for phosphorylation by protein kinases such as protein kinase C, tyrosine kinase and casein kinase II. Isolated muscle fibers react to many neurotransmitters and neuropeptides that may be linked to the modulation of the SKv1.1 channel. This indicates that SKv1.1 may modulate the excitability of cells in combination with other receptors. SUMMARY 1) A cDNA (SKv1.1) encoding a Shaker-related K5 channel has been isolated from an adult cDNA library of the human parasitic trematode Schistosoma mansoni. 2) The deduced amino acid sequence (512 aa, 56.5 kDa) contains structural features that are common to other voltage-gated K+ channels. 3) Dendrogram analysis shows that SKv1.1 is classified in the Shaker family. The presence of a Shaker channel in Schistosoma supports the conclusion that the gene duplication event that generated four Sh subfamilies happened before the emergence of Schistosoma. 4) SKv1.1 shows significant sequence identity with most other Shaker channels, with 64-74% identity in the core region (81-86), but not at the N- and C-terminal ends. 5) Evidence for the presence of alternative transcripts of SKv1.1 was not found. Northern blot analysis detected a single primary transcript of 2.8 kb. Southern blot analysis indicated that SKv1.1 is present as a single copy in the genomic DNA of S. mansoni. 6) Expression of SKv1.1 in Xenopus oocytes produced a rapidly activating and inactivating outward K? current which is highly sensitive to 4-AP, but is insensitive to external TEA, MCDP, DTX and CTX. 7) Immunohistochemistry on isolated muscle fibers using 113 114 antibody specific to SKv1.1 demonstrated the expression of SKv1.1 gene in frayed fibers and crescent fibers where A- type currents have been measured. In combination with the results of the electrophysiological characterization, A-type currents in the isolated muscle fibers appear to be directed by SKv1.1 gene. 8) Immunohistochemistry on paraffin sections of S. mansoni has demonstrated the presence of SKv1.1 proteins in neurons as well as muscle cells. 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