Ml \ \ W ‘I WNWlHlNHlWNUIIHWWWIN (ON THESIS ' J. a A an; l:hwunr'CHI a! [3.35.35 an... ‘::t L--—-= arm“ ..:V ‘0515 3 This is to certify that the thesis entitled Biochemical Characterization of the H-Diazepam Binding Site in an American Cockroach, Periplaneta americana, Head.Membrane Preparation presented by Kevin Leigh Blair has been accepted towards fulfillment of the requirements for M.S. Entomology degree in K \’ £ 5’! /_ ( A. \ RA ‘\ t: L&\’ ‘v ‘l1‘\—" ‘ (\ ‘\ \/’\ Major professor August 2, 1985 I)ate 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution MSU LIBRARIES n RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. BIOCHEMICAL CHARACTERIZATION OF THE 3H-DIAZEPAM BINDING SITE IN AN AMERICAN COCKROACH, PERIPLANETA AMERICANA, HEAD MEMBRANE PREPARATION By Kevin Leigh Blair A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Entomology 1985 ABSTRACT BIOCHEMICAL CHARACTERIZATION OF THE 3H-DIAZEPAM BINDING SITE IN AN AMERICAN COCKROACH, PERIPLANETA AMERICANA, HEAD MEMBRANE PREPARATION by Kevin Leigh Blair A 3H-diazepam binding assay was used to characterize the benzodiazepine binding site in membranes isolated from the head of the American cockroach, Periplaneta americana. The binding sites are one of the “peripheral type" since ROS-4864 was the most potent benzodiazepine tested and clonazepam and its 3-methyl analog, R011-3128, were the least potent. In agreement with this conclusion, GABA did not stimulate 3 H-diazepam binding. Titration curves demonstrated that the specific binding was predominately to low affinity (u! range) sites. Most all of the drugs tested, that exhibited significant competitive activity are known to block voltage sensitive sodium channels through the batrachotoxin sensitive site. Chlordimeform was the most potent pesticide tested. DDT and pyrethroids had no statistically significant effect on binding, indicating that they interact at a site different from diazepam and Chlordimeform. Calcium chelation by EGTA irreversibly altered the conformation of the binding site. Thus diazepam and Chlordimeform may be exerting their actions (at least in part) through voltage sensitive sodium channels. Zu BOBO, dem Ersten ii ACKNOWLEDGEMENTS I would like to express my sincere appreciation to the Lord God for a challenging project and a curiosity to match. Also, for providing me with an excellent teacher, Dr. Fumio Matsumura. Your suggestions and advice were most beneficial. I also wish to thank Dr. Jim Miller, Dr. Ralph Fax, and Dr. Matt Zabik for the ideas, encouragement and knowledge that you have conveyed to me. TABLE OF CONTENTS LIST OF TABLES... .................. . ............................. v LIST OF FIGURES ................................................ vi INTRODUCTION ........ ....... . ................................... 1 MATERIALS AND METHODS . ......................................... 4 Materials . ................................ . .................. 4 Animals .................................................... 4 Chemicals ............................................ . ..... 4 Methods ............ ....................................... ...8 Tissue Preparation ...... ....... . ....................... ....8 American Cockroach .................................... ...8 Rabbit .. ............................... . .............. ...9 Binding Assay .................... . ......................... 9 Analysis ................ . ................................. 10 RESULTS . ....................... . ..... . ......... . ............. ..11 DISCUSSION ............ . ..................................... ...25 REFERENCES ............... . ........... . ...... .. .............. ...30 iv LIST OF TABLES Reduction of 3H-Diazepam Specific Binding to Membranes from the Head of American Cockroach by various Benzodiazepines.. Reduction of 3H-Diazepam Specific Binding to Membranes from the Head of American Cockroach by various Neuroactive Agents .0... ........ O 000000000000 00.... ........... 00.... ...... Reduction of 3H-Diazepam Specific Binding to Membranes from the Head of American Cockroach by various Pesticides .... Reduction of 3H-Diazepam Specific Binding by 100 uM Chlordimeform to Membranes from Rabbit Brain and Kidney ...... .12 ...15 ...16 ...18 LIST OF FIGURES Chemical Structures of the Various Neuroactive Agents StUdied .0... 0000000000 O ......... .0... 00000000000000 5 Titration Curves of Diazepam and ROS-4864 ................. 13 Effect of Nucleotide Phosphates (1 MM) on H-Diazepam (3.6 MM) Specific Binding .. ................... 19 Effect of Denaturation on 3H-Diazepam (3.6 mM) SpECific Binding 00.00.0000... ........ 00...... 0000000000000 21 Effect of Ions on 3H-Diazepam (3.6 nM) Specific Binding to EGTA Treated Membranes ................ 24 vi INTRODUCTION The benzodiazepines are a class of neuroactive drugs developed for their anxiolytic, anticonvulsant, sedative, and Inuscle relaxant properties in mammals (1, 2). Stereospecific, high affinity (3, 4) and low affinity (5) receptors have been identified in mammalian brain and are termed as "CNS type". Another high affinity receptor, pharmacologically distinct from the above type, has also been identified. It has been demonstrated in several mammalian peripheral tissues: e.g., lung, liver, and kidney (6), as well as in the brain (7), and is termed as "peripheral type". The "CNS type" and "peripheral type“ high affinity receptors are differentiated by their different affinities for the benzodiazepines clonazepam and ROS-4864. The high affinity "peripheral type" receptor has low affinity for clonazepam and high affinity for ROS-4864 (see 8 for review). The low affinity "CNS type" receptor has only a marginally higher affinity for clonazepam (5). Only the high affinity ”CNS type" receptor correlates well with the efficacy of benzodiazepines to potentiate the action of gamma-amniobutyric acid (GABA), their major molecular and pharmacological action (see 9, 10 for reviews). Indeed, the GABA receptor and high affinity "CNS type" benzodiazepine receptor are on the same protein (11). GABA is the major inhibitory neurotransmitter in the mammalian brain. GABA mediated synapses may comprise as much as 30-50 percent of the total synapses in the brain (12). GABA is also an inhibitory transmitter at the arthropod neuromuscular junction (13, 14). GABAergic function has also been demonstrated in the central nerve cord of the American cockroach (15, 16). GABA's inhibitory action is predominately through the activation of chloride channels. This GABA induced conductance change can be antagonized competitively by bicuculline and noncompetitively by picrotoxinin (17). While the importance of GABAergic systems in mammals and the arthropod neuromuscular junction has long been recognized, the role of GABA in insect CNS functioning has not received extensive investigation. It has been reported that the picrotoxinin receptor is the major site of action for the cyclodiene type pesticides and gama-hexachlorocyclohexane Cr-HCH) in insects (see 18 for review). These pesticides also interact with the picrotoxinin receptor in the mammalian brain (19, 20). Avermectin Bla, a microbially derived pesticide, has been found to potentiate GABAergic actions in preparations of lobster neuromuscular junction (21), nematode nerves (22), and rat brains (23, 24). These interspecies similarities indicate evolutionary conservation of these components in the GABA system. Type II pyrethoids act as partial antagonists to the 35S-t-butylbicyclophosphorothionate (3SS-TBPS) binding site in the mamalian brain (25). TBPS is a picrotoxinin receptor agonist (26). 3H-R05-4864 is also displaced by type II pyrethroids in the mamalian brain (27). Diazepam delayed the onset of type II pyrethroid toxicity in mouse and American cockroach, but not that of type I pyrethroids (28). Benzodiazepine receptors were thought to be nonexistent in invertebrates (29), but have recently been demonstrated in the housefly thorax (30) and housefly head (31). Binding to the thoracic receptor was enhanced by GABA, but the ligand specificity was quite different from that of the mamalian GABA/benzodiazepine complex. Type II pyrethroids, at high UM concentrations, enhanced 3H-flunitrazepam binding in the housefly head. Chlordimeform, an acaricide, displaced 3H-diazepam in an American cockroach head preparation at 100 uM, Diazepam was also observed to cause a transient excitation, then a block of movement in the German cockroach (32). Thus there is impetus to better understand the benzodiazepine receptor(s) in insects and its (their) potential interactions with a GABA related system. The purpose of this study is to examine the basic biochemical characteristics of the benzodiazepine receptor(s) in the head of the American cockroach. Special attention has been paid to the specificity of interactions with a wide range of neuroactive agents. MATERIALS AND METHODS Materials Animals and Chemicals Animals The American cockroaches (Periplaneta americana L.) were taken from cultures maintained by this laboratory for several years. New Zealand albino rabbits were obtained from a licensed dealer in Grand Rapids, Michigan. Chemicals Chemicals were obtained from the following sources: ROS-4864, Dr. J. Bennett (Pharmacology, Michigan State University); praziquantel and R011-3128, Dr. R. Pax (Zoology, Michigan State University); all other benzodiazepines, Dr. H. Moehler (Hoffman La Roche and Co.); Guthion ®, Dr. M. Zabik (Michigan State University); cis and ‘trans-permethrin, Environmental Protection Agency; cypermethrin, FMC; decamethrin, Roussel UCLAF; “Y-HCH, Hooker Chemical company; Baygon ®, Chemagro Corporation. Chlordimeform was synthesized in this lab by the condensation of 4-chloro—o-toluidine and dimethyl formamide. 3H-diazepam (78.9 Ci/mmole) was purchased from New England Nuclear. All other chemicals were purchased from commercial sources. The structures of the chemicals studied are shown in Figure 1. H b O z-n ’9 ..o’... .6 ROS-£864 ©s Flunitracham 9%,", R011- 312 8 Flurlcham Figure 1. ...o ”5 ©c. Clanozepnn Chlordtszepoxidc Chemical structures of the neuroactive agents studied. GABA Picrotoxin1n cud H2 H,N/\r° OH "0W Glycine Octopanine [,f‘\‘N‘:l N (3:50 DY (*5 Ddltiazcn ‘ Nifedipine "s“ Afi©ffl /\ C": /\ Hibd) Verapanil — H 0 © © ‘ ’ xk/ A a, 0"“: Carbamazepine Lidocaine Figure 1 Cont. Pentylenetctrazol I Phenytodn (”37° 3-isobuty1-1-ncthy1 aunthinc J" (J Prathunntcl O “W“Aicre) CI “Inns-p: m th tin ZWA©A© Cit-pemthdn 2W°fl@“‘@ Cypemthrin :'>-/:7*°*©‘*@ hcmthdn ‘N 5%? 5/ OCH; R. Cuthim Figure 1 Cont. N1 co tine Chlordiufom o @dzcwa o x Baygon R' METHODS Tissue Preparation American Cockroach The heads of adult male American cockroaches were homogenized (1 ml/head) in ice-cold modified Van Harrevelds saline (205 mM NaCl, 5.4 mMKCl, 13.6 mM_CaCl2, 2.6 mM.MgClz, and 5 mM_Tris-HCI, pH 7.7) (33) plus 0.3 mM_ phenylmethylsufonyl floride (except where noted) with a glass-glass homogenizer (PyreiED. The homogenate was further homogenized with a TeflorIE-glass homogenizer (Thomas type C) at 800-1000 RPM, 6-8 strokes. This homogenate was then centrifuged at 10009 for 10 minutes at 44 C. The supernatant was collected, filtered through glass wool and stored for later use. The pellet was resuspended in 0.6 ml/head of the same buffer and homogenized with glass-glass (Pyre>®) and then TefloriE glass (Hheaton 30 ml) homogenizers as before. This homogenate was centrifuged and collected as before. The supernatants were combined and centrifuged at 100.0009 for 30 minutes. Thereafter, the supernatant was discarded and the inside of the centrifuge tube was wiped with tissue paper. The pellet was resuspended with a TefloIQ-glass homogenizer (Nheaton 30 ml) as before in 5 mM Tris-HCl buffer (pH, 7.1) (except where noted) to give the equivalent of 1 head/500 ul. The preparation was made fresh on the day of the experiment. Rabbit Rabbits were killed by over-etherization and cardiac puncture. The brains and kidneys were removed, chopped, and stored in 0.32 M sucrose at -800 On the day of the experiment, the samples of brains or kidneys were thawed, minced, and homogenized in 20 volumes of 0.32 M sucrose with a Teflodfilglass homogenizer (Nheaton 30 ml) as before. The homogenate was centrifuged at 10009 for 10 minutes. The supernatant was recentrifuged at 144,0009 for 45 minutes. The supernatant was discarded and the pellet was suspended in 50 mM Tris-HCl (pH, 7.4), with a TeflodELglass homogenizer (Hheaton 30 ml) as before, to give a protein concentration of approximately 4 m9/500 ul. Binding Assay Experiments were conducted in 5 ml glass culture tubes in triplicate, in an ice bath. The tissue homogenate was allowed to stabilize for 30-40 minutes. To 200 ul of 5m_M_ Tris-HCl (pH 7.1) (or buffer plus treatment), 500 ul of the homogenate was added, and the system was allowed to stabilize for another 30-40 minutes. An unlabeled ligand of interest in 1 ul of dimethyl sulfoxide (DMSO for control) was added and the radiolingand binding was initiated by the addition of 3H-diazepam in 300 ul of buffer to give a final concentration of 3.5-3.7 3 nM. H-diazepam. The assay was terminated after 40 minutes (except where I noted) by dilution with 4 ml chilled buffer and rapid filtration through 10 Nhatman GF-B filters. The filter was washed once with 5 ml of the chilled buffer. The filters were then dried, the protein solubilized in 350 ul 0.2 M NaOH over night, and the radioactivity determined by conventional liquid scintillation spectroscopy. Protein was determined by the Lowry method (34). Analysis Specific binding is defined as the difference between total binding and binding in the presence of 100 UN unlabeled diazepam. Percent displacement is the binding in the presence of the ligand of interest divided by the specific binding of the diazepam control. The data were analyzed by the Rank Sum Test or by Random Analysis of Variance. The arc sin V'Y'conversion was used to normalize the data. The Student Newman Keal multiple comparisons procedure was used to determine significance in the Random Analysis of Variance. RESULTS Transmission electron micrographs of the American cockroach head preparation showed it to be comprised predominantly of vesiculated membranes, 50-200 nm in diameter (micrograph not shown). Very few mitocondria were observed. Specific binding typically comprised 30-40 percent of the total binding. The contribution of CNS and muscular tissues to the specific binding was studied. The specific binding to the superoesophageal and suboesophageal ganglia was 8.2 1 2.0 fmol. 3 3 H-Diazepam/head. The muscular components contributed 26.0 1 8.2 fmol H-Diazepam/head. Thus on a per head basis, the musculature contributes the majority of the binding. However, on a mg protein basis, the CNS components bind more, 61.0 1 14.9 fmol 3H-Diazepam/mg protein and 33.4:p 10.5 fmol 3H-Diazepam/mg protein for CNS and musculature respectively. The filter accounted for 50-60 percent of the nonspecific binding. Several benzodiazepines were tested for their ability to displace 3H-diazepam (Table 1). The most potent was ROS-4864 with a reduction of 107 1,5.5x. Medazepam was quite potent with a reduction of 78 :_14%. Chlordiazepoxide, flunitrazepam, and flurazepam were equally potent with a reduction of 45 :_4.9, 44.1 6.5, and 42 :_6.7% respectively. Clonazepam was rather weak with only 17‘:_9.4%. The antischistosomal 3 drug R011-3128 actually increased H-diazapam binding by 24 :_7.6%. 11 12 TABLE 1. Reduction of H-Diazepam Specific Binding to Membranes from the Head of American Cockroach by Various Benzodiazepines Benzodiazepine Concentaation Reduction, % of Controla u Diazepam 100 100b A ROS-4864 100 107 i 5.5 B Medazepam 100 78 :_14 C Chlordiazepoxide 100 45 :_4.9 D Flunitrazepam 100 44.: 6.5 D Flurazepam 100 42 :_6.7 D Clonazepam 100 17 :_9.4 E R011-3128 100 ~24‘:_7.6 F a Each mean is of three separate experiments performed in triplicate ‘1 standard deviation. Values followed by the same letter are not significantly different by Student Newman Keal multiple comparison procedures at p<.05. b Normalized control. 13 100 so 3H-diazepam displaced. (‘70) of control Figure 2. mommm . (omosaaea ' I d J j n _1 9 a a s 4 - Log conc. (M) Titration curves of (o) diazepam and (o) ROS-4864. Each symbol and verticle bar is the mean and standard devgation of three replicates of one experiment. H-diazepam concentration was 3.6 nM, 14 Titration curves of diazepam and ROS-4864 were then obtained (Figure 2). The curve for diazepam indicates the presence of a small saturable component below 100 nM. Above 100 mM, the curve indicates no saturation up to the limit of solubility (100 Mfl) of diazepam in this preparation. ROS-4864 displaced 3H-diazepam binding at similar concentrations. It appears to be a little less potent in the nM_range and a little more potent in the u! range. This was typical of three separate experiments. 3H-diazepam To explore the biochemical characteristics of the binding representatives from various classes of neuroactive agents were studied (table 2). GABA was without effect at 100 n! and 10 uM. The chloride channel antagonists picrotoxinin and pentylenetetrazol were 3H-diazepam binding also without effect. Conversely, glycine enhanced by 50‘:_14%. Octopamine, 3-isobutyl-1-methyl xanthine, and nifedipine were also without statistically significant effect. The most potent compounds tested were the calcium channel antagonists diltiazem (46 i; 6.4%) and verapamil (45 ‘1_ 5.7%); the sodium channel antagonists phenytoin (55‘: 8.5%), carbamazepine (40 :_6.4), and lidocaine (52 1 4.2%); and the antischistosomal drug praziquantel (51: 14%). The displacements by phenytoin and verapamil were not additive. None of the pesticides except Chlordimeform, statistically significantly displaced 3H-diazepam (Table 3). To further characterize the CNS/peripheral nature of these 15 TABLE 2 Reduction of 3H-Diazepam Specific Binding to Membranes from the Head of American Cockroach by Various Neuroactive Agents a Agent Concentration Reduction, % of Control (NM) Diazepam 100 100b A GABA 10 8.5 :_20 B C GABA 0.1 -8.0‘:'4.2 B Picrotoxinin 100 ~11“: 7.1 B Pentylenetetrazol 100 -8.5 1 4.9 B Glycine 0.1 -50':’14 D Octopamine C 100 22 1 8.5 c E 3-isobutyl-l-methyl xanthine 100 13.5 :_.01 C Nifedipine 100 24‘: 9.9 C E F Diltiazem 100 46 i 6.4 E F Verapamil 100 45 1 5.7 E F Phenytoin 100 55‘:_8.5 F Carbamazepine 100 40.: 6.4 E F Lidocaine 1000 52 1 4.2 E F Phenytoin plus 100 53.: 1.1 E F Verapamil 100 Praziquantel 100 51 + 14 E F a Each mean is of two separate experiments performed in triplicate :_standard deviation. Values followed by the same letter are not significantly different by Student Newman Keal multiple comparison procedure at p< .05. b Normalized control. c Assays terminated at 20 minutes. 16 TABLE 3 Reduction of 3H-Diazepam Specific Binding to membranes from the Head of American Cockroach by Various Pesticides Pesticide Concentration Reduction, % of control a 041) Diazepam 100 100 b A DDT 100 0 i 27 8 DOT 10 -6.6 i B Cis-permethrin 100 4.5 :_ B Trans-permethrin 100 2.4 3.19 B Cypermethrin 100 -20 i 57 B Decamethrin 1 26 1 1.5 B Decamethrin .01 -16 i 33 B Baygon 100 10‘: 15 B Lindanec 100 44 1 24 B Guthion 100 25 :_9.9 B Nicotine 100 36 :_13 B Chlordimeform 100 52 :_17 C a Each mean is of two or three separate exeperiments performed in triplicate + standard deviation. Values followed by the same letter are fiot significantly different by Student Newman Keal multiple comparison at p< .05. b Normalized control. c Gamma-hexachlorocyclohexane. 17 interactions, Chlordimeform was tested in rabbit brain and kidney preparations (Table 4). Chlordimeform was moderately potent in the kidney preparation (35 ‘1' 12%), but had no effect in the brain preparation. Phosphonucleotides are often involved 'hi kinase mediated receptor-message transuction. The nucleotides 6 N ,2-0-dibutyryladenosine 3':5'-cyclicmonophosphate (dibut-C-AMP), N2 ,2'-0-dibutytylguanosine 3':5'-cyclicmonophosphate (dibut-C-GMP) guanosine-5'-diphosphate (GDP), and guanosine-5'-triphosphate (GTP) were tested (Figure 3). Only the guanosine cyclicmonophosphate analog had a significant effect. It increased specific binding by 50‘:_12%. To determine the proteinaceous nature of the interactive sites of the binding, various denaturation procedures were used (Figure 4). Treatments with sodium dodecyl sulfate (1% w/v), heating at 900 C for 5 minutes, or bovine serum albumin (1% w/v) reduced specific binding by 95 :_18%, 66 :_38%, and 36 1_14%, respectively. The results of trypsin (1% w/v) was variable and the specific binding was not significant. 0n the other hand, milder treatments of the same agents or methods: sodium dodecyl sulfate (0.1% w/v); trypsin (0.01% w/v); bovine serum albumin (0.01% w/v); and heating the preparation to 900 C increased the specific binding by 64 i 38%, 51 i 24%, 61 i 24%, and 141 i 20%, respectively. 18 TABLE 4 Reduction of 3H-Diazepam specific binding by 100 uM_Chlordimeform to membranes from rabbit brain and kidney Tissue Reduction, % of Control a Brain 8.8 1 7.7 Kidney 35 1 12b a Each mean is of three separate experiments performed in triplicate :;standard deviation. Control specific binding was determined with 100 uM.R0 5-4864. b p< .05 by Ranked Sum Test. 19 ouaznmosaocoe u«~u>ut.nu.n oc«.oc¢:m~xu>~:a«urctNu 2 any oun:Ano:uo:oe ouuuauw.n..n caduceus-nauauaa«vIOIN. swap saw cascag »a mo. vav #9. A43 mo. vav Ho. Ase .au~u_Fa_Lu =_ cascoccmg acmswcmaxm one do :o_uow>wu ugmccmum can came mgu m? m:_P use can scam .mcruom_u== as» do cowamucm wza Emuwm muche wco umpmcw2Lmu we: ammmc ugh .cmumpupcP mm: m:_c=wn m>puaupumaeou mg» sauce mmuacps cc umcmw mew: mmupuompuzc one Jm=_uewa o_cpumam A2: o.mv aaam~a_u-= co A2: Hy mmamgamosa mnwuow_u== co uuwuwm Ouagauosauuur.ntocuuocnao any ouucaaoga«VI.nloc«uocuau any oz Acv .m mczmvu cow can. C Iozzuoa 30 z 'Surputg atgtaads medazlra.u Figure 4. 20 Effect of denaturation on 3H-diazepam (3.6 nM) specific binding. The isolation buffer was replaced with 0.25 M sucrose. Sodium dodecyl o sulfate (A,B) was added to the membranes at 25 C, then placed on ice. Trypsin ($2,700 units/mg) (C,D) was incubated for 1 hour at 25 C with the membranes prior to initiating the assay, th n placed on ice. Membranes were heated t8 90 C in 5 minutes (E,F). Thenomaintained at 90 for 5 minutes (F only) at 90 C for 5 minutes, then placed on ice. Bovine serum albumin 86,H) was incubated with the membranes for 1 hour at 25 C prior to initiating the assay, then placed on ice. Each bar and line is the mean and standard deviation of one experiment performed in triplicate. (*) .01 \3 w~o.o .:PE:a_< Eacmm m:_>om >\3 a“ .cwssn_< Eacmm mcw>om u com cu and: .:WE m cow u com an “no: >\3 RHo o .cwmaaLb >\3 aw .cwmuxcp >\z x~.o .mumm_=m _xumccu E:_nom >\z xH .mumc_=m .aUmcou Eamuom sz Aw Adv Amy Acv .uv Amy Aqv .e mesmpu cap cow can Ioaauog ;o z 'Suxpuza 31;}:ads cedazrza a: 22 The effects of various ions on 3H-diazepam specific binding was studied in the presence and absence of the calcium chelator ethyleneglycol-bis-(B-aminoethyl ether)-N,N,N',N',-tetraacetic acid (EGTA) (Figure 5). specific binding in the controls of EGTA treated preparations was typically 3-4 times that of the non EGTA controls. CaCl2 added after this treatment did not reverse this increase. All of the salts were added to give a 200 mM cation final concentration except CaCl which was tested at 10 mM. All of the salts tested, except CaCl 2 2. increased the specific binding to EGTA treated membranes: NaCl, 70 1 315; KC], 94 1 27%; NH N0 163 1 71%; (NH 215 1 45%; Na so 4 3’ 4’2504’ 2 4’ 239 1 73%; and choline chloride, 143 1 71%. CaCl2 enhanced specific binding in the absence of the EGTA treatment by 31 1 26%. No other salts enhanced specific binding in the absence of the EGTA treatment. Sucrose (200 mM) decreased specific binding in the non EGTA treated membranes by 511 18%. Figure 5. 23 Effect of ions on 3H-diazepam (3.6 nM) specific binding to EGTA treated membranes. Final cation concentration were 10 nM for CaCl and 200 mM_for all others. Sucrose concgntration was 200 mM. Van Harrevelds saline was replaced with 0.25 M_sucrose. The final membrane suspension was made in 5 mM_Tris-HCl (pH, 7.1) plus 2 mM_EGTA. Specific binding to untreated controls was typically 2000-5000 DPM. Specific binding to treated controls was 10,000-15,000 DPM. Specific binding enhancement by NaCl and KCl were concentration dependant. Each bar and line is the mean and standard deviation of one experiment performed in triplicate. (*) (.01