Separate Subunits for Agonist and Benzodiazepine Binding in the Y- Aminobutyric Acida Receptor Oligomer*

Separate Subunits for Agonist and Benzodiazepine Binding in the y- Aminobutyric AcidA Receptor Oligomer*

(Received for publication, June 24,1986)

Stefano O. CasalottiJ, F. Anne Stephenson§, and Eric A. Barnard From the Medical Research Council Molecular Neurobiology Unit, Medical Research Council Centre, Cambridge CB2 2QH, Great Britain

The 7-aminobutyric acidA (GABAa) agonist muscimol benzodiazepine-binding site as shown by its photoaffinity can be photoactivated by 254 nm illumination to affin labeling by [3H]flunitrazepam applied to the pure receptor ity label its binding site in the GABAa receptor. We protein (1,4). These results have now been confirmed in other have conducted this reaction on the pure receptor from laboratories (Ref. 5 and reviewed in Ref. 6). bovine cerebral cortex in detergent solution, showing Recently, two groups have employed the G ABA agonist, that [3H]muscimol can produce then a specific saturable muscimol, as another photoaffinity reagent to label a GABA- labeling. In the detergent solution, the receptor alone binding site in membranes from rat cerebellum (7, 8). We is sensitive to 254 nm irradiation; this reduces the have adapted this method to label the pure GABA a receptor efficiency of incorporation to below that in the mem from bovine cortex and have hence identified the high-affinity branes, but the competing photoreaction with [3H]muscimol is sufficient and occurs at a representative set of GABA-binding subunit of the receptor. the muscimol-binding sites, such that it can be em MATERIALS AND METHODS ployed for the photolabeling of those sites. The affinity of [3H] muscimol displayed in this irreversible reaction [iV-met/iy(-3H]Flunitrazepam (85 Ci/mmol) and [methylene-3H] is indistinguishable from that of its reversible binding. muscimol (9 Ci/mmol) were from Amersham Int. Flunitrazepam and Ro 7-1986/1 were kindly supplied by Dr. H. Mohler, Hoffmann-La 7-Aminobutyric acid and bicuculline compete in the Roche (Basel, Switzerland), and chlorazepate was a gift from Boeh- photolabeling reaction according to their known affin ringer Ingelheim (Bracknell, United Kingdom). Reduced Triton X- ities at the 7-aminobutyric acid-binding site. 100 was from Aldrich, and all other materials and methods not The labeling is shown to occur at the /9-subunit (ap specified were as given previously (1, 2). parent Mr 57,000) in the pure receptor. The binding Membrane Receptor Preparations—Bovine brain was dissected and sites for 7-aminobutyric acid agonists, on the /9-sub frozen at —80 °C within a maximum of 1 h after removal. Thoroughly units, and the benzodiazepine binding sites, on the a- washed twice frozen-thawed brain membranes were prepared (9), and subunits, are linked allosterically so that a strongly membrane-bound receptor activity was determined using [3H]musci- cooperative hetero-oligomeric structure of this recep mol for the measurement of GABA binding in a centrifugation assay tor is deduced. or using [3H] flunitrazepam for the measurement of benzodiazepine binding in a filtration assay (1). Purification and Analysis of the GABAA-Benzodiazepine Receptor— The receptor was purified from bovine cerebral cortex by Ro 7-1986/ 1-agarose affinity chromatography with 0.2% Triton X-100 in the Earlier studies in this laboratory have resulted in the puri final media. For some experiments, a reduced form of Triton X-100 fication to homogeneity of the G A BA a* receptor complex of was used in the equilibration and elution of the ion-exchange chro bovine (1, 2) or rat (3) brain cortex in media containing matography column. Soluble receptor activity was measured by the polyethylene glycol precipitation-filtration assay (1). Triton X-100, with co-retention of the specific binding sites Samples prepared for SDS-PAGE were first concentrated either for G ABA agonists (recognized by [3H] muscimol) and antag by precipitation with 12% (w/v) trichloroacetic acid, washing twice onists and for the brain-type benzodiazepines and for /3- with acetone, and air drying (1) or by precipitation and washing with carbolines. Furthermore, if the media throughout are instead MeOH/CHCb (3:1) (v/v) (10). The samples were dissolved in sample prepared in CHAPS/phospholipid solution, the same complex buffer containing 1 % SDS, denatured instantly at 100 ®C for 5 min is obtained with retention also of the active sites for the in the presence of 10 m M dithiothreitol, and subjected to SDS-PAGE under reducing conditions in 10% (w/v) polyacrylamide slab gels, all chloride ion channel-regulatory ligands and of the allosteric as described by Sigel and Barnard (2). interactions between all of the afore-mentioned sites (2). In For samples which were photoaffinity-labeled, following SDS- both types of preparation, SDS-polyacrylamide gel electro PAGE either (i) the slab gels were sliced at 1.5-mm intervals and phoresis revealed only two types of subunit, a (apparent M r extracted and counted as described in Ref. 1 or (ii) the proteins were 53,000) and /9 (apparent M r 57,000). The a-subunit carries the transferred to nitrocellulose by Western blotting as described in Ref. 11. The nitrocellulose was impregnated for 15 min at room tempera * The costs of publication of this article were defrayed in part by ture with Autofluor (National Diagnostics, Aylesbury, United King the payment of page charges. This article must therefore be hereby dom), and the labeled bands were visualized by fluorography on marked “advertisement" in accordance with 18 U.S.C. Section 1734 preflashed Fuji x-ray film. solely to indicate this fact. Photoaffinity Labeling—Thoroughly washed twice frozen-thawed $ Supported by a graduate studentship from Fidia Laboratories. membranes from bovine brain cortex were diluted to 0.6 mg of § Holder of a Royal Society University research fellowship. To protein/ml with 10 m M HEPES (pH 7.4), 1 m M EDTA, 1 m M whom correspondence should be addressed: MRC Centre, Hills Rd., benzamidine HC1, 1 mg/ml soybean trypsin inhibitor, 1 mg/ml ovo Cambridge CB2 2QH, Great Britain. mucoid trypsin inhibitor, 1.25 m M dithiothreitol, 0.02% NaN3. Ali 1 The abbreviations used are: GABA, 7 -aminobutyric acid; CHAPS quots (2.35 ml) were incubated for 20 min on ice with [ 3H]muscimol 3 -[(3 -cholamidopropyl)dimethylammonio]-l-propanesulfonate; (60 n M final concentration). Similarly, the purified receptor (0.3 ml HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; SDS- containing 2 pmol of [3H] flunitrazepam-binding sites) was incubated PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. with either [3H] flunitrazepam or [3H] muscimol (each at 100 n M final

15013 15014 Subunits Binding GABA and Benzodiazepines concentration) for 30 min on ice with continuous shaking. These pM muscimol) was much lower in the pure protein than in the samples were irradiated (in 8 -cm diameter flat-bottom vials) with membranes, averaging only 4% of the total irreversible bind ultraviolet light from a Camag Universal ultraviolet lamp (peak ing, after 30 min of reaction on ice. wavelength 254 nm) at a distance of 6 cm, again on ice with continuous shaking. The irradiation was interrupted for 2 min at 10-min intervals For the pure receptor alone in Triton X-100 solution, the to avoid overheating of the samples. illumination at 254 nm caused a time-dependent loss of the At the end of the stated total incubation time, the irradiated agonist sites, with the loss of 78% of the initial reversible samples were incubated on ice for 90 min with excess nonradioactive agonist-binding sites with 30 min of illumination. The sites ligand, 10 pM flunitrazepam, or 10 pM muscimol, respectively. For remaining (after 30 min of illumination) then showed the the membrane preparation, the unbound 3H-labeled ligand was re same relative susceptibility to the [3H] muscimol photolabel moved from the samples by centrifugation at 8000 X g followed by four washes with 10 m M HEPES (pH 7.4) where the first wash ing as the original population. When [3H]muscimol (100 nM) contained also 10 p M muscimol; for the purified receptor, the protein was irradiated for 30 min, it lost 75% of its capacity to bind was precipitated with 12% trichloroacetic acid and washed as de reversibly to the pure receptor; on the rat membranes, an 80% scribed above. Control experiments in the dark showed that these loss of its photolabeling capacity upon 10 min of irradiation treatments were sufficient to remove all of the free 3H-labeled ligand. of [3H]muscimol was reported (8). Due to these two competing The acetone-washed pellets were either dissolved for counting of radioactivity with the scintillant described in Ref. 1 or dissolved in reactions, as well as the intrinsically low efficiency of irrever sample buffer and subjected to SDS-PAGE under reducing conditions sible attachment of the acylnitrene that has been supposed to as above. form on the photolysis of muscimol (8), the photolabeling of the pure receptor reached only 7.5 ± 0.8% of the sites present RESULTS AND DISCUSSION which can bind [3H] muscimol reversibly. The U V absorption When bovine brain cortex membranes were incubated with of the 0.1% Triton X-100 used as the solvent was also consid [3H]muscimol and subsequently irradiated with U V light, it ered as a possible contributory factor to this low efficiency, was found, in agreement with the results of Cavalla and Neff but this detergent could be replaced by a reduced, non-ab (8) on rat cerebellar membranes, that tritium was irreversibly sorbing form of Triton X-100 (see “Materials and Methods”) incorporated into the membrane fraction. The maximum spe with identical values for the maximum receptor photolabeling cific photolabeling of the GABA a agonist-binding sites that attainable. Dithiothreitol had no effect on the irreversible and was achieved thus corresponded to 107 fmol of [3H] muscimol reversible total and nonspecific reaction with [3H] muscimol binding sites/mg of protein. This irreversible incorporation (Table I). into the membrane-binding sites was time-dependent during Despite the rather low efficiency of the photolabeling re a 90-min illumination on ice (Table I). Similar irradiation (30 action, it appears to proceed at binding sites representative min) in the absence of [3H]muscimol resulted in no significant of the high-affinity [3H]muscimol sites (1, 2) of the purified loss of the sites of reversible muscimol binding, confirming in receptor in Triton X-100 medium. Thus, the dependence upon this species the results (7,8) with rat cerebellar synaptosomal the concentration of [3H] muscimol showed saturation behav membranes. Treatment of the membranes with 1.25 mM ior similar to that of the reversible binding of that ligand to dithiothreitol prior to the reaction gave no significant differ the pure receptor (1) and yielded a Scatchard plot showing a ence in the specific irreversible incorporation of [3H]musci single set of sites with apparent K D = 15 ± 5 nM (Fig. 2A). mol; the ratio of total to nonspecific incorporation of radio With such an irreversible reaction, the Scatchard equation is activity was, however, then increased. not strictly applicable; but the error in using it will be small When the purified receptor was illuminated similarly in the when the irreversible phase is extremely fast, which should presence of [3H] muscimol, the ligand was specifically and be the case with such a photoradical (12). With the use of the irreversibly incorporated into the isolated protein. This reac natural photoaffinity ligand employed here, however, the U V tion reached its maximum extent after 30 min of U V irradia irradiation time is 30 min. During this extended incubation tion (Fig. 1). Nonspecific labeling (i.e. in the presence of 10 period, it is further possible that there are more deviations

T a ble I [ 3HJMuscimol photoaffinity labeling of bovine cortical membranes and GABAA-benzodiazepine receptor purified from bovine cortex Thoroughly washed twice frozen-thawed membranes from bovine brain cortex were diluted to 0.6 mg of protein/ ml and were photoaffinity-labeled with [3H]muscimol as described under “Materials and Methods.” Similarly, the purified receptor was diluted to 4.4 pmol of [3H]muscimol-binding sites/ml, and the photoaffinity labeling reaction was conducted on 0.3-ml samples. The results are expressed as the mean ± S.D. of triplicate determinations; t denotes the period of UV irradiation, always carried out on ice at 6 -cm separation of source and sample. Total Nonspecific Sample UV irradiation conditions irreversible irreversible incorporation incorporation cpm/ml cpm/ml Bovine cerebral cortical membranes t = 10 min + 1.25 mM 349 ± 41 234 ± 26 dithiothreitol t = 30 min + 1.25 mM 1208 ± 200 497 ± 34 dithiothreitol t = 90 min + 1.25 mM 2086 ± 240 875 ± 37 dithiothreitol t = 30 min 2385 ± 381 1878 ± 150

Purified bovine GABAA-benzodiazepine receptor t = 10 min 415 ± 38 67 ± 2 t = 30 min 1320 ± 23 88 ± 14 t = 30 min + 1.25 mM 1385 ± 23 55 ± 12 dithiothreitol Subunits Binding GABA and Benzodiazepines 15015

o E u o in E 9 a E ■o e X 9 « o £> V -D H3 in u v > u V « a o in

Irradiation time (min) Fig. 1. Time dependence for photoaffinity labeling of the purified bovine receptor with [3H]muscimol. Purified receptor (48 pmol of reversible [3H]muscimol-binding sites/ml) was diluted 10-fold in 20 mM potassium phosphate, 1 mM EDTA, 0.1% Triton X-100 (pH 7.4), and samples (0.3 ml) were incubated for 20 min on ice in the presence of 100 nM [3H] muscimol. Samples were UV- irradiated on ice for increasing periods of time, following which each sample was further incubated for 90 min on ice in the presence of 10 fiM muscimol. The protein was precipitated, washed with acetone, and counted. All procedures were as described under “Materials and Methods.” The circles denote the specific irreversible incorporation of [3H]muscimol (mean ± S.D. for three determinations). The non specific incorporation was determined by similar irradiation in the presence of 10 jiM muscimol and was subtracted; this corresponded to 4% of the total irreversible incorporation at 30 min of illumination. from the equilibrium conditions which are assumed in the Scatchard analysis. For example, it was described above that Fig. 2. A, Scatchard plot of [3H]muscimol irreversible binding to [3H]muscimol is itself inactivated with U V irradiation; and the purified bovine GABAA-benzodiazepine receptor. Purified recep its concentration and the fractional occupancy of the receptor tor (1 pmol of reversible [3H] muscimol-binding sites in 0.3 ml) was population, therefore, by [3H] muscimol must alter during the incubated for 20 min on ice with [3H] muscimol in the concentration range 5-120 nM. Samples (0.34 ml) were UV-irradiated for 30 min on illumination procedure. The purified receptor is also suscep ice and further incubated for 90 min in the presence of 10 fiM tible to U V irradiation. Nevertheless, despite these occur muscimol. The protein was precipitated, washed with acetone, and rences, an equivalence of the apparent KD value here for counted. All procedures were as described under “Materials and irreversible binding with that (KD = 12 ± 3 nM) for the Methods.” Nonspecific irreversible incorporation was determined by reversible binding to the pure receptor in the same medium parallel UV irradiations in the presence of 10 fiM muscimol. The (1) was found. Furthermore, the pharmacological specificity Scatchard plot shown is representative of two experiments and is best fit with a single site of apparent KD = 15 nM. B, pharmacological of the photolabeled sites is that of the agonist sites in the specificity of the irreversible [3H] muscimol-binding site. The purified receptor; the agonist G ABA or the antagonist bicuculline bovine receptor (1 pmol of reversible [3H] muscimol-binding sites/ml methobromide inhibited the irreversible labeling competi in 0.3 ml) was incubated for 20 min on ice with increasing concentra tively (Fig. 2B), with respective IC 50 values of 53 ± 4 nM and tions of either GABA (•) or bicuculline methobromide (A) and [3H] 6 ± 0.1 /iM, values very close to their affinities (1, 5) for the muscimol (50 nM final concentration). Samples were UV-irradiated purified receptor. It is known that in vertebrates there exist for 30 min on ice, and the assays were completed as in A to give the mean ± S.D. The apparent ICW values determined were 53 ± 4 nM at least two classes of GA BA receptors. These are the GABA a (n = 2 ) for GABA and 6 ± 0.1 fiM (n = 2) for bicuculline methobro receptor which is discussed herein and the GABA b receptor mide. which differs from the A-type receptor in both its pharmaco logical specificity and in that GABA b responses are mediated afore-mentioned [3H]flunitrazepam photolabeling, which oc via the interaction with a GTP-binding protein (4). The curs primarily on the a-subunit of the pure receptor (1, 4). GABA b receptor agonist baclofen, in the presence of 2.5 mM Ca2+, which is requisite for binding activity, did not affect the When a low concentration (5 nM) of [3H]flunitrazepam was used in a 5-min period of that photoaffinity reaction, only the irreversible [3H] muscimol binding, showing that GABA b re ceptor sites are not also labeled. a-subunit became labeled in the pure receptor, and this was SD S-PA G E under reducing conditions showed that the /3- clearly smaller in Afrthan the subunit photolabeled with [3H] subunit of M r 57,000 was specifically labeled by the [3H] muscimol (Fig. 3). The respective positions agreed with those muscimol photoreaction (Fig. 3). This was seen both by fluo- (a = M t 53,000 and /3 = M r 57,000) found after Coomassie rography (Fig. 3) and by slicing gels (results not shown) and Blue and silver staining after SD S-PA G E of the pure receptor determining the profile of radioactivity within them. The preparation, as illustrated previously (1, 2). labeling in the /3-subunit, but not the a-subunit, of the pure The evidence presented here shows that [3H] muscimol per receptor was confirmed by using the marker provided by the forms a photoaffinity reaction on the purified GABA a recep- 15016 Subunits Binding GABA and Benzodiazepines ABC D Mr values, and the apparent molecular masses of membrane polypeptides in these gels can easily vary by several thousand n daltons according to the conditions of sample preparation and 330 kDa electrophoresis. A gel pattern showing both of the GABAa receptor subunits as standards was not employed in those investigations on the membranes (7, 8). Furthermore, we have found that when the purified receptor labeled with 125I is subjected to SDS-PAGE under the conditions identical to 97kDa those described above and the 125I-labeled a- and /3-subunits are detected by either gel slicing or autoradiography, the resolution of the polypeptides in this range is such that the gel slicing and counting of 125I gave a single band. Autoradiog 6 7 k Da raphy in a parallel lane showed, however, clearly resolved a- and /3-subunits (data not shown). We conclude, therefore, that there is no real contradiction with the previous results at the membrane level. There is electrophysiological evidence that channel opening in the GABAa receptor requires the simultaneous binding of two agonist molecules (13). Our studies on the pure protein have suggested the subunit structure a2^ 2 (4), which now implies one GABA site (at least) on each of two /3-subunits. It is of particular interest that the GABA site and the ben 36kDa zodiazepine site are now found to be located on different subunits since these two sites are closely linked allosterically (14). This denotes a close steric relationship in the oligomeric structure of parts of the a- and /3-subunits in a zone of the receptor on the external side of the membrane concerned in the recognition of these two types of ligand. F ig . 3. [3H]Muscimol photoaffinity labeling of the purified REFERENCES bovine receptor. The purified receptor (55 pmol of [3H] muscimol binding sites/ml) was photoaffinity-labeled with 100 nM [3H]flunitra- 1. Sigel, E., Stephenson, F. A., Mamalaki, C., and Barnard, E. A. zepam (100-^1 aliquot) or [3H]muscimol (600-/d aliquot) as described. (1983) J. Biol. Chem. 2 5 8, 6965-6971 After the trichloroacetic acid precipitation samples were prepared for 2. Sigel, E., and Barnard, E. A. (1984) J. Biol. Chem. 259, 7219- SDS-PAGE under reducing conditions and the protein was finally 7223 transferred to nitrocellulose membranes in Western blots. A fluoro- 3. Stephenson, F. A., Sigel, E., Mamalaki, C., Bilbe, G., and Barnard, graph of the nitrocellulose after 26 days of exposure at -80 °C is E. A. (1984) Dev. Neurosci. 17, 437-442 shown: lane A, receptor photoaffinity-labeled with [3H]muscimol; lane 4. Stephenson, F. A., Mamalaki, C., Casalotti, S. O., and Barnard, B, receptor photoaffinity-labeled with [3H]muscimol in the presence E. A. (1986) Biochem. Soc. Symp., in press of 10~4 M bicuculline methobromide; lane C, receptor photoaffinity- 5. Schoch, P., Haring, P., Takacs, B., Stahli, C., and Mohler, H. labeled with [3H]flunitrazepam; lane D, molecular mass standards. (1984) J. Recept. Res. 4, 189-200 6. Stephenson, F. A., and Barnard, E. A. (1986) in Receptor Meth odology and Biochemistry (Olsen, R. W., and Venter, J. C., eds) Vol. 5, pp. 261-274, Alan R. Liss, Inc., New York tor in Triton solution as it does in the membranes and that 7. Asano, T., Sakakibara, J., and Ogasawara, N. (1983) FEBS Lett. this reaction occurs at the high-affinity GABA agonist site. 1 5 1, 277-280 The site of this reaction is on the /3-subunit, and hence we 8. Cavalla, D., and Neff, N. H. (1985) J. Neurochem. 44, 916-921 9. Stephenson, F. A., Watkins, A. E., and Olsen, R. W. (1982)Eur. locate the GABA agonist site on that subunit. J. Biochem. 1 2 3, 291-298 In the photoactivated reaction of [3H] muscimol with rat 10. Wessel, D., and Fliigge, U. I. (1984) Anal. Biochem. 138, 141- cerebellar membranes, Cavalla and Neff (8) found that a 143 subunit of apparent MT 52,000 was labeled; whereas Asano et 11. Stephenson, F. A., Casalotti, S. O., Mamalaki, C., and Barnard, al. (7) reported (using a gel slicing method) that the labeling E. A. (1986) J. Neurochem. 46, 854-861 occurred on the same subunit as the benzodiazepine photoaf 12. Chowdry, V., and Westheimer, F. H. (1979)Annu. Rev. Biochem. 4 8, 293-325 finity labeling. Both of those observations appeared to indi 13. Sakmann, B., Hamill, O. P., and Bormann, J. (1983) J. Neural cate that the a-subunit reacts in the membranes. However, Transm. 1 8, (suppl.) 83-95 the a- and /3-subunits differ only by 4,000 in their apparent 14. Olsen, R. W. (1982) Annu. Rev. Pharmacol. Toxicol. 22, 245-277 Journal o f Neurochemistry Raven Press, New York © 1986 International Society for Neurochemistry

Antibodies Recognising the GABAA/Benzodiazepine Receptor Including Its Regulatory Sites

F. Anne Stephenson, Stefano O. Casalotti, Cleanthi Mamalaki, and Eric A. Barnard

Department of Biochemistry, Imperial College of Science and Technology, London, England

Abstract: Polyclonal antibodies have been raised against binding (TBPS), the barbiturate-enhanced [3H]fluni- the G A B A A/benzodiazepine receptor purified to homo trazepam binding, and the GABA-enhanced [3H]fluni- geneity from bovine cerebral cortex in deoxycholate and trazepam binding were all removed together into the Triton X-100 media. Radioimmunoassay was applied to immunoprecipitate. Western blot experiments showed measure specific antibody production using the ,25I-la- that these antibodies recognise the a-subunit of the belled y-aminobutyric acid (GABA)/benzodiazepine re purified GABA/benzodiazepine receptor. These results ceptor as antigen. The antibodies specifically immuno- further support the existence in the brain of a single pro precipitated the binding sites for [3H]muscimol and for tein, the GABA a receptor, containing a set of regulatory [3H]flunitrazepam from purified preparations. In addi binding sites for benzodiazepines and chloride channel tion, when a 3-[(3-cholamidopropyl)dimethylammonio] 1- modulators. Key Words: G A B A — Benzodiazepine— propanesulphonate (CHAPS) extract of bovine brain t-Butylbicyclophosphorothionate— Antibodies. Ste membranes was treated with the antibodies, those sites phenson F. A. et al. Antibodies recognising the G A B A a/ as well as the [3H]propyl-p-carboline-3-carboxylate benzodiazepine receptor including its regulatory sites. J. binding, the [35S]/-butylbicyclophosphorothionate Neurocltem. 46, 854-861 (1986).

The GABA a receptor from mammalian brain be plexity in the binding sites of this receptor is indi longs to the class of receptors having ligand-gated cated by the finding that three different types of ion channels, abundant evidence being available to benzodiazepine receptor ligands, i.e., benzodiaze show that it is an integral membrane protein with pine agonists, antagonists, and inverse agonists, both a recognition site for the natural neurotrans can modulate GABA a receptor function (Braestrup mitter and an intrinsic channel for chloride ions, et al., 1982). both of which are modulated by various allosteric The G A B A A/benzodiazepine receptor has been binding sites (reviewed by Olsen, 1982). Thus, it has solubilised successfully with certain nondenaturing been shown that, in the membrane-bound state, the detergents such that the aforementioned regulatory GABA a receptor protein is associated with (i) a binding properties persist in the extracts, in partic high-affinity binding site for the anxiolytic drugs, ular with use of the zwitterionic detergent 3-[(3- the benzodiazepines (Chang and Barnard, 1982); (ii) cholamidopropy l)dimethy lammonio] 1 -propanesul- a related high-affinity binding site for the (3-carbo- phonate (CHAPS) (Stephenson and Olsen, 1982). line ligands (Braestrup et al., 1980); (iii) a specific Furthermore, the G A B A A/benzodiazepine receptor binding site for the channel ligands, picrotoxinin, or has been purified to homogeneity from bovine and the convulsant cage compound f-butylbicyclophos- rat cerebral cortex using a benzodiazepine affinity phorothionate (TBPS) (Squires et al., 1983); and (iv) column (Sigel et al., 1983; Schoch et al., 1984; Sigel a barbiturate allosteric binding site that is closely and Barnard, 1984). In these purified receptor prep related to the channel ligand site (Leeb-Lundberg arations, all the binding sites can be found on the et al., 1980; Ticku and Maksay, 1983). Further com isolated protein (Sigel et al., 1984). However, it is

Received June 24, 1985; accepted September 24, 1985. Abbreviations used: CHAPS, 3-[(3-cholamidopropyl)dimeth- Address correspondence and reprint requests to Dr. F. A. Ste ylammonio] 1-propanesulphonate; GABA, y-aminobutyric acid; phenson at MRC Molecular Neurobiology Unit, MRC Centre, HEPES, 4-(2-hydroxyethyl)-l-piperazine ethanesulphonic acid; Hills Road, Cambridge CB2 2QH, U.K. SDS-PAGE, polyacrylamide gel electrophoresis in the presence The present address of Drs. Casalotti, Mamalaki, and Barnard of sodium dodecyl sulphate; TBPS, f-butylbicyclophosphoro- is MRC Molecular Neurobiology Unit, MRC Centre, Hills Road, thionate. Cambridge CB2 2QH, U.K.

854 GABA!BENZODIAZEPINE RECEPTOR ANTIBODIES 855 only when brain membranes are extracted and pu rod gels was carried out according to Stephenson et al. rified in the presence of CH APS and exogenous (1981), focussing for 5 h at 4°C at 1 mA/tube; the gels phospholipid that the channel-related binding sites, were sliced at 1-mm intervals and the I25I content was i.e., the T B P S binding activity and the barbiturate measured in a gamma-counter. stimulation of benzodiazepine binding activity, can Iodination of the GABAA/benzodiazepine receptor be found (Sigel and Barnard, 1984). The purified receptor (1 ml) was dialysed for 2 h at 4°C We used the bovine G A B A A/benzodiazepine re against 20 mA/ K + phosphate (pH 7.4)/0.1% Triton X-100 ceptor, in the form obtained by initial extraction (2 L) to remove NaN-, and sucrose. The dialysed receptor (100 p.1, 10 pmol [3H]muscimol binding sites) was iodin- into N a +-deoxycholate and purification in Triton X- ated by the mild chloramine T method of Froehner et al. 100 media (Sigel et al., 1983), to raise polyclonal (1977) using 0.1-0.2 mCi N al25I. Bound and free l25I were antibodies. We describe here the radioimmu separated by gel filtration, on a Sephadex G75 column (18 noassay that we used to measure antibody produc x 0.5 cm), equilibrated and eluted with 20 mA/ K + phos tion and the characterisation of the antibodies. The phate (pH 7.4)/0.1 mA/ EDTA/0.1 mA/ phenylmethylsul- evidence obtained in this characterisation further phonyl fluoride/1 mA/ benzamidine HCl/0.02% demonstrates the existence of a single protein com NaNV0.1% Triton X-100. plex in the brain in which all of the allosteric reg Immunisation procedure and radioimmunoassay ulatory properties of the G A B A A/benzodiazepine The purified G A B A A/benzodiazepine receptor was dia receptor reside. lysed against 20 mA/ K + phosphate (pH 7.4)/0.1% Triton X-100 for 4 h at 4°C to reduce the salt concentration. The MATERIALS AND METHODS dialysed receptor (25-30 |xg protein in 1.5 ml) was emul [A-/n?//7y/-3H]Flunitrazepam (sp act 85 Ci/mmol), sified with an equal volume of Freund’s complete adju vant and was injected at six to eight sites intradermally [wer/iy/ene^Hlmuscimol (sp act 9 Ci/mmol), and Na l25I into New Zealand White rabbits. All subsequent injec were purchased from Amersham International (U.K.). [35S]TBPS and [prapv/-2,3-3H]propyl-p-carboline-3-car- tions were in Freund’s incomplete adjuvant at six to eight boxylate were from New England Nuclear. Flunitra- sites subcutaneously. Rabbits were bled at 3-day intervals following the second and subsequent immunisations. zepam and Ro 7-1986/1 were gifts from Hoffmann-La The l25I-labelled receptor was diluted in 20 mA/ K + Roche (Basel, Switzerland) and chlorazepate was a gift phosphate (pH 7.4)/0.1 m from Boehringer Ingelheim (Bracknell, U.K.). The im M EDTA/0.1% Triton X-100 to munochemicals were from Amersham International a final concentration of 1 nA/. It was incubated for 5 h at (U.K.) and Sewards Laboratories. Other materials and 4°C with increasing amounts of antiserum diluted in preimmune serum to give a fixed amount of serum (1 p.1 methods not specified were as given by Sigel et al. (1983) total) in the assay (60 p.1 total assay volume). Nonspecific and Sigel and Barnard (1984). immunoprecipitation was measured by incubation with Membrane and solubilised receptor preparations preimmune serum only (1 |xl). Anti-(rabbit IgG Fc) (10 Thoroughly washed, two times frozen-thawed bovine julI) was added and an immunoprecipitate allowed to form brain membranes were prepared by the method of Olsen overnight. The precipitate was collected by centrifuga et al. (1981). N a+-deoxycholate and C HAPS extracts of tion, washed superficially with 0.2 M NaCl, and the l25I bovine brain membranes were prepared by the method was measured in a gamma-counter. of Stephenson et al. (1982). Endogenous G A B A was re In an alternative radioimmunoassay, the purified re moved from the preparations by the passage of extracts ceptor was reversibly labelled with either [3H]muscimol through a Sephadex G-25 column as previously described (30 nA/ final concentration) or with [3H]flunitrazepam (30 (Stephenson et al., 1982) except that the column was nM final concentration). The assay was then carried out equilibrated and eluted with buffer that contained 0.5% as described above except that the assay volume was 120 CHAPS. |xl and the precipitates were dissolved and counted in liquid scintillation fluid containing 9% (vol/vol) Soluene- Purification and characterisation of the 350 (Packard). GABAA/benzodiazepine receptor The G A B A A/benzodiazepine receptor was purified Purification of IgG The IgG fraction of the rabbit anti-bovine receptor an from bovine and rat cerebral cortex by the method of tiserum was purified essentially by the method of Ey et Sigel et al. (1983). Soluble receptor activity was measured al. (1978). Protein A-Sepharose was washed with 0.1 by a polyethylene glycol precipitation-filtration assay as M citrate, pH 3.0, and equilibrated with 10 mA/ 4-(2-hy- previously described (Sigel et al., 1983). Protein concen droxyethyl)-l-piperazine ethanesulphonic acid (HEPES) tration was determined by the method of Lowry et al. (pH 8.0). Rabbit antiserum, buffered with 10 mA/ H EP ES (1951) with bovine serum albumin as the standard. (pH 8.0) (8 ml), was applied to the column with contin Samples analysed by polyacrylamide gel electropho uous circulation overnight at 4°C. The column was resis in the presence of sodium dodecyl sulphate (SDS- washed with the 10 mM H EP ES (pH 8.0) (50 ml) and PAGE) were first concentrated by precipitation with 12% eluted with 0.1 M citrate (pH 3.0) in 1-ml fractions, each (wt/vol) trichloroacetic acid, washed twice with acetone, of which was immediately quenched with 1 Tris • HC1 dissolved in sample buffer, and denatured, and subjected M (pH 8.0; 0.6 ml). Purified IgG was dialysed against phys to SDS/PAGE under reducing conditions in 10% (wt/vol) iological buffered saline and stored in aliquots at -20°C. acrylamide slab gels, with silver staining where stated, all as described by Sigel and Barnard (1984). The 125I- Western blotting labelled receptor was visualised by autoradiography on Western blotting was performed according to the pro Fuji x-ray film. Isoelectric focussing in polyacrylamide cedure of Burnette (1981), except that the transfer buffer

J. Neurochem., Vot. 46, No. 3, 1986 856 F. A. STEPHENSON ET AL.

contained 0.1% SD S and electroblotting was carried out at 60 V for 4 h at 4°C. Following the transfer of protein to the nitrocellulose sheet, the SD S gel was silver-stained for residual protein and the nitrocellulose sheet was quenched by incubation with (i) 50 m M Tris-HCl (pH 7.4)/ 0.9% (wt/vol) NaCl (buffer A) for 10 min at 4°C; (ii) 50 mM Tris-HCl (pH 7.4)/0.9% (wt/vol) NaCl/5% (wt/vol) bovine serum albumin/0.5% Triton X-100 (buffer B) for 1 h at 4°C. The nitrocellulose sheet was incubated over night at 4°C with either the rabbit antiserum diluted 1:50 or 1:100 with buffer B or the appropriate purified IgG fraction diluted 1:1,000 or 1:2,000 with buffer B. The fil ters were washed (4 x 15 min each) with buffer B and incubated with biotinylated anti-(rabbit IgG) diluted 1:500 in buffer B for 1 h at room temperature and were washed B (4 x 15 min each) with buffer B and further incubated with streptavidin peroxidase diluted 1:400 in buffer B for Pa 20 min at room temperature. Filters were washed 3x15 i m min each with buffer B and 3 x 5 min each with buffer A and developed with 2',5'-diaminobenzidine (50 mg) dis solved in 25 mAf K + phosphate (pH 7.4)/0.2 M a b e d KCl/0.03% CoCU (100 ml) which also contained 0.015% (wt/vol) H;0 ;. The developing solution was prepared im mediately prior to use. FIG. 1. The properties of the antigen used in the radioim munoassay. A: Isoelectric focussing of purified bovine 125l- GABAA/benzodiazepine receptor. The purified receptor was iodinated as described in Materials and Methods and 86 fmol RESULTS [3H]muscimol binding sites applied to the isoelectric fo cussing gel. Focussing was carried out as described in Ma terials and Methods and subsequently the gel was sliced at GABAA/benzodiazepine receptor characterisation 1-mm intervals and counted in a gamma-counter. B: Results The bovine G A B A A/benzodiazepine receptor of SDS-PAGE of the 125l-receptor in a 10% polyacrylamide was purified to a maximum specific activity of 4.3 gel. Lane 1 corresponds to 25 fmol [3H]muscimol binding nmol [3H]muscimol binding sites/mg protein and 1.2 sites of 125l-GABAA/benzodiazepine receptors. Lane 2 con nmol [3H]flunitrazepam binding sites/mg protein. tains 125l-standard proteins: a, bovine serum albumin, Mr 67,000; b, catalase, M r 60,000; c, ovalbumin, M r 43,000; d, These values were those found consistently (Sigel lactate dehydrogenase, M r 36,000. et al., 1983) for purification in the media used here, and as previously it was found that the receptor protein contained two major subunits of M r 53,000 of the immune serum of 1:1,000 (rabbit 1), 1:300 (a) and M r 57,000 ((3). When the purified receptor (rabbit 2), or 1:300 (rabbit 3) where the dilution was iodinated, a specific activity of 1,750 Ci/mmol factor refers to the dilution of the immune serum in [3H]muscimol binding sites (mean value) was ob the total assay volume. In an alternative solid-phase tained, which corresponds to 406 mCi/g protein. enzyme-linked immunosorbent assay (ELISA), The 125I-labelled G A B A A/benzodiazepine receptor where the receptor was immobilised on polyvinyl was shown to have the same subunit structure as chloride plates (data not shown), half-maximal op unlabelled receptor (Fig. 1) and in addition was tical density was measured at lower dilutions of im shown to possess a single, symmetrical peak in mune sera and the ratio of the total to nonspecific narrow-range isoelectric focussing gels, at pi 5.6. binding was not as favourable as in the radioim munoassay. Antibody production The G A B A A/benzodiazepine receptor is a good Antibody specificity antigen in rabbits and high antibody titres were ob The polyclonal antibodies specifically coimmu- tained in all the animals used, after two immunisa noprecipitated purified G A B A A/benzodiazepine re tions. The antibody production was measured using ceptor labelled reversibly with either [3H]fluni- a radioimmunoassay with 125I-receptor as antigen. trazepam or [3H]muscimol (Fig. 3). The immuno Typical titration curves are shown in Fig. 2 for the precipitation was concentration-dependent and the antibodies produced in each of three rabbits. N on ratio of the precipitated binding sites for [3H]- specific immunoprecipitation was low, being 3% of flunitrazepam to those for [3H]muscimol was the total radioactivity of the incubation mixture. In 2.0 ± 0.3 (n = 3). This value was that found after general, repeated immunisations did not change sig correction to 100% labelling of both sites using the nificantly the antibody titre or the antibody affinity values that had been previously determined (Sigel for 125I-receptor and Fig. 2 is therefore representa et al., 1983) for the ligand dissociation constants in tive of all antisera obtained. The half-maximal point solution, i.e., KD 12 nM (muscimol) and KD 10 nM of the immunoprecipitation was seen at a dilution (flunitrazepam). Control experiments showed first

J. Neurochem., Val. 46, No. 3, 1986 GABA!BENZODIAZEPINE RECEPTOR ANTIBODIES 857

In a parallel experiment the purified receptor was incubated with preimmune or immune serum, immunoprecipitation was carried out as described above, and the supernatants after centrifugation were assayed for binding activities. A parallel de pletion of specific [3H]flunitrazepam and [3H]- muscimol binding activity in the immunoprecipitation was found (Fig. 3). The antisera also precipitated specifically the re ceptor labelled reversibly either with [3H]flunitrazepam or with [3H]muscimol, from both N a+- deoxycholate and C H A PS extracts of bovine brain membranes (Table 1). In addition we were able to show in the C H A P S crude extracts, where the bar biturate regulation of G A B A and of benzodiazepine binding activities and the [35S]TBPS specific log^antiserum (pi) binding sites are preserved, that specific immuno- FIG. 2. Radioimmunoassay to measure anti-(GABAA/benzo- precipitation of receptor reversibly labelled by diazepine receptor) antibodies. 125l-GABAA/benzodiazepine [35S]T B P S occurred (Table 1). It was shown in con receptor (1 nM) was incubated for 5 h at 4°C with increasing trol experiments that [35S]T B P S was not immuno amounts of antiserum diluted in preimmune serum to give a precipitated in the absence of the C H A P S extracts fixed amount of serum (total 1 |xl) in the assay (assay volume 60 m-I). Anti-(rabbit IgG Fc) (10 p.l) was added and the assay and that the immunoprecipitated radioactivity was completed as described in Materials and Methods. The re displaced by 100 [iM picrotoxin. Specific immuno- sults are expressed as specific cpm precipitated minus cpm precipitation from the CH A PS extract was carried precipitated by preimmune serum (1 (xl). (•— •), Rabbit 1; out for each radioactive ligand, at a single concen (O— O), rabbit 2; (A— A), rabbit 3. tration and these were [3H]flunitrazepam 30 nM, [3H]muscimol 60 nM, [3H]propyl-0-carboline-3-carboxylate 30 nM, and [35S]TBPS 35 n M , respec that the radioactive ligands were not precipitated tively. The number of binding sites immunoprecip by the immune serum in assays where the purified itated was corrected to 100% labelling of all sites receptor was omitted, and second that the [3H]- using K d values that had been previously deter flunitrazepam or [3H]muscimol immunoprecipitated mined for C H A P S extracts of bovine brain or in the was completely displaced in the presence of 10 |xM case of [3H]propyl-p-carboline-3-carboxylate, for flunitrazepam or 10 |xM muscimol, respectively. GABA/benzodiazepine receptor purified in the

FIG. 3. Immunoprecipitation of specific li gand binding activity by rabbit anti-(bovine GABAA/benzodiazepine receptor) anti bodies. In (A) and (B), purified GABAA/ben E C zodiazepine receptor (2 pmol [3H]mus- 10 cimol binding sites) was incubated with ei ther [3H]flunitrazepam (30 nM) or [3H]muscimol (30 nM), respectively, and with in creasing amounts of antiserum diluted in preimmune serum to give a fixed amount of serum (total 1 fil) in the assay. Immuno precipitation was carried out as described in Materials and Methods and the results Immune serum (||l) .01 .1 1 are expressed as fmol 3H-binding sites im Control Immune serum (|JI) munoprecipitated per assay. In (C) and (D) serum purified bovine GABAA/benzodiazepine re ceptor (1 pmol [3H]muscimol binding site) was incubated with increasing amounts of antiserum diluted as described above and immunoprecipitation carried out as de scribed in Materials and Methods. The su pernatants obtained were assayed for [3H]flunitrazepam and [3H]muscimol binding activity using a bovine 7-globulin/ polyethylene glycol precipitation filtration Immune serum (p i) .01 .1 1 assay. The results are expressed as ligand Control Immune serum (|J|) binding activity remaining in the superna serum tant versus antiserum dilution.

J. Neurochem., Vol. 46, No. 3, 1986 858 F. A. STEPHENSON ET AL.

T A B LE 1. Coimmunoprecipitation o f [3HJmuscimol, activity to the brain membranes and this was ac [3HJfhmitrazepam, [3H]propyl-$-carboline-3-carboxylate, counted for in the controls that were used. and [35S]TBPS binding sites from CHAPS crude Furthermore, it was found that in the desalted extracts of bovine cerebral cortex CH APS extracts, immunoprecipitation carried out Specific binding sites in the presence of 1 mM pentobarbital or 100 p.M immunoprecipitated G A BA for receptor labelled reversibly with [3H]- (pmol/ml serum) flunitrazepam (10 n M final concentration) and 1 m M Ligand Rabbit 1 Rabbit 2 pentobarbital for receptor labelled reversibly with [3H]muscimol (30 n M final concentration) showed [3H]Flunitrazepam 109 ± 1 66.2 ± 1 immunoprecipitation of both barbiturate-stimulated [3H]Muscimol 101.6 ± 5 75.6 ± 9 G ABA and benzodiazepine binding activity and [3H]Propyl-p-carboline- 3-carboxylate 38 ± 4 22 ± 3 GABA-stimulated benzodiazepine binding activity [35S]TBPS 55.6 ± 20 31.8 (Table 2).

Bovine brain membranes were extracted with 1.69£ (wt/vol) Immunoblotting CHAPS, the soluble fraction collected by centrifugation at In Western blot experiments, i.e., when the pu 100,000 g, and the endogenous GABA removed from the extract rified G A B A A/benzodiazepine receptor was sub by the passage through a Sephadex G-25 column as described jected to SDS-PAGE under reducing and nonre in Materials and Methods. The desalted CHAPS extract (100 p.1, which contained 150 fmol [3H]flunitrazepam binding sites) was ducing conditions and subsequently transferred to incubated with either [3H]flunitrazepam. [3H]muscimol, nitrocellulose, the polyclonal antibodies recognised [3H]propyl-(3-carboline-3-carboxylate, or [35S]TBPS at 30 nM, 60 only the a-subunit (Fig. 4). Some staining was also nM, 30 nM, and 35 nM final concentrations, respectively, and apparent at the top of the gel in lane 3 of Fig. 4 and either preimmune or immune serum (2 |xl in each case). Immu- noprecipitation was carried out as described in Materials and it is thought that this was a reaction with aggregated Methods and the results are expressed as specific pmol radio receptor that had not entered the gel. The same active ligand binding sites precipitated/ml serum where the staining pattern, i.e., on the a-subunit, was ob number of ligand binding sites was corrected to 1009rc labelling served for the specific antiserum and the respective of all sites using the values that had been previously determined purified IgG fractions. This applied to receptor ex for the ligand dissociation constants in either CHAPS bovine brain extracts, i.e., KD 8 nM (flunitrazepam, Stephenson and tracted and purified either by the N a +-deoxycho- Olsen, 1982), KD 54 nM (muscimol, Stephenson and Barnard, late/Triton X-100 method or by the C H A PS/aso- unpublished), KD 59 nM (TBPS, Stephenson et al.. 1984) or for lectin procedure. In addition, when G A B A A/ben- GABA/benzodiazepine receptor purified in Na~-deoxycholate/ zodiazepine receptor purified from rat cerebral Triton X-100 media, KD 9 nW (propyl-|3-carboline-3-carboxylate. Barnard et al., 1984). Nonspecific immunoprecipitation was cortex was used as antigen in the Western blots, the taken as the radioactivity precipitated with preimmune serum. rabbit anti-(bovine G A B A A/benzodiazepine re The results are expressed as the means ± the standard deviation ceptor) antibodies specifically recognised the a- and are representative of three separate experiments. subunit of the rat receptor (Fig. 4).

DISCUSSION presence of Na + -deoxycholate and Triton X-100. In this paper, we have described the production These were KD 8 nM, flunitrazepam (Stephenson and Olsen, 1982); KD 54 nM, muscimol (Stephenson T A B L E 2. Imnumoprecipitation of GABA and and Barnard, unpublished); KD 54 n M TBPS (Ste barbiturate-enhanced benzodiazepine binding activity phenson et al., 1984); and KD 9 nM , propyl-0-car- and barbiturate-enhanced muscimol binding activity boline-3-carboxylate (Barnard et al., 1984). The from CHAPS crude extracts ratio of the [3H]flunitrazepam binding sites immu [3H]Flunitrazepam [3H]MuscimoI noprecipitated to those for [3H]muscimol was 1.1 immunoprecipitated immunoprecipitated ± 0.3 (n = 4); [3H]flunitrazepam binding sites im Assay conditions (cpm) (cpm) munoprecipitated to those for [35S]TBPS was 2.1 ± 0.2 (n = 3), and [3H]flunitrazepam binding sites im Preimmune serum 183 ± 42 187 ± 68 Antiserum 723 ± 62 554 ± 29 munoprecipitated to those for [3H]propyl-(3-carbo- Antiserum + 10 5 M line-3-carboxylate was 2.9 (n = 2). GABA 949 ± 38 — The antibodies produced are not directed at the Antiserum + 10 3 M high-affinity G A B A or the benzodiazepine binding pentobarbital 1,732 ± 165 628 ± 55 site of the G A B A A/benzodiazepine receptor. This Immunoprecipitation was carried out as described in Materials was shown by the lack of inhibition of both and Methods using a CHAPS-desalted extract as the source of [3H]flunitrazepam and [3H]muscimol binding ac antigen at a concentration of 80 fmol benzodiazepine binding tivity to thoroughly washed, two times frozen- sites per assay. [3H]Flunitrazepam and [3H]muscimol final con nM nM, thawed bovine brain membranes, in the presence of centrations were 10 and 30 respectively. Results are expressed as the means ± the standard deviation and are rep increasing amounts of antisera. Normal rabbit resentative of three separate experiments using two different serum itself slightly inhibited [3H]muscimol binding rabbit anti-(bovine GABA/benzodiazepine receptor) antibodies.

J. Neurochcm., Vol. 46, No. 3, 1986 GABA /BENZODIAZEPINE RECEPTOR ANTIBODIES 859

1 2 3 4 5 determined for [3H]flunitrazepam photoaffinity-labelled crude detergent extracts (Tallman et al., 1981). Sucrose density gradient centrifugation of 93K the 125I-receptor has shown, however, that the size 66K _ of the native l2T-receptor is smaller than that of the m fl M r58K unlabelled GABAA/benzodiazepine receptor (re *■ M r 53K sults not shown) and it is thought that the iodination 43K mm process modified some subunit-subunit interac tions. 31K _ All of the antisera tested after two immunisations with purified GABAA/benzodiazepine receptor spe cifically immunoprecipitated the l2>I-receptor as 21.5K shown in Fig. 2. However, it was also of interest to show that the antibodies could immunoprecipitate specifically both the flunitrazepam and the mus cimol binding activities. This was found to be the case for both purified preparations (Fig. 3) and for Na + -deoxycholate and CHAPS extracts (Table 1). FIG. 4. Immunoblotting with rabbit anti-(bovine GABAA/ben-The ratio of the muscimol to benzodiazepine zodiazepine receptor) antibodies. Western blotting was carbinding sites immunoprecipitated was slightly lower ried out as described in Materials and Methods. Lane 1 con tains marker proteins with molecular weights as indicated.(i.e., 2) than found for the ratio for the binding sites Lane 2 is a silver-stained 10% polyacrylamide gel of GABAa/determined directly by binding assays in Na+-de- benzodiazepine receptor purified from bovine cerebraloxycholate/Triton X-100-purified receptor and cortex. Lanes 3 and 4 are immunoblots with purified bovinecrude Na + -deoxycholate detergent extracts where GABAA/benzodiazepine receptor as antigen and the rabbit 3 antiserum as antibody (lane 3) or the purified IgG fraction theof endogenous GABA had been removed (Ste rabbit 3 antiserum as antibody (lane 4). Lane 5 is an immu-phenson et al., 1982; Sigel et al., 1983). This may noblot with purified rat GABAA/benzodiazepine receptorbe explained by the fact that normal rabbit serum as antigen and the antibody used was the same as thatwas for shown to inhibit |3H]muscimol binding activity lane 4. and, therefore, the number of |3H jmuscimol binding sites immunoprecipitated is an underestimation. On of polyclonal antibodies to the bovine GABAA/ben- the other hand, in experiments where the depletion zodiazepine receptor. The antigen used was this re of ligand binding activity remaining in the super ceptor extracted from bovine brain membranes with natant after immunoprecipitation was measured the ionic detergent Na + -deoxycholate and subse (Fig. 3), there was always an exact correlation be quently purified on a benzodiazepine affinity tween the percentage decrease of l3H]flunitrazepam column in the presence of Triton X-100. This puri and the percentage decrease of l3H]muscimol fied receptor had high-affinity binding sites for both binding activity. This is additional experimental ev GABA and benzodiazepine ligands and an alloster- idence to support the hypothesis that the two ically linked low-affinity GABA site, i.e., showing binding sites are located on the same single entity. GABA enhancement of benzodiazepine binding ac When the GABAA/benzodiazepine receptor is ex tivity (Sigel et al., 1983). The purified receptor has tracted and purified in the presence of CHAPS and a molecular weight of 230,000 as determined by asolectin, a high-affinity TBPS binding site is puri H20/D20 sucrose density gradient centrifugation fied with the GABAA/benzodiazepine receptor and gel filtration (Barnard et al., 1984) and contains (Sigel and Barnard, 1984). Thus, we tried to im two subunits, Mr 53,000 (a) and Mr 57,000 ((3). The munoprecipitate specific TBPS binding activity a-subunit is specifically photoaffinity-labelled with from purified receptor preparations with the poly [3H]flunitrazepam. clonal antibodies. However, the TBPS binding site The assay that was developed to measure the spe is unstable and is rapidly inactivated during the pu cific antibody production was a radioimmunoassay rification procedure and with storage at either using 125I-labelled GABAA/benzodiazepine receptor — 20°C or 4°C on the completion of isolation (Sigel as antigen. The properties of the l25I-receptor were and Barnard, 1984; Stephenson et al., 1984). In the examined and it was shown (Fig. 1) that the labelled time taken to complete the radioimmunoassay, the receptor had the same subunit composition as found TBPS binding site was inactivated and no positive in both Coomassie- and silver-stained gels of the results were obtained. However, the binding site is purified receptor (Fig. 4). Also, the l2T-receptor fo more stable in crude extracts, presumably due to cussed as a single sharp symmetrical peak in iso the presence of endogenous phospholipid, and electric focus gels (Fig. 1), which confirmed the ho when a crude CHAPS extract of bovine cerebral mogeneity of the preparation; the isoelectric point cortex was used as antigen, specific TBPS binding that was found, pi 5.6, agrees well with the values sites were immunoprecipitated (Table 1) in parallel

J. Neurochem., Vol. 46, No. 3, 1986 860 F. A. STEPHENSON ET AL. with [3H]flunitrazepam and L3H]muscimol binding (Schoch et al., 1985) where it has been shown that sites (Table 1). The ratio of the flunitrazepam to these monoclonal antibodies immunoprecipitated TBPS specific ligand sites immunoprecipitated from the high-affinity muscimol and benzodiazepine C H A P S brain extracts was 2 (Table 1). This is in binding sites and low-affinity G ABA site. Immu contrast to the determination of the ratio by binding noprecipitation of channel ligands was not shown. studies where we found that the ratio of flunitra At least a proportion of the antibodies recognise zepam to T B P S binding sites was 1.3 in membranes nonconformational dependent epitopes as demon and 1 in CHAPS extracts of bovine brain (Ste strated by the Western blots (Fig. 4). However, in phenson et al., 1984). The higher ratio found in the all of the antiserum tested so far we have been able immunoprecipitation experiments described herein to immunoblot only the a-subunit (Fig. 4). The may be explained by the inactivation of the T B P S reason for this is unclear. It is possible that the a- binding site during the time taken to complete the subunit contains the major immunogenic region and radioimmunoassay. The ratio of the benzodiazepine that the (3-subunit is not accessible for antibody pro to G A B A specific binding sites immunoprecipitated duction. However, Schoch et al. (1985) have dis from CHAPS crude extracts is lower (i.e., 1.1, criminated four epitopes of the GABAA/benzodi Table 1) than found for the ratio of these binding azepine receptor with monoclonal antibodies. Two sites immunoprecipitated from purified receptor of these are nonconformational dependent epitopes preparations that were isolated in the presence of with the recognition of either the a- or (3-subunit in N a+-deoxycholate and Triton X-100. However, this Western blots (Schoch et al., 1985). We have found is consistent with the lower GABA-to-benzodiaze- that the (3-subunit is more susceptible to proteolytic pine binding site ratio found for receptor isolated degradation during the isolation procedure. Indeed, and assayed in the presence of C H A P S and aso- in an initial publication, the stoichiometry of the lectin (Sigel and Barnard, 1984). Seifert and Casida subunits was given as 2:1 a:(3 (Sigel et al., 1983). (1985) have also shown that C H A P S has G A B A - But, if more stringent precautions are taken to pre mimetic properties and thus, in the presence of vent proteolysis occurring during the purification CHAPS, the G ABA receptor concentration is un with the inclusion of both soybean trypsin inhibitor derestimated. The ratio of flunitrazepam to propyl- and chicken egg white ovomucoid containing ovoin- (3-carboline-3-carboxylate binding sites specifically hibitor, the stoichiometry is 1:1 (unpublished re immunoprecipitated from CHAPS brain extracts sults). Thus, it is more likely that following immu was 2.9 (Table 1). We had previously shown for nisation, the (3-subunit is rapidly proteolytically di GABA/benzodiazepine receptor purified from bo gested in vivo with the a-subunit remaining as the vine cerebral cortex in the presence of Na + -deoxy- predominant antigenic determinant. We have cholate and Triton X-100 that the ratio of benzodi shown that the bovine and the rat GABAA/benzo azepine to propyl-|3-carboline-3-carboxylate sites diazepine receptor share at least one epitope since was 1.25 (Stephenson et al., 1984). The reason for the antibodies also recognise the a-subunit of the the discrepancy between the two ratios is unclear G A B A A/benzodiazepine receptor from rat cerebral but it is possible that in C H A P S extracts, the af cortex. Shared epitopes have been demonstrated finity of the receptor for the (3-carboline ligands is between the G A B A A/benzodiazepine receptor from reduced, leading to an apparent increase in the bovine and from human brain (Schoch et al., 1985) ratio. Also, in the CHAPS extracts the G ABA and this would indicate that the GABAA/benzodi binding sites immunoprecipitated are stimulated in azepine receptor structure is highly conserved in the presence of pentobarbital (Table 2) and both mammals. To what extent this homology is apparent GABA-enhanced benzodiazepine and pentobar will be shown once the complete amino acid se bital-enhanced benzodiazepine binding activities quence of each subunit has been determined. The were immunoprecipitated. production of specific antisera to the individual With a polyclonal antibody, coprecipitation may polypeptides will be an invaluable tool in that task. not be definitive proof of coexistence of sites on one protein, since conceivably different antibodies Acknowledgment: This work was supported by the could be present each of which independently Medical Research Council Molecular Neurobiology Re reacts with different binding proteins. However, search Group at Imperial College. F.A.S. holds a Royal this cannot be the explanation here since (i) the dif Society University Research Fellowship. We thank Liz ferent activities are removed in parallel throughout, Ashton and Alison Bartlett for typing this manuscript. and (ii) the antibodies recognise only one polypep tide (as seen in the Western blots). Hence eith er the REFERENCES various binding activities seen are all due to sites on the a-subunit or they are due to sites on different Barnard E. A., Stephenson E A., Sigel E., Mamalaki C., Bilbe subunits within one protein oligomer. Similar re G., Constanti A., Smart T. E., and Brown D. A. (1984) Structure and properties of the brain GABA/benzodiazepine sults have been reported using monoclonal anti receptor complex, in Neurotransmitter Receptors: Mecha bodies to the G A B A A/benzodiazepine receptor nisms of Action and Regulation (Kito S., Segawa T., Ku-

J . Neurochem., Vol. 46, No. 3, 1986 GABA!BENZODIAZEPINE RECEPTOR ANTIBODIES 861

riyama K., Yamamura H. I., and Olsen R. W., eds), pp. helin T., Haefely W., and Mohler H. (1985) Co-localisation 235-254. Plenum Press, New York. of GABAa receptors and benzodiazepine receptors in the Braestrup C., Nielsen M., and Olsen C. E. (1980) Urinary and brain shown by monoclonal antibodies. Nature 314, 168— brain 0-carboline-3-carboxylates as potent inhibitors of 171. brain benzodiazepine receptors. Proc. Natl. Acad. Sci. Seifert J. and Casida J. E. (1985) Solubilization and detergent USA 77, 2288-2292. effects on interactions of some drugs and insecticides with Braestrup C., Schmiechen R., Neef G., Nielsen M., and Pe the f-butylbicyclophosphorothionate binding site within the tersen E. N. (1982) Interaction of convulsive ligands with y-aminobutyric acid receptor-ionophore complex. J. Neu- benzodiazepine receptors. Science 216, 1241-1243. rochem. 44, 110-116. Briley M. S. and Langer Z. L. (1978) Influence of GABA re Sigel E. and Barnard E. A. (1984) A y-aminobutyric acid/ben- ceptor agonists and antagonists on the binding of zodiazepine receptor complex from bovine cerebral cortex: [3H]diazepam to the benzodiazepine receptor. Ear. J. Phar improved purification with preservation of regulatory sites macol. 52, 129-132. and their interactions. J. Biol. Chem. 259, 7219-7223. Burnette W. M. (1981) “ Western blotting.” Electrophoretic Sigel E., Stephenson F. A., Mamalaki C., and Barnard E. A. transfer of proteins from sodium dodecyl sulfate-polyacryl (1983) A y-aminobutyric acid/benzodiazepine receptor com amide gels to unmodified nitrocellulose and radiographic de plex of bovine cerebral cortex. J. Biol. Chem. 258, 6965- tection with antibody and radioiodinated protein A. Anal. 6971. Biochem. 112, 195-203. Sigel E., Stephenson F. A., Mamalaki C., and Barnard E. A. Chang L.-R. and Barnard E. A. (1982) The benzodiazepine/ (1984) The purified GABAA/benzodiazepine barbiturate re GABA receptor complex: molecular size in brain synaptic ceptor complex: four types of ligand binding sites and the membranes and in solution. J. Neurocltem. 39, 1507-1518. interactions between them are preserved in a single isolated Ey P. L., Prowse S. J., and Jenkin C. R. (1978) Isolation of pure protein complex. J. Recept. Res. 4, 175-188. IgG, IgG2a and IgG2b immunoglobulins from mouse serum Squires R. F., Casida J. E., Richardson M., and Saederup E. using Protein A-Sepharose. Immunochemistry 15, 429-436. (1983) [35S]r-Butylbicyclophosphorothionate binds with high Froehner S. C., Reiness C. T., and Hall Z. W. (1977) Subunit affinity to brain specific sites coupled to y-aminobutyric structure of the acetylcholine receptor from denervated rat acid-A and ion recognition sites. Mol. Pharmacol. 23, 326- skeletal muscle. J. Biol. Chem. 252, 8589-8596. 336. King R. G. and Olsen R. W. (1984) Solubilisation of convulsant/ Stephenson F. A. and Olsen R. W. (1982) Solubilization by barbiturate binding activity on the y-aminobutyric acid/ben- CHAPS detergent of barbiturate-enhanced benzodiazepine- zodiazepine receptor complex. Biochem. Biophys. Res. GABA receptor complex. J. Neurochem. 39, 1579-1586. Commitn. 119, 530-536. Stephenson F. A., Harrison R., and Lunt G. G. (1981) The iso Leeb-Lundberg F., Snowman A. M., and Olsen R. W. (1980) lation and characterisation of the nicotinic acetylcholine re Barbiturate receptors are coupled to benzodiazepine recep ceptor from human skeletal muscle. Ear. J. Biochem. 115, tors. Proc. Natl. Acad. Sci. USA 77, 7468-7472. 92-97. Lowry O. H., Rosebrough N. J., Farr A. L., and Randall R. J. Stephenson F. A., Watkins A. E., and Olsen R. W. (1982) Phys (1951) Protein measurement with the Folin phenol reagent. icochemical characterisation of detergent-solubilised y-ami J. Biol. Chem. 193, 265-275. nobutyric acid and benzodiazepine receptor proteins from Olsen R. W. (1982) Drug interactions at the GABA receptor- bovine brain. Eur. J. Biochem. 123, 291-298. ionophore complex. Anna. Rev. Pharmacol. Toxicol. 22, Stephenson F. A., Sigel E., Mamalaki C., Bilbe G., and Barnard 245-277. E. A. (1984) The benzodiazepine receptor complex. Purifi Olsen R. W., Bergman M. 0., Van Ness P. C., Lummis S. C., cation and characterisation. Dev. Neurosci. 17, 437-442. Watkins A. E., Napias C., and Greenlee D. V. (1981) y-Ami- Tallman J. F., Mallorga P., Thomas J. W., and Gallager D. W. nobutyric acid receptor binding in mammalian brain. Het (1981) Benzodiazepine binding sites; properties and modu erogeneity of binding sites. Mol. Pharmacol. 19, 217-277. lation, in GABA and Benzodiazepine Receptors (Costa E., Schoch P., Haring P., Takacs B., Stahli C., and Mohler H. (1984) DiChiaraG.,andGessaG. L.,eds), pp. 9-18. Raven Press, A GABA/benzodiazepine receptor complex from bovine New York. brain: purification, reconstitution and immunological char Ticku M. K. and Maksay G. (1983) Convulsant/depressant site acterisation. J. Recept. Res. 4, 189-200. of action at the allosteric benzodiazepine-GABA receptor Schoch P., Richards J. G., Haring P., Takacs F., Stahli C., Stae- ionophore complex. Life Sci. 33, 2263-2375.

J. Neurochem., Vol. 46, No. 3, 1986 ■Lt-

HOLECULAR CHARACTERIZATION

OF THE GABA/BENZODIAZEPINE RECEPTOR

Stefano Oscar CASALOTTI

A thesis submitted for the degree of Doctor of Philosophy

of the University of London and for the Diploma of Membership

of the Imperial College of Science and Technology.

Department of Biochemistry Imperial College of Science and Technology, London SW7 2AZ

and

Molecular Neurobiology Unit

Medical Research Council Cambridge CB2 2QH

August 1988

i ABSTRACT

The GABA-benzodiazepine receptor is a complex multi subunit integral membrane protein containing binding sites for a number of endogenous and exogenous ligands of pharmacological importance. The purification of this protein from the mammalian central nervous system has led to the proposal of a tetrameric model for this receptor with two a (Mr 53,000) and two 0 (Mr 57,000) subunits. The main object of these studies was the further characterization of the molecular structure of the GABA- benzodiazepine receptor.

The distribution of the binding sites for the two classes of ligands, the GABA agonists and the benzodiazepines, was investigated at the receptor subunit level. Photoaffinity labelling experiments using the ligands [3H]muscimol and [3H]flunitrazepam were carried

. . , , • * 5 . out. The irreversible incorporation of [JH]muscimol m both bovine brain membranes and in the purified

GABA/benzodiazepine receptor was analysed in detail. It was found that [3H]muscimol preferentially specifically photolabels the 0 subunit as opposed to [ H]flunitrazepam which preferentially photolabels the a. subunit.

The GABA/benzodiazepine receptor structure was also studied with specific antibodies. Polyclonal antibodies against the purified receptor were raised in rabbits and used in Western blots to analyse the subunit properties of

ii the protein. The production of monoclonal antibodies against the receptor was also pursued. Due to the low immunogenicity of the bovine GABA/benzodiazepine receptor in mice, an extensive investigation of a variety of immunization and fusion methods were carried out in order to isolate the desired specific monoclonal antibodies. Two monoclonal antibodies specific for the GABA/benzodiazepine receptor were obtained and used for the characterization of the receptor. Monoclonal and polyclonal antibodies were also raised against a synthetic peptide, the C-terminal region of the newly identified a 3 subunit, to further investigate the receptor structure.

The cloning, sequencing and functional expression of both the a and p polypet ides of the protein complex, permitted further experiments on the ligand binding properties of the receptor. Linearized plasmid vectors containing the cDNA clones of the receptor al and p subunits were used for the in vitro synthesis of pure

RNA which was micro-injected into Xenoous oocytes and ligand binding assays were performed on the membrane fractions of the latter. Aknowledcnnents

I would like to thank first of all Dr. Anne F.

Stephenson for patiently teaching and advising me on all aspects of this work. My thanks go also to Prof. E.A.

Barnard for allowing me to be part of his research team and for constructive criticism given during the project. I thank Dr. G. Wilkin for taking interest in my work.

Among my collegues in the laboratory I would particularly like to thank for their useful advices Drs.

K. Demouliou-Mason, C. Mamalaki and K. Tsim and everybody else for their friendship.

During this work I was financially supported by Fidia

Reseach Laboratories (Abano Terme, Italy) to whom I am very grateful.

Finally I would like to thank my family for their support and encouragemnt throughout and also my thanks go to Boi (now my wife) with whom I shared the ups and down of a Ph.D.

IV CONTENTS

Abstract U

Acknowledgment iv

Contents y

List of figures xvj List of tables ';?vxrx

List of abbreviations xx

Chapter 1 General Introduction 1

1.1 Introduction 2

1.2 Electrophysiological studies of the GABA-R 5

1.3 Biochemical properties of the GABA-R 6

1.4 Solubilization of the GABA-R 11

1.5 Purification of the GABA-R 13

1.6 Molecular characterization of the GABA-R 15

1.6.1 Molecular size of the GABA-R 15

1.6.2 Subunit size and composition of the GABA-R 16

1.7 Photoaffinity labelling of the GABA-R 17

1.7.1 Photoaffinity labellinig of the GABA-R

with [3H]flunitrazepam 17

1.7.2 Characterization. . of the [ i H]flunitrazepam

labelled proteins 19

1.7.3 Other ligands that photoaffinity label the

GABA-R 22

1.8 Analysis of the GABA-R with antibodies 23

1.9 Molecular biology of the GABA-R 24

v 1.10 The objectives of this study 26

Chapter 2 GABA-R purification and photoaffinity

labelling. 30

2.1 Introduction 31

2.1.1 The objectives of photoaffinitylabelling 31

2.1.2 Properties of the photoaffinity ligands 33

2.1.3 Specificity of photoaffinity labelling

reactions 34

2.1.4 The mechanism of the photoaffinity

labelling reaction of [3H]flunitrazepam 36

2.1.5 The mechanism of the photoaffinity

labelling reaction of [3H]muscimol 37

2.2 Materials and Methods 42

2.2.1 Materials 42

2.2.2 The preparation of membranes from bovine

cerebral cortex 43

2.2.3 The synthesis of the Ro 7-1986/1 affinity

column 44

2.2.4 The purification of the GABA-R 45

2.2.5 The assay of ligand binding assay 48

2.2.5. A The measurement of membrane-bound receptor

activity 48

2.2.5. B The measurement of soluble receptor

activity 49

2.2.6 The preparation of SDS-polyacrylamide gels 49

2.2.6. A 10% polyacrylamide gels 50 2.2.6. B SDS polyacrylamide gradient gels 51

2.2.7 Sample preparation for SDS-PAGE 51

2.2.7. A Tricarboxilic acid (TCA) prepcipitation 52

2.2.7. B Methanol/chloroform precipitation 52

2.2.8 The running conditions for SDS-PAGE 53

2.2.9 SDS-PAGE detections methods ‘ 53

2 .2.9. A Coomassie blue staining 53

2.2.9. B Silver staining 53

2.2.9. C Detection of radioactivity 54

2.2.10 Western blotting 54

2.2.11 Ligand binding to GABA-R immobilized to

Zetabind filters 55

2.2.12 Photoaffinity labelling of the membrane-

bound GABA-R 55

2.2.13 Photoaffinity labelling of purified GABA-R 56

2.2.14 Tryptic digestion of photoaffinity

labelled brain membranes 57

2.2.15 Cyanogen bromide cleavage of the photo

affinity labelled GABA-R 57

2.2.16 High pressure liquid chromatography of the

cyanogen bromide cleavage products 58

2.2.18 The determination of protein concentration 58

2.2.18. A The method of Lowery et al. (1951) 58

2.2.18. B The biuret method 59

2.3 Results 60

2.3.1 The purification of the GABA-R 60

vii Ligand binding to GABA-R after Western blotting 64 The UV absorption spectra of GABA-R ligands 64

Photoaffinity labelling of bovine brain membranes with [3H]muscimol 64

Time course of the membrane photoaffinity labelling reaction 65

Determination of the efficiency of the

[3H]muscimol photo-labelling reaction 66

The effect of DTT on the [3H]muscimol photoaffinity labelling reaction 67

The effect of barbiturates on the [3H]- muscimol photoaffinity labelling reaction 68 Photoaffinity labelling of membrane-bound

GABA-R with [35S]TBPS. 69

Photoaffinity labelling of the purified

GABA-R with [3H]muscimol 70

Time course of the photoaffinity labelling reaction 70

Pharmacological specificity of the [3H]- muscimol photoaffinity labelling reaction 71

Ligand concentration dependance of [3H]- muscimol photoaffinity labelling 71

SDS-PAGE analysis of the [3H]muscimol photoaffinity labelled GABA-R 72

VIII Purified receptor 72

Membrane bound receptor 74 Trypsin digestion of photoaffinity

labelled membranes 75

Cyanogen bromide cleavage of [3H]muscimol

photoaffinity labelled GABA-R 76

Discussion 102

GABA-R purification 102

The study of the distribution of ligand

binding sites within the macromolecular

structure of the GABA-R 105

3 The production of antibodies against

the GABA-R 119

Introduction 120

The production of monoclonal antibodies 120

Immunization of the spleen donor animal 121

The fusion of the spleen cells with

myeloma cells 124

The selection, screening and cloning of

the hybrodomas 125

The localization of neuroreceptors with

monoclonal antibodies 127

The structural characterization of neuro

receptor using monoclonal antibodies 128

IX 3.1.5 Immunopurification of neuroreceptors by

monoclonal antibodies 130

3.1.6 Strategies in the production of mono

clonal antibodies 132

3.2 Materials and Methods 134

3.2.1 Materials 134

3.2.2 Immunization protocols 135

3.2.2. A Immunization with native GABA-R 135

3.2.2. B Immunization with heat denatured GABA-R 135

3.2.2. C Immunization with SDS denatured GABA-R 135

3.2.2. D Immunization with carboxymethylated GABA-R 136

3.2.2. E Immunization with alum precipitated GABA-R 136

3.2.2. F Immunization with intrasplenic injections

of GABA-R 137

3.2.2. G Immunization by implantation of nitro

cellulose blotted with GABA-R 137

3.2.3 The collection of immune sera 138

3.2.4 The cell culture condition 138

3.2.5 Liquid nitrogen storage of cell lines 139

3.2.6 The preparation of macrophages 139

3.2.7 The fusion of the myeloma and spleen cells 140

3.2.7. A The 33% PEG fusion method 140

3.2.7. B The 50% PEG fusion method 141

3.2.8 In vitro immunization of spleen cells

with soluble antigen 142

x The selection of spleen cells by the panning method 143

Screening of the hybridoma cell lines 143

The enzyme linked immunosorbent assay

(ELISA) 143 The measurement of ligand binding to

GABA-R coated on ELISA plates 145

Solid phase radioimmunoassay 145

The Dot blot screening procedure 146

The immunostaining of Western blots 147

The cloning of hybrodoma cell lines 147

Hybrodoma cell line cloning by the limiting dilution method 148

Hybrodoma cell line cloning by the serial and the "safe” serial dilution methods 149

Hybrodoma cell line cloning by the soft agar method 149

The production of ascites fluids 150

Antibody purification by the protein A method 151

Methods for the precipitation of immunoglobulines 151

The ammonium sulphate precipitation method 151

The boric acid precipitation method 152

The euglobulin precipitation method 152

Purification of IgM by FPLC 153 3.2.17 Purification of IgM by DEAE Sephacel ion-

exchange chromatography 153

3.2.18 Purification of IgM by gel filtration 154

3.2.19 The production of antibodies in the absence

of FCS 154

3.2.20 The immunocytochemical staining of brain

tissue 155

3.2.21 The immunoprecipitation of the GABA-R with

monoclonal antibodies 156

3.2.22 The synthesis of immunoaffinity purifi

cation columns 157

3.2.22. A The coupling of immunoglobulins to cyanogen

bromide Sepharose 4B 157

3.2.22. B The coupling of immunoglobulins to Affigel 158

3.2.22. C The coupling of immunoglobulin to protein A

Sepharose 4B 159

3.2.23 The biotinylation of immunoglobulins 160

3.2.24 Immunoaffinity purification of the GABA-R 160

3.2.25 The coupling of a synthetic peptide to KLH 161

3.3 Results

3.3.1 The characterization of anti-native-GABA-R

polyclonal antibodies 163

3.3.2 Anti-carboxymethylated-GABA-R antibodies 164

3.3.3 Monoclonal antibodies studies 164

3.3.4 The titres of the mice injected with

GABA-R 165 3.3.5 The screening procedures of anti-GABA-R

monoclonal antibodies 168

3.3.5. A The ELISA screening assay 168

3.3.5. B The Dot blot screening assay 169

3.3.5.C The screening of monoclonal antibodies

with a soluble antigen assay 170

3.3.6 Cells fusion for the production of mono

clonal antibodies against the GABA-R 171

3.3.6. A Fusions carried out with the 36% PEG

method 171

3.3.6. B Fusions carried out with the 50% PEG

method 172

3.3.7 In vitro technioues for the oroduction of

monoclonal antibodies against the GABA-R 174

3.3.8 The studies of the production of anti-

idiotypic monoclonal antibodies 178

3.3.9 The cloning of hybridomas cell lines 178

3.3.10 The production of ascites fluid 179

3.3.11 The immunoprecipitation of the GABA-R 179

3.3.13 The purification of the monoclonal

antibodies 181

3.3.14 The construction of the immunoaffinity

columns 184

3.3.15 The effect of detergent on the monoclonal

antibody binding to the GABA-R 185

XIII 3.3.16 Immunocytochemical staining of brain

tissue 186 3.3.17 Western blot analysis of GABA-R with

monoclonal antibodies 187

3.3.18 The characterization of anti-a3-

C-Tenninal-peptide antibodies 188

3.4 Discussion 212

3.4.1 The production of polyclonal antibodies

against the GABA-R 212

3.4.2 The production of monoclonal antibodies

against the GABA-R 215

3.4.3 Alternative methods for the production of

monoclonal antibodies against the GABA-R 223

3.4.4 Evaluation of the techniques of hybrodoma

production 226

3.4.5 Characterization of the anti-GABA-R mono

clonal antibodies 228

3.4.6 The production of antibodies against

synthetic peptides of the GABA-R 235

Chapter 4 Biochemical studies of the GABA-R

expressed in Xenopus oocytes 238

4.1 Introduction '239

4.1.1 The Xenopus oocyte expression system 239

xiv 4.2 Materials and Methods 242 4.2.1 Materials 242

4.2.2 GABA-R al RNA and 0 RNA synthesis 242 4.2.3 Reticulocyte lysate translation of al

RNA and 0 RNA 244

4.2.4 Translation of al RNA and 0 RNA of the GABA-R in Xenoous oocytes 244

4.2.5 Assay of ligand binding activity to GABA-R expressed in Xenopus oocytes 245

4.3 Results 246

4.3.1 Synthesys of the al RNA and 0 RNA of the GABA-R 246 4.3.2 Ligand binding assay on injected oocytes 246

4.4 Discussion 4.4.1 Characterization of the GABA-R ligand

binding sites expressed in Xenopus oocytes 245

Chapter 5 General discussion 256

References 264 LIST OF FIGURES

Figure Page

1.1 Chemical structure of the GABA-R ligands 28 2.1.1 The photoaffinity labelling reaction

equilibria 39 2.1.2 The photoaffinity labelling mechanism of

[3H ]flunitrazepam 4 0 2.1.3 The photoaffinity labelling mechanism of

[3H]muscimol 41

2.3.1 Na-deoxycholate purification of the GABA-R 78 2.3.2 Triton-urea purification of the GABA-R 79 2.3.3 Excitation spectra of muscimol, flunitrazepam and TBPS 80

2.3.4. A Time course of [3H]muscimol photo

affinity labelling of brain membranes 81 2.3.4. B Effect of UV on bovine brain membranes 82

2.3.5 Effect of DTT on [3H]muscimol re

versible and irreversible binding 83

2.3.6. A Pentobarbital and GABA enhancement of reversible and irreversible binding 84

2.3.6. B Effect of UV irradiation on membranes

pentobarbital enhancement 85

2.3.7. A Time course of [3H]muscimol specific

photoaffinity labelling of purified GABA-R 86

2.3.7. B Effect of UV irradiation on the purified GABA-R 87

x v i 2.3.7.C Effect of UV irradiation on [3H]-

muscimol 88

2.3.8 Pharmacological specificity of [3H]-

muscimol photoaffinity labelling reaction 89 2.3.9. A Concentration dependance of specific

irreversible [3H]muscimol binding 90

2.3.9. B Scatchard plot of specific irreversible

[3H]muscimol binding 91

2 .3.10. A SDS-PAGE of photolabelled GABA-R: gel

slicing 92 2.3.10. B SDS-PAGE of photolabelled GABA-R: fluo-

rography 93 2.3.11 SDS-PAGE of photolabelled membranes 94

2 .3.12 . A Trypsin dependent release of radioactivity from photolabelled membranes: trypsin concentration dependance 95

2.3.12. B Trypsin dependent release of radioactivity from photolabelled membranes: time course 96

2.3.13. A HPLC separation of trypsin-cleaved photo

labelled purified GABA-R 97

2.3.13. B SDS-PAGE of trypsinized purified photo labelled GABA-R 98

3.1.1 Diagrammatic representation of the pro

duction of monoclonal antibodies. 133

3.3.1 Immunoblot with rabbit anti-GABA-R poly

clonal antibodies 190

x v jj 3.3.2 Diagrammatic representation of the different approaches employed for the production of monoclonal antibodies. 191

3.3.3 Antibody titre of mouse anti-GABA-R poly clonal antibodies 192

3.3.4 Diagrammatic representation of the screening procedures 193 3.3.5 Dot blot chequerboard test 194

3.3.6. A Purification attempts of monoclonal anti body 1A6 195 3.3.6. B Purification of IgM and IgG monoclonal antibodies 196

3.3.7 Attempts of GABA-R immunopurification 197 3.3.8 Dot blot test of the effect of detergent

on monoclonal antibody binding 198 3.3.9. A Immunoblot of purified GABA-R 199

3.3.9. B Immunoblot of bovine brain regions

membranes 200

3.3.9. C Immunoblot of brain membranes from

different species 201 4.3.1 Autoradiograph of rabbit reticulocyte

lysate in vitro translation of

GABA-R RNA 249

x v i i i LIST OF TABLES

Table Page 1.1.1 Solubilization of the GABA-R 29 2.3.1 Yields of Na-deoxycholate GABA-R purification 99

2.3.2 Yields of Triton-urea GABA-R purification 100

2.3.3 Photoaffinity labelling of bovine membranes

with [3H]muscimol 101 3.3.1 Immunization procedures and titres obtained 203 3.3.2 Ligand binding assay of the GABA-R immo bilized on the ELISA plates 204 3.3.3 A summary of the fusions performed 205 3.3.4.A The effect of antigens and mitogens on

the growth of spleen cells in culture 206 3.3.4.B A summary of fusions carried out with in vitro cultured spleen cells 207

3.3.4.C Immunoprecipitation of purified GABA-R 208

3.3.5 . A Protein A Sepharose chromatography 209

3.3.5.B Purification of the IgM monoclonal 1A6 210

3.3.6 Synthesis of immunopurification columns 211

3.4.1 A summary of the techniques used for the production of anti-GABA-R monoclonal antibodies 237

4.3.1 Binding assay of oocyte injected with GABA-R RNAs 250 LIST OF ABBREVIATIONS

BSA Bovine serum albumin CHAPS 3((3-Cholamidopropyl)dimethyl ammonio)-1- propanesulphate CDNA complementary DNA

DMEM Dulbecco's modified Eagles minimal essential medium DTT Dithiothreitol

EDTA Ethylene diamino trtaacetic acid

EGTA Ethylene glycol bis (0-aminoethyl ether) N,N'-tetraacetic acid ELISA Enzyme linked immunosorbent assay FPLC Fast protein liquid chromatography GABA Tf-aminobutyric acid

GABA-R GABAa receptor

HEPES 4-(2-Hydroxyethy1)-1-piperazineethane-sulphonic acid HPLC High pressure liquid chromatography MDP Muryldipeptide PEG Polyethylene glycol

PBS Phosphate buffered saline

PMSF Phenylmethylsulphonylfluoride

SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophresis

TBPS t-Butyl bicyclophosphorothionate TCA Trichloroacetic acid

TEMED N,N,N/,N#-tetramethylene diamine

UV ultraviolet. CHAPTER 1

GENERAL INTRODUCTION

1 1.1 Introduction

The understanding of the properties of the central nervous system requires the characterization of the mechanisms of neuronal activity. Such characterization must include the identification of the molecular entities

that are involved in the physiological activities of the

neurons. The discovery of the 7 aminobutyric acid (GABA) in the mammalian brain was of great importance for the study of the inhibitory mechanisms of the central nervous system (Roberts and Frankel, 1950). The many studies that have followed have provided a wealth of information on both GABA and its receptors (reviewed by Olsen and Venter 1986).

GABA is present in the mammalian brain in micromolar concentrations and it has been shown to be an inhibitory neurotransmitter of the central nervous system by a variety of experimental approaches that include microiontophoretic applications of GABA and electrophysiological recording, ligand binding studies, autoradiographic studies and immunological studies

(reviewed by Roberts, 1986). The search for a receptor molecule that mediates the effects of GABA, have led to the identification of at least three membrane-bound proteins to which GABA can bind: the GABA membrane transport system, the GABA^ receptor and the GABAfi receptor. GABA binding to the membrane transport system

2 was shown to be Na+-dependent (Enna and Snyder, 1975) and can therefore be subtracted from the total GABA binding activity, by using Na+-free buffers. The GABAB receptor is coupled to the adenylate cyclase system and, in the presence of Ca++ or Mg++ ions, it is bound by its specific ligand (-) baclofen (Bowery et al. . 1983) . The GABAA receptor is instead specifically bound by the GABA antagonist bicuculline (Greenlee et al., i978). The work here presented will deal exclusively with the GABAA receptor which henceforth will be referred to as the GABA-R. The GABA-R is a ligand-gated Cl" ion channel and it plays an important role in the GABAergic inhibitory system of the central nervous system. The GABA-R is widely distributed in the vertebrate brain (Schoch et al., 1985), and it is believed to be localized both on the post- synaptic and on the pre-synaptic terminals (Nicoll, 1975).

The GABA-R is found also peripherally e.g. in the adrenal chromaffin cells (Bormann and Clapham, 1985) and in invertebrates (Beadle et al., 1986). The binding of GABA to the GABA-R causes the channel to open and allows Cl” ions driven by the chemical gradient across the neuronal cell membrane, to enter the cell and as a result, the cell is hyperpolarized. The GABA-R thus mediates an inhibitory effect induced by GABA. The GABA-R is also the target of other classes of compounds. The depressant barbiturates bind to an allosteric site of the GABA-R and modulate the binding of

3 GABA agonists (Haefely et al., 1979). The benzodiazepines

also bind to an allosteric site of the GABA-R and they have been divided into three classes according to their physiological properties (Richards and Mohler, 1984): the agonists (e.g. flunitrazepam and diazepam) potentiate the inhibitory effect of GABA, the antagonists reverse the effect of the benzodiazepine agonists (e.g. Ro 15-1788)

and the inverse agonists (e.g. CGS 9896) decrease the inhibitory effect of GABA. A third major class of

molecules that bind to the GABA-R are the convulsants

(e.g. dihydropicrotoxinin and t-butyl bicyclophosphorothionate (TBPS)) which are believed to bind to the Cl“ ionophore (Ticku et al., 1978; Squires et al.. 1983).

Other classes of molecules that interact with the

GABA-R include: (i) the p carbolines which were originally

thought to be endogenous ligands for the benzodiazepine

sites and some show benzodiazepine inverse agonist binding properties (Richards and Mohler, 1984); (ii) the anthelminthic avermectins (Olsen and Snowman, 1985); (iii)

some steroids such as alfaxalone (Harrison and Simmonds,

1984) and (iv) the endogenous peptides GABAmodulin

(Toffano et al., 1978) and diazepam binding inhibitor

(DBI) (Costa and Guidotti, 1985).

The evidence for the presence of the binding sites

for all of the above-described compounds in a single macromolecular structure has come from

4 electrophysiological, pharmacological and biochemical studies of the GABA-R, which will be here described.

1.2 Electrophvsioloaical studies of the GABA-R

The first direct evidence for the inhibitory effects of GABA in the mammalian central nervous system dates back to the work of Krnjevic and Schwartz (1967), who showed that iontophoretic applications of GABA to cat brain cerebral cortical neurons produced inhibitory post- synaptic potentials. It was later shown by Curtis et al.

(1971) that bicuculline selectively inhibited GABAergic responses whilst having little effect on the action of another inhibitory amino acid, glycine. A more detailed analysis of the ion channels was performed after Katz and Miledi (1972) realized that the fluctuations in the pharmacologically stimulated membrane currents, were a complex biological signal rather than instrument artefact.

These "noise" analyses of GABA induced currents allowed the estimation of the channel duration (Barker and Owen,

1986). The study of ion channels has recently benefited from the development of the patch clamp techniques that allow single channel measurements. When these techniques were used for the study of GABA-induced Cl" currents, it was shown that the ‘GABA-R ionophore has a major conductance state of 20-30 pS (Sakmann et al., 1983, Hamill et al..

1983) and a Hill coefficient of circa 2 was obtained from dose response curves of GABA-induced whole cell currents

5 (Sakmann et al., 1983). The latter finding together with the burst-like appearance of the GABA-induced currents have led to the proposal of a model where two ligand molecules successively bind the closed receptor. The

latter, after isomerization, opens for 15-40 milliseconds with 2-3 interruptions per burst. Shorter-lived openings of the channel were also detected, and are believed to represent opening of the monoliganded state of the receptor (Sakmann et al., 1983).

1.3 Biochemical properties of the GABA-R

The initial biochemical characterization of membrane- bound receptors is usually carried out by binding studies to particulate fractions using the radioactive analogues of the receptor-specific ligands. The affinity of a ligand for a receptor is expressed by its KD (dissociation constant) and the total number of sites for the ligand are referred to as B ^ ^ The KD of a ligand is effectively the concentration of the ligand at which half the binding sites are occupied.

Initial studies of GABA binding sites in cerebral cortex membrane preparations were complicated by the presence of GABA in the tissue. However it was shown that the endogenous ligand could be removed by treating the membranes with multiple freeze-thaw cycles (Enna and

Snyder, 1977) or with Triton X-100 (Grenlee et al., 1978).

In multiply-washed, freeze-thawed membranes, two binding

6 sites for [3H]GABA ( Olsen et al., 1981) were found. The high affinity binding sites (KD = 1 3 + 6 nM) are less abundant = 0.33 pmol/mg protein ) than the low affinity sites (KD = 300 + 150 nM, B ^ x = 1.8 pmol/mg protein) (Olsen et al., 1981). Although the two binding sites show the same pharmacological characteristics, it has not been possible to determine whether they represent distinct receptor types, or different conformational states of the same receptor. The functional significance of the high affinity sites is also difficult to understand, because these sites should be occupied at the concentration of GABA that is known to occur in the brain. The endogenous ligand could however be compartmentalized and thus be, in the synaptic cleft, at an effectively lower concentration. The electrophysiological studies however, have shown that channel opening occurs at micromolar GABA concentration (Simmonds, 1981), which indicates that it is the binding to the low affinity sites that is responsible for the physiological events.

The GABA binding sites have also been characterized with the GABA agonist [3H]muscimol (Beaumont et al. .

1978), which has a higher affinity and a slower off rate than GABA itself, but otherwise shows the same pharmacological characteristics. Other GABA agonists which have been used are piperidine-4-sulphonic acid

(Krogsgaard-Larsen et al., 1981), isoguvacine (Morin and

Wasterlain, 1980) and 4,5,6,7-tetrahydroisoxalozolo (5-4-

6) pyrjdin-3-ol (THIP) (Falch and Krogsgaard-Larsen,

7 1982). The competitive antagonist bicuculline-methobromide displaces the binding of [3H]GABA and its agonists (Olsen and Snowman, 1983) and also blocks GABA-induced currents by eliminating the Cl" ion activation (Curtis et al..

1971). The barbiturate pentobarbital has been shown to enhance the binding of GABA and its agonists in thoroughly-washed membranes (Haefely et al., 1979? Olsen and Snowman, 1982). This enhancement is dependent on the presence of Cl” ions (200 mM) and can be reversed by the Cl" channel blockers such as picrotoxin and TBPS and by the competitive antagonist bicuculline. The enhancement of GABA binding by barbiturates appears to involve changes in both the Kq and Bmax (Olsen and Snowman, 1982) . The observed enhancement could also be due to an increase in affinity of normally undetected very low affinity binding sites (Kd > 1 uM) (Olsen and Snowman, 1982) . Electrophysiologically micromolar concentrations of the barbiturates prolong the GABA-R channel burst duration (Study and Barker, 1981) without affecting the conductance. At higher concentrations (>50 uM) however, these drugs can directly activate Cl” channels (Jackson et al., 1982).

Most of the in vitro binding studies carried out with benzodiazepines have used either [3H]flunitrazepam (Speth et al. . 1977 and 1978) or [3H]diazepam (Squires and

Braestrup, 1977? Mohler and Okada, 1977). These studies

8 have shown the presence of a single high affinity binding site with a KD in the nanomolar range, a Bmax of approximately 1 pmol/mg protein and a Hill coefficient close to unity (Squires and Braestrup, 1977; Mohler and

Okada, 1977 a,b). Benzodiazepine binding is however displaced by the triazolopyridazines (e.g. CL 218,872) with a Hill coefficient, nH < 1 (Squires et al., 1979).

This finding together with the similar type of binding affinity of the alkyl derivatives of 0-carboline-3- carboxylate (Nielsen and Braestrup, 1980) has led to the interpretation of the existence of two sub-populations of benzodiazepine sites, referred to as Type I and Type II benzodiazepine receptors. The proportions of the two binding sites varies between different brain regions, which was shown also by histochemical autoradigraphy (Young et al., 1981).

Benzodiazepine binding to the GABA-R is increased by GABA agonists in a reversible and dose-dependent manner with typical EC50 values of 1.6 uM and 0.5 uM for the enhancement by GABA and muscimol respectively (Tallman et al.. 1978). The increase in binding is due to an increase in affinity (KD), and the concentration of GABA required for the enhancement approaches the values required for the occupancy of the low affinity binding sites (Tallman et al., 1978). Benzodiazepines do not themselves induce an increase in the Cl“ current but if benzodiazepine agonists are applied together with GABA they cause a potentiation of

9 the Cl" ion current by increasing the frequency of opening of the GABA-R ion channel (Study and Barker, 1981). GABA binding is also increased in the presence of benzodiazepines such as diazepam, CGS 9896 and quazepam which has preferential affinity for Type I benzodiazepine receptors (Guidotti et: al., 1979; Meiners and Salama, 1985 and Corda et al., 1986)• All compounds were effective at micromolar concentrations. This enhancement was due to an increase in the of GABA low affinity binding sites and was blocked by the antagonist Ro 15-1788, which itself produced only a small enhancement (Corda et al., 1986). Benzodiazepine binding to brain membrane preparations is also increased by barbiturates (Leeb-Lundberg et al., 1980). The increase which is dose-dependent, stereospecific and halide anion dependent (Cl”,Br",I”), is due to a shift in affinity for the benzodiazepines with no apparent change in the number of binding sites. This facilitation was reversed by picrotoxin (Supavilai and

Karobath 1981? Olsen, 1981).

The convulsants picrotoxin and TBPS were shown to occupy the same binding sites on the GABA-R, which are distinct from the GABA, benzodiazepine or barbiturate binding sites (Squires et al., 1983, Maksay and Ticku,

1985). These ligands are thought to block Cl" current by directly binding to the Cl” channel ionophore (Van

Renterghem et aT., 1987). The ligand [35S]TBPS is of particular interest for the study of this binding site of

10 the GABA-R, because it has an affinity constant KD = 20 nM higher than that of the picrotoxin derivative [3H]a- dihydropicrotoxinin (Squires et al., 1983). TBPS was also shown to enhance in a dose-dependent manner the binding of [3H] propyl 0-carboline-3-carboxylate to the Type I benzodiazepine receptors. This enhancement was antagonized by micromolar concentrations of GABA, which was reversed by bicuculline. Furthermore, benzodiazepine agonists inhibited [3®S]TBPS binding while inverse agonists had the opposite effect (Gee et al., 1986).

To summarize, in vitro binding studies of thoroughly- washed cerebral cortical membranes have identified four specific distinct binding sites associated with the GABA- R. These are the sites for GABA and its agonists and antagonists, the benzodiazepines, the barbiturates and the convulsants. The fact that these sites were shown to allosterically interact with each other led to the suggestion that they were all present on the same macromolecular structure (HaefflLy et al., 1979, Squires et al., 1982? Olsen, 1981)

1.4 Solubilization of the GABA-R

An essential step in the further characterization of the GABA-R was the solubilization of the protein from the membrane lipid bilayer in a manner in which all of the above-mentioned binding sites could be preserved. Table

1 . 1 shows a variety of detergents that have been used for this purpose. The detergent Na-deoxycholate is the one

11 that gave the highest yield of soluble benzodiazepine sites at near-physiological salt concentration (Sigel et al., 1983), while Triton X-100 together with 1 M KC1 was shown to solubilize about 90% of the benzodiazepine sites present in the membranes (Stauber et al.., 1987). The receptor solubilized with either of these two detergents did not possess all the ligand allosteric interactions present in the membrane-bound receptor, nor the binding site for TBPS or barbiturate regulation. The zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio]-1- propanesulphonate (CHAPS) has been shown to solubilize less benzodiazepine binding sites, but the solubilized product maintained its TBPS binding sites (Sigel and

Barnard, 1984? King et al., 1987), and the barbiturate and GABA potentiation of benzodiazepine binding were also preserved (Stephenson and Olsen, 1982; Stephenson and Olsen, 1983). The CHAPS solubilized receptor was however unstable and lost, upon storage, TBPS binding and the allosteric interactions between the barbiturates and the benzodiazepine binding sites (Stephenson and Olsen, 1982).

Recently however it has been shown that the long term stability of the allosteric ligand interactions can be maintained by the inclusion of a total brain lipid preparation in the solubilization mixture (Bristow and Martin, 1987).

12 1.5 Purification of the GABA-R

The low abundance of the GABA-R makes conventional purification methods inadequate for the isolation of this protein. The GABA-R has instead been successfully purified by means of benzodiazepine affinity columns where a benzodiazepine, e.g. Ro 7-1986/1, (Sigel et al., 1983) was bound to a spacer arm attached to a solid matrix. The purification procedure was accomplished by the application of the brain membrane soluble extract to the benzodiazepine affinity column. The latter was extensively washed to remove non-specifically bound proteins. The receptor was bio-specifically eluted with a water soluble benzodiazepine e.g. chlorazepate, (Sigel et al., 1983), which was itself removed by ion-exchange chromatography

(Sigel et al. . 1983), gel filtration (Olsen et al., 1984) or diafiltration (Tallman, 1984). The purification procedure yielded a highly purified product (Sigel et al.. 1983? Martini et al., 1982? Olsen et al.. 1984? Taguchi and Kuriyama, 1984) in essentially one chromatographic step. The percentage yield of purified receptor was generally however only 5-10% of the starting material (Sigel et al., 1983? Kirkness and Turner, 1986a and this work), with the major loss occurring at the benzodiazepine column elution step. Recently, Stauber et al.. (1987) have reported a high yield purification method of the GABA-R from rat cerebral cortex using a benzodiazepine affinity column where Ro 7-1986/1 was

13 attached to Affigel 202 via l-ethyl-3-(3- dimethylaminopropyl) carbodiimde, and the chlorazepate elution was performed in the presence of 4 M urea. Milligram quantities of GABA-R were reported to have been obtained, which is a ten-fold improvement compared to the yields obtained by the previous methods (Sigel et al.. 1983? Kirkness and Turner, 1986a). The purified receptor showed the same pharmacological properties of the crude soluble extract from which it was isolated. Therefore where the detergent Triton X-100 or

Na-deoxycholate were used, the purified receptor contained benzodiazepine and GABA binding sites (e.g. Sigel et al.. 1983? Stauber et al.. , 1987). Where throughout the purification procedure CHAPS and asolectin (Sigel and Barnard, 1984) , or CHAPS and L-a-phosphatidylcholine

(Kirkness and Turner, 1986a) or CHAPS alone (Stephenson et al. . 1986b) were used, TBPS specific binding and barbiturate (Sigel et al.., 1983) and anaesthetic propanidid facilitation (Kirkness and Turner, 1986b) of both benzodiazepine and GABA binding were also retained.

These latter properties were best stabilized by the inclusion of total brain lipid preparation throughout the purification procedure (D. R. Bristow, personal communication). The stoichiometry of the binding sites for the various ligands has still not been exactly determined. The presence of endogenous ligands in the tissue and of the ligands used for the biospecific elution of the receptor from the affinity column and the different

14 stability of binding sites, all contribute to complicate the accurate assessment of this ratio. Values from 1 to 4 for the ratio of GABA to benzodiazepine binding sites and of 1 for the ratio of TBPS to benzodiazepine binding sites, have been reported (Sigel et al., 1983? Sigel and

Barnard, 1984)

1.6 Molecular characterization of the GABA-R

1.6.1 Molecular size of the GABA-R

The size of the receptor has been estimated in situ by irradiation inactivation studies, but different results have been reported. The calculated molecular sizes ranged from 50 kDa (Paul et al., 1981; Nielsen et al., 1985), 90- 100 kDa (Doble and Iversen, 1982) to 200-220 kDa (Chang et al., 1981; Chang and Barnard, 1982) when the GABA or benzodiazepine binding sites were assayed after the irradiation, and up to 548 kDa when the inactivation of the TBPS binding sites was included in the calculation of the total size of the GABA-R (Nielsen et al., 1985). Chang and Barnard (1982) and Schwartz et a_l. , (1985) have however shown that some of the variation is due to the preparation of the sample before it is irradiated. The molecular size of the solubilized receptor has also been estimated by sucrose density gradient centrifugation and gel filtration. In both cases, the calculation of the exact molecular size is complicated by the fact that the receptor is incorporated into the

15 detergent micelle whose size must be accounted for (Tanford and Reynolds, 1976). Again, a variety of molecular sizes have been reported ranging from 200 - 240 kDa to 600 kDa (Yousufi et al., 1979; Asano and Osagawara,

1981? Chang and Barnard, 1982; Schoch and Mohler, 1983? King et al., 1987? Mamalaki e£ al., 1988).

1.6.2 Subunit size and composition of the GABA-R

The purified GABA-R has been shown to migrate as a single band in an iso-electric focusing gel (Stephenson et al., 1986a), which indicates that the purified product was homogeneous. When the receptor preparation was analysed under reducing conditions by sodium dodecyl sulphate- polyacrylamide gel electrophoresis (SDS-PAGE) it showed two bands of molecular weight 53 kDa ( a subunit) and 57 kDa (the p subunit) (Sigel et al., 1983, Stephenson et al., 1986). Other groups have detected bands of similar molecular weight in purified GABA-R preparations of different species (Kirkness and Turner 1986a? Taguchi and

Kuriyama, 1984). Deng et al. (1986) have also reported four minor bands in a bovine preparation, and Olsen et al., (1984) a total of four bands in preparations from rat brain. It is not clear at this time if the additional subunits observed are components of the GABA-R.

Quantitative densitometric scans of Coomassie Blue stained

SDS-PAGE gels of purified bovine receptor have shown that the a and the (3 subunits appear to be present in equimolar

16 amounts (Mamalaki et al.., 1987), which has led to the suggestion that the subunit stoichiometry of the GABA-R is a2^2 • Both subunits are glycosylated as shown by carbohydrate staining of SDS-PAGE of the receptor (Sigel and Barnard, 1984). When the purified GABA-R was treated with endoglycosidase F, the molecular weights of the deglycosylated a and p subunits were shown to be 44 kDa and 55 kDa respectively (Mamalaki et al., 1987).

1.7 Photoaffinitv labelling of the GABA-R

1.7.1 Photoaffinitv labelling of the GABA-R with r—HIflunitrazepam

Mohler et al., (1980) were the first to report that the ligand, [3H]flunitrazepam, could upon UV irradiation specifically irreversibly label a polypeptide in a synaptosomal preparation of rat brain. Initial photolabelling experiments with [3H]flunitrazepam (Mohler et al., 1980? Sieghart and Karobath, 1980? Mohler, 1982) were performed by irradiating membrane preparations, in the presence of [3H]flunitrazepam, at A = 366 nm for 10-30 min. The results obtained from these experiments (Mohler et al. . 1980, Mohler, 1982) showed that: 1)

[3H]flunitrazepam photoaffinity labelled a maximum of 25% of the reversible binding sites, 2) if the photoaffinity labelling was carried out with unlabelled flunitrazepam and the remaining sites were assayed for reversible binding, the latter were reduced by a number nearly four

17 times as great as the number of sites photoaffinity labelled, 3) the inactivation of the non-photolabelled sites was a characteristic of the benzodiazepine agonists and not the antagonists. From these observations the authors suggested that the benzodiazepine binding sites were in clusters of 4, and that the ability of a benzodiazepine to bind to the remaining sites of a photoaffinity labelled membrane sample could be used to classify the ligand as an agonist or antagonist. It was however later shown that firstly, the non photoaffinity labelled sites were not inactivated but it was the affinity of the benzodiazepines for the receptor that was reduced and the sites were detectable at higher ligand concentrations (Gibbs et al., 1985). Secondly, when the irradiation was carried out at X = 254 nm, 40% of the reversible [3H]flunitrazepam binding sites were photoaffinity labelled (Herblin and Mechem, 1984). Thirdly, when a wider range of benzodiazepine agonists and antagonists were tested, it was shown that the ligand affinity changes for photoaffinity labelled membranes could not be used to predict the ligand efficacy (Brown and Martin, 1983).

Electrophysiological recordings of neurons photolabelled with [3H]flunitrazepam showed that GABA- induced Cl” currents were not constituently increased in these neurons and, that while the binding affinity for benzodiazepines was decreased, the coupling mechanism to the GABA sites was not affected (Gibbs et al., 1985?

18 Brown and Martin, 1984). Herblin (1985) found that while photoaffinity labelling reduced the affinity of benzodiazepines it did not affect the binding of the p carbolines which suggested that the latter might have a separate, though related, binding site within the GABA-R.

In summary, pharmacological studies of the photolabelling of the GABA-R with [3H]flunitrazepam have shown that up to 40% of the receptor population can be irreversibly labelled with flunitrazepam upon UV irradiation (Herblin and Mechem, 1984). The photoaffinity labelled receptor does not show any alterations of either its allosteric properties nor its channel function, but does show a reduced affinity for some benzodiazepine ligands (Gibbs et al., 1985) . The fact that p carbolines are unaffected by the labelling with flunitrazepam may indicate that they do not share the same sites as the benzodiazepines (Brown and Martin, 1984, Herblin 1986).

1.7.2 Characterization of the r—HIflunitrazepam

labelled proteins

Following the first report by Mohler et al. (1980) of the photolabelling properties of [3H]flunitrazepam, the study was extended to membrane preparations from different brain regions which were also photolabelled with

[3H]flunitrazepam and then analysed by SDS-PAGE. Sieghart and Karobath (1980) confirmed the finding of Mohler et al. (1980) that a protein of molecular weight 51 kDa (p51) was

19 specifically labelled in membranes from cerebellum. Sieghart and Karobath (1980) however also found that in other brain regions, e.g. cerebral cortex, striatum and hippocampus, other proteins of molecular weights 53 kDa

(p53) , 55 kDa (p55) and 59 kDa (p59) were additionally labelled. Labelling of all the above bands could be increased by the addition of 10 uM GABA, an effect that was reversed by bicuculline (Sieghart and Karobath, 1980). The Type I benzodiazepine receptor-specific ligand CL 218

872 inhibited labelling of p51 more strongly than the labelling of p55 (Sieghart and Karobath, 1980). This result together with the absence of p55 in cerebellum indicated that the Type I and the Type II benzodiazepine receptors, as defined pharmacologically, are associated with different polypeptides.

The SDS-PAGE profile of [3H]flunitrazepam photoaffinity labelled polypeptides from rat brain membranes changed during the first 30 post-natal days (Eichinger and Sieghart, 1985). While additional labelled polypeptides decreased with the age of the animal, the labelling to the p51 increased in parallel with the development of reversible binding to Type I benzodiazepine receptors

(Eichinger and Sieghart, 1986)

When photolabelled membranes were subjected to proteolytic degradation, about 40% of the counts were removed by trypsin treatment (Klotz et al., 1984? Eichinger and Sieghart, 1985). The reduction was time- and trypsin- concentration dependent. SDS-PAGE analysis of the

20 proteolytic products showed that p51 was totally reduced to two polypeptides, p39 and p25, whereas p55 was only partially reduced to p45 and p42. The latter finding suggested that there might be three proteins of molecular weight 55 kDa that co-migrate on SDS-PAGE.

Photoaffinity labelling of avian brain membrane preparations (Hebebrand gfc a l . , 1986) showed that two major bands (53 kDa and 54 kDa) were labelled. Ontogenic and phylogenic studies on a variety of avian and reptile photolabelled membrane preparations (Hebebrand et a_l., 1987) have shown that different [3H]flunitrazepam labelled proteins are present in the different phyla and also that these proteins follow different developmental profiles within the same species.

Photoaffinity labelling of the purified GABA-R from bovine cerebral cortex with [3H]flunitrazepam has shown that the a subunit (53 kDa) is predominantly labelled although some labelling also occurred in the p subunit (Sigel et al., 1983? Kirkness and Turner, 1986a? Sato and

Neale, 1987). When the photolabelled purified receptor was subjected to trypsinization, the time course of the trypsinization reaction showed the disappearance of the a and the p subunit and the transient appearance of radioactively labelled intermediates, 48 kDa and 46 kDa, followed by degradation to very small fragments ( < 10 kDa) (Sigel et al., 1984).

21 1.7.3 Other ligands that photoaffinitv label the GABA-R

The agonist [3H]clonazepam and the partial inverse agonist [3H]Ro 15-4513 have also been shown to photoaffinity label the benzodiazepine binding site of the

GABA-R. (Mohler et al., 1980; Sieghart et al., 1987).

Similar polypeptide labelling patterns were obtained with these two photoaffinity ligands as compared to those obtained with [3H]flunitrazepam. However, [3H]Ro 15-4513 was irreversibly bound to nearly 100% of the reversible binding sites (Sieghart et al., 1987).

The GABA agonist muscimol was also reported to irreversibly photolabel the GABA-R (Asano et al., (1983); Cavalla and Neff, 1985a). When rat cereb ellar membranes were irradiated in the presence of [3H]muscimol, the ligand was irreversibly incorporated in a time- and ligand concentration-dependent manner. The labelling was totally blocked by GABA agonists and antagonists but not by the

GABAg receptor agonist baclofen. SDS-PAGE analysis of the [3H]muscimol photolabelled membranes revealed a protein of molecular weight 50 kDa which was originally thought to correspond to the a subunit of the GABA-R (Cavalla and Neff, 1985a) . It is shown in this report and in Deng et al. (1986) that in the purified state of the GABA-R it is the p subunit that is predominantly specifically photoaffinity labelled with [3H]muscimol.

22 1.8 Analysis of the GABA-R structure with antibodies

Polyclonal and monoclonal antibodies have been raised against the purified GABA-R (Schoch et al., 1985? Haring et al.. 1985; Stephenson et a l . , 1986a? Mamalaki et al..

1987? Stauber et al., 1987? Vitorica et al., 1988 and this work). The polyclonal antibodies raised by Stephenson et al.. (1986a) were shown to immunoprecipitate both purified 125I-receptor, and [3H]muscimol , [3H]flunitrazepam, and [35S]TBPS binding sites from a crude CHAPS soluble extract. This provided further evidence that all of the above binding sites are present on a macromolecular structure. Western blot analysis of the purified receptor with polyclonal antibodies showed that the a subunit was more immunogenic than the p subunit as the former was always specifically or preferentially labelled by the antibodies (Stephenson et al., 1986a? Stauber et al.. 1987; Vitorica et al., 1988 and this work).

Monoclonal antibodies that specifically recognised either the a or the p subunits were raised by Schoch et al., (1985) and recently by Vitorica et al., (1988). Both a and p subunit-specific antibodies raised by Schoch et al. (1985) immunoprecipitated, in the same ratio,

[3H]muscimol and [3H]flunitrazepam binding sites from a purified receptor preparation (Schoch et al., 1985). The monoclonal antibodies raised by Vitorica et al., (1988) were reported to immunoprecipitate [3H]flunitrazepam,

[3H]muscimol and [35S]TBPS binding sites also from a CHAPS

23 crude soluble extract which is consistent with the model of the association of these sites in a single macromolecule. Monoclonal and polyclonal antibodies have been shown to cross-react between different mammalian and avian species (Haring et al., 1985; Stephenson et al., 1986a?

Mamalaki et al., 1987), which indicated that the GABA-R is conserved evolutionally. A degree of homology between the two subunits was shown by a monoclonal antibody that on Western blots could recognise both the a and the p subunits (Mamalaki et al.. 1987).

Immunocytochemical studies with both a and p subunit specific monoclonal antibodies have shown an equivalent staining pattern in rat and bovine tissue sections.

Similar staining, with the exception of the molecular layer of the cerebellum, was obtained from autoradiograms of tissue sections incubated with [3H]flunitrazepam or

[3H]muscimol (Schoch et al., 1985), which indicated that the epitopes recognised by the monoclonal antibodies raised against the purified receptor are common to the total population of the GABA and benzodiazepine binding sites.

1.9 Molecular biology of the GABA-R

Recombinant DNA techniques have been applied to elucidate the primary amino acid sequences of the a and p polypeptides of the GABA-R and to express the in vitro synthesized subunit specific RNAs in Xenopus oocytes

24 (Schofield et al., 1987). Partial amino acid sequence was obtained from non-contiguous cyanogen bromide cleaved peptides. Best guess oligonucleotide probes were then synthesized and used to screen calf and bovine complementary DNA (cDNA) libraries. Three clones encoding for the a subunit (named al, a2 and <*3) and one for the p subunit have to date been identified (Levitan et al..

1988). The i a and p subunits have an amino acid composition homology of 57% including conservative substitutions. Hydropathy plots of the amino acid sequences revealed that all subunits have four potential transmembrane regions, M1-M4, and it has been proposed that both the C-terminus and the N-terminus of the protein are extracellular. Sequence homology between the subunits is highest within the trans-membrane regions and lowest between M3 and M4. This is also the region which in the p subunit, the potential phosphorylation site is found. Both subunits also contain potential glycosylation sites, two and three in the a-^ and p subunits respectively. It was found that the GABA-R subunits have a significant homology with all the cloned subunits of the acetylcholine receptor. The homology reaches 62% in the transmembrane region and parts of the N-terminal region, including conservative substitutions. Additionally, the 48 kDa subunit of the glycine receptor (Grenninglogh et al.,

1987) showed 56% homology with the a subunit of the GABA-R (Barnard et al., 1987). The homology is also observed at

25 the secondary and tertiary level as all of these receptor subunits have four potential transmembrane regions, and they also all contain a predicted extracellular p - structural loop which can be formed by the disulphide bonding of two cysteine residues. The putative amphip.atic transmembrane regions of the nicotinic acetylcholine receptor subunits are however not found in the GABA-R or in the glycine receptor. These results indicate that these three ligand-gated ion channel receptors belong to a super-family of chemically-gated ion channels that may have a common ancestral gene that has diversified in the course of evolution (Barnard et a l ., 1987). The specific RNAs for the GABA-R subunits have been expressed in the Xenopus oocyte translation system. It was found that when both the a l and the p subunits were expressed in the oocyte, a GABA induced Cl” current was formed. The current was blocked by bicuculline and potentiated by barbiturates and the benzodiazepine chlorazepate, which indicated that all the functional binding sites of the GABA-R had been successfully expressed (Schofield et al., 1987).

1.10 The objectives of this study

When this work was started in October 1984, the GABA-

R had been characterized in its membrane-bound and solubilized state. A method for its purification to homogeneity had allowed the characterization of the pure receptor and the determination of its subunit composition.

26 Polyclonal antibodies and some monoclonal antibodies had been raised against the purified GABA-R. Many questions however about the receptor structure remained unknown. One of these was the distribution of the ligand binding sites within the receptor subunits. Thus the technique of photoaffinity labelling was carried out with the ligands [3H]flunitrazepam, [3H]muscimol and [35S]TBPS.

A second aim of the work was to find an alternative, high yield purification procedure for the GABA-R. The two sets of monoclonal antibodies that were available (Schoch et al., 1985 and Mamalaki et al., 1987) had not been shown to immunoprecipitate the receptor from a crude soluble extract. Thus novel monoclonal antibodies were raised primarily with the aim of using one or more of these for the synthesis of an immunoaffinity column. The molecular cloning of the GABA-R (Schofield et al., 1987) has opened other possibilities for the study of the molecular structure of the receptor. One approach carried out in this work has been the pharmacological and biochemical study of the expression of the cloned GABA-R subunits in Xenopus oocytes. This work, in conjunction with site-directed mutagenesis studies and electrophysiological measurements, had the long term aim of a more detailed knowledge of the structure/function relationship of the GABA-R.

27 Figure l.i

GABA Muscimol Bicuculline Methyl Chloride

Flunitrazepam

Pentobarbital TBPS

Chemical Structure of some of the ligands that bind to the

GABA-R. Reproduced from Stephenson (1988), Mamalaki (1986), Sieghart et al.(1987).

28 Table 1.1.1

Solubilisation Recovery Barbiturate TBPS Ref conditions (a) (b) (c)

Na-deoxycholate (0.5%) 73% ND ND (1) Triton X-100 (2.5%) 8% ND ND (1) Triton X-100 (2.5%)+lM KC1 90% ND ND (1) CHAPS(1.5%) + asolectin ND ND + (2) CHAPS (20 mM) 67% + + (3) CHAPS (100 mM) 18% + ND (4) OCTG (100 mM) 70% - ND (4) CHAPS (1.5%) ND ND + (5) Na-Deoxycholate (0.5%) ND ND - (5) Triton X-100(2%)+CHAPS(5mM) 55% - ND (6) CHAPS (25 mM) + a Lecithin ND + ND (6) Na-Deoxycholate (0.5%) 53% -- (7) CHAPS(1.5%) + asolectin 35% + + (7)

(a) = the recovery of [3H]flunitrazepam binding after solubilization

(b) = the presence of barbiturate enhancement of GABA and/or benzodiazepine agonist binding (c) = the presence of [35S]TBPS binding sites

ND = not determined OCGT = Octylglucopyranoside Ref = references: (1) Stauber et al. (1987)? (2) Stephenson et al. (1986b); (3) Stephenson and Olsen (1982)? (4) Hammond and Martin (1986) ? (5) King et al.. (1987) ? (6) Kirkness and Turner (1987); Sigel and Barnard (1984).

Solubilization yields and pharmacological properties of the solubilized GABA-R.. (See text 1.4)

29 CHAPTER 2

GABA-R PURIFICATION AND PHOTOAPFINITY LABELLING

30 2.1 INTRODUCTION

This chapter will describe the routine purification and the photoaffinity labelling with [3H]flunitrazepam and

[3H]muscimol of the GABA-R. In this introduction some of the basic objectives and the criteria of specificity of the photoaffinity labelling reactions will be described.

2.1.1 The objectives of photoaffinitv labelling experiments

Most of the known ligands of central nervous system receptors bind to their respective binding sites in a reversible manner. The characterization of the receptor would however, be aided if these binding sites could be occupied by ligands in an irreversible manner. This can be achieved either with affinity labels that covalently bind the receptor binding sites, or with photoaffinity labels whose reversible binding to the receptor is changed to an irreversible covalent binding by the irradiation with UV light of the ligand-receptor complex. The advantages of the latter system are that the ligand molecules are allowed to bind to the active sites before they are activated and that the ligand molecules not bound to the active site can be neutralized by the buffer system. The

31 specificity of the ligand incorporation by photoaffinity labelling is therefore greater than that of affinity labelling.

Nearly every known neurotransmitter receptor has

been photolabelled (for review see Fedan et al., 1984) and

the technique has been exploited in different ways. Photoaffinity labelling has been of use in the development

and monitoring of the purification procedures of neuroreceptors as for example for the ^-adrenergic receptor (Stiles et al., 1983). The irreversible covalent nature of the reaction allows the photolabelled protein also to be analyzed under denaturing conditions such as SDS-PAGE• This has been particularly useful for the

characterization of the molecular size of the protein or

its photolabelled subunit(s), in most cases before the

receptor itself waspurified, e.g. the glycine receptor (Graham et al..1983). SDS-PAGE of photolabelled receptors can also be used to monitor their developmental changes and tissue specificity. One example is that of the

benzodiazepine receptor studied extensively by Eichinger

and Sieghart (1986). The photolabelled receptor can be

subjected to both enzymatic and chemical digestion which

permits the isolation and identification of the peptide to which the ligand is irreversibly bound (Tallman, 1985). Information on the structure/function relationship of the

nicotinic acetylcholine receptor were obtained by the use

of the natural photoaffinity label chlorpromazine which is

32 believed to bind to the ionophore (Cox et al., 1984). Photoaffinity labelling of receptors can thus yield useful information about their structure however, it requires the availability of ligands with the appropriate characteristics.

2.1.2 Properties of the photoaffinitv liaands

A ligand used for photoaffinity labelling should satisfy several criteria. These are: 1) The ligand should have high affinity for its specific binding sites. 2) The ligand must be stable in aqueous solution.

3) UV irradiation should yield a highly reactive intermediate which should not rearrange to a less reactive compound or to one of lower affinity before it forms the covalent bond with the receptor of interest.

4) The ligand should not be photoexcitable following covalent bond formation.

5) The ligand should preferably be photoexcitable at

A > 300 nm, at which wavelengths the protein is less susceptible to denaturation.

Most of the ligands that bind neuroreceptors reversibly do not fulfill the above requirements for the use as photoaffinity labels. In general therefore, ligands have been specifically derivatised to contain known

33 photoexcitable groups such as an azide residue. A large number of such chemical groups are known and have been reviewed by Bayley and Knowles (1977).

Some ligands however have been successfully used as photoaffinity labels even though they do not contain recognisable photoreactive residues. Indeed, it is not possible at the present time to predict from the chemical structure whether the ligand can be used as a photoaffinity label. The two ligands used in this work, flunitrazepam and muscimol were not specifically derivatized.

2.1.3 Specificity of ohotoaffinitv labelling reactions

Irradiation with UV light of the photoligand causes some of its electrons to be excited to a higher energy state. The ligand can then form a covalent bond with any nucleophile or chemical bond that can react with the ligand in the excited state. Figure 2.1.1 depicts the various reactions and their relative equilibrium constants that characterize the photolabelling process. Before irradiation, the ligand molecules are in equilibrium between a bound and a free state. Upon irradiation the whole ligand population is excited. The ligand present at its binding site can either form a covalent bond with an accessible residue which is defined as specific irreversible binding, or it could leave the binding site and react with another residue of the same or of a

34 different protein. The latter is defined as non-specific irreversible binding. Alternatively, the excited ligand could react with the solvent. The following factors are therefore important to obtain high specific photolabelling (see Figure 2.1.1): 1) The receptor sites should be maximally occupied at the time of the UV irradiation, while the ligand concentration in the solution should be relatively low in order to reduce non-specific binding. Therefore ligands with Kd < 10”7 M are poor photolabels. 2) The rate of reaction (kSp)of the ligand with the residue in the active site must be faster then the rate

(kr) at which the excited ligand leaves the binding site,

(i.e. ksp > kr). 3) The rate of the ligand binding with the solvent

(ksoi) and/or the scavenger must be faster than with the binding to non-specific sites (knSp) i.e. ksoi + ksc » v ^nsp*

Scavengers are generally small organic molecules such as amines, mercaptans and aminomercaptans which can play an important role in the reduction of non-specific labelling. Their general properties must be characterized:

1) The scavenger should not affect the rate of reversible binding by the alteration of the ligand or of the receptor structure.

2) UV light should not be absorbed by the scavenger as

35 it is usually used at high concentrations. 3) The ligand in the binding site should be oriented in such a way that it will not react with the scavenger.

4) The scavenger must not react with the ligand before irradiation.

The total number of sites photolabelled are calculated after the reversibly bound ligand has been removed. The specific irreversible labelling is calculated by the subtraction of the non-specific irreversible binding from the total irreversible binding. The former is calculated in samples incubated and irradiated in the presence of excess unlabelled ligand. Where possible, the unlabelled ligand should be a different agonist which is not itself a photolabel, and it should be used at a concentration that will inhibit the specific binding but not non-specific irreversible binding.

2.1.4 The mechanism of the photoaffinitv labelling reaction of flunitrazeoam

Sherman-Gold (1983) proposed that the irradiation of flunitrazepam induces a resonance in the ligand molecule which results in the formation of a positively charged carbonyl carbon at position 2 (See Figure 2.1.2). Such resonance cannot occur if hydrogen or halogen atoms are substituted at position 7. The positively charged atom may then interact with a nucleophile group of the receptor.

36 This interaction results in the opening of the 7 membered ring of flunitrazepam and the formation of the covalent bond between the nucleophile and the carbonyl atom. This proposed mechanism is corroborated by the fact that clonazepam which is also a photoaffinity label (Mohler et al.. 1980) and the recently developed photolabel Ro 15-

4513 (Sieghart et al., 1987) have respectively an N02 and an N3 group at position 7. A particular feature of flunitrazepam is that if it does not react with a nucleophile, it returns to its original conformation and can be re-photoactivated (Sherman-Gold, 1983).

2.1.5 The mechanism of the photoaffinitv labelling

reaction of muscimol

Cavalla and Neff (1985 a and b) have proposed (Figure 2.1.3) that upon UV irradiation of [3H]muscimol, an aryl nitrene is formed. This aryl nitrene can undergo Curtius rearrangement to form an isocyanate whose role as an affinity label is unclear. Both the isocyanate and the aryl nitrene have few of the characteristics of GABA-R ligands and probably have low affinities and high dissociation rates for the muscimol binding sites.

Photoaffinity labelling with [3H]muscimol is complicated further by the fact that the photodecomposition produces a ketone group adjacent to the tritium label of the aryl nitrene. This structure is maintained in the photolabelled

37 protein. The tritium label is in an enolisable position and can be exchanged with water especially under acidic conditions.

38 Ficrure 2.1.1

z \ fcac ^ zxn

Key Hi; <3 O Receptor photolabel solvent scavenger

The photoaffinity labelling reaction equilibria.

The diagram shows that before irradiation the ligand is at equilibrium between the bound and non-bound state. During irradiation the ligand is excited; this can alter the binding equilibrium. As a consequence of the UV irradiation the ligand is irreversibly bound to either the receptor active site, a receptor non-specific site, the solvent or the scavenger. The diagram was adapted from Ruoho et al. (1984). (See text 2.1.3)

39 Figure 2.1.2

nucleophilic attack

The UV light - induced resonance in flunitrazepam and the possible interaction with a nucleophile. [3H]Flunitrazepam is tritiated in its N-methyl group. Reproduced, from Sherman-Gold (1983) (see text 2.1.4)

40 Figure 2.1.3

The proposed UV light-induced rearrangement of the muscimol molecule. The putative reactive reagents are (III) the acyl nitrene and (IV) the isocyanate. The former is the most reactive. [3H]Muscimol is tritiated in the carbon adjacent to the amine group. Reproduced from Cavalla and Neff (1985a), (see text 2.1.3)

41 2.2 Material and Methods

2.2.1 Materials

[Methylene-3H]muscimol (7-20 Ci/mmol), [methyl-3H]- flunitrazepam (80-85 Ci/mmol) were purchased from Amersham

International (UK). [35S]TBPS (30-98 Ci/mmol) was from New England Nuclear (USA). Na-deoxycholate was from Koch-Light Laboratories (UK). GF/C glass fibre filters were from Whatman. Chlorazepate and Ro 7-1986/1 were gifts of Boehtinger Ingelheim (U.K.), and Dr. H. Mohler (Switzerland)respectively. Zetabind filters were from BioRad (USA), nitrocellulose filters (pore size 0.1 - 0.45 um) were from Schleicher and Schuell (West Germany) .

Autofluor was from National Diagnostics (UK). All other chemicals were of analytical grade from various commercial sources. The PLRP-S reverse phase HPLC column, 8 um particle size, 300 Angstrom particle size was manufactured by Polymer Laboratories UK.

Phosphate Buffer Saline (PBS) 8.0 g NaCl, 0.2 g KC1, 1.15 g Na2HP04, 0.2 g KH2P04 in 1 1 H20. (pH 7.5)

Soluene/Toluene scintillant: 100 ml Soluene (Koch Light), 7 g 2,5—diphenyloxole (PPO), 0.6 g 1 1,4,-di-[2-(5- phenyloxazolyl)]-benzene (POPOP) in 1 1 toluene.

42 Alternatively Optiphase ”Hi Safe" from National Diagnostics (U.K) was used.

Molecular weight markers were purchased from Pharmacia

(Sweden) and were: Phosphorylase B (93 kDa), Bovine Serum Albumin (66 kDa), Ovalbumin (45 kDa), Carbonic Anhydrase (31 kDa) and Soybean Trypsin Inhibitor (21 kDa).

2.2.2 The preparation of membranes from bovine cerebral

cortex

Bovine brains were collected from the slaughterhouse and kept on ice during transportation (<60 min). Cortices were dissected out, frozen immediately on solid C02 and kept at -80°C until use. Membranes were prepared by the method of Sigel et al., (1983). The cortex was thawed, chopped and homogenized in a Waring blender at 4°C in 10 mM 4-(2-Hydroxyethyl)-1-piperaz ine-ethane-sulphonic acid (HEPES) , pH 7.4, 320 mM sucrose, 1 mM ethylene diamino tetraacetic acid (EDTA), 1 mM benzamidine, 0.02% (w/v) sodium azide, soybean trypsin inhibitor (10 mg/1), ovomucoid trypsin inhibitor (10 mg/1), 0.3 mM phenylmethylsulfonyl fluoride (PMSF), (Buffer A) . The homogenate was centrifuged at 1,000 g for 12 min at 4°. The supernatant was collected and centrifuged at 27,000 g for 45 min at 4°C. The pellet (P^) was resuspended, using a glass Teflon homogenizer, in the same buffer as above except that sucrose and PMSF were omitted (Buffer

43 B) . The homogenate was centrifuged at 27,000 g for 45 min at 4°C and the pellet (P2) resuspended in buffer B. This membrane suspension was either used directly or for membrane protein solubilization. In some experiments,in order to carry out ligand binding assays, it was essential to remove endogenous GABA from the membrane preparations. This was done by the following freeze-thaw cycle (Olsen et al., 1981). The membranes were frozen for 12 h at -20°C, thawed, centrifuged at 27,000 g for 30 min and resuspended in buffer B. The freeze-thaw cycle was repeated twice. This preparation was diluted to the appropriate protein concentration and was used for ligand binding studies and also photoaffinity labelling studies.

2.2.3 The synthesis of the Ro 7-1986/1 agarose affinity column

The synthesis of the affinity column was performed according to the method of Sigel et al. (1983). Adipic dihydrazide agarose (30 ml) was washed on a sintered glass funnel with ice cold H20 (10 vol). Sodium iodoacetate (4.2 g in H20) was added, and the pH adjusted to pH 5.0 with

HC1 (final volume 40 ml). To catalyze the reaction, 1- ethyl-3-(3-diaminopropyl)carbodiimide (1 g) was added. The pH was readjusted to pH 5.0, and the reaction allowed to continue with gentle shaking at room temperature for 3 h. The gel was then washed with ice cold H20 (3 vol), and 100

44 mM Na-carbonate (3 vol) , pH 9.0. A control gel sample to which no ligand was added was taken at this stage. Ro 7- 1986/1 (216 mg) was suspended in ethanol (3 ml) and completely dissolved by dilution to a final volume of 30 ml with Na-carbonate, pH 9.0. The ligand solution was added to the above prepared gel and shaken gently for 19 h at room temperature. The pH was maintained at pH 9.0. The gel was allowed to settle and the unreacted ligand was aspirated. The gel was resuspended in Na-carbonate, pH 9.0 (30 ml) containing 0.3% (v/v) 0 mercaptoethanol, for 2 h at room temperature to reduce any unreacted groups on the gel. After synthesis, the gel was washed at 100 ml/h with (i) 5 mM HEPES , pH 7.4, 500 mM NaCl, 0.5 mM EDTA, 2% (w/v) Triton X-100, 0.02% (w/v) NaN3, (250 ml)? (ii) 50 mM

Na-acetate, pH 5.5, 6 M urea, 1% (w/v) Triton X-100, (250 ml); (iii) 5 mM HEPES, pH 7.4, 150 mM KCl, 0.1 mM EDTA, 0.1% (w/v) Triton X-100, 0.02% NaN3, pH 7.4, (100 ml).

This washing procedure was also carried out after each use of the affinity column.

2.2.4 The purification of the GABA-R

The method used was that of Sigel et al., (1983).

The P2 membrane pellet (2.2.2) was resuspended in Buffer B

(2 .2 .2) at 12-16 mg protein/ml (180 ml), to which the following were added in this order: 3.5 M KCl (8.1 ml),

45 bacitracin (18 mg), 20% (w/v) Na-deoxycholate (DOC) (4.8 ml) to final concentrations respectively of 150 mM, 1 mg/ml and 0.5% (w/v). Solubilization was continued for 10 min at 4°C and the extract was centrifuged for 1 h at

100,000 g. The supernatant was degassed and applied to the

Ro 7-1986/1 agarose affinity column at 60 ml/h. The column was washed overnight with 10 mM K-phosphate, pH 7.4, 50 mM KC1, 10% (w/v) sucrose, 0.02% (w/v) Na azide, 0.1 mM ethylene glycol bis(^-aminoethyl ether) N,N'-tetraacetic acid (EGTA), 2 mM Mg-acetate, 0.2% (w/v) Triton X-100 at

40 ml/h. The column was further washed with 20 mM K- phosphate, pH 7.4, 10% (w/v) sucrose, 2 mM Mg-acetate, 0.1 mM EGTA, 0.2% (w/v) Triton X-100 (20 ml). The receptor was eluted at 20 ml/h with 20 ml of the above buffer containing 10 mM chlorazepate. The pooled fractions (fractions 5-12, 5 ml each) from the affinity chromatogra phy step were adjusted to pH 6.5 with 100 mM H3P04 and applied at 40 ml/h to a DEAE-Sephacel column pre equilibrated with 20 mM K-phosphate, pH 7.4, 2 mM Mg acetate, 10% (w/v) sucrose,, 0.2% (w/v) Triton X-100. The column was washed with 100 ml of the above equilibration buffer at 40 ml/h. The receptor was eluted at 10 ml/h with the same buffer but also containing 0.8 M KC1 and fractions 3-6 (1 ml each) were stored at -20°C until use. In some cases, the receptor was purified by the method of Stauber et al. (1987). Membranes were prepared as above (2 .2 .2) and the resuspended membrane pellet P2

46 (2.2.2) in Buffer B (180 ml) was solubilized by addition of an equal volume of 2 M KC1, 5% (w/v) Triton X-100 and

0.2 mg/ml bacitracin (180 ml) to final concentrations respectively of 1 M, 2.5% (w/v), 0.1 mg/ml. The suspension was stirred gently for 30 min at 4°C and centrifuged at

100,000 g for 1 h. The supernatant was applied to the Ro

7-1986/1 agarose affinity column at 50 ml/h. The column was washed overnight at 40 ml/h with 10 mM K-phosphate, pH 7.4, 2 mM Mg-acetate, 200 mM KC1, 0.1 mM EGTA, 10% (w/v) sucrose, 0.2% (w/v) Triton X-100. The column was further washed with 20 mM K-phosphate, pH 7.4, 2 mM Mg- acetate, 10% (w/v) sucrose, 1 M urea, 0.2% (w/v) Triton X-

100 (30 ml). The receptor was eluted with the same buffer also containing 10 mM chlorazepate. The pH of the pooled receptor-containing fractions was adjusted to pH 6.5 with

100 mM H3PO4, and applied to a DEAE Sephacel column that had been pre-equilibrated with 20 mM K-phosphate, pH 6.5,

2mM Mg acetate, 10% (w/v) sucrose, 1 M urea, 0.2% (w/v) Triton. The column was washed and eluted as described above except that 1 M urea was present throughout. The eluted material (fractions 3-6, 1ml each) was dialysed against 20 mM K-phosphate, pH 7.4, 10% (w/v) sucrose 0.02%

(w/v) Triton X-100 (2x 11). All operations were carried out at 4°C and the dialysed material was stored at -20°C until use.

47 2.2.5 The assay of licrand binding activity.

2.2.5.A The measurement of membrane-bound receptor activity.

Brain membranes (2.2.2) were resuspended to 0.6 mg protein/ml in 20 mM K-phosphatef pH 7.4, 0.1 mM EDTA.

Membranes (160 ul) were incubated in triplicate with the radioactive ligand (20 ul), whose final concentrations, unless stated otherwise, were 10 nM, 10 nM and 50 nM for

[3H]flunitrazepam, [3H]muscimol and [35S]TBPS respectively. To parallel sets of samples, either buffer

(20 ul) or the same ligand but unlabelled (1000 x the concentration of radioactive ligand, 20 ul) was added. Incubations were carried out for 30 min at 4°C for

[3H]muscimol, 45 min at 4°C for [3H]flunitrazepam, and 90 min at 37°C for [35S]TBPS. Bound and free ligands were separated by either centrifugation or filtration. In the centrifugation assay, samples were centrifuged in an Eppendorf centrifuge at 10,000 g for 10 min and superficially washed with 20 mM K-phosphate pH 7.4.

Soluene (20 ul) was added and incubated at room temperature for at least 3 h. The tubes were cut and placed into scintillation vials into which 4 ml of toluene/Soluene scintillant were added. Samples were counted in a scintillation counter. For the filtration assay the samples were filtered under vacuum onto Whatman GF/C filters? the filters were

48 washed with 20 mM K-phosphate, pH 7.4 (3 x.,4 ml), dried under an infrared lamp, and counted as above.

2.2.5.B The measurement of soluble receptor activity

Soluble or purified receptor preparations were appropriately diluted in triplicate in 20 mM K-phosphate, pH 7.4, 0.1 mM EDTA, 0.1% (v/v) Triton X-100, (160 ul) .

The radioactive ligands, either [3H]flunitrazepam or

[3H]muscimol, both 30 nM final concentration, (20 ul) were added. To parallel sets of samples either buffer (20 ul) or the same ligand, but unlabelled, (1000 x the concentration of radioactive ligand, 20 ul) were added. Incubations were carried out for 1 h at 4°C. Bovine gamma globulin (15 ul, 33% (w/v)) and 36% (w/v) polyethylene glycol (PEG) (85 ul), respectively 1.65 mg/ml and 10% (w/v) final concentration, were added. The mixture was vortexed thoroughly, incubated for 15 min on ice, and filtered on Whatman GF/C filters. The filters were washed with 20 mM K-phosphate, pH 7.4, 10% (w/v) (PEG), 0.1 mM

EDTA (3x 4ml), dried and counted as in 2.2.5.A.

2.2.6 The preparation of SDS-Polvacrvlamide gels

Polyacrylamide gels were prepared according to the method of Douglas and Butow (1976).

49 2.2.6.A 10% SDS-Polvacrvlamide gels

The following stock solutions were prepared: (i) acrylamide solution: 30% (w/v) acrylamide, 0.8% (w/v)

N-N'-methylene-bis-acrylamide.

(ii) running gel buffer: 1.5 M Tris/HCl, pH 8.8, 8 mM

EDTA, 0.4% ( W / V ) SDS.

(iii) stacking gel buffer: 0.5 M Tris/HCl, pH 6.8, 8 mM

EDTA, 0.4% (w/v) SDS.

(iv) electrode buffer: 50 mM Tris, pH 8.8, 384 mM glycine,

2 mM EDTA, 1% (w/v) SDS. To make 10% polyacrylamide slab gels (30 ml) or rod gels, the following were mixed: acrylamide solution (10 ml), H20 (12.4 ml), running gel buffer (7.5 ml),

N , N , N ' ,N '-tetramethylene diamine (TEMED) (15 ul) , 10%

(w/v) ammonium persulphate (150 ul). The solution was mixed and immediately poured into glass plates assembled with spacers which were either 0.75 mm or 1.5 mm thick, or into hollow rods. The gel was allowed to polymerize for at least 30 min at room temperature. The stacking gel was prepared by mixing: acrylamide solution (1.3 ml), H20 (4.6 ml) , stacking gel buffer (2 ml) , TEMED (4ul), 10% (w/v) ammonium persulphate (160 ul). The solution was mixed and poured on top of the polymerized running gel and a well

forming comb was inserted. The gel was allowed to polymerize for 15 min at room temperature.

50 2.2.6.B SDS-polvacrvlamide gradient gel

The following stock solutions were prepared:

(i) acrylamide solution: as in 2 .2.6.A.

(ii) running gel buffer : 3 M Tris/HCl pH 8.8.

To make a 12.5%-20% gradient gel the two following solutions were prepared:

A) 12.5% solution (20 ml): acrylamide solution (8 ml), running gel buffer (2.5 ml), 10% (w/v) SDS (0.2 ml), 1.5% (w/v) ammonium persulphate 0.47 ml, H20 (8.83 ml), TEMED

(7 ul). B) 20% solution (20 ml): acrylamide solution (13.34 ml), running gel buffer (2.5 ml), 10% (w/v) SDS (0.2 ml), 1.5% (w/v) ammonium persulphate (0.47 ml), H20 (1.83 ml), TEMED

(7 ul), sucrose (3 g).

The two solutions were poured in two separate chambers of a gradient maker with the outlet leading to the top of a set of assembled glass plates. The gradient gel was completely poured within 5 min of adding TEMED and .ammonium persulphate. The gel was allowed to polymerize for at least 2 h and then was overlayered with stacking gel as described in 2 .2.6.A.

2.2.7 Sample preparation for SDS-PAGE

Samples analysed on a polyacrylamide slab gel were prepared by one of the two following methods:

51 2.2.7.A Tricarboxylic acid (TCA) precipitation

The samples were incubated with 24% (w/v) TCA (1 vol) on ice for 15 min and centrifuged at 10,000 g for 15 min. The supernatant was discarded and the pellet washed with 12% (w/v) TCA (500 ul) and centrifuged at 10,000 g for 15 min. The pellet was washed with acetone (2 x 500 ul) and dried under vacuum

2.2.7.B Methanol/Chloroform precipitation

Samples were precipitated also by the

methanol/chloroform method (Wessel and Flugge, 1984). Methanol (4 vol) was added to the sample which was vortexed and centrifuged for 10 sec at 10,000g. Chloroform

(1 vol) was added and centrifuged as above, followed by

H20 (3 vol). The mixture was vortexed vigorously and centrifuged at 10,000 g for 2 min. The upper phase was removed, and methand^(3 vol) was added. The samples were

centrifuged for 4 min a v 10,000 g, the supernatant was re moved and the pellets were dried under vacuum. All volumes

are with respect to the original sample volume.

The pellets obtained from either precipitation method

were resuspended in 0.25 M Tris/HCl pH 6.5, 4 mM EDTA, 7% (w/v) SDS, 10% (v/v) glycerol, 0.25% (w/v) bromophenol

blue, 1% (w/v) dithiothreitol (DTT). The samples were

boiled for 5 min, and loaded into the wells of the

52 stacking gel described in 2.2.6.

2.2.8 The running conditions for SDS-PAGE

Electrophoresis was carried out at constant voltage (200 V) for 4 h or at constant voltage (50 V) overnight, until the dye front reached the bottom of the gel.

2.2.9 SDS-PAGE detections methods

2.2.9. A Coomassie Blue Staining

After electrophoresis the gel was incubated in 50%

(v/v) methanol, 10% (v/v) acetic acid, 0.2% (w/v)

Coomassie blue R in H20, heated in a microwave oven for 2 min, and incubated on a shaker at room temperature for at least 1 h. The gel was destained with several changes of

20% (v/v) methanol, 5% (v/v) acetic acid in H20.

2.2.9. B Silver staining

After electrophoresis the gel was fixed with two changes (15 min each) of 50% (v/v) methanol, 10% (v/v) acetic acid in H20. The gel was washed with three changes of distilled water each time by heating it in the microwave oven for 2 min. The gel was incubated in 0.05%

(w/v) DTT for 2 min in the microwave oven. The gel was incubated for 30 min at room temperature with 0.1% (w/v)

AgN03. The stain was developed with 3% (w/v) Na2C03 ,

0.02% (v/v) formaldehyde. The reaction was stopped by the

53 addition of 10% (w/v) citric acid.

2.2.9.C Detection of radioactivity

To detect radioactive samples, gel lanes were cut into 1 mm slices and placed into scintillation vials.

Soluene (100 ul) was added and the vials were capped and incubated overnight. Toluene/Soluene scintillant (4ml) was added and the radioactivity counted. Alternatively, after electrophoresis the gel was fixed in 50% (v/v) methanol, 10% (v/v) acetic acid in H20 for 1 h. The gel was washed with H20 (3x 200 ml), and incubated for 30 min in Autofluor. The gel was dried in a gel drier, placed in a light-tight cassette and exposed to

X-ray film at -70°C. The film was developed in an X-O-Mat machine.

2.2.10 Western Blotting

Western blotting was carried out according to the procedure of Burnette (1981). Nitrocellulose filters (pore size 0.1 um) or nylon membrane filters (Zetabind) were pre-soaked in transfer buffer which was 25 mM Tris, pH

8.8, 192 mM glycine, 0.1% (w/v) SDS, 20% (v/v) methanol.

Methanol was omitted for transfer to Zetabind filters. The filter was placed on top of the electrophoresed gel. Contact between the gel and the filter was maintained by placing them in a sandwich made of 4 sheets of blotting paper and a sponge on either side. The sandwich was

54 clamped and placed in a blotting tank containing the above transfer buffer. Transfer was performed for either 4 h at constant voltage, (60 V), or overnight at constant voltage (30 V), and 1 h at 60 V, in all cases at 4°C.

2.2.11 Liaand binding to GABA-R immobilized on Zetabind filters.

The Zetabind filters were quenched in phosphate buffered saline (PBS , see Materials 2.2.1) containing 1% (w/v) bovine serum albumin (BSA) for 1 h at room temperature. The filters were incubated with either [3H] flunitrazepam (10 nM) or [3H] muscimol (10 nM) for 2 h at room temperature. The filters were washed rapidly (3 sec) 1-3 times with PBS by immersion, incubated in Autofluor for 15 min, dried and exposed to X-ray film at -70°C

2.2.12 Photoaffinitv labelling of membrane-bound GABA-R.

Membranes, prepared as in 2.2.2, were resuspended at

0.6 mg protein/ml in 50 mM Tris/citrate, pH 7.1. When the ligand [3H]muscimol was used, the buffer also contained

1.25 mM DTT. To triplicate membrane samples (400 ul) either [3H]flunitrazepam (10-100 nM, 50 ul) , [3H]muscimol

(10-100 nM, 50 ul) or [35S]TBPS (50 nM, 50 ul) were added. Flunitrazepam (50 ul) , GABA (50 ul) or TBPS (50 ul), all at 1000 x the concentration of the radioactive ligand were added to a set of tubes containing

55 [3H]flunitrazepam, [3H]muscimol, [35S]TBPS respectively to measure non-specific binding. All samples were irradiated with UV light at X = 254 nm for 0-90 min, from a distance of 6 cm at 4°C. During irradiation, the incubation mixture was continuously shaken and kept on ice. Irradiation was paused for 2 min every 10 min to avoid overheating of the incubation mixture. The irradiated samples were incubated with the respective unlabelled ligands for 90 min at 4°C, centrifuged at 10,000 g for 10 min and washed in the above assay buffer (4x 500 ul) . The pellet was counted as described in 2.2.5.A, or analysed by SDS-PAGE (2.2.6).

2.2.13 Photoaffinitv labelling of the purified GABA-R

Purified receptor was prepared as described in 2.2.4 except that in some cases a reduced form of Triton X-100

(Aldrich) was used at the ion-exchange chromatography stage. The receptor was diluted 1:10 in the assay buffer, as described (2.2.5.B) but containing the reduced form of Triton X-100. To triplicate receptor samples (250 ul each), either [3H]flunitrazepam (10-100 nM, 25 ul) or

[3H]muscimol (10-100 nM, 25 ul) were added. To parallel sets of tubes either assay buffer (25 ul) or the unlabelled flunitrazepam or GABA, at 1000 x the

[ 3H]flunitrazepam and [3H]muscimol concentration respectively, were added. The samples were incubated as described in 2.2.5.B and irradiated as described in

56 2.2.12. The samples were Incubated with the respective unlabelled ligands for 1 h at 4°C, and TCA precipitated (2.2.7. A). The tips of the tubes were cut and counted in scintillant (2.2.5) or the samples were analysed by SDS- PAGE (2.2.6).

2.2.14 Trvotic digestion of photoaffinitv labelled brain

m e m b r a n e s

Membranes photolabelled with [3H]flunitrazepam or

[3H]muscimol (2.2.12) were incubated at 37°C with trypsin (0.1 - 5.0 mg/ml) . The reaction was stopped by cooling to 4°C, and by the addition of soybean trypsin inhibitor and ovomucoid trypsin inhibitor (both 5 mg/ml final concentration). Samples were centrifuged in an Eppendorf centrifuge at 10,000 g for 15 min. The supernatants were collected and counted; the pellets were washed with the same assay buffer, centrifuged as above and counted after solubilization with Soluene (as described 2.2.5.A).

2.2.15 Cvanoaen bromide cleavage of the photoaffinitv

labelled GABA-R

Purified receptor was photolabelled and TCA precipitated as described in 2.2.7.A. The pellet was resuspended in 70% (v/v) formic acid (500 ul) . Cyanogen bromide (250 ug/ml in 70% formic acid, 25 ul) was added followed by 70% formic acid (500 ul). The reaction mixture was incubated for 24 h at room temperature in the dark

57 then lyophilized.

2.2.16 High pressure liquid chromatography (HPLC) of the cvanoaen bromide cleavage products.

The cyanogen bromide cleaved photolabelled receptor

(2.2.15) was resuspended in 6 M guanidine thiocyanate and injected on a PLRP-S reverse phase HPLC column. The column was washed for 10 min with 5% (v/v with water) acetonitrile 0.1% (v/v) trifluoroacetic acid (TFA). Peptides were eluted for 40 min with a 5-80% (v/v with water) 0.1% (v/v) acetonitrile gradient. A dual wave length detector (A = 214 nm and A = 280 nm) was used. Fractions (1 ml) were collected manually or whenever a peak was registered by the detector. The relative time of the run was recorded for each collection tube. A sample from each tube (200 ul) was counted for radioactivity. The sample containing the radioactivity peak was analysed by gradient SDS-PAGE (2.2.6.B).

2.2.18 The determination of protein concentration

2.2.18.A The method of Lowrv et al. (1951)

The assay reagent was prepared by mixing 1% (w/v)

CuS04 (1 ml) , with 2% of (w/v) Na-tartrate (1 ml) and 2%

(w/v) Na2C03 in 0.1 M NaOH, 0.5% (w/v) SDS (98 ml).

Samples were TCA precipitated (2.2.7.A), and resuspended in 200 ul H20. The assay reagent (1 ml) was added and

58 incubated for 10 min. Folin Ciocalteu reagent (100 ul diluted 1 :1 with water) was added and each tube vortexed immediately. The reaction was allowed to continue for 30 min and the absorbance was read at A - 750 nm. The protein content of each sample was calculated with reference to a standard curve determined with bovine serum albumin

(BSA).

2.2.18. B The Biuret , method.

Samples were TCA precipitated (2.2.7.A) and resuspended in H20 (200 ul), to which 9% (w/v) NaOH ,0.4% (w/v) cholic acid (700 ul) were added, followed by 1% (w/v) CuS04 (100 ul) . The samples were mixed immediately and the reaction left to develop for 15 min and the absorbance was read at

A = 540 nm. The protein content was estimated as in

2.2.18. A against a BSA standard curve.

59 2.3 Results

2.3.1 Purification of the GABA-R

The preparation of purified GABA-R was carried

out according to the method of Sigel et al. (1983) or by a modification of that of Stauber et al. (1987) (2.2.4).

Although a considerable amount of time was invested in this exercise, the ligand affinity purification of the receptor performed for the accomplishment of this project should be considered routine work. Table 2.3.1 shows the results of one typical preparation according to the method of Sigel et al. (1983) . Using the detergent Na-deoxycholate, 43% of the

[3H]flunitrazepam binding sites present in the membranes were recovered in the soluble fraction. When the soluble extract was applied to the affinity column, 90% of the

[3H]flunitrazepam binding sites which had been solubilized, were bound to the affinity column. The overnight wash of the column was found to contain 12% of the [3H]flunitrazepam binding sites which had been applied to the affinity column. Following the elution from the ion 's exchange column, 10.6% [JH]flunitrazepam binding sites of the initial soluble extract applied to the affinity column were recovered. It was found that 21.7% of the receptor

60 binding activity was lost during the ion-exchange step, as calculated from the recovery of [3H]muscimol binding sites from the DEAE-Sephacel column. The ratio of the Bmax of

[3H]muscimol to [3H]flunitrazepam binding sites, calculated from the reported KD values (Sigel et al., 1983), was 2.7 in this preparation and varied between 2 and 4 in other preparations. The protein yield of this preparation was 107 ug. SDS-PAGE analysis (Figure 2.3.1) showed two bands of apparent molecular weight 53 kDa and

57 kDa which have been named respectively the a subunit and the 0 subunit (Sigel et al., 1983). In general, the a subunit was more strongly stained and sharper than the

0 subunit. The latter however was more susceptible to degradation, indicated by the fact that the same preparation analysed in subsequent SDS-PAGE experiments showed no variation in the at subunit, but showed a decrease in the staining of the 0 subunit (results not shown). The above-described preparation was representative of the initial purification work but, although the purification procedure was very reproducible in terms of the purity of the GABA-R obtained, a decrease in the yield of active receptor was observed for subsequent isolations.

Throughout this project 159 purification procedures were carried out and while the average yield of the initial purifications was 365 + 62 pmol [3H]muscimol binding sites

(n=20), these decreased to 203 + 36 (n=25) in the la t er

61 preparations. The protein yield of the later preparations was calculated to be approximately 80 ug. This loss of yield was first believed to be due to the ageing of the affinity column, but when two new affinity columns were synthesized, the original yields were not restored. A detailed analysis of the purification procedure revealed that no decrease of efficiency of binding to the newly- synthesized columns occurred, while there was a decrease in the apparent efficiency of elution. Furthermore, a decrease of recovery (from 80% to 60%) from the ion- exchange column was also observed. In order to try to increase the yield of the purification procedure the following modifications were tried: a) larger volumes of soluble extract were loaded onto the column. b) the chlorazepate concentration in the elution buffer was increased to 20 mM. c) the chlorazepate elution rate was decreased to 5 ml/h.

None of the above modifications of the original procedure resulted in an increase in the yield of purified GABA-R.

Table 2.3.2 shows the yield of a representative GABA- R purification experiment according to the method of

Stauber et a_l. (1987) . The major differences in this method are that the membranes are solubilized with high salt and Triton X-100 concentration, and that 1 M urea was included in the chlorazepate elution buffer (2.2.4). This

62 preparation is referred to as the Triton/urea preparation. The solubilization efficiency of the GABA-R with Triton X-

100 in the presence of high salts was higher than that found with Na-deoxycholate solubilization, and was 89.5 +

8.2% (n=5) of the [3H]flunitrazepam sites present in the membranes. When this soluble extract was applied to the

affinity column, 78.3 + 6.1% (n=5) of the

[3H]flunitrazepam sites present in the soluble extract bound to the column. The overnight wash and the urea wash both contributed to a loss of 12% and 0.2% respectively of the initial [3H]flunitrazepam binding sites applied. The overall yield of the Triton/urea preparation was within the same range of the original yields obtained from the Na-deoxycholate preparation. In order to try to increase this yield, the chlorazepate concentration was again increased to 20 mM and the urea to 2 H, but no increase in the yield of purified GABA-R was obtained.

SDS-PAGE analysis of the purified product (Figure

2.3.2) showed the 2 bands a (53 kDa) and 0 (57 kDa) .

Another band of apparent molecular weight 65 kDa was also visible, but this band was present in the urea-wash eluate and is probably a non-receptor polypeptide. The subunit stoichiometry, as determined from the gel staining was not significantly different from that observed with the Na- deoxycholate preparation. The ratio of the [3H]muscimol to

[3H]flunitrazepam binding sites was = 2.6 + 0.2, (n=5), as

63 calculated from a single point determination adjusted to Bmax as described above. The protein yield was about 100 ug per preparation.

2.3.2 Liaand binding to GABA-R after Western blotting

Nitrocellulose sheets onto which purified GABA-R had been blotted were incubated with radioactive ligands as described in 2.2.11., No signal was obtained frgm the fluorography of the filters incubated with either

[3H]flunitrazepam or [3H]muscimol .

2.3.3 The UV absorption spectra of GABA-R ligands

In order to establish the photochemical properties of the ligands that were used for photoaffinity labelling studies, the excitation and emission spectra of flunitrazepam, muscimol and TBPS were recorded in a spectrofluorimeter (Figure 2.3.3). The emission spectra were first detected then the excitation spectra were recorded at the wavelength that gave maximum emission, i.e. A = 370 nm, 300 nm and 290 nm for flunitrazepam, muscimol and TBPS respectively. All three excitation spectra showed a peak at A = 260 nm.

2.3.4 Photoaffinitv labelling of bovine brain membranes with T—HImuscimol

Photoaffinity labelling of rat brain membranes had been previously reported by Asano et al. (1983) and

64 Cavalla and Neff (1985a). The following experiments were done in order to establish the experimental conditions for the photolabelling with [3H]muscimol of bovine membranes and to further investigate the nature of the irreversible reaction. Membranes were used at 0.6 mg protein/ml which was the concentration that gave the best signal to noise ratio for UV photolabelling.

2.3.4. A Time course of the membrane photolabelling reaction

A time-dependent irreversible incorporation of [JHJmuscimol into bovine cortex membranes was found. Figure 2.3.4.A shows however that the photolabelling reaction seems to be composed of 2 mechanisms. There is a fast photolabelling rate which occurs during the first 30 min of irradiation and a second, slower rate that does not plateau. In Figure 2.3.4.B is shown the effect of irradiation on the ligand binding properties of the membrane preparation. Membranes were irradiated under the same conditions described above but in the absence of

[3H]muscimol. Membrane aliquots were then incubated with

[3H]muscimol (20 nM) and assayed for reversible ligand binding by the centrifugation method (2.2.5.A). Figure

2.3.4. B shows that following an initial apparent increase in binding sites, after 30 min there is a decrease in the number of [3H]muscimol binding sites. After 90 min, a 90%

+ 7% (n = 3) loss of the reversible [3H]muscimol binding

65 sites was observed. The initial increase of the number of binding sites was observed in all the inactivation time courses performed. It will be shown later (2.3.6.A and

Figure 2.3.7.C) that [3H]muscimol was affected by the UV irradiation and it lost75% + 5% (n = 2) of its capacity of binding to purified GABA-R after 30 min irradiation.

2.3.4.B Determination of the efficiency of the r—HImuscimol photoaffinitv labelling reaction

The maximum irreversible [3H]muscimol binding obtained represented 10.5 + 1.2% (n=7) of the initial reversible binding sites. In order to understand the low efficiency of the photolabelling, a series of experiments were performed (see Table 2.3.3): a) membranes were first photolabelled with [3H] or unlabelled muscimol, washed with assay buffer (4x 600 ul), and further photolabelled with [3H]muscimol. b) membranes were first photolabelled with [3H] or unlabelled muscimol, washed as above, and assayed for

[3H]muscimol reversible binding activity. c) membranes were UV irradiated, then either photolabelled with [3H]muscimol or assayed for

[3H]muscimol reversible binding activity.

The results in Table 2.2.3 show that UV treatment decreased reversible and irreversible [3H]muscimol binding by 6% + 0.4% and 20% + 1.7% (n = 2) with respect to non-

66 treated membranes. Photolabelling with unlabelled muscimol reduced by 80% + 7.5% (n = 2) the number of sites available for reversible binding, while only 8.9% + 0.9%

(n = 2) of the reversible sites were photolabelled with

[3H]muscimol after 20 min UV irradiation. The difference between the reversible binding to membranes previously photolabelled with [3H]muscimol compared to membranes previously photolabelled with unlabelled muscimol, is equal to the number of sites that were irreversibly photolabelled with [3H]muscimol. Initial photolabelling with unlabelled muscimol followed by photolabelling with

[3H]muscimol suggested that there are still sites available for photolabelling. However, two successive photolabelling treatments both with [3H]muscimol yielded a number of photolabelled sites equivalent to those obtained from the photolabelling with [3H]muscimol of previously irradiated membranes.

2.3.4.C The effect of DTT on the r—HImuscimol

photoaffinitv labelling reaction

All [3H]muscimol membrane photolabelling experiments were carried out in the presence of DTT (1.25 mM) which acts as a scavenger (Cavalla and Neff, 1985a). To establish the effect of DTT on reversible and irreversible

[3H]muscimol binding, photolabelling and reversible binding assays were carried out in the presence and absence of DTT. Figure 2.3.5 shows that DTT had no effect

67 on reversible binding, but it decreased both the total and the non-specific irreversible binding. The fact that the actual specific irreversible binding was the same in the presence and in the absence of DTT indicated that the scavenger did not interfere with any [3H]muscimol binding sites but did reduce the non-specific irreversible incorporation of radioactivity.

2.3.4.D The effect of barbiturates on the r—HI muscimol photoaffinitv labelling reaction

The effect of the barbiturate pentobarbital on reversible (Olsen et al., 1981) and irreversible binding was tested, by incubating bovine cortex membranes with

[3H]muscimol (4nM) in the presence of increasing concentrations of pentobarbital (0-1 mM, final concentration). Figure 2.3.6.A shows that pentobarbital (1 mM) enhanced by 50% + 8 (n = 3) the reversible

[3H]muscimol binding to bovine brain cortex membranes while no enhancement of irreversible [3H]muscimol binding was found. A similar experiment was performed to test the effect of pentobarbital and GABA on reversible and irreversible [3H]flunitrazepam binding. Figure 2.3.6.A shows that reversible binding was increased 21.5% + 1 .2%

(n = 3) and 18.9% + 6 (n = 3) in the presence of GABA and pentobarbital respectively, while again no increase in irreversible binding was observed. One explanation for

68 these results is offered by the following experiment. Membranes were first irradiated for 15 min under the standard conditions, and subsequently the capacity of pentobafbital and/or GABA to enhance the reversible

[3H]muscimol and [3H]flunitrazepam binding was tested.

Figure 2.3.6.B shows that the enhancement by either GABA or pentobarbital of both [3H]muscimol and

[3H]flunitrazepam reversible binding was decreased by the UV treatment.

2.3.5 Photolabellino of membrane-bound GABA-R with T— SI TBPS

Attempts were made to use the ligand [35S]TBPS as a photoaffinity label. Membranes were resuspended in the described buffer (2.2.12) which additionally contained 250 mM KC1. They were incubated with [35S]TBPS (50 nM final concentration) for 90 min at 37°C. Initial results indicated that apparent irreversible binding was obtained. However when the post-irradiation washes of the membrane samples were further increased (up to 7 washes with 5 h incubations at room temperature) it was shown that all the apparent specific irreversibly bound [35S]TBPS could be removed and that there was no difference between the irradiated and non-irradiated membranes. This slow rate of dissociation of [35S]TBPS allowed the following 2 experiments to be attempted.

1) The membranes after incubation with [35S]TBPS were

69 centrifuged and resuspended in ligand-free buffer before the irradiation was carried out. This procedure was done to reduce the non-specific irreversible binding but did not result in any specific photolabelling.

(2) Membranes after incubation with [35S]TBPS were washed once as above, UV irradiated and directly solubilized with SDS sample buffer and subjected to 10% polyacrylamide SDS-

PAGE (2.2.6). The gel was sliced and counted. The top 6-8 slices contained radioactivity, but no difference was found between the total and non-specific lane where the excess unlabelled TBPS was added in the original incubation mixture.

2.3.6. Photoaffinitv labelling of the purified GABA-R

with r—HImuscimol

2.3.6. A Time course of the photolabelling reaction

Figure 2.3.7.A shows that maximum labelling was obtained after 30 min UV irradiation. Maximum irreversible binding represented 7.5% + 0.8% (n = 3) of the total initial specific reversible binding sites.

The [3H]muscimol binding sites of the purified receptor preparation were more susceptible to UV irradiation than those of the membrane-bound receptor. It was found that 78% + 7 (n = 3) of the reversible

[3H]muscimol binding sites were lost after 30 min UV

70 irradiation (see Figure 2.3.7.B) The remaining sites could however still be photolabelled in a time-dependent manner similar to those of non-irradiated receptor preparations.

The ligand [3H]muscimol itself is irreversibly inactivated by UV irradiation. Figure 2.3.7.C shows that after 30 min irradiation [3H]muscimol had lost 75% + 5% (n = 2) of its capacity to bind to the GABA-R.

2.3.6. B Pharmacological specificity of the r—H]muscimol photoaffinitv labelling reaction

In order to test the pharmacological specificity of the photoaffinity labelling reaction, purified receptor was incubated with [3H]muscimol (50 nM) as described in 2.2.13, but in the presence of increasing concentrations of GABA or bicuculline methobromide. Irradiation and counting was carried out as described in 2.2.13. The two competitive ligands reduced the irreversible [3H]muscimol binding as depicted by the two sigmoid curves seen in

Figure 2.3.8. The apparent IC50 values were 53 + 4nM (n =

2) and 6 + 0.1 uM (n=2) for GABA and bicuculline methobromide respectively.

2.3.6. C. Ligand concentration dependence of r—HImuscimol

photolabelling

Purified receptor was diluted 1:10 in assay buffer and incubated for 30 min with increasing concentrations of

71 [3H]muscimol. The samples were then irradiated and treated as described above. Figure 2.3.9.A shows that maximum labelling was obtained at about 100 nM [3H]muscimol. The binding data were also analyzed by a Scatchard plot and an apparent Kq of 12+3 nM (n=2) was determined (Figure 2.3.9.B)

2.3.7 SPS-PAGE analysis of the r—HImuscimol photolabelled receptor

2.3.7.A Purified receptor

Purified receptor (800 ul of the DEAE-Sephacel eluate

2.2.4 ) was photolabelled with [3H]muscimol as described in 2.2.13 and applied to a 10% SDS-PAGE (2.2.7. A). Parallel lanes were loaded with purified receptor (200 ul of the DEAE-Sephacel eluate 2.2.4) photolabelled with

[3H]flunitrazepam (20 nM) which was used as an internal marker for the a subunit. The gel was analysed for radioactivity by either gel slicing (2.2.9.C), fluorography (2.2.9.C), or blotted to Zetabind and fluorographed (2.2.10). Figure 2.3.10.A shows a plot of the counts obtained from a gel slicing experiment. The graph shows that the incorporation of the two ligands occurred at two bands with different molecular weights.

Since it has been shown that [3H] flunitrazepam labels preferentially the a subunit (Sigel et al., 1983), and

72 that silver staining of the same receptor preparation showed two bands, the a and p subunits, the [3H]muscimol labelling was assigned to the p subunit. This result was not reproducible mainly because the separation of the two bands was not always clear in the gel slicing. When gel fluorography was carried out, no signal from the

[3H]muscimol photolabelled receptor was obtained even after the film was exposed for 2 months. This duration of exposure time should have been sufficient to visualize the radioactivity that had been loaded onto the gel (6- 8,000 specific cpm) . This problem had in fact been reported earlier by Cavalla and Neff (1985a,b) who suggested that some of the tritium label was lost during the gel fixation procedure. In order to overcome this problem, the gel fixation procedure was avoided by carrying out a Western blot and exposing the Zetabind filter to X-ray film as described in 2.2.10. Figure

2.3.10.B shows that while [3H]flunitrazepam predominantly specifically labels the a subunit, [3H]muscimol predominantly labels the p subunit. However, both ligands also label to a minor extent the p and the a subunit respectively. This result was very reproducible (n=8), where the only variation was the extent of labelling by

[3H]muscimol on the a subunit and of [3H]flunitrazepam on the p subunit. However, in all cases the p subunit was the one predominantly labelled by [3H]muscimol and the

73 a subunit by [3H]flunitrazepam.

2.3.7.B Membrane-bound receptor Bovine brain membranes were photolabelled with

[3H]muscimol and [3H]flunitrazepam and analysed by SDS-

PAGE. It has however proven impossible to obtain a satisfactory result with either gel slicing or gel and

Zetabind fluorography, even after 1 year of exposure to films, from samples photolabelled with [3H]muscimol. A signal was visible after two weeks exposure from samples labelled with [3H]flunitrazepam. The experiments were hampered by the fact that the maximum specific incorporation of [3H]muscimol into the membranes was 2000 cpm/mg protein. It was found that on gels 1.5 mm and 0.75 mm thick, not more then 400 ug or 200 ug of protein respectively could be loaded without compromising the quality of the gel. Western blotting from 1.5 mm gels was also found to be unsatisfactory because it yielded diffuse and distorted bands. Membrane preparations from different bovine brain regions, cerebellum, hippocampus and striatum, were also photolabelled with [3H]muscimol and [3H]flunitrazepam. It was found that the maximum specific incorporation of [ H]muscimol was obtained with cortex membranes (see legend Figure 2.3.11). When analysed by fluorography

(Figure 2.3.11' after transfer to Zetabind filters, no signal was obtained from the samples labelled with

74 [3H]muscimol. However, [3H]flunitrazepam labelled the a subunit of all the the brain membrane regions and the

0 subunit of cortex and hippocampus.

2.3.8 Trypsin digestion of photoaffinitv labelled

membranes

Membranes were photolabelled with [3H]flunitrazepam and [3H]muscimol as described in (2.2.12) and then subjected to trypsin digestion. In order to establish the appropriate conditions, photolabelled membranes were incubated with increasing amounts of trypsin. Figure 2.3.12.A shows that after a 3 h incubation with trypsin at 37°C, the peak of release of radioactivity into the supernatant was obtained for both [3H]muscimol and

[3H]flunitrazepam photolabelled membranes at a trypsin concentration of 0.1 mg/ml. A higher percentage of the incorporated radioactivity was released, after 3 h, from membranes labelled with [3H]muscimol (62% + 7%, n = 2) than with [3H]flunitrazepam (49% + 6%, n = 2). In order to establish if all the incorporated radioactivity could be released, an extended time course of tryptic digestion was performed. Figure 2.3.13.B shows that even after 12 h incubation with trypsin at 1 mg/ml, 82% + 7 and 72% ± 8 (n

= 2) respectively of the [3H]muscimol and

[3H]flunitrazepam irreversibly bound was released from the photolabelled membranes. Figure 2.3.12.B also shows that there is an endogenous "trypsin-like" activity present in

75 the membranes because radioactivity was released from the membranes in the absence of trypsin. In a control experiment, trypsin was added to [3H]flunitrazepam and [3H]muscimol photolabelled membrane samples which already contained trypsin inhibitors at 5 mg/ml concentration. It was found that, while there was no release of radioactivity into the supernatant from membranes labelled with [3H]flunitrazepam under these conditions, 44% of the radioactivity was released after 12 h from membranes photolabelled with [3H]muscimol.

2.3.9 Cvanoaen bromide cleavage of r—HImuscimol photoaffinitv labelled purified GABA-R

Purified receptor was photolabelled with [3H]muscimol and cleaved with cyanogen bromide (2.2.15). Figure 2.3.13. A shows the elution profile at A = 214 nm. Several peaks representing cleaved peptides are seen. All fractions were measured for radioactivity and only one major peak of radioactivity was obtained (Figure

2.3.13. A). This result was obtained from three HPLC runs of two independently prepared and photolabelled GABA-R preparations. The fraction containing maximum radioactivity was loaded onto a gradient SDS polyacrylamide gel (2.2.6.C) which was then silver stained. Figure 2.3.13.B shows that the lane loaded with this HPLC fraction contains one major broad band of

76 approximate molecular weight 12 kDa. The gel was scanned for radioactivity and although the signal was very weak it corresponded to the stained peptide.

77 Figure 2.3.1

Na-deoxycholate purification of the GABA-R.

Silver stained 10% polyacrylamide SDS-PAGE. Lanes A and B are respectively 200 uiHnd 100 u (H f purified GABA-R precipitated with the TCA method. Lanes C and D are respectively 200 ul and 100 ul of purified GABA-R precipitated with the methanol/chloroform method (see text

2.3.1).

78 Figure 2.3.2

ABC

Triton/urea purification of the GABA-R.

Silver stained 10% polyacrylamide SDS-PAGE. Lane A and B: purified GABA-RMfrom two independent Triton/urea preparations. Lane C: urea eluate of the Ro 7-1986 affinity column carried out before the chlorazepate/urea elution.

79 Figure 2.3.3

Excitation spectra of muscimol, flunitrazepam and TBPS: The emission of the excitation spectra were measured at the indicated wavelength: A: Muscimol (Fixed emission 300 nm)

B: Flunitrazepam (Fixed emission 370 nm)

C: TBPS (Fixed emission 290 nm)

80 ie ore f h ^lucml pcfc photoaffinity specific ^Hlmuscimol the of course Time vrgs ftilct sas+SMad ersnaie f 4 of representative and SEMassaystriplicate+ ofaverages xeiet. (Seeexperiments.2.3.4.A).text are Values membranes. cortex cerebral bovine of labelling [3H]muscimol irreversibly bound Figure 2.3.4. A 81 raito ie (min) time Irradiation

Figure 2.3.4.B

c .g '© 52 o >© Q. S o> o E E o 8 E 3 "D E, C X 3 co 2

0 20 40 60 80 100

Irradiation time

(min)

The effect of UV irradiation on bovine cortex membranes.

The membranes were UV irradiated for the indicated length of

time and assayed for specific [3H]muscimol reversible

binding. Values are averages of triplicate assays + SEM and are representative of 3 experiments. (See text 2.3.4.A)

82 Ficrure 2.3.5

3000 r

-oc ^ 3 C 2 £s 2000 o

ci CD“ ■ U)o ^ E t 1 1000 5 ~

total non-specific specific □ - DTT H + DTT

□ -DTT ea + DTT The effect of DTT on [3H]muscimol reversible and irreversible binding.

A: [3H]muscimol reversibly bound to bovine cortex membranes in the presence (shaded bars) or absence (open bars) of DTT. B: [3H]muscimol photoaffinity labelled to bovine cortex membranes in the presence (shaded bars) or absence (open bars) of DTT. Values are averages of triplicate assays + s.d. and representative of 2 experiments. (See text 2.3.4.C). Figure 2.3.6,A

200 r

-ac 5 T 3 Ca _D> ® rgO) 100 c aa> SL

M +P F+G F + P F + G + P

□ reversible 0 irreversible

Pentobarbital (P) and GABA (G) enhancement of reversible (open bars) and irreversible (shaded bars) [3H]muscimol (M) and [3H]flunitrazepam (F) specific binding. Values are averages of triplicates + s.d. and representative of 3 experiments. (See text 2.3.4.D)

84 Figure 2.3.6.B

200 r

150 -o c i n 3 J3o T3 CaJ o> ® O) 100 ISc o® 8.

0 F+G F + P

The effect of UV irradiation on bovine cortex membranes. Pentobarbital (P) and GABA (G) enhancement of reversible

[3H]muscimol (M) and [3H]flunitrazepam (F) specific binding to control membranes (open bars) and UV-irradiated (15 min) membranes (shaded bars). The values are averages of triplicate assays + s.d. and representative of three experiments. See text 2.3.4.D.

85 SM n rpeettv o 3 xeiet. Se text (See experiments. 3 of representative and SEM + iecus f [3H]muscimolcourseTimeof specificlabellingphotoaffinity ofpurifiedGABA-R. are Valuesaverages oftriplicateassays 2.3.6.A)

specifically bound (pmol/ml) 1 2 3 4 5 60 50 40 30 20 10 0 Figure2.3.7.A 86 Irradiation time (min) time Irradiation

Figure 2.3.7.B

Effect of UV irradiation on purified GABA-R.

Purified GABA-R was irradiated for the indicated length of time and was assayed for specific [3H]muscimol reversible binding. Values are averages of triplicates + SEM and are representative of three experiments. (See text 2.3.6.A.)

87 Figure 2.3.7.C

Irradiated [3H]muscimol

Irradiation time of [3H]muscimol

(min)

Effect of UV irradiation on [3H]muscimol. [3H]muscimol was UV irradiated for the indicated length of time and used in a reversible binding assay of purified GABA-R. Values are averages of triplicates + SEM and

representative of two experiments. (See text 2.3.6.A)

88 % irreversible [3H] muscimol photoaffinity labellingreaction.photoaffinity rpiae sas n rpeettv o to experiments. two of representative and assays triplicate h prfe GB- ws nuae wt increasing with incubated was GABA-R purified The hraooia seiiiy f h [3H]muscimol the of specificity Pharmacological n [Hmsio ad V raitd Vle ae vrgs of averages are irradiated. Values UV [3H]muscimol and and ( bicuc;ullinemethobromide □) (♦), GABA ofconcentrations (See2.3.6.B)text ■o .o (A & 3 c >* o 9 7 -5 -7 -9 Fig2.3.8 89 o 0 [drug] log 10

90 Figure 2.3.9.A Figure [3H]muscimol concentration (nM) Irradiation was carried out for 30 min. Values are [3H]muscimol binding. [3H]muscimol averages of triplicate assays + SEM and representative two of representative + and SEM assays triplicate of averages Concentration dependence of specific irreversible experiments. 2.3.6.C). text experiments. (See A|qisj0AejJ! |oiupsniu[He] Bound [3H] muscimol/free Values areaveragesValuesof triplicate assaysand representative purifiedGABA-R. cthr po o [3]ucml reesbe idn to binding [ irreversible of 3H]muscimol plot Scatchard f w eprmns Te paet D a 1 ±n. Se text (See ±5nM. 15 was KD apparent The experiments. two of 2.3.9). 3]ucml on (pmol/ml) bound [3H]muscimol iue 2.3.9.BFigure 91

Figure 2.3.10.A

Photoaffinity labelling of purified GABA-R: gel slicing of

10% polyacrylamide SDS-PAGE. A: purified GABA-R photolabelled with l3H] flunitrazepam

(total cpm loaded = 12,000 cpm). B: purified GABA-R photolabelled with [3H]muscimol (total cpm loaded = 7,500). (See text 2.3.7.A). Figure 2.3.10.B

A B C D

Photoaffinity labelling of purified GABA-R: fluorography of

Western blot of 10% polyacrylamide SDS-PAGE.

Lane A: purified GABA-R photolabelled with [3H]muscimol in the presence of 0.1 mM bicuculline methobromide. Lane B: . . . ^ , purified GABA-R photolabelled with [H]muscimol. Lane C: purified GABA-R photolabelled with [3H]flunitrazepam in the presence of 0.1 mM chlorazepate. Lane D: purified GABA-R photolabelled with [3H]flunitrazepam. (See text 2.3.7.A). Figure 2.3.11

kOa

ABCD E f G H I JK

Photoaffinity labelling of bovine brain membranes: fluorography of Western blot of 10% polyacrylamide SDS-PAGE.

Lane Ligand Brain Irreversible specific region binding (fmol/mg protein)

A [ H]flunitrazepam + cortex — chlorazepate B [3H]flunitrazepam cortex 420 + 21 C [3H]muscimol + cortex GABA D [3H]muscimol cortex 212 + 16 F [3H]muscimol cerebellum 177 ± 12 G [3H]flunitrazepam cerebellum 271 ± 15 H [3H]muscimol hippocampus 135 ± 16 I [3H]flunitrazepam hippocampus 384 ± 21 J [3H]muscimol striatum 115 ± 13 K [3H]flunitrazepam striatum 230 ± 22

Lane E contains molecular weight markers . The film was exposed for 5 months. (See text 2.3.7.B).

94 Figure 2*3.12.A

Log10 trypsin concentration

(ug/ml)

Trypsin concentration dependence of the release of radioactivity from bovine cortex membranes photolabelled with [3H]muscimol (□) and [3H]flunitrazepam (♦). Values are averages of triplicate assays (s.d. < 10%) expressed as percentage of the specific radioactivity irreversibly bound after 30 min irradiation. The results are representative of two experiments (See text 2.3.8).

95 Ficrare 2.3.12.B

■o

_> O A o05 •O 05

"O CD CO O05 2 6 . >

o B ■O 05

Release of radioactivity from membranes photoaffinity labelled with [3H]muscimol (A) and [3H]flunitrazepam (B).

The radioactivity was measured at the indicated times in the supernatants of the samples to which trypsin (1 mg/ml) had been added (0)and in control samples of photoaffinity labelled membranes ( + ) . Values are averages of triplicate assays (s.d. < 10%) and are representitive of two experiments. (See text 2.3.8).

96 O.D.C214 nm) : rfl o te bobne A=24 m o te HPLC the of nm) (A 214= absorbance the of GABA-R CTFr c] profile ecivec! purified A: of separation HPLC htafnt aeld ih [3H]muscimol. with photoaffinitylabelled lae B poie f h rdociiy esrd n the in radioactivity measured the of profile B:eluate. fractionsof (Seethe HPLCeluate. text 2.3.9). Figure2.3.13.A 9 7

Figure 2.3.13.B

A B C

SDS-PAGE of CFFr cjfcvrf purified GABA-R photolabelled with

[ H]muscimol.

Silver stained gradient gel (12.5%-20% polyacrylamide). Lane

A: whole purified GABA-R. Lane B: trypsinized GABA-R. Lane

C: HPLC elution fraction that contained the highest radioactivity (see Figure 2.3.13.A.). The arrow indicates where the peak of radioactivity was detected from the gel scan. (See text 2.3.9).

98 Table 2.3.1

Sample [3H]flunitrazepam [3H]muscimol binding sites binding sites (pmol) (pmol)

Membranes 3080 (a)

Deoxycholate 1323 (a) extract

Wash through 133 (a) column

Overnight wash 158 (a) Chlorazepate (b) 438 elution

Purified receptor 127(c) 348(d)

(a) not detectable because of the presence of endogenous GABA.

(b) not detectable because of the presence of chlorazepate.

(cJ) Specific activity = 3.48 n m o l/m g

The deoxycholate GABA-R purification procedure.

Total amount of [3H]flunitrazepam and [3]muscimol binding activity recovered at various stages of the GABA-R purification performed according to the method of Sigel et al. (1983), starting with 100 g of bovine cerebral cortex. (See text 2.3.1).

99 Table 2.3.2

Sample [3H]flunitrazepam [3H]muscimol binding sites binding sites (pmol) (pmol)

Membranes 3426 (a) Triton extract 3084 (a) Wash through 678 (a) column

Overnight wash 370 NA

Urea wash 6 NA

Chlorazepate wash (b) 548 Purified receptor 163(c) 318(d)

(a) not detectable because of the presence of endogenous GABA.

(b) not detectable because of the presence of chlorazepate. NA = not assayed

(0) Specific activity - ?.(?5 nmo]/no

The Triton/Urea GABA-R purification method. Total amount of [3H]flunitrazepam and [3H]muscimol binding sites recovered at the various stages of the purification procedure according to the method of Stauber et al. (1987) starting with 100 g of bovine cerebral cortex. (See text

2.3.1).

100 Table 2.3.3

Pre-treatment [3H]muscimol Pre-treatment [3H]muscimol of membranes reversible of membranes photoaff assay labelli (cpm) (cpm)

None 6461 None 577

UV 6162 UV 449 irradiation irradiation Photolabelling 1334 Photolabelling 274 with muscimol with muscimol

Photolabelling 1843 Photolabelling 451 with [3H]muscimol with [3H]muscimol

Photoaffinty labelling of bovine cortex membranes with

[3H]muscimol (specific binding).

Membrane samples (500 ul) were treated as described above and assayed by either reversible binding or the photoaffinity labelling reaction. All irradiations were for

20 min. Values are the average of triplicates (SEM < 10%), and representative of two experiments. (See text 2.3.4.B)

101 2.3 Discussion

2.3.1 GABA-R purification

The purification procedure of Sigel et al. (1983) has yielded a highly purified GABA-R preparation, which on

SDS-PAGE analysis showed two subunits of 53 kDa and 57 kDa. The recovery of the [3H]flunitrazepam binding sites eluted from the affinity column was 10% of the sites that were applied and this low yield of the routine purification procedure of the GABA-R has been a limiting step in the progress of this work. The majority of the loss of the receptor during the purification procedure occurred between the application and the elution of the soluble protein from the affinity column. Two major reasons may account for this finding. The receptor could be denatured during the overnight wash or, it may not have eluted from the column when the chlorazepate containing buffer was applied. It is difficult to test for the presence of denatured GABA-R in the overnight wash because the receptor is not active and it is in presence of other contaminating proteins. It was instead evident that not all the receptor was eluted from the affinity column. SDS-PAGE analysis of the urea regeneration wash of the affinity column showed bands that

102 corresponded to the GABA-R a and 0 subunits as well as others, however, this receptor containing sample, was neither pharmacologically active nor pure. The low yield of the purification procedure is therefore partly due to the inability of the free ligand to displace the receptor from the immobilized ligand. No improvement in the yield of purified GABA-R was detected when the elution was carried out more slowly or at higher chlorazepate concentrations. A decrease in yield was experienced in the later purification procedures, but this may be explained by (i) ageing of the original column, (ii) decreased elution from the newly synthesized columns or (iii) a reduction in yield of the ion-exchange column. The original affinity column did show a decrease in its capacity of binding of the soluble receptor. New affinity columns were thus synthesized which bound soluble receptor with the same efficiency as - the original column. These new columns however, yielded reduced quantities of purified GABA-R compared to the original column. This was not due to the fact that a higher concentration of Ro 7- 1986/1 was bound to the new affinity columns, which may require different elution conditions, because an affinity column that had been derivatized with 50% less ligand than described in 2.2.3, bound 20-30% less and yielded 30-40% less receptor than any of the previous columns (2.3.1).

There was also variability in the recovery of the receptor from the ion-exchange column. Stauber et al.

103 (1987) also found this variability and suggested it might reflect heterogeneity in the detergent-protein micelle aggregation. However, while Stauber et al.. (1987) described variations in the percentage of binding of the receptor to the DEAE-Sephacel column in this work, there were fluctuations in the recovery of the GABA-R ligand binding activity following salt elution. Stauber et al. (1987) have also shown that a higher percentage of bound receptor can be eluted from the affinity column if urea is added to the elution buffer. It is thought that urea increases the effective free-ligand concentration in the elution buffer by reducing the non specific binding of the free-ligand to the column (Stauber et al., 1987). In this work, in contrast to the results reported by Stauber et al. (1987) , an increase in yield was not observed when urea was i;i the purification procedure as described in 2.2.4. This difference may stem from the fact that Stauber et al. (1987) used a column with a different spacer arm which may affect the accessibility of the free eluting ligand to the bound receptor. SDS-PAGE analysis of the purified receptor performed in this work according to the method of Stauber et al. (1987), showed that the GABA-R was composed of the a and the p subunit but a 65 kDa subunit was also present

(Figure 2.3.2.B). SDS-PAGE analysis of the urea wash carried out before the chlorazepate/urea elution, also

104 showed the 65 kDa protein band, and a band of the same molecular weight as the p subunit. This indicates that the 65 kDa band is non-specifically eluted by the urea and that the band of the same molecular weight of the

0 subunit is either a contaminant or the GABA-R p subunit which has been dissociated by the urea from the a subunit bound to the affinity column.

2.3.2 The study of the distribution of- ligand binding sites within the macromolecular structure of the GABA-R

The characterization of the macromolecular structure of the GABA-R must include the determination of the location of its various ligand binding sites, initially at the subunit level.

It has been demonstrated in other systems that the specific ligand binding properties of individual polypeptide chains can be retained following SDS-PAGE. For example, the a subunit of the nicotinic acetylcholine receptor has been shown to specifically bind to

[125I]ce bungarotoxin in Western blots (Gershoni et al.,

1983). Similarly GTP binding proteins have been identified in Western blots probed with [32P]guanidine triphosphate (GTP) (Bhullar and Hasham, 1987). Therefore such an iritial approach was used here to localize the GABA-R ligand binding sites. The blotted GABA-R subunits after transfer to Zetabind filters were not labelled by either

105 [ JH]muscimol or [JH]flunrtrazepam. This is probably due to the fact that the conformation of the GABA-R receptor ligand binding sites is not maintained in the blotted polypeptides. It should also be noted that Gershoni et al.

(1983) reported a decrease in affinity in the binding of a-bungarotoxin to the nicotinic acetylcholine receptor of several orders of magnitude. Additionally, both Gershoni et al. (1983) and Bhullar and Hasham (1987) used ligands with high specific radioactivity (125I and 32P), therefore tritiated ligands may not be suitable for these experiments.

A second approach that has been used for the localization of the neuroreceptor ligand binding sites is photoaffinity labelling (Fedan et al., 1984). In this work, the photochemical properties of the ligands that were used for photoaffinity labelling studies were first analysed and the excitation spectra of flunitrazepam, muscimol and TBPS (Figure 2.3.3) showed that all these ligands can fluoresce. This means that the ligand molecules, upon UV irradiation, absorbed energy and then re-released it. This ability of the ligands to shift to a higher energy state is a characteristic of photoligands. However, it is not possible to predict whether the intermediate product of the UV irradiation can form a covalent bond with the protein. The ability to fluoresce is a necessary but not sufficient property of the ligands

106 for the formation of a specific irreversible bond between them and the receptor. This was highlighted by the photoaffinity labelling experiments carried out with

[35S]TBPS. The latter was shown to be photoexcitable but it did not photoaffinity label bovine cerebral cortex membranes. TBPS is however an example of a ligand that could be modified as a photoaffinity ligand by chemical derivitization. Milbrath et al. (1979) and Bowery et al. (1976) have analysed a series of bicyclic phosphates, of which TBPS is one, and have shown that the structure within the ligand molecule that is responsible for the toxic and convulsant effects is the tetracarboxy.lic group (see Figure 1.1). They have also observed that at the position occupied by the sulphur in the TBPS molecule, a photolabile group could be substituted without significantly affecting the binding of the ligand.

Chemical synthesis of bicyclic cage compounds may thus be worthy of'further: investigation.

The photoaffinity labelling of rat cerebellar membranes with [3H]muscimol had been reported by Asano et al. (1983) and Cavalla and Neff (1985a). In this work, photoaffinity labelling of bovine cerebral membranes with

[3H]muscimol was performed to establish the conditions of the reaction in this tissue and to further investigate the polypeptides that are specifically labelled.

The rate of the photoaffinity labelling of

[3H]muscimol sites was found to be slower than that

107 reported by Cavalla and Neff (1985a) and Deng et al. (1987). Both groups obtained maximum photolabelling after 10 min irradiation whilst 30 min were required in this work (Figure 2.3.4.A). Deng al« (1986) also showed that photolabelling continued at a different rate after the first apparent plateau was reached. The results of Cavalla and Neff (1985) are not directly comparable here, because they only showed data for the first 10 min of irradiation. In this work, two apparently different rates of photolabelling of the membranes were observed (Figure

2.3.4.A). The second slower rate of photolabelling coincided with a rapid rate of inactivation of the reversible binding sites, in membranes irradiated in the absence of [3H]muscimol (Figure 2.3.4.B). This apparently contradictory result could be explained by a photoactivation of the receptor itself, additional to that of the ligand. However, in order to simplify the interpretation of the results, all irradiations were limited to 30 min. From the assay of the inactivation by UV irradiation of reversible binding sites, it was observed that there was an initial increase of binding sites in the first 15 min of irradiation (Figure 2.3.4.B).

It is conceivable that the irradiation directly affected the membrane structure making cryptic receptors available for ligand binding. The fact that the rate of photolabelling was slower than that reported by Cavalla

108 and Neff (1985a) may be attributed to the different UV lamp used.

A major limitation in the use of [3H]muscimol as a photoaffinity label has been its low efficiency of labelling. The percentage of sites photolabelled as compared to the reversible binding sites was 10%. This was due to several factors which are the inefficiency of covalent binding of acyl nitrenes, the inactivation of the receptor and of the ligand molecules themselves. Cavalla and Neff (1985a) reported an efficiency of photolabelling of 20% as calculated from the of the reversible and irreversible reactions. Again, the quantitative difference between these results could be attributed to the UV lamp used.

When membranes which had been photolabelled with unlabelled muscimol were assayed for reversible binding sites, the latter were found to be reduced by 80%. A discrepancy between the number of sites photolabelled and the number of sites inactivated was observed also with the flunitrazepam photoaffinity labelling (Mohler et a_l. , 1980). The latter was attributed to a reduction of affinity for the ligand of the photolabelled receptors, a similar mechanism probably occurs with the muscimol photoaffinity labelling. A further possible explanation is that a certain percentage of the photolabelled sites was lost because tritium label of the bound ligand was exchanged with the medium (Cavalla and Neff, 1985b).

109 Multiple photoaffinity labelling with [3H]muscimol did not increase the total number of labelled sites. However, if the membranes were first photolabelled with unlabelled muscimol and then with [3H]muscimol, some labelling was observed. These latter two findings taken together would suggest that some of the ligand photolabelled in the first irradiation is replaced by the other ligand in the second irradiation. This could have been proven by performing the photolabelling experiment in the reverse order. However, the most important fact was that the amount of radioactivity incorporated in the membranes could not be increased to above 10% of the reversible binding sites.

The effect of DTT on both reversible and irreversible binding was tested and results have shown that DTT did not have any effect on reversible [3H]muscimol binding, but it did reduce the non-specific binding of irreversible

[3H]muscimol labelling. DTT therefore fulfills the requirements for a scavenger described in 2.1.3. Its use is particularly important in SDS-PAGE autoradiography for the detection of the [3H]muscimol specifically bound polypeptides.

The effect of barbiturates on the [3H]muscimol photoaffinity labelling reaction was also studied.

Enhancement of photolabelling of [3H]flunitrazepam by GABA was shown in rat brain membranes by Sieghart and Drexler

110 (1983) . In this work however, no enhancement of either

[3H]muscimol or [3H]flunitrazepam by GABA and/or pentobarbital was obtained. It was instead found that UV irradiation of the membranes reduced the enhancement by pentabarbital and/or GABA of the reversible

[3H]flunitrazepam and [3H]muscimol binding. This discrepancy with the previously published results might again be attributed to the apparatus used. It is possible that only some of the light emitted by the lamp with which the irradiations were carried out had a wavelength of A = 254 nm. Additionally, the lamp may have emitted light at shorter wavelengths which could have denatured the protein.

The initial characterization of the polypeptide that contains the [3H]muscimol binding site was carried out in photoaffinity labelling experiments of the purified GABA- R. Again the photolabelling reaction to this GABA-R preparation was assessed. The maximum number of sites photolabelled were obtained after 30 min of UV irradiation

(Figure 2.3.7.A), and a secondary rate of photolabelling comparable to that of membranes was not seen. The inactivation of purified receptor by UV irradiation was faster than for membranes and occurred from the beginning of the irradiation. The efficiency of photolabelling expressed as the percentage of reversible sites irreversibly labelled, was 7.5% + 0.8 or 23% + 2.8% (n = 3) respectively if calculated with reference to the total

111 number of sites available before and after the irradiation. This calculation however does not take into consideration the inactivation of the ligand, whose effective concentration was decreased by both inactivation and irreversible labelling. The decrease in the concentration of active ligand alters the equilibrium of the reversible binding and consequently can affect the extent of photoaffinity labelling.

The photoaffinity labelling reaction of [3H]muscimol was shown to be dependent on the ligand concentration (Figure 2.3.9.A). The results were also analyzed in a

Scatchard plot and a KD of 15 + 5 nM (n = 2) was found, which was similar to that for [3H]muscimol reversible binding to the purified receptor (Sigel et al., 1983). Scatchard analysis cannot strictly be applied to irreversible binding studies unless the rate of reaction of the ligand is very fast and the number of sites and the concentration of the ligand is not significantly affected during the photoaffinity labelling reaction. Thus

Scatchard analysis of photolabelling is valid if it actually represents the percentage site occupancy at equilibrium before the irradiation. It was here shown that the ligand binding sites and the concentration of active ligand decreased during the photolabelling reaction.

Therefore, the fact that the K q calculated from the irreversible binding reaction was not significantly

112 different from that of reversible binding indicates that the ligand molecules and the binding sites were inactivated in a directly proportional manner. Alternatively, it may be that despite the inactivation observed, the major factor that determines the amount of sites photolabelled is the percentage of reversible sites occupied by the ligand at the beginning of the irradiation. The pharmacological specificity of the photoaffinity labelling reaction of [^H]muscimol was demonstrated by competition binding studies with GABA and bicu^'ulline (Figure 2.3.8). The IC50s calculated for the inhibition of irreversible binding by the . two ligands were not significantly different from those determined for reversible binding (Sigel et al., 1983? Schoch et al.,

1984). It should be realized that the photoligand, after the covalent binding has formed cannot be displaced, while the competitive ligand can be displaced during the photolabelling reaction. Therefore, the finding that the IC50s for the reversible and irreversible binding sites were similar is further evidence that the [3H]muscimol photoaffinity labelled sites represent the population of binding sites occupied at equilibrium before irradiation.

The purified GABA-R photolabelled with

[JH]flunitrazepam and [JH]muscimol was analysed by SDS-

PAGE and it was shown that [3H]flunitrazepam predominantly specifically photoaffinity labelled the a subunit and

113 [3H]muscimol predominantly specifically photoaffinity labelled the p subunit (Figure 2.3.10.B). This result was also shown by a gel slicing experiment (Figure 2.3.10.B) and was confirmed by the concomitant publication of the results of Deng et al. (1986). They have also shown by gel slicing of SDS-PAGE of purified photoaffinity labelled

GABA-R that [3H]muscimol preferentially labelled the p subunit. The result of the SDS-PAGE fluorography of the purified GABA-R photoaffinty labelled with [3H]muscimol was obtained only when the labelled protein was transferred to Zetabind filters. This procedure thus avoided the acidic gel fixation step, which according to Cavalla and Neff (1985a,b) can cause the photolabelled

[3H]muscimol molecule to exchange its tritium label with the free H+ atom present in the fixing solutions. The SDS-PAGE analysis of the photoaffinity labelled

GABA-R indicates that [3H]flunitrazepam binding sites are present on the a subunit and the [3H]muscimol binding sites are present on the p subunit. The minor labelling of the other respective subunitsby the two ligands may be due to non-specific binding or alternatively, it is conceivable that the binding site might be present at the interface of the two subunits. However, the possibility that both subunits could have active sites for both ligands, but that the irreversible covalent bond is formed with only one subunit cannot be excluded. Kirkness and

114 Turner (1986c) have also carried out photoaffinity labelling experiments with [3H]muscimol of purified GABA-R from porcine but have found a different polypeptide labelling pattern (E. Kirkness, personal communication) and further investigations are necessary to clarify these differences. Recently it has been shown by recombinant DNA techniques that Xenopus oocytes injected with a subunit RNA alone can elicit GABA-gated responses (Blair et al..

19 88-). However, while the electrophysiological responses are measured at micromolar concentrations of GABA, the affinity of the photolabelling reaction was in the nanomolar range. It was instead shown here that the

[3H]muscimol irreversible binding had the same pharmacological characteristics of the reversible binding. Additionally it was found that in membranes irreversible binding reduced further reversible binding. These two findings argue in favour of the idea that [3H]muscimol photoaffinity labels a representative proportion of the total population of the reversible high affinity

[3H]muscimol binding sites, which the SDS-PAGE experiments indicate to be predominantly present on the p subunit. The fluorographic analysis of SDS-PAGE of membranes photolabelled with [3H]muscimol was also carried out, but it was not possible to obtain a signal in the fluorograph despite the fact that maximum amounts of labelled protein were loaded onto the gel, and the gel was exposed to a X- ray film for up to 1 year. The aim of these experiments

115 was to further test the presence of the [3H]muscimol binding site on the p subunit and to compare [3H]muscimol and [3H]flunitrazepam photoaffinity labelled polypeptides in preparations of membranes of different brain areas and different brain ages. The visualization of the polypeptides labelled by [3H]flunitrazepam was readily obtained in all brain regions within 2-3 weeks of film exposure. The inability to obtain similar results with

[3H] muscimol can be attributed to the lower specific activity of [3H]muscimol, the relative inefficiency of the photoaffinity labelling reaction, the maximal amount of proteins that can be loaded onto a slab gel and the instability of the tritium label in the photolabelled

[3H]muscimol molecules. Cavalla and Neff (1985a) were able to see a signal from gel fluorography and their success was probably due to a higher efficiency of labelling and a more sensitive fluorographic system.

To test the properties of the [3H]muscimol irreversible binding component, membranes photolabelled with [3H]muscimol and [3H]flunitrazepam were cleaved with trypsin. Both the rate and the extent of the removal of the radioactivity differed for the membranes photoaffinity labelled by the two ligands which indicates that the two sites must be in a non-transmembrane receptor region at a distance from each other of at least one trypsin cleavage site. These experiments were also done to analyze the

116 trypsinized membrane-bound receptor under SDS-PAGE, but the same problems experienced with non-trypsinized photoaffinity labelled membrane preparations were encountered.

It is not clear why all the radioactivity incorporated into the membranes is not removed by trypsinization. If the photolabels were bound to only one specific residue, then it would be expected that either all or none of the bound label would be removed after trypsinization. The results obtained may suggest therefore the presence of either a heterogeneity of receptors, multiple residue labelling of the same receptor molecule, or alternatively that the photolabelled receptors are in different conformations or in different lipid environments which may affect the efficacy of trypsin.

The instability of [3H]muscimol labelling was shown by the release of radioactivity from the photolabelled membrane in the presence of trypsin inhibitors. This indicates that either the [3H]muscimol photoaffinity labelling site is subject to non-trypsin-like proteases, or that some of the ligand or its tritium label is released from the membranes after photoaffinity labelling.

Cyanogen bromide cleavage of the purified GABA-R photoaffinity labelled with [3H]muscimol was carried out to isolate the peptide that contained the covalently bound label after chromatographic separation of the peptides. The HPLC profile showed the presence of several

117 polypeptide fragments but only one contained radioactivity. The latter when analysed by silver stain of SDS-PAGE showed only one major broad band of approximate molecular weight 12 kDa. This peptide can now be subjected to microsequencing and the elucidation of a 5-6 amino acids sequence should be sufficient to establish the location of the [3H]muscimol photoaffinity labelled peptide within the known amino acid sequence of the GABA-R p subunit. It will be of interest to establish whether this peptide contains arginine residues whose chemical modification has been shown to preferentially reduce

[3H]muscimol binding to the GABA-R (Widdows et al., 1987).

The identification of the GABA binding site will be of use in the molecular characterization of the GABA-R and will allow comparisons to be made with the putative binding sites for acetylcholine in the nicotinic receptor

(reviewed by Stroud and Finer-Moore, 1985) and the binding sites of the glycine receptor (Grenninglogh et al., 1987), as both these two receptors, together with the GABA-R, are believed to belong to an ion-gated receptor super-family

(Barnard et al., 1987).

118 CHAPTER 3

THE PRODUCTION OF ANTIBODIB8 AGAINST THE GABA-R

119 3.1.1 Introduction

This chapter will deal with the production and

characterization of polyclonal and monoclonal antibodies against the GABA-R. In this Introduction, monoclonal antibody production techniques and the use of monoclonal

antibodies for the molecular characterization of receptors will be described.

3.1.2 The production of monoclonal antibodies

Antibodies are produced by the B cells of the spleen. Each B cell precursor undergoes rearrangement at its immunoglobulin gene in such a way that the mature B cell will produce only one type of monospecific antibody. Kohler and Milstein (1975) devised a method by which the antibody-producing B cell is immortalized. This was achieved by the fusion of dissected spleen cells with cultured cells of a myeloma cell line. The fusion produced hybridoma cells capable of continuous duplication and antibody secretion. The introduction of this technique has been a major breakthrough and hundreds of monoclonal antibodies against a variety of different antigens have been produced. However, 13 years after the first success, many difficulties are still encountered by researchers who

120 seek to produce monoclonal antibodies against specific antigens. The production of monoclonal antibodies can be divided into three main parts: immunization of the spleen- donor animal, the cell fusion, and the screening of the hybridomas (See Figure 3.1.1).

3.1.2.A Immunization of the spleen donor animal

Only a small percentage of the spleen cells is fused with myeloma cells (about 1 in every 106 cells) and therefore, in order to ensure that at least one of the B cells that is producing the antibody of interest fuses successfully, a large number of these specific cells must be present in the spleen. A variety of antigen injection protocols have been tried to obtain the highest possible stimulation and proliferation of the required specific antibody producing B cells. Most protocols involve multiple subcutaneous and/or intraperitoneal injections at 3-4 week intervals with antigen mixed with an adjuvant. The most common adjuvant used is Freund's adjuvant which when "Complete" also contains inactivated bacteria and is generally used only for the first immunization. The adjuvant's role is to stimulate non-specifically the immune system and to prevent the antigen from dispersal immediately after the injection. The immune response, the antibody titre, is generally assayed by the measurement of anti-antigen specific circulating antibodies in the

121 injected animal. A blood sample is taken 6-8 days after the last injection, and is tested with the same system that is to be used to screen the hybridoma culture media.

The titres obtained depend principally on the immunogenic properties of the antigen itself. However, some guidelines to maximize the immune response have been described. These are: 1) It has been shown that the response can be dependent on the amount of antigen injected although high doses of some antigens can lead to immunosuppression (Stahly et al., 1980). 2) Intervals shorter than 2 weeks between injections can yield low affinity and low titre antibodies. 3) Essential to the success of the production of the desired monoclonal antibody is the last injection which should be carried out 3-4 days before the fusion. The treatment of the antigen before it is injected can affect the type of antibody that is obtained.

Therefore, the antigen is generally injected in the form in which its immuno-characterization will be carried out.

Thus for example, for immunopurification studies the antigen should be injected in the native form and for

Western blot studies in a denatured form. However, the present knowledge of the triggering of the immune response indicates that the antigen is first processed by the antigen presenting cells which retain fragments of the

122 antigen on their surface (Roitt et al., 1985). T and B cells then act on these fragments which have probably lost their original conformations.

Alternative methods of immunization have been devised. In one such method the antigen is injected directly into the spleen and the fusion carried out 4 days later (Spitz et al., 1984). This method requires reduced amounts of antigen and, if successful, is very fast. It is not clear however how the normal stimulation of the immune system is by-passed. Following a different method, antigen has also been bound to nitrocellulose filters which were implanted intraperitoneally (Sternik and Stunner, 1984).

Again, reduced amounts of antigen are required and the solid support of nitrocellulose should in theory extend the time the antigen is present in the recipient animal. Monoclonal antibodies have also been produced by stimulating the B cells in vitro (Reading, 1986). The advantages of this technique are again economy of antigen, the reduced amount of time required to stimulate a response and the enhanced ability to overcome some of the problems of in vivo immunization such as suppression and tolerance. The use of adjuvants and mitogens can be very important in in vitro immunization. In this study the synthetic muramyl dipeptide (MDP) N-acetyl-muramyl-L- alanyl-D-isoglutamine and lipolysaccharides (LPS) have been used. MDP has been shown to be the minimal unit required to duplicate the immunostimulating effects of the

123 mycobacteria in Freund's complete adjuvant, and in in vitro has been shown to potentiate the immune response of murine spleen cells (Leclerc et al., 1979). LPS stimulates the immune system by enhancing the release of proteins from the macrophages (Morrison and Ulevitch, 1978).

3.1.2.B The fusion of spleen cells with myeloma cells

The spleen cells are fused to myeloma cells by means of polyethyleneglycol (PEG). This compound is most effective in its small molecular weight form (1.5 - 4.0 kDa) , which is also the form in which it is most toxic to the cells. The time, the temperature, and the concentration of PEG to which the cells are exposed, are all important factors for the success of the fusion. Most of the recent reports favour short, 0.5-1 min, incubations with 50% PEG and neutral pH at 37°C (reviewed by Langone and Vunakis, 1986) In a standard PEG fusion, all the cells can indiscriminately fuse with one another. Lo et al., (1984) have developed an alternative system whereby the B cells that are producing the antibody of interest are preferentially fused. This was achieved by the formation of a bridge between biotinylated-myeloma cells and the avidin-labelled antigen which had been bound to the specific antibody on the surface of the B cells. The cells were exposed to a high intensity electrical field which

124 preferentially caused the fusion between those cells joined by the antibody-antigen-avidin-biotin bridge. This method has the advantage that only a few hybridomas are produced, but a high percentage of these are producing the antibodies of interest.

One important choice in the fusion experiment is the myeloma cell line to be used. A number of these cell lines have been developed (reviewed in Galfre and Milstein,

1981) . It is preferable to use a cell line that itself does not secrete antibodies. The two cell lines used in this work were the SP2/0Ag8 and the NSO. The former is a clone of the SP2 cell line which is itself a hybridoma and it is thus a tetraploid, a factor that may confer the cell line with a certain degree of instability when it acquires another set of chromosomes from the spleen cells. NSO cells are diploid and are reported to be more stable

(Galfre and Milstein, 1981).

3.1.2.C The selection, screening and cloning of the

hvbridomas

After the fusion, all the non-fused myeloma cells must be eliminated because they grow faster and outnumber the hybridomas. During the selection of the hybridomas, the main pathways for the synthesis of nucleotides are specifically blocked and nucleotide precursors are added as substrates for the salvage pathways. Under these conditions the myeloma cells, which have been selected to

125 be defective in the nucleotide synthesis salvage pathways will die, while the hybridomas which contain the genes for the salvage pathways of the parent spleen cell will survive. The Hypoxanthine-Aminopterin-Thymidine (HAT) medium is the most commonly used for this selection. Aminopterin blocks the main nucleotide synthesis pathways and hypoxanthine and thymidine are substrates of the salvage pathways. The effect of aminopterin may continue even after the selection is discontinued, thus hypoxanthine and thymidine need to be supplemented for long periods during the culturing of the cells. An alternative selection method is to use hypoxanthine and asazerine. Asazerine inhibits only one of the enzymes of the nucleotide synthesis major pathway, and its effects are readily reversible when the cells are returned to non- selective medium.

After the fusion, cells are aliquoted into separate wells (0.1 - 1.0 ml) and the resultant supernatants are later assayed. The screening system must be very sensitive in order to detect the low amount of antibodies that are produced during the early stages of the cell culture. The antigen is used in the screening assay either in a form that will maximise the number of specific antibody producing clones that can be selected and/or should be in the same form in which the antigen is intended to be studied. The screening is followed by the separation of

126 the specific antibody-producing hybridomas from non producing hybridomas which can overgrow the former. The pH, temperature and C02 content of the medium are carefully monitored because pluriploids are unstable and can lose some of their chromosomes and thus their ability to produce the antibodies.

3.1.3 The localization of neuroreceptors with monoclonal antibodies

The monospecificity of monoclonal antibodies has been exploited for the localization of neuroreceptors in tissue sections (e.g. Schoch et al., 1985; Swanson et al., 1983). Receptors can also be detected by their specific radiolabelled ligands (e.g. Marangos et al., 1987), however the detection by monoclonal antibodies has certain advantages. These are:

1) The visualization of the antibody stain is carried out directly on the tissue rather than on an autoradiograph and it is thus quicker to carry out and it is easier to localize the staining.

2) Antibodies often have higher affinities for their antigens than the ligands for their receptors which allows more extensive washes to be carried out in order to remove non-specific binding. 3) The staining signal of antibodies can be amplified with secondary antibodies.

4) Antibodies can be used for the staining of fixed

127 tissues where the receptor may have lost its native properties.

5) Antibodies can be readily visualized in the electron microscope and gold labelled antibodies are particularly useful for this application (e.g. Horisberger and Rosset,

1977). The characteristics required for monoclonal antibodies to be used in immunolocalization of receptors are (i) high affinity, (ii) an ability to recognise the antigen in a native form, (iii) to be directed against exposed epitopes of the membrane-embedded protein.

3.1.4. The structural characterization of neuroreceptors using monoclonal antibodies

Monoclonal antibodies have been used to characterize the structure of neuroreceptors such as the nicotinic acetylcholine receptor (reviewed in Tzartos, 1984). Monoclonal antibodies have been used to show antigenic similarities between the receptor subunits of the nicotinic acetylcholine receptor (Mehraban et al., 1982) and between the same receptor of different species

(Tzartos et al.., 1981). In Western blots, monoclonal antibodies can identify the receptor, its subunits or its proteolytic fragments. Immunoprecipitation studies with monoclonal antibodies can indicate the presence of multiple ligand binding sites on the same receptor (Haring

128 ft

et al., 1985).

The ligand binding sites of the receptors can be studied by monoclonal antibodies directed against this

portion of the receptor. However, the statistical probability of obtaining such antibodies from a panel of

antibodies directed against the whole receptor is low. An

alternative method which has been used to raise this kind of antibody has been to develop anti-idiotypic antibodies to anti-ligand antibodies. This can be achieved by raising

either anti-ligand antibodies which are then used to immunize a second animal (e.g. Farid and Lo, 1985) or alternatively, by screening for anti-idiotypic antibodies directly in the animal immunized with the ligand

(Cleveland and Erlanger, 1986). The first approach is hindered by the statistical probability of first raising an antibody against the part of the ligand that

recognises the receptor and then to raise a second antibody directed against the epitope recognition site of the first antibody. The second approach exploits the fact

that the immune system naturally produces anti-idiotypic

antibodies (Jerne, 1974) . These antibody producing cells

are however, less abundant and follow a different developmental profile which complicates the choice for the

timing of the fusion. The use of molecular cloning in the neuroreceptor

field has opened the way for the development of monoclonal

129

ft antibodies against synthetic peptides constructed from the known amino acid sequence of cloned receptors (e.g. Neumann et al., 1985)• This method has the advantage that one can probe for specific parts of the protein such as putative active sites and intracellular or extracellular domains.

The properties required for monoclonal antibodies to be used for Western blots are high affinity and the ability to recognise the receptor in a denatured state. For immunoprecipitation of the receptor the monoclonal antibodies should have high affinity and the ability to recognise the receptor in a native soluble form in the presence of detergent.

3.1.5 Immunopurification of neuroreceptors bv monoclonal antibodies

Most neuroreceptors have been purified to date by ligand affinity chromatography. Immunoaffinity purification works on the same principles but has the following potential advantages.

1) Most antibodies can be attached to a column without losing affinity for the receptor, while many ligands are not suitable for covalent attachment to a solid support e.g. GABA. 2) A higher yield of purified protein can be obtained because the elution of the antigen from the column is

130 carried out by the alteration of the ionic conditions rather than by the use of a competitive ligand (e.g. Mamoi and Lennon, 1982).

3) A monoclonal antibody affinity column could selectively purify subtypes of receptors that have identical ligand recognition properties.

4) The eluate from an immunoaffinity column will not contain, as in ligand affinity chromatography, high concentrations of the eluting ligand which can be difficult to remove and can desensitize the receptor.

One of the major problems of immunoaffinity purification is that the extreme pHs or chaotropic agents which are required for the elution step i.e. the dissociation of the antigen-antibody complex, can affect the ligand binding properties of the purified protein

(Mamoi and Lennon., 1984)

The properties required for monoclonal antibodies to be used for immunopurification of membrane bound proteins are: (i) the ability to recognise the receptor in a soluble form in the presence of detergents, (ii) a relatively low affinity for the antigen to facilitate dissociation and (iii) to recognise the receptor in a conformational-dependent manner only. If the antibody does not have one of the last two properties, difficulties will be encountered in the elution of the receptor from the column.

131 3.1.6 Strategies in the production of monoclonal antibodies

It has been described above that the various applications of monoclonal antibodies require antibodies with specific properties. The two major procedures that will influence the type of antibody produced are the treatment of the antigen before immunization and the screening system of the hybridoma colonies. It should be realized however that at the present time it is not possible to dictate the properties of the antibodies that will be produced in the animal and those which will be isolated by the screening system. This is indicated by the variety of methodologies that have been carried out in the production of monoclonal antibodies irrespective of the intended use of the antibodies. The approach of first developing a panel of monoclonal antibodies and then using each one according to its properties is still the one that is the most widely used.

132 Figure 3.1.1

bleed

meyloma cells Tit re

culture N/

remove spleen

dissect cells

clone

Diagrammatic representation of the production of monoclonal antibodies. (See text 3.1.2)

1 33 *

3.2 Materiala and Methods

Lbed (2.2.4) .1. Materials the eluting Tissue culture medium and bovine foetal serum were e receptor fhased from Flow Laboratories (UK). Sterile plastic- losphate, pH e for tissue culture and microtitre assay plates for preparation ELISA assay were from Falcon (UK). Polyethylene glycol Lsified with 00) from two different sources was used. These were i injected tish Drug House (UK) and Bathesda Research Ln mice, and >oratories. The Freund's Complete and Incomplete .n rabbits, uvant were from Difco (UK). All biotinylated species- * ites at 2-3 scific antibodies and the streptavidin-biotinylated h Freund's •oxidase complex were from Amersham International (UK). duction of L other species-specific antibodies were from Dakko juvant was enmark). The MonoQ™ ion-exchange column was from animal was larmacia (Sweden). The DotBlot apparatus was from BioRad

JSA). The adjuvant muryl dipeptide (MDP) was from BioLabs

SA). Goldstain for the total protein stain of proteins zR ^nsferred to nitrocellulose was from Janssen (Belgium). n 3.2.2 . A mbrane cell concentrators with molecular weight cut off

i ected with 30 kDa were from the Amicon Corporation (USA). >.2. A. All other chemicals and reagents were of

talytical grade from different commercial sources.

in 3.2.2.A

134 3 .2.2 Immunization protocols

3.2.2. A Immunization with native GABA-R

Purified GABA-R was prepared as described (2.2.4) except that the Triton X-100 concentration in the eluting buffer was reduced to 0.02% (w/v). . The receptor preparation was dialyzed against 10 mM K-phosphate, pH 7.4, 0.02% (w/v) Triton X-100 (2x2 1). The preparation was lyophilized, resuspended in H20 and emulsified with Freund's Complete adjuvant (1 vol) and injected intraperitoneally (200 ul, 40-200 ug protein) in mice, and intramuscularly (300 ul, 100 ug protein) in rabbits. Subsequent injections were given at the same sites at 2-3 week intervals with antigen emulsified with Freund's incomplete adjuvant (1 vol). For the production of monoclonal antibodies, antigen without adjuvant was injected intraperitoneally 4 days before the animal was sacrificed.

3.2.2. B Immunization with heat denatured GABA-R

The receptor sample was prepared as in 3.2.2. A resuspended in H20, boiled for 30 min and injected with

Freunds'Complete adjuvant and injected as in 3.2.2.A.

3.2.2. C Immunization with SDS-denatured GABA-R

The receptor preparation was dialyzed as in 3.2.2.A

135 and concentrated 10 fold to 200-300 ul with an Amicon membrane concentrator (molecular weight cut-off = 3 0 kDa). SDS was added to a final concentration of 0.1% (w/v) and the mixture was boiled for 3 min. The samples were

injected with Freund's Complete adjuvant as in 3.2.2.A.

3.2.2. D Immunization with carboxvmethvlated GABA-R.

Purified GABA-R was prepared as in 2.2.4. To a sample of this preparation (1.5 ml), 8 M Urea (500 ul) and 1 M DTT (10 ul) were added and incubated for 30 min at room temperature. Iodoacetamide, 1 M (20 ul) was added and the

mixture incubated for a further 30 min at room temperature in the dark. This preparation was dialyzed, lyophilized and injected with Freund's Complete Adjuvant as in 3.2.2. A.

3.2.2.E Immunization with alum-precipitated GABA-R

Alum precipitation of the GABA-R was carried out

according to the method of Norman et a_l. (1986) .

Purified receptor preparation (4 ml) (3.2.2.A) was

concentrated to 600 ul in an Amicon 30 cell concentrator. Alum (10% (w/v) A1KP04) (1 vol) was added, and the

solution mixed. KOH (1 M) was added dropwise until pH 6

was obtained. After 30 min incubation, the mixture was

centrifuged at 10,000 g in an Eppendorf centrifuge for 15 min, and the pellet was washed with PBS (see Materials

2.2.1, 3x 500 ul) . The pellet was resuspended in PBS and

136 injected intraperitoneally (100 ul, 40 ug protein).

3.2.2. F Tmrnunination by intrasplenic injections of GABA-R

Intrasplenic immunizations were carried out as described by Spitz et al., (1984). Mice were anaesthetized in an ether chamber, the abdominal hair was moistened with ethanol and shaved. A small incision was made first in the skin then in the inner muscle layer. With a sterile pair of forceps, the spleen was gently pulled out of the abdominal cavity. The needle of a loaded .Hamilton syringe was inserted along the length of the spleen, and the antigen (50 ul) was slowly injected while the needle was being withdrawn. The spleen was replaced in the body cavity, the muscle layer and the skin were sutured and the animal allowed to recover from anaesthesia.

3.2.2. G Immunization bv the implantation of nitrocellulose

blotted with GABA-R

Purified receptor was electrophoresed and transferred to nitrocellulose filters by Western blotting as described in 2.2.10. The receptor bands were identified by the staining of an adjacent nitrocellulose strip with Goldstain. Pieces of nitrocellulose filter (circa 4 mm2) containing the receptor bands were cut out, briefly immersed in Freund's Complete adjuvant and placed in the body cavities of anaesthetized mice. The operation was

137 repeated after 3 weeks with the use of Freund's incomplete adjuvant.

3.2.3 The collection of the immune sera.

Mice were placed under an infrared lamp for 15 min. They were immobilized in a Falcon tube (50 ml) with air

holes. The tail was greased with vaseline, and a small

nick in the vein was made with a scalpel blade. Blood (approximately 500 ul) was collected in Eppendorf tubes and was allowed to coagulate for 1 h at 37°C, and for 12 h at 4°C. The tubes were centrifuged at 10,000 g for 5 min, the serum collected and stored at -20°C until use. Rabbits were wrapped in a cotton blanket, one ear was

rubbed and greased with vaseline. A small nick was made in

the vein and the blood (2-5ml) was collected in Falcon tubes. The serum was collected as described above.

All animal operations and handling were performed with strict adherence to the current "Prevention of

Cruelty to Animals" laws.

3.2.4 The cell culture conditions

Hybridoma and myeloma cell lines were grown in Dulbecco

Modified Eagle's Medium (DMEM) containing 15% foetal calf serum (FCS), penicillin (100 unit/ml) and streptamycin (100 unit/ml). This medium will be referred to as complete

DMEM. Whenever the cell concentration was less than 1000

cells/ml, macrophages were added to the culture medium

138 (see 3.2.6). Cultures were grown in an incubator at 37°C and at a C02 content of 8% at a subconfluent cell concentration. After the colony had sufficiently expanded, the cells were allowed to grow to overconfluency and

eventually death. The cells were removed by centrifugation

at 3000 g for 10 min; the supernatant, i.e. spent medium,

was collected and stored at 4°C with the addition of 0.02% (w/v) NaN3.

3.2.5 Liquid nitrogen storage of cell lines

For long term storage, the cells were resuspended in complete DMEM containing 10% DMSO (106 cells/ml) and aliquoted (0.5 ml) into freezing vials. These were placed in a foam-box lined with cotton wool and stored at -70°C

for 12 h before transfer to liquid nitrogen. When re

quired, the vials were quickly thawed, the cells were precipitated by centrifugation (1500 rpm for 5 min), the supernatant discarded and the cells resuspended in complete DMEM.

3.2.6 The preparation of the macrophages

An adult mouse was killed by cervical dislocation.

The arimal was washed extensively with 70% ethanol and brought to the sterile lamina flow hood. The abdominal skin was cut and pulled back in order to expose the whole peritoneal wall. Cold DMEM (3 ml) was injected at the

139 midline with a 25G needle. The mouse carcass was massaged to disperse the medium. A 21G needle was inserted through the peritoneal wall and the medium was drawn out with a syringe. The medium was centrifuged (1500 rpm for 5 min) ? the supernatant discarded and the pellet containing macrophages resuspended in complete DMEM (50 ml), which was the final concentration at which the macrophages were used.

3.2.7 The fusions of the myeloma and the spleen cells

3.2.7.A The 33% PEG fusion method

A myeloma cell line (SP2/0Ag8 referred to as SP2, or NSO) was grown at exponential growth rate for at least one week. The day before the fusion was carried out, the cells were resuspended in fresh complete DMEM. A mouse which had received the final injection 4 days earlier was sacrificed, thoroughly sprayed with ethanol and brought to the sterile flow hood. The spleen was removed using sterile dissecting instruments and placed in DMEM (2 ml) in a culture dish. A cell suspension was made by teasing the spleen with two large needles. The free cells were transferred to a Falcon tube (50 ml) and diluted with DMEM

(18 ml). The medium and cells from two subconfluent 90 mm culture dishes of myeloma cells (approximately 107 cells) were also transferred to a Falcon tube (50 ml) and both tubes were centrifuged at 1500 rpm for 5 min. The

140 supernatants were aspirated and the two pellets resuspended in DMEM (20 ml/tube) and centrifuged at 1500 g for 5 min. The supernatants were aspirated and both pellets resuspended in DMEM (2 ml) and mixed in the same round bottom Falcon tube and centrifuged at 1500 g for 5 min. The supernatant was carefully removed and the pellet resuspended in 33% (v/v) PEG from BDH in DMEM (0.3 ml), which had been previously autoclaved and kept at 37° C.

The tube was vigorously tapped for three minutes to ensure complete resuspension of the pellet. The tube was centrifuged at 1500 g for 5 min and the supernatant aspirated. The pellet: was gently resuspended in selection medium (100 ml) which was complete DMEM containing asazerine (1 ug/ml), hypoxanthine (1 ug/ml) and macrophages (as described in 3.2.6). The medium was then distributed to 96-well plates (100 ul/well). Two days

later the plates were inspected for infection and death of myeloma cells. Seven days after the fusion each well was

supplemented with complete DMEM (100 ul). When clones were occupying more then 10% of the well surface they were

assayed. Unfused myeloma cells were also cultured under the same conditions to check the efficacy of the selective medium.

3.2.7. B The 50% PEG fusion method

Spleen cells and myeloma cells were prepared as in

3.2.7. A and centrifuged together at 1500 g for 5 min. To

141 the pellet 50% PEG (BRL) (0.5 ml) was added over 15 sec and the pellet resuspended by vigorously tapping the tube. After 1 min, DMEM (1 ml) was added over a period of 1 min while stirring with the same pipette. This last step was repeated 6 times. At all times the temperature was maintained at 37°C by holding the tube immersed in a beaker containing 37°C water. Complete DMEM (2 ml) was added and the tube centrifuged at 1500 g for 5 min. The supernatant was aspirated and the pellet resuspended in selection media and aliquoted to 96-well plates as in

3.2 .7 .A.

3.2.8 In vitro immunization of spleen cells bv soluble

antigens

Spleens of either adult or new born mice (2 days old) were dissected as in 3.2.7.A and washed twice with complete DMEM. Cells were resuspended at a concentration of 106 cells/ml in complete DMEM also containing 10”5 M p- mercaptoethanol. Different cocktails of adjuvant (MDP, 5 ug/ml), mitogens (lipolysaccharide LPS, 5 ug/ml), dextran sulphate (25 ug/ml) antigens (GABA-R or ovalbumin 0.3 ug/ml) were filter-sterilized through 0.45 um sterile filters and added to the culture dishes. After 4 days the cells were counted and used for fusion with a myeloma cell line as described in 3.2.7.

142 3.2.9 The selection of the spleen cells bv the panning method

The antigen (ovalbumin or purified GABA-R) was diluted to 10 ug/ml in sterile PBS and coated onto plastic culture plates (30 mm or 90 mm diameter) for 12 h at 37°C.

The plates were washed with sterile PBS (3 x 10 ml) . Spleen cells, freshly dissected from an immunized mice, or harvested after 3 days of in vitro immunization (3.2.8) were transferred to the coated plates at a concentration of 106 cells/ml. The cells were allowed to bind to the antigen coated on the plates for 5 h at 37°C with gentle agitation every 30 min. Then ”non-attached cells” were collected with two gentle washes of complete DMEM. The attached cells were removed by either (i) incubation with DMEM pH 3.0 (10 min), or (ii) incubation with 0.05% (w/v) trypsin, 0.8% (w/v) sucrose, 0.025% EDTA, (10 min), or (iii) mechanical scraping. All were fused to myeloma cells as described in 3.2.7.

3.2.10 Screening of the hvbridoma cell lines

3.2.10.A The enzvme linked immuno-sorbent assay (ELISA)

The GABA-R was prepared as in 2.2.4 except that the

Triton X-100 concentration of the ion exchange elution buffer was reduced to 0.02% (w/v). It was added to

143 microtitre 96 well plates (60 ul/well, 1:10 in PBS), and incubated at least 14 h at 4°C. The receptor was aspirated and the wells washed with PBS containing 0.25% (w/v) gelatin (3 x 200 ul/well). All wells were then incubated with PBS-gelatin for 45 min at 37°C. The PBS-gelatin was removed and the antibodies to be tested were added (100 ul/well) , and incubated for 1 h at 37°C. The wells were washed with PBS/gelatin (4 x 200 ul) and incubated with PBS/gelatin for 10 min at 37°C. After removal of the latter, biotinylated species-specific biotinylated anti immunoglobulin antibodies (1:750 dilution in PBS/gelatin, 100 ul/well) were added and incubated for 90 min at 37°C. All wells were washed with PBS/gelatin (4 x 200 ul/well), and the streptavidin-horseradish-peroxidase complex was added to each well (1:1000 dilution in PBS/gelatin, 100 ul/well) and incubated for 45 min at 37°C. The wells were then washed with PBS-gelatin (3 x 200 ul/well) and with PBS only (200 ul/well) . The substrate was prepared immediately prior to use and was 4 mg orthophenyldiamine,

1 ul 40% (v/v) H202, 10 mg ammonium carbonate in H20 (10 ml) . It was added to each well (80 ul/well) and the reaction was allowed to proceed for up to 30 min. It was terminated by the addition of 20% (v/v) sulphuric acid.

The absorbance of each well was measured with a multi channel spectrophotometer at A = 492 nm.

144 3.2.10.B The measurement of liaand binding to GABA-R coated on ELISA plates

GABA-R purified as in 3.2.10.A was diluted 1:10 in

PBS and coated for 16 h at 4°C onto ELISA plates. The

receptor was removed and assayed for [3H]flunitrazepam binding. The plates were washed with PBS (3 x 200 ul/well) and were incubated for 45 min at 37°C in PBS-gelatin as for the ELISA assay (3.2.10.A) or in PBS alone at room

temperature. The wells were incubated with 3 0 nM [3H]flunitrazepam in soluble assay buffer (2.2.5.B) for 45 min at 4°C. The ligand was removed and the wells were washed with PBS (1-2 x 200 ul/well). The wells were cut and counted in scintillant (2.2.5.B).

3.2.10.C Solid-phase radioimmunoassay

Rabbit anti-mouse immunoglobulins (10 ug/ml in PBS,

100 ul/well) were coated onto ELISA plates overnight at

4°C followed by incubation with 0.25% (w/v) gelatin in PBS at 37°C for 45 min. The wells were washed with PBS-gelatin

(3 x 200 ul/well), and incubated with the primary antibody to be tested (100 ul/well) for 1 h at 37°C. The wells were washed with PBS-gelatin (3 x 200 ul/well) and the soluble extract of bovine brain membranes (2.2.4) was added (100 ul/well), and incubated for 3 h at room temperature. The supernatant was removed and assayed for [3H]flunitrazepam binding activity (2.2.5.B). The wells were also assayed

145 for the presence of ligand binding (3.2.10.C).

3.2.10.D The Dot Blot screening procedure

A sheet of nitrocellulose paper (0.45 urn pore size) was incubated for 30 min in PBS and placed in a 96 well BioRad dot blot apparatus. Purified receptor diluted Is 10 (100 ul/well) in PBS was added to the wells and allowed to filter through the nitrocelluose by gravity for 45 min. All wells not required for the experiments were blocked

with 3% (w/v) gelatin (100 ul/well). BSA (1%, w/v) in PBS (200 ul/well) was allowed to filter through the nitrocellulose filter by gravity. The wells were washed with PBS-BSA (3 x 200 ul/well) under vacuum. The antibodies to be tested (100 ul/well) were allowed to filter through the nitrocellulose filter by gravity. The

wells were washed with PBS-BSA (3 x 200 ul/well) under

vacuum, and the species-specific biotinylated anti immunoglobulin antibody (1:500 dilution in PBS-BSA, 100

ul/well) was allowed to filter through the nitrocellulose filter under gravity. The wells were washed under vacuum with PBS-BSA (3 x 200 ul/well) and the streptavidin-

horseradish peroxidase complex (100 ul/well, 1:500

dilution in PBS-BSA) was allowed to filter through the nitrocellulose filter by gravity. The wells were washed

under vacuum with PBS (3x 200 ul/well). The nitrocellulose

filter was removed from the apparatus and developed with

146 50 ug 2,5,diaminobenzidine, 3 ml 1% (w/v) CoCl2, 50 ul 40% (v/v) H202 freshly prepared in PBS (97 ml) . The reaction was stopped by washing the filter with water (3x5 min).

3.2.11 The immunostaininq of Western blots

Proteins were transferred to nitrocellulose filters

(0.1 urn pore size) by Western blotting (2.2.10). The filter was quenched in wash buffer which was: 50 mM Tris HC1 pH 7.4, 0.9% (w/v) NaCl, 10% (w/v) BSA or 10% (w/v) low fat dry milk, 0.5% (v/v) Tween 20 for 1 h at 37°C. The filter was incubated with an antibody preparation which was either neat spent culture medium or serum diluted in detergent-free wash buffer. The incubation was carried out for 1-2 h at room temperature or overnight at 4°C. The

filters were washed with wash buffer (4 x 15 min) and incubated with species-specific biotinylated anti immunoglobulin antibodies (1:500 dilution in detergent-

free wash buffer) for 1 h at room temperature. The filters were washed with wash buffer (4 x 15 min) and incubated with the streptavidin-peroxidase complex (1:400 dilution

in detergent-free wash buffer) for 2 0 min at room temperature and washed with wash buffer (3 x 10 min) then with PBS (1 x 10 min) . The nitrocellulose filters were developed as described in 3.2.10.D.

3.2.12. The cloning of hvbridoma cell lines

Hybridoma cells from wells whose supernatant was

147 shown to be positive by ELISA (3.2.10.A), were transferred to 24-well plates containing complete DMEM (1 ml /well) and assayed 2-3 days later by ELISA. The cells from wells that were positive were used for cloning.

3.2.12.A Hvbridoma cell line cloning bv the limiting dilution method

The cells were expanded into two 30 mm culture plates and the cells of one dish were frozen for long term storage (3.2.5). The cells from the other dish were resuspended and 2 aliquots were added (50 ul and 100 ul respectively) to 2 Falcon tubes (50 ml) containing complete DMEM with macrophages (10 ml each) . A 96 well plate was prepared by the addition of sterile H20 (150 ul) to all outer wells, and complete DMEM with macrophages (150 ul) to all other wells. The 2 above cell suspensions

(50 ul/well) were added to the DMEM containing wells. Cells were allowed to grow to subconfluency and the supernatant of each well was tested by ELISA. The cells from the four most positive wells of the initially lower concentration of cells were transferred to 24 well plates and supplemented with 2 ml of complete DMEM with macrophages. The cells were transferred to 30 mm dishes and the cells from the most positive well were used for another cycle of cloning. All other cells were frozen for long term storage. The cloning was repeated 3 times or

148 until all the wells were positive. The cells were then subjected to serial dilution as described below.

3.2.12. B Hvbridoma cell line cloning bv the serial dilution and the wsafew serial dilution methods

Complete DMEM with macrophages (150 ul) was added to all the wells of a 96-well plate. To the first column of wells, resuspended hybridoma cells (< 106 cells/well ,50 ul/well) were added. After mixing by pipetting, medium (50ul) from each well of the first column was added to the corresponding well of the second column. This was repeated for all columns. After five days, some of the medium (100 ul/well) was removed and complete DMEM (100 ul) was added. Wells which visibly contained only one colony were screened by ELISA and the cells from the most positive well were considered monoclonal, if the cells had already been cloned by limiting dilution as above. If serial dilution was performed as a first cloning step, the "safe” serial dilution was carried out. The wells from the third or fourth column, which contained more than one clone, were assayed and expanded while the most diluted cells were allowed to grow. The "safe" serial dilution was carried out twice for each clone.

3.2.12. C Hvbridoma cell line cloning bv the soft agar method

Soft agar cloning was performed according to the

149 method of Galfre and Milstein (1981). An equal volume of sterile 2% (w/v) agar was added to 2 x concentrated DMEM, supplemented with 15% (v/v) FCS. This solution was further diluted with DMEM (1 vol) (final agar concentration = 0.5% (w/v)). This medium was poured into 90 mm culture dishes (15 ml/dish) and allowed to set for at least 15 min. Cell suspensions (101 - 106 cells/ml in DMEM, 1 ml) were mixed with the 0.5% (w/v) agar-DMEM medium (1 ml) and immediately poured over the above-described agar culture plate. The agar was allowed to set for 15 min. The plates were kept in the incubator and when colonies were visible (approximately 10 days later) these were picked and expanded in 24-well plates and later assayed. If this was the first cloning step it was repeated. Positive clones from the most diluted plates were considered monoclonals.

3.2.13 The production of ascites fluid

The production of ascites fluid was carried out according to the method of Hoogenraad et a^. (1983) .

Adult BALB/c mice were injected intraperitoneally with pristane (2,6,10,14-tetramethylpentadecane) (0.3 ml).

After 6-10 days, the mice were injected intraperitoneally with hybridoma cells resuspended in DMEM (106 cells/mice). After 6-12 days the mice were sacrificed and the ascitic fluid was collected from the abdominal cavity.

150 3.2.14 Antibody purification bv the protein A method

Sepharose 4B protein A matrix (0.5 g) was swollen to 1.5 ml in water for 15 min. The gel was transferred to a

10 ml disposable column and pre-eluted with the elution buffer which was 50 mM glycine, pH 3.0. The spent medium of a colony of antibody-producing hybridomas was buffered

to a final concentration of 10 mM Tris/HCl, pH 7.0 and supplemented with 0.02% (w/v, final) NaN3. The medium was

applied and circulated for 12 h on the protein A column which had been re-equilibrated with PBS. The column was washed with PBS (100 ml) and eluted with the above elution buffer at 10 ml/h and fractions were collected (2 ml/fraction) into tubes containing 1 M Tris/HCl, pH 7.5 (1 ml) . The amount of antibodies obtained was estimated by reading the absorbance at A = 280 nm:

[antibodies](mg/ml) = Absorbance2go / 1-13

3.2.15 Methods for the precipitation of immunoglobulins

3.2.15.A The ammonium sulphate precipitation method

Saturated ammonium sulphate was prepared by the addition of ammonium sulphate (1 Kg) to distilled water (1

1). The solution was stirred for 1 h, boiled and allowed to cool. Saturated ammonium sulphate was added step-wise, to a final concentration of either 30% (v/v) or 50% (v/v), to spent medium and stirred gently for 30 min at room

151 temperature. The solution was centrifuged at 20,000 g for 30 min and the supernatant was discarded. The pellet was resuspended in the same final concentration of ammonium sulphate, centrifuged at 20,000 g for 30 min and the pellet was resuspended in PBS. This sample was dialysed against PBS (2x 2 1) or the buffer system that was used subsequently.

3.2.15. B The boric acid precipitation method

The spent culture medium of a hybridoma cell line was added to 0.5% (w/v) boric acid (20 vol). The mixture was stirred gently for 1 h at 4°C and centrifuged at 10,000 g for 15 min at 4°C. The supernatant was discarded and the pellet resuspended in PBS. To this, 0.5% (w/v) boric acid (20 vol) was added and the above procedure repeated. The final pellet was resuspended in PBS or the buffer required for the subsequent use of this immunoglobulin preparation.

3.2.15. C The euqlobulin precipitation method

The spent culture medium of a hybridoma cell line was concentrated approximately ten fold with an Amicon membrane concentrator (molecular weight cut off = 30 kDa).

The concentrated media was dialysed against 0.1 mM K- phosphate, pH 7.0 (4 x 100 vol) at 4°C. The dialysed solution was then centrifuged at 20,000 g for 20 min and the pellet resuspended in PBS.

152 3.2.16 Purification of IaM bv fast protein liquid chromatography (FPLC)

The following buffers were prepared to set up an ionic strength gradient. Buffer A: 1:10 dilution of Buffer B. Buffer B: 26.12 g/1 K2HP04, 13.205 g/1 NaH2P04, pH 6.5; Spent culture medium, or the product of any of the precipitation methods described in 3.2.15, were extensi vely dialysed against buffer A and a maximum of 25 mg protein in 10 ml buffer A was loaded by means of a superloop onto a Pharmacia Mono Q ion-exchange colummn at a rate of 1.5 ml/min. The column was washed with Buffer A (10 ml) at 1.5 ml/ min. The immunoglobulins were eluted with a 0 - 100% (v/v) gradient of buffer B (15 ml) at a rate of 1.5 ml/min, and fractions (2 ml) were collected. Protein elution was monitored at A = 280 nm. All fractions were assayed by ELISA (3.2.10.A).

3.2.17 Purification of IaM bv DEAE Seohacel

ion-exchange chromatography

A DEAE Sephacel ion-exchange column (4 x 30 cm) was packed and equilibrated in Buffer A as in 3.2.16. All procedures were carried out at room temperature. The antibody containing sample was dialysed against Buffer A

(2 x 2 1) and a maximum of 0.5 g protein were loaded onto the column. The column was washed with Buffer A (400 ml) .

The immunoglobulins were eluted with a step gradient (0.05

153 M, 0.1 M, 0.15 M, 0.20 M, 0.25 M, 0.30 M K-phosphate pH 7.0). Fractions (5 ml) were collected and assayed by ELISA (3.2.10.A).

3.2.18 Purification of IaM bv ael filtration

Ultragel ACA 34 was used to pack a gel filtration column (2 x 1000 cm). The column was equilibrated in 20mM K-phosphate pH 7.4 overnight at 20 ml/h. The antibody containing sample (2ml) was loaded and run through the column at 20 ml/h. Fractions (5 ml) were collected and assayed by ELISA (3.2.10.A).

3.2.19 The production of antibodies in the absence

of FCS

Cells of a hybridoma cell line producing the antibody of interest were grown and expanded in complete DMEM. After the cell cultures were expanded to the final required medium volume, the latter was centrifuged at 1500 rpm for 5 min. The supernatant was completely removed and the pellet was resuspended in the same volume of DMEM without FCS. The cells were allowed to grow for 3-4 days or until they had all died. The dead cells were removed by centrifugation at 3000 g for 15 min and the supernatant filtered through a 0.45 urn filter.

154 3.2.20 The immunocvtochemical staining of brain tissues Fresh bovine brain tissue was cut into 1 cm2 cubes and either fixed in 4% (w/v) paraformaldehyde for 24 h or not treated. Fixed and non-fixed tissues were glued to metal blocks immersed in dry ice and allowed to freeze for

30 min. The blocks were transferred to the -20°C chamber of a cryostat and left to equilibrate for 2 h. Sections (5-10 urn thick) were cut with the cryostat and placed on “gelatinized slides” which had been immersed in 0.3% (w/v) gelatin, 0.25% (w/v) chromium aluminium in water and dried. The tissues on the slides were allowed to dry for 1 h at room temperature and were subsequently stored at - 70°C until further use. When required, the slides were equilibrated to room temperature and were treated by one of the following methods: (i) no treatment

(ii) acetone fixation (20 sec or 5 min),

(iii) methanol fixation (2 min), (iv) paraformaldehyde fixation (2 min)

(v) acetic acid fixation (2 min).

The latter two fixing conditions were also followed by incubation with 0.1% (w/v) Triton X-100 in PBS. The slides were placed horizontally in a humid chamber, 10% (v/v) FCS in PBS (200 ul) was added and incubated for 30 min. The FCS-PBS was removed and the slides were incubated with undiluted spent medium or 1:100 ascites fluid for 1 h at room temperature or overnight at 4°C. The slides were

155 washed with PBS (3 x 200 ul) and incubated with secondary antibodies which were either biotinylated species-specific anti-immunoglobulins (1:100 dilution in PBS-FCS), or a

chromophore-conjugated species-specific anti immunoglobulin (1:40 dilution in PBS-FCS), for 1 h at room temperature. The slides were then washed with PBS (3x 200 ul) and if biotinylated second antibodies had been used, the slides were further incubated with streptavidin horseradish peroxidase (1:300 dilution in PBS-FCS) for 1 h at room temperature. The slides were washed with PBS (3x 200 ul) and developed with diaminobenzidine as substrate as described in 3.2.10.D. All slides were mounted in 50% (v/v) glycerol in PBS, which also contained antifading agent for the slides stained with fluorescent antibodies.

The coverslips were sealed with nail varnish.

3.2.21 The immunoprecioitation of the GABA-R with monoclonal antibodies

Soluble receptor samples (50 ul, purified or undiluted

crude extract) were incubated with a range of dilutions of

antibodies (50 ul) for 3 h at room temperature on a rotatory shaker. Immunobeads (50 ul) were added and

incubated as above for 1 h. Assay buffer (200 ul, see 2.2.5.B) was added and the tubes centrifuged in an

Eppendorf centrifuge at 10,000 g for 5 min. The supernatants were aliquoted into triplicate assay tubes.

156 The pellets were washed with assay buffer (2 x 500 ul), resuspended in assay buffer (300 ul) and aliquoted into triplicate assay tubes. Both the supernatants and the pellets were assayed for [3H]flunitrazepam and [3H]muscimol binding activity as described in 2.2.5.B.

3.2.22 The synthesis of Immunoaffinitv purification columns

3.2.22.A The coupling of immunoglobulins to cyanogen bromide Seoharose 4B

Immunoglobulins were bound either to a freshly prepared cyanogen bromide Sepharose column or to a preactivated cyanogen bromide Sepharose 4B column. Sepharose 4 B gel was placed in a measuring cylinder and allowed to settle overnight. Cyanogen bromide (0.75 g) was dissolved in dry acetonitrile (0.75 ml) and stirred for 30-45 min until completely dissolved. The Sepharose gel (15 ml equivalent settled volume) was extensively washed with H20 on a sintered glass funnel and resuspended in 2 M N^C03 (15 ml). Cyanogen bromide/acetonitrile (0.75 ml) prepared as described above, was added, and the mixture was stirred for 3 min before the gel was washed in sequence under vacuum with: a) 0.1 M NaHC03/Na2C03 pH

9.5 (200 ml); b) H20 (200 ml); c) as in a).

Alternatively pre-activated cyanogen bromide Sepharose 4 B freeze-dried powder (4 g) was swollen in 1 mM HC1 for

157 15 min to yield 14 ml gel. This was washed with 1 mM HCl (500 ml) on a sintered glass filter, followed by coupling buffer (5 ml) which was 0.1 M NaHCC^, pH 8.3, 0.5 M NaCl.

Either of the two above-described gels were transferred immediately to the antibody containing sample

(7 ml) which had previously been dialysed against coupling buffer and whose optical density at A = 280 nm had been measured. The coupling reaction was allowed to continue for 2 h at room temperature or overnight at 4°C with continuous rotation. The gel was allowed to settle, the supernatant was decanted and its optical density determined as above. The gel was resuspended in 0.2 M glycine, or 1 M ethanolamine, pH 8.0 (20 ml), and rotated for 2 h at room temperature. The gel was washed with the coupling buffer (5 x 30 ml) followed by 0.1 M glycine, pH 2.5 (3 x 30 ml). Prior to use, the gel was washed with the elution buffer (50 ml) (3.2.24), followed by the buffer (50 ml) which was used to apply the sample to the column.

3.2.22.B The coupling of immunoglobulins to Affioel

Affigel (8ml) was washed on a sintered glass funnel under vacuum with ice cold water (200 ml) followed by coupling buffer (20 ml) which was 20 mM K-phosphate pH

8.0. The gel was transferred to the antibody containing solution (10 ml) which had been dialysed against coupling buffer and its optical density measured at A = 280 nm. The gel was incubated overnight at 4°C on a rotatory shaker.

158 The gel was allowed to settle, the supernatant was removed and its optical density was measured at A = 280 nm. The gel was resuspended in 1 M ethanolamine (pH 8.0) and incubated on a rotatory shaker for 1 h at room temperature. The gel was transferred to a column and, before use, it was pre-eluted and regenerated as described in 3.2.22.A.

3.2.22. C The coupling of immunoglobulins to protein A Sepharose 4B

Protein A Sepharose matrix was prepared as in 3.2.14. For the coupling of IgM monoclonal antibodies, anti-mouse immunoglobulins (40 mg in PBS) were first applied to the protein A Sepharose column. The column was washed with PBS (100 ml) and spent hybridoma medium (500 ml) was circulated through the column for 16 h at 4°C . The column was washed with 0.1 M borate pH 8.0 (100 ml) and the gel was transferred to a tube containing borate buffer (10 ml) . Dimethyl-pimelimidate (50 mg) was added immediately to the gel which was rotated for 1 h at room temperature.

The gel was washed with borate buffer (20 ml) , and incubated with 1 M ethanolamine pH 8.0 for 1 h at room temperature. The gel was pre-eluted and regenerated as in

3.2.22. A.

159 3.2.23 The biotinylation of immunoglobulins

This procedure was carried out with the aim of constructing an avidin-biotin-immunoglobulin affinity- column . Immunoglobulins from ascites fluid (3 ml) (3.2.13)

were partially purified by euglobulin precipitation

(3.2.15.C). Biotin-H-hydroxysuccinimide ester was

dissolved in dimethylformamide (1.7 mg/ml) and added to the above antibody preparation (20 ul dimethylformamide/ml

antibody preparation), and incubated for 4 h at room temperature. The solution was dialysed against PBS (2 x 500 ml) and stored at -20°C until use.

3.2.24 Tmmunoaffinitv purification of the GABA-R

Bovine brain cortex membranes prepared as in 2.2.2 were solubilized as described in 2.2.4 with Na- deoxycholate or Triton X-100. In either case, the soluble extract was diluted 1:10 with wash buffer which was 10 mM

K-phosphate, pH 7.4, 200 mM KC1, 10% (w/v) sucrose, 0.2%

(w/v) Triton X-100. The diluted soluble extract (200 ml) was applied to one of the above described immunoaffinity

columns at a rate of 40 ml/h at room temperature. The

column was washed with the above wash buffer (200 ml) at 60 ml/h and then eluted with one or more of the following

elution buffers a) 50 mM glycine, pH 11, 0.2 M KCl, 10%

(w/v) sucrose, 0.2% (w/v) Triton X-100, b) same as in a)

except at pH 3.0. c) 10 mM K-phosphate, pH 7.4, 3.5 M K-

160 thiocyanate, 10% (w/v) sucrose, 0.2% (w/v) Triton X-100. All elutions were carried out at 10 ml/h. High and low pH eluates were neutralized with 1 M Tris pH 7.4. K- thiocyanate eluates were dialysed against wash buffer. The eluates were assayed for [3H]flunitrazepam binding

activity (2.2.5.B) and were analysed by SDS-PAGE

(2 .2 .6 ).

3.2.25 The coupling of a synthetic peptide to keyhole limpet haemocvanin

The C-terminus (14 amino acids) of the a3 subunit of

the GABA-R (Levitan et al., 1988) was synthesized with an additional N-terminal cysteine (AFRC, Babraham). The sequence was CVNRESAIKGMIRKQ. Keyhole

limpet haemocyanin (KLH) was dissolved in 10 mM K- phosphate, pH 7.2, (20 mg/ml) and was dialyzed against the same buffer (2x 1 1). The above phosphate buffer (110 ul) was added to the KLH solution (200 ul). Maleimido benzoic

acid N-hydroxysuccinamide ester (MBS) (3 mg/ml in

dimethylformamide, 170 ul) was slowly added to the KLH

solution and gently agitated for 1 h at room temperature.

The sample was applied to a P30 column (20 ml) that had been pre-equilibrated with 50 mM K-phosphate, pH 6.0.

Fractions (1 ml) were collected and measured at A = 280 nm. Protein containing fractions (typically fractions 6 - 8) were pooled. The lyophilized peptide (10 mg) was

resuspended in the above 10 mM K-phosphate buffer (1.5 ml)

161 and added to the above pooled fractions. The mixture was incubated for 3 h at room temperature with gentle agitation, and stored at -20°C until use.

162 3.3 Results

3.3.1 The characterization of anti-native-GABA-R

polyclonal antibodies

The anti-native-GABA-R polyclonal antibodies raised in rabbits by Dr. Stephenson (Stephenson et al., 1986a), were used in Western blot analyses of the purified receptor. It can be seen in Figure 3.3.1 and in Stephenson et al. (1986a), that the sera of the two immunized rabbits tested (rabbit 1 and rabbit 3) stained the a subunit of the bovine GABA-R. The sera were used at dilutions in the range of 1:2 - 1:200 and all gave qualitatively similar results. At dilutions > 1:200, no signal was obtained.

Protein A purified IgG from rabbit 3 serum (Stephenson et al., 1986a) gave an identical staining pattern (see Figure 3.3.1) at dilutions of 1:2000. Western blots were also performed on Zetabind filters and again, only staining of the a subunit was detected. The background stain was higher in Zetabind filters compared to nitrocellulose filters.

In order to establish the minimum amount of receptor that could be detected by the respective antibodies, parallel lanes of an SDS-PAGE gel were loaded with decreasing amounts of purified receptor which ranged

163 between 0.03 - 3.0 ug protein. These were electrophoresed, blotted and immunostained with purified IgG from rabbit 3 serum (1:1000 dilution). The least receptor that could be detected was 0.1 ug protein (results not shown).

3.3.2 Anti-carboxvmethvlated-GABA-R (cam-GABA-R) polyclonal antibodies

The antigen (cam-GABA-R) was prepared as described in 3.2.1.D and it showed the same subunit composition as the native receptor when analysed by silver staining on SDS- PAGE. Figure 3.3.1 shows the results of a Western blot carried out with the serum from the bleed of the third injection of cam-GABA-R. This polyclonal serum also recognised only the a subunit. The serum was tested in immunoprecipitation studies but it did not precipitate any of the receptor ligand binding activities.

3.3.3 Monoclonal antibody studies

At the beginning of this part of the project there was one anti-GABA-R monoclonal antibody, 1A6, available in the laboratory (Mamalaki et al., 1987) and three cell lines which originally produced antibodies against the GABA-R before but not after the final cloning steps (Dr.

Mamalaki, personal communication). Also available were six further cell lines that had given a weak positive signal in the initial screening of the fusion performed by Dr.

Mamalaki (Mamalaki et al., 1987) which had been frozen

164 prior to cloning. The following sections deal with the production of new monoclonal antibodies and the characterization of both the latter and those that were already available.

3.3.4 The titres of the mice injected with GABA-R

The immune response of the mice injected with GABA- R was measured by the ELISA assay (3.2.10.A) and was expressed in the form of antibody titres. The titre is defined here as the serum dilution that gives the half- maximal optical density measurement in the ELISA assay.

The determination of a representative antibody titre is shown in figure 3.3.3.

Table 3.3.1 shows the antibody titres obtained with a variety of different immunization strategies described in 3.2.1. The titres shown are of the sera collected 6-7 days after the second and third boosters. When possible the serum was also collected from the animal after sacrifice and removal of its spleen prior to monoclonal antibody production. These latter titres are not shown in Table 3.3.1 because they are not directly comparable since they were determined only 4 days after the last immunization. Some of these titres will, however, be referred to in the text.

Table 3.3.1 shows that mice injected with 40 ug of purified GABA-R per injection (mice 3-6) obtained antibody

165 titres of 1:100 with the exception of mouse 3 which had an antibody titre of 1:1000. Mice 5 and 6 received the last booster prior to antibody production intrasplenically and

intravenously respectively. However these procedures did

not improve their antibody titre. Consistently higher antibody titres (1:800) were obtained in mice that were injected with 200 ug of purified GABA-R per injection (mice 9-13 and 17).

Purified GABA-R was also injected in a heat- denatured state (mice 7,8) and SDS-denatured state (mice 14,21,22) and both antigens induced, in the recipient mice, titres < 1:50. The titres were measured with the described ELISA assay (3.2.10.A) except that the

microtitre plates were coated with purified GABA-R treated

in the same way as for the injection. Mice injected with 50 ug of alum-precipitated GABA-R

per injection (mice 19, 20) developed a relatively high antibody titre (1:600) when compared to mice injected with

similar amounts of native GABA-R (mice 3-6). One of the mice injected with alum precipitated GABA-R (mouse 18) developed the highest titre against GABA-R recorded in

this study (1:3000). This mouse together with mouse 3 are

the only examples, from all the mice tested, where

individual mice developed antibody titres higher than that developed by the other mice injected with the same antigen.

166 The purified GABA-R was also presented to the mice bound to DEAE-Sephacel (mice 15,16) and to nitrocellulose filters (mice 23-28), as described in 3.2.1. However in both cases the antibody response was < 1:50 when measured in the ELISA assay or in Western blots. Mice injected with ovalbumin or with KLH-coupled peptide produced titres > 1:10,000 after the second

injection. These titres were measured using the ELISA assay described in 3.2.10.A except that the microtitre plates were coated with the same antigen that had been injected in the mice. Mice that were injected with Agarose-Ro 7-1986 (150 mg dry weight) (mice 29-31) developed antibody titres of 1:600. The object of this experiment was the production of anti-idiotypic antibodies, therefore the sera from these mice were collected 12 days rather than 6-7 days

after the immunization, and were tested against GABA-R by the ELISA assay (3.2.10.A). The same sera were also used in [3H]flunitrazepam inhibition binding assays to the

receptor and to bind directly to the [3H]flunitrazepam

ligand itself. It was shown that no [3H]flunitrazepam

could be bound to the antibodies as detected in the

PEG/filtration binding assay (2.2.5.B). However when

[3H]flunitrazepam binding to a soluble crude preparation was assayed, it was shown that the sera could specifically reduce by 25% + 3% (n = 2) [3H]flunitrazepam binding, but only if the sera had been pre-absorbed with RO 7-1986/1

167 agarose for 1 h at room temperature. Table 3.3.1, as stated above, shows the antibody titres of the sera taken after the second and third injection.

Several mice (mice 7, 11, 12, 15, 19, 20) received further injections but their antibody titres against their respective antigen did not increase.

3.3.5 The screening procedures of anti-GABA-R

monoclonal antibodies

The monoclonal antibody 1A6 was identified by using the

ELISA screening assay described in 3.2.10.A. However in order to obtain monoclonal antibodies with a large range of binding properties several assays systems were tested. These are described in diagrammatical form in Figure

3.3.4.

3.3.5.A The ELISA screening assay

The monoclonal antibody 1A6 was used in every ELISA

performed as a positive control for the assay. The sensitivity of the assay was improved by the reduction of

the Triton X-100 concentration to 0.02% (w/v) in the

DEAE-Sephacel column eluting buffer (2.2.4) and by the

increase in the incubation time of the receptor on the

ELISA plates to 48 h. Under these conditions the spent medium from a 1A6 culture gave at a 1:500 dilution, a

signal 30% higher than to GABA-R coated for 12 h or coated in the presence of 0.05% (w/v) Triton X-100.

168 The receptor bound to the ELISA plates was assayed for ligand binding activity as described in 3.2.10.B in

order to determine whether the immobilized receptor

retained its ligand binding properties. Table 3.3.2 shows

representative results obtained from two such experiments.

It can be seen in Table 3.3.2 that storage of the receptor at 4°C and in the PBS diluted form, caused a 18% ± 2 % (n - 2) loss of the [3H]flunitrazepam binding activity compared to receptor stored at -20°C. After the overnight incubation, 37% ± 4 % (n = 2 ) of receptor had bound to the plates as calculated from the reduction of

[3H]flunitrazepam binding in the supernatant removed from the wells. When the wells were assayed, it was found that [3H]flunitrazepam could specifically bind to the wells

coated with receptor as shown in Table 3.3.2. Nearly 100% of the [3H]flunitrazepam sites could be accounted for.

3.3.5.B The dot blot assay

The dot blot assay was tested as an alternative

screening system to the ELISA assay with the aim of developing a more sensitive screening assay. Figure 3.3.5 shows a chequerboard test designed to establish the lowest possible concentration of GABA-R required to obtain a signal from the highest possible antibody dilution. The monoclonal antibody 1A6 was diluted up to 1:1000 and the

receptor up to 1:100. The results show that a clear signal

169 above background was obtained at a dilution of 1:100 and 1:15 of the antibody and the GABA-R respectively. This was not an improvement on the ELISA assay for either the sensitivity or the amount of receptor needed.

3.3.5.C The screening of monoclonal antibodies with a soluble antigen assay

The establishment of an immunoprecipitation screening assay (3.2.21) was hindered by the fact that the only monoclonal available at the time, 1A6 did not immunoprecipitate the crude soluble receptor. Thus it could not be used to test the sensitivity or the viability of the assay. One fusion was screened with this assay but no positive hybridoma colony was detected although a monoclonal antibody was isolated from the same fusion using the ELISA screening assay. An alternative method used to screen the hybridomas for the production of antibodies that can recognise the receptor in a soluble form, was the solid phase radio immunoassay (3.2.10.C). However no decrease of

[3H]flunitrazepam binding activity could be detected in crude soluble samples applied to wells coated with either

1A6 or an anti GABA-R polyclonal antibody (3.3.1); nor could any [3H]flunitrazepam binding activity to these wells be detected.

170 3.3.6 Cell fusions for the production of monoclonal antibodies against the GABA-R

3.3.6.A Fusions carried out with the 36% PEG method

Table 3.3.3 shows the results o£ the fusion experiments performed. The first two fusions were carried out with animals which had been injected intrasplenically. The main reason for the choice of this method was that the fusion experiment can be carried out 4 days after the single injection whereas normal immunization protocols take 2-3 months. The spleen cells of mice 1 and 2 were fused to

SP2 myeloma cells with the 33% PEG method (3.2.7.A). This was the method currently used in the laboratory and it had given positive results for antibodies raised against acetylcholinesterase (Tsim et al., 1988). The fusions yielded 230-250 hybridomas and in each case positive wells showing a signal 2-3 times above background in the ELISA assay, were detected upon the first screening of the 96- well plates. The cells from these wells were expanded into

24-well plates and re-screened after two days. Most of these wells were not positive. Three that were positive initially, also reverted when the cells were further expanded.

This problem of instability of the hybridoma cell lines was persistent. It is shown in Table 3.3.3 that while all the fusions carried out with the 33% PEG method produced initially positive hybridomas, no stable hybridoma cell

171 line could be isolated. The antibody titres of the mice that were used for these fusion experiments varied between 1:100 - 1:1000 (see Table 3.2.1) but had no effect on

either the number of monoclonal antibodies or on the number of initially positive wells that were obtained

(Table 3.3.3). The production of more stable hybridoma cell lines was pursued by the use of the myeloma cell line NS0 as a fusion partner for the spleen cells of mice 5 and 6. The spleen cells of mouse 6 and the NS0 cells were, after the PEG fusion, distributed into 24-well plates. The reason for this was that newly formed hybridomas tend to be more stable if grown in an environment where other cells are

growing (De Bias et al., 1981) . However the number of

hybridomas produced was not increased. Difficulties were

experienced in the handling of the NS0 cell line? these cells adhered to the culture plates which complicated the maintenance of the cells in an exponential growth rate before the fusion.

3.3.6.B Fusions carried out with the 50% PEG method

In order to improve the fusion technique, it was modified as described in 3.2.7.B. The major changes were

(i) PEG was purchased in a sterile form and thus did not require autoclaving, (ii) PEG was used at a concentration of 50% (w/v) . (iii) The cells were exposed to PEG for 1

172 min before the latter was gradually diluted with DMEM. As it can be seen in Table 3.3.3 these changes increased up to 4 fold the number of wells containing growing

hybridoma colonies. According to the Poisson distribution

the average number of hybridomas per well is 0.5, 1, 2, 3 respectively for when 39%, 63%, 86% and 95% of the wells show growing colonies (De Bias et al., 1981). Therefore, as the cells were distributed after the fusion in 1000 wells and approximately 80% of the wells contained

hybridomas, it is estimated that up to 1500 hybridomas

were produced in every fusion with the 50% PEG method, as compared to the 200-300 produced with the earlier 33% PEG method. After the 50% PEG fusion method was tested on an uninjected animal (mouse T4), it was used to fuse the

spleen cell of a mouse (mouse 14) injected intrasplenically with SDS-denatured receptor. About 1500

hybridomas were produced and the culture media of 18 wells

were positive on the first screen. The ELISA screen used was that described in 3.2.10.A except that the microtitre plates were coated with SDS-denatured receptor. Two hybridoma cell lines that were giving signals 5-6 times

above background were successfully cloned by two rounds of the "safe” serial dilutions (3.2.12.B). These two monoclonal antibodies however did not recognise the

receptor in Western blots and showed a weak reaction against a band of approximately 21 kDa which is sometimes

173 visible in the silver stain of receptor preparations following SDS-PAGE. The monoclonal antibodies were therefore tested in the ELISA assay where soybean trypsin

inhibitor was coated as the antigen. Both antibodies were positive in this test. Furthermore, when ELISA plates were coated with GABA-R purified in the absence of the trypsin inhibitors, the monoclonals were no longer positive in the ELISA assay. All the evidence therefore indicated that these antibodies were directed against soybean trypsin inhibitor. The spleen cells of mice 17 and 18, injected with

native receptor over long intervals and alum precipitated receptor respectively (3.2.2.E), were fused to SP2 cells with the 50% PEG method. The number of hybridomas obtained

were 1400-1500 and from the fusion of mouse 17 a relatively high number (21) of initially positive wells

were detected. From each of the two above fusion experiments, an anti-GABA-R antibody was succesfully

cloned. The anti-native-GABA-R was named 86G and the anti-

alum-precipitated-GABA-R was named 102D.

3.3.7 In vitro techniques for the production of

monoclonal antibodies against the GABA-R

In order to increase the rate of isolation of new

monoclonal antibodies against the GABA-R it was decided

that new strategies for the production of monoclonal

174 antibodies had to be tried. Therefore while new immunization methods like nitrocellulose implantation, alum precipitation, SDS denaturation, were being carried out, the production of monoclonal antibodies by in vitro immunization and in vitro selection of the appropriate antibody producing speen cells, known as "panning", was attempted. Preliminary studies for the design of a suitable in vitro procedure needed to be carried out, thus three mice (Tl, T2 and T3) were injected with ovalbumin and titres > 1:10,000 were obtained (see Table 3.3.1). Four days after the final boost, the animals were sacrificed, and the spleens incubated as described in 3.2.8. The effects of ovalbumin, LPS, dextran sulphate, Triton X-100, and cholate, used at the concentration described in 3.2.8, were tested on the spleen cell culture. The number of viable cells present in the cultures before and after 4 days of culture were measured with a haemocytometer. Table

3.3.4.A shows that the addition of ovalbumin to the culture produced an increase in the number of spleen cells from ovalbumin-immunized mice. Table 3.3.4.A also shows that the mitogens LPS and dextran sulphate increased to the same extent the recovery of the number of cells in the cultures of ovalbumin-immunized and non-immunized animals. This increase was higher than that induced by ovalbumin. It was also shown that while Triton X-100 (0.002% w/v) was tolerated in the cultures, cholate

175 (0.005% w/v) had a lethal effect. After four days, cells 4-8 x larger than spleen cells were visible in the cultures. These were believed to be blast cells which are antibody producing cells. When these cells were counted (see Table 3.3.4.A) it was shown that these were most abundant in cultures supplemented with LPS and dextran sulphate. Also their numbers were increased by the addition of ovalbumin to the spleen cell cultures from ovalbumin-immunized mice. Spleen cells from mice immunized with native GABA- R (mice 11, 12 and 13, Table 3.3.1) were also cultured in vitro. Table 3.3.4.A shows that in contrast to the results obtained with cells from ovalbumin-immunized mice, the addition of GABA-R did not increase either the number of spleen cells recovered, nor the number of blast cells observed. A further approach used for the isolation of monoclonal antibodies was to select the spleen cells by the panning technique (3.2.9). Initial experiments were carried out with spleen cells from ovalbumin-immunized mice, and it was found that the most efficient method of recovering the cells attached to the antigen-coated culture plates was to remove them with a sterile scraper as opposed to the use of trypsin or acidic medium as described in 3.2.9. The experiment was then repeated using GABA-R as antigen and the selected cells were used

176 in fusion experiments. Table 3.3.4.B shows the results of the fusion experiments of in vitro cultured cells. The cells were fused to SP2 cells by the 33% PEG method because the 50% method had not been developed at this time. Spleen cells from the immunized mice were pooled and the number of cells equivalent to one spleen were used for each panning or fusion experiment. The number of hybridomas produced was lower than that obtained in the previous fusion experiments (see Table 3.3.3 and Table 3.3.4.B). Where the panning experiments were performed, the number of cells recovered was approximately 106. Hybridoma colonies secreting anti-ovalbumin antibodies were obtained but no monoclonal antibodies against the GABA-R could be produced when the experiment was repeated using the purified GABA-R as antigen. After the 50% PEG fusion method was developed, further in vitro experiments were carried out. Spleen cells from new-born BALB/c mice (2 days old) were immunized in vitro with purified GABA-R in the presence of mitogens and the adjuvant MDP (3.2.8). More than 800 hybridomas were obtained by the fusion of the spleen cells of 6 new-born mice with SP2 cells by the 50% PEG method (see Table 3.3.4.B). Among these, two hybridoma colonies were initially positive but subsequently lost their ability to produce anti-GABA-R antibodies.

177 3.3.8 The studies of the production of anti-idiotypic monoclonal antibodies

The spleen cells of mouse 29 that had been injected with Ro 7-1986/1 agarose, were fused using the 50% PEG method. The estimated 1000 hybridomas obtained were

screened by the ELISA assay with purified GABA-R as antigen (3.2.10.B). One positive hybridoma colony was initially detected but again it lost its antibody producing capacity in the initial cloning steps. The supernatant of all the hybridoma colonies were also tested in a competition binding assay in order to test whether the supernatants were able to displace [3H]flunitrazepam binding to a crude soluble GABA-R preparation. No positive wells were detected with this method.

3.3.9 The cloning of hvbridomas cell lines

The limited serial dilution method that had been used for the cloning of 1A6 (Mamalaki et al., 1987) was used here also for the cloning of the hybridomas that had been produced in the same fusion experiment (Mamalaki et

al., 1987 and 3.3.3). From nine hybridomas six were not positive upon thawing and the others lost their ability to give a positive signal after one or two limiting dilution

steps. One particular cell line was subjected to cloning three times by restarting each time from a positive stage. However with each attempt, it became negative in the ELISA

178 assay after the same number of cloning steps. Two of the above cell lines were subjected to soft agar cloning (3.2.12.C) but no stable positive clones were obtained by this method either. The hybridomas produced in this study, 102D and 866, were screened by two cycles of the "safe" serial dilution (3.2.12.B) which allowed the isolation of the clones within 2—3 weeks. Less purified GABA-R was required to coat the microtitre plates by the use of the "safe" serial dilution cloning method. This is because a smaller number of hybridoma supernatants were assayed here than with the limiting dilution or the soft agar cloning methods.

3.3.10 The production of ascites fluid

Hybridoma cells of the 1A6, 102D and 86G cell lines were all injected into pristane-primed mice for the production of ascites fluid containing the respective antibodies (3.2.13). Ascites fluids with titres of above 1:10,000 were obtained in all cases as determined by

ELISA. The ascites fluids were used for immunoprecipitation, Western blots and in immunocytochemical studies.

3.3.11 The immunoprecipitation of the GABA-R

Immunoprecipitation studies of the purified receptor and the crude receptor were carried out with the two

179 monoclonal antibodies 102D and 86G. In all cases both the presence of [3H]muscimol and [3H]flunitrazepam binding sites in the pellet and the decrease of the same sites from the supernatant were measured. Initial results showed that the purified receptor was immunoprecipitated by both antibodies in an antibody concentration-dependent manner. Table 3.3.4.C shows the percentage of [3H]flunitrazepam and [3H]muscimol binding sites precipitated by a serial dilution of 102D and 86G ascites fluids. Control assays were carried out in which either the receptor, the primary antibodies or the immunobeads were omitted. Some immunoprecipitation (5% of total binding sites) occurred

only in the case where the primary antibodies were omitted. The addition of 1% BSA to the reaction mixture did not change either the non-specific immunoprecipitation nor the total number of sites precipitated. Immunoprecipitation of Triton X-100 or Na- deoxycholate crude soluble extracts however did not precipitate any GABA-R binding activity. Furthermore, the

antibodies did not immunoprecipitate GABA-R binding

activity from a soluble extract which had been partially purified by ion-exchange chromatography as in the receptor purification (2.2.4). Also, a non-relevant anti

acetylcholinesterase monoclonal antibody (courtesy of Dr.

Tsim) showed an identical immunoprecipitation profile to that obtained with both the anti-GABA-R antibodies.

180 3.3.13 The purification of the monoclonal antibodies

Monoclonal antibodies produced by the tissue culture method (3.2.4) must be purified before they can be used in the construction of an immunoaffinity column.

The protein A purification of the monoclonal antibodies

102D and 86G was performed as described in 3.2.14. Table 3.3.5.A shows that after overnight recirculation of 500 ml of spent medium, higher yields of purification were obtained for 86G than for 102D. The culture media were reapplied twice more to the protein A Sepharose column and more immunoglobulin molecules were thus isolated. The media containing the monoclonal antibodies were assayed by

ELISA before and after their application to the protein A Sepharose column. Table 3.3.5.A shows that the optical absorbance obtained from the assay of a 1:500 dilution of the media before and after purification was reduced more in the 86G medium than in the 102D one. The lower binding affinity of 102D to the protein A column is an indication that 102D belongs to the IgG3 subclass, which has the lowest affinity for protein A of the IgG subclasses. The purified 102D and 86G antibody preparations showed on the ELISA assay titres of 1:500 and 1:800 respectively. These antibody preparations were analysed by SDS-PAGE and were shown to be pure and the heavy chains molecular weights were 52 kDa and 58 kDa for 86G and 102D respectively. A typical purified preparation of 86G is shown in Figure

181 3.3.6.

The purification of the 1A6 monoclonal antibody was more difficult because IgM molecules do not bind to protein A. It was necessary therefore to utilize other methods of purification. A standard protocol has not been developed as most of the methods reported in the

literature aim at the separation of IgM from other

immunoglobulins present in the serum and not at achieving purification of IgMs (Sampson et al., 1984.) One initial approach that was used was the precipitation of the IgM from the medium. The methods used were ammonium sulphate, boric acid, and euglobulin precipitation (3.2.2). Each method was tested for its efficiency by measuring with the ELISA assay the amount of

antibody left in the culture media after the precipitation, and for its specificity by SDS-PAGE of the precipitated product. Table 3.3.5.B shows that maximum precipitation was obtained with 50% ammonium sulphate and that a purer product was obtained using the euglobulin precipitation. It was however evident from the extent of purification achieved, that precipitation procedures could only be used as the first step for the isolation of these antibodies. Ion-exchange chromatography of the 50% (w/v) ammonium sulphate precipitation product of the spent 1A6 medium was carried out with both an FPLC column (3.2.16) and a 500

182 ml DEAE-Sephacel column (3.2.17). The FPLC column which had a maximal capacity of 25 mg protein, allowed faster and more reproducible partial purifications whilst the DEAE-Sephacel column allowed a larger scale purification to be carried out. Purity to homogeneity of the IgM molecules was however not obtained with either method (see Figure 3.3.6.A). IgMs were further purified by gel filtration

(3.2.18). The positive fractions from the ion-exchange chromatography were concentrated with an Amicon membrane concentrator and applied to a column of Ultragel ACA 34 . The fractions that were positive by the ELISA assay, however showed on SDS-PAGE a similar profile to that of the ion-exchange purification (Figure 3.3.6.A). As most of the non-immunoglobulin proteins in culture medium derive from the FCS, the cells were grown in its

absence as described in 3.2.19. The cells, after transfer to the FCS free medium, died within three days but IgM could still be detected by ELISA. The IgM molecules produced in FCS-free medium were then precipitated by the

euglobulin method (see Table 3.3.5.B). SDS-PAGE analysis

of the precipitate showed that IgM was the predominant protein and only a few contaminating proteins were still present (Figure 3.3.6.B). This method proved to be the best and fastest for obtaining partially purified IgMs. Approximately 10 mg of IgM protein from 1 1 of culture medium were obtained. These partially purified IgM

183 monoclonal antibodies were used for the construction of an affinity column.

3.3.14 The construction of t h e -immunoaffinitv c o l u m n s

Immunopurification columns were constructed with all the three anti-GABA-R monoclonal antibodies available, 1A6, 102D and 86G, according to the methods described in

3.2.22. Table 3.3.6 summarises the results obtained. For each column the percentage of immunoglobulins present in the medium and absorbed by the matrix was measured either by monitoring the change in OD at A = 280 nm of the antibody containing coupling medium, or by titration curves of the latter in the ELISA assay. Several immunoaffinity columns were thus constructed which varied both in the percentage of antibody that had been absorbed by the matrix from the medium, and in the concentration of coupled immunoglobulins per ml of matrix. These are two independent parameters that can affect the functioning of the immunoaffinity column. Bovine cerebral membrane soluble extracts were applied to the columns as described in 3.2.24, Table 3.3.7 however shows that none of the antibody columns constructed were able to remove a detectable amount of [3H]flunitrazepam binding activity from the soluble extracts applied to them. The columns were eluted by one or more of the methods described in 3.2.24 and the eluates were analysed by SDS-PAGE. None of

184 the eluates assayed showed a polypeptide pattern similar to that of the GABA-R. In one experiment where no salts were present in any of the buffers used for the immunopurification experiments, 20% of the [3H]flunitrazepam binding activity was absorbed by the immunomatrix and it was recovered in the eluate (Table 3.3.7). The recovered ligand activity was however due to non-specific protein binding to the column. This was shown by the fact that the same SDS-PAGE protein profile was observed in the eluate and the soluble extract applied to the column (Figure 3.3.7). An IgM preparation was biotinylated as described in

(3.2.23) with the object of coupling it to a streptavidin matrix. However when the biotinylated IgMs were assayed by ELISA they were shown to have lost their ability to recognise the GABA-R.

In order to investigate the possibility that the 102D and 86G antibodies could have been affected by the protein

A purification treatment, crude ascites fluid preparations were directly coupled to Affigel 10 but again no receptor activity was removed from the soluble extract with this matrix.

3.3.15 The effect of detergent on the monoclonal antibody

binding to the GABA-R

The following experiment was carried out in order to try to understand why the monoclonal antibodies 1A6, 102D

185 and 86G were able to recognise the receptor in the ELISA but not in solution. A dot blot assay was done as described (3.2.10) except that the purified GABA receptor was diluted either in PBS containing 0.1% (w/v) Triton X- 100 or in PBS alone and bound to the nitrocellulose filters. Washing of the wells was also carried out using PBS with or without Triton X-100 respectively. Spent culture media were added either neat or supplemented with 0.1% (w/v final) Triton X-100. Figure 3.3.8 shows that the presence of the detergent blocked almost completely the antibody binding to the receptor. In a control experiment

(not shown) it was demonstrated that the detergent does not affect the binding of the GABA-R to the nitrocellulose filters.

3.3.16 Immunocvtochemical staining of brain tissue

Cryostat sections of bovine cortex, cerebellum, and hippocampus were used unfixed or fixed as described in 3.2.20. No staining was seen with the monoclonal antibodies 102D, 86G and 1A6 which resembled the GABA-R staining observed by Schoch et al. (1985) and De Bias et al. (1988). Fixed sections were also partially solubilized with Triton X-100 but again no immunostaining was observed.

186 3.3.17 Western blot analysis of GABA-R with monoclonal antibodies

The monoclonal antibodies 102D, 86G and 1A6 were tested initially in Western blots with purified bovine

GABA-R as antigen. Figure 3.3.9.A shows that 102D primarily labels the a subunit. Monoclonal 1A6 recognised both the a and the ft subunit although the a subunit was more heavily stained, as shown by Mamalaki et al., (1987). Figure 3.3.9.A also shows that 86G did not recognise the blotted receptor. The inability of 86G to recognise denatured receptor was confirmed in the earlier described dot blot experiment (see 3.3.16 and Figure 3.3.8) where 86G could not recognise receptor to which SDS had been added. The monoclonal antibodies 1A6 and 102D were used in Western blots to determine the antibody cross-reactivity between different brain areas, and between different species. Each gel lane was loaded with 200 ug of protein of the respective membrane sample prepared as in 2.2.1. The [3H]flunitrazepam binding activity of the membrane samples varied between 0.9 - 13.1 fmol/mg protein as shown in the Figure 3.3.9 legend. It can be seen from Figure

3.3.9.B that both 1A6 and 102D recognise a polypeptide of the same molecular weight as the a subunit of the GABA-R in membrane preparations of bovine cortex, cerebellum, hippocampus and striatum. The intensity of the stain of

187 4

each membrane preparation was not significantly different. On the same Western blot, the immunoreactivity of calf (12 days old) cortex and cerebellar membranes were tested but no significant difference in the staining pattern was seen between the adult and the young animal.

Figure 3.3.9.C shows the staining pattern of 102D and 1A6 of brain membranes from different species. The a subunit was stained by 102D in brain membrane preparations of cow, pig, rat, 1 day old chick, but not from the bovine adrenal medulla and dove brain. The monoclonal 1A6 gave similar results to 102D with the exception of pig cortex membranes which were not stained by 1A6.

3.3.18 The characterization of anti-a3 C-terminal- peotide antibodies

The a3 C terminal peptide was prepared as described in 3.2.25 and was injected into rabbits (500 ug/injection) and in mice (200 ug/injection) which were all bled after the second injection. A mouse immunized with the peptide was used for a fusion experiment adopting the 50% PEG method. Approximately 700 hybridomas were formed (see

Table 3.3.3) and from these 11 stable clones against the

KLH complex were isolated. However ten of these also recognised KLH alone, and only one (75B) produced antibodies specific to the peptide. In order to test whether the anti-peptide monoclonal antibody 75B could

188 also recognise the GABA-R, the spent culture medium of a 75B culture was tested in an ELISA assay with purified GABA-R as antigen. It was also used in a Western blot of purified receptor, bovine adult and young (12 days) cortex membranes and bovine cerebellum membranes. The monoclonal 75B was not positive in either of the two above assay systems.

The sera of the first bleed of the rabbits injected with the KLH-peptide showed titres > 1:10,000 in the ELISA assay with the KLH-peptide used as antigen. Figure 3.3.10 shows that when the same serum was tested in the ELISA assay against purified GABA-R, no immunoreactivity could be detected. However an antibody titre against the purified GABA-R was obtained in the animal after a further booster (Figure 3.3.10).

189 Figure 3.3.1

Immunoblotting with rabbit anti-GABA-R polyclonal antibodies.

Lane A: molecular weight markers as indicated. Lane B: silver stained purified GABA-R. Lane C and Lane D immunoblots of bovine purified GABA-R stained with antiserum and protein A purified immunoglobulins from rabbit 3. Lane E immunoblot of purified rat GABA-R stained as in D. (See text Figure 3.3,2

I ntra peritoneal injections 2 -3 boosters

bleed

Tit re

in vitro intrasplenic immunization immunization

Fusions

Diagrammatic representation of the different approaches

for the production of monoclonal antibodies tried in this

work. (See text 3.3.3).

191 Antibody titre oftitreAntibody mouse polyclonal anti-GABA-Rantisera. h ir hw s :00 (SeetitreisTheshown 1:3000. 3.3.3).text netd ih lm rcpttd AAR se al 3.3.1). Table (see GABA-R precipitated alum with injected Absorbance Units ELISA assay of the second bleed of mouse 18 which had been 18had whichsecondof mousethe bleed of assay ELISA 192 Figure3.3.3 Log10 dilution factor dilution Log10

Figure 3.3.4

ELISA

Solid phase radlolmmuno assay

Immunoprecipitation r Key x antilmouse Immunoglobulin] anti [GABA-R]

GABA-R Biotin Streptavidin- ®ea<^ Ligand horse radish peroxidase

Diagrammatic representation of the screening procedures carried out. (See text 3.3.5.).

193 Figure 3.3.5

Dot blot chequer board test. Dilutions of purified GABA-R were added to the rows, and dilutions of the monoclonal 1A6 were added to the columns.

The GABA-R dilution were A: no receptor, B: 1:5 dilution, C:

1:10 dilution, D: 1:15 dilution, E: 1:20 dilution, F: 1:50 dilution, G: 1:100 dilution, H: no receptor. The antibody dilutions were 1: no antibody, 2: neat culture medium, 3:

1:10 dilution, 4: 1:100 dilution, 5: 1:1000 dilution. (See text 3.3.5.B) Figure 3.3.6.A

A B C D

Purification attempts of the IgM monoclonal antibody 1A6.

Silver stained 10% polyacrylamide of the elution fractions that were most positive in the ELISA assay. Lane A: FPLC elution fraction. Lane B: DEAE-Sephacel elution fraction.

Lane C: Gel filtration elution fraction. Lane D commercial

IgM preparation. (See text 3.3.13).

195 Figure 3.3.6.B

kDa

3 1 —

A B

Purification of IgG and IgM monoclonal antibodies.

Lane A: protein A purified IgG antibody 102D. Lane B: euglobulin precipitation of FCS-free culture medium containing the IgM antibody 1A6. (See text 3.3.13).

196 Figure 3.3.7

kDa

68 — 60 —

45 —

31 —

A B

Attempt of immunopurification of the GABA-R with monoclonal

1A6 coupled to cyanogen bromide Sepharose-4B. Coomassie stained 10% polyacrylamide SDS-PAGE. Lane A: crude soluble extract applied to the antibody column. Lane B: pH 11 eluate from the same antibody column. (See text 3.3.14).

197 Figure 3.3.9 . A

kDa

68 — •

60 —

31 —

ABC

Immunoblot of purified GABA-R with the monoclonal antibodies.

Each lane contained 200 ul of purified GABA-R preparation and were stained with: Lane A: 1A6, Lane B

102D, Lane C: 86G. See text 3.3.17.

199 Figure 3.3.9.R

102D 1A6

kOa

68 —

CD EFABCD Q A B

Immunoblot of bovine membrane regions with monoclonal antibodies 1A6 and 102D.

All lanes contained 200 ug total protein except for lane G that contained purified GABA-R.

, , o , Lane Brain specific [ ]flunitrazepam region binding activity (pmol/mg protein)

A cortex 3.6 ± 0.4 B cerebellum 1.2 ± 0.3 C hippocampus 4.3 ± 0.3 D striatum 1.2 ± 0.4 E calf cortex 13.1 ± 0.7 F calf cerebellum 2.9 + 0.4 G purified receptor

The lanes were stained with the monoclonal antibodies as indicated. (See text 3.3.17).

200 Figure 3.3.9.C

102 D 1*6

kDa

68 —

3 1 —

ABC D E F G A B C D E F G

Immunoblots of brain cortex membranes from different species.

Each lane was loaded with 200 ug protein except for lane G that contained bovine purified GABA-R.

Lane Species Specific [3H]flunitrazepam binding activity (pmol/mg protein)

A Cow 3.6 ± 0.4 B Dove 0.9 ± 0.4 C Cow 1.3 ± 0.3 (adrenal medulla) D Rat 4.8 + 0.5 E Pig 3.9 ± 0.4 F Chick 6.6 + 0.6 (1 day old)

The lanes were stained with the indicated monoclonal

antibodies. (See text 3.3.17).

201 Figure 3.3.9.C

102 D______( |______1A*. 1

kDa >

88—

bo—

45—

31—

ABC D E F O A BCOEF G

Immunoblots of brain cortex membranes from different species. Each lane was loaded with 200 ug protein except for lane G that contained bovine purified GABA-R.

Lane Species Specific [3H]flunitrazepam binding activity (pmol/mg protein)

A Cow 3.6 ± 0.4 B Dove 0.9 + 0.4 C Cow 1.3 ± 0.3 (adrenal medulla) D Rat 4.8 ± 0.5 E Pig 3.9 ± 0.4 F Chick 6.6 + 0.6 (1 day old)

The lanes were stained with the indicated monoclonal antibodies. (See text 3.3.17).

201 Titres ofrabbitantiseraTitres against <*3 peptide. LS sa o irtte lts otd ih AAR f the of GABA-R with coated plates miarotitre on assay ELISA rabbit. ea f h second the ofsera absorbance units '( e ) thirdand Figure3.3.10 202 +) (+ le o te immunized the of bleed

Table 3.3.1 Mouse Antigen and site Amount of antigen Titres of injection injected (ug) (a)

1,2 Native GABA-R 30 — — i.spleen. 3 Native GABA-R 40 1:1000 i.p. 4 Native GABA-R 40 1:100 i.p 5 Native GABA-R 40 1:50 1:100 i.p. + i.spleen. 6 Native GABA-R 40 1:50 1:100 i.p. + i.v. 7,8 Heat-denatured GABA-R 40 1?30 1:30 i.p. 9.10 Native GABA-R 200 1:300 1:800 i.p. T1,T2,T3 Ovalbumin 10 >1?10,000 i.p. 11.12,13 Native GABA-R 200 1:300 1:800 i.p. 14 SDS-denatured GABA-R 30 i.spleen. 15,16 DEAE-bound GABA-R 40 1:80 1:80 i.p. 17 Native GABA-R 200 1:700 i.p. 18 Alum-ppt GABA-R 50 1:500 1:3000 i.p. 19,20 Alum ppt GABA-R 50 1:300 1:600 i.p. 21,22 SDS-denatured GABA-R 100 1:50 i.p. 23,24,25 GABA-R a subunit - (b) NC implant 26,27,28 GABA-R p subunit - (b) ** NC implant 29,30,31 Agarose Ro 7-1986 0. 15 g 1:600 i.p. 33,34,35 KLH-cr3 peptide 100 >1?10,000 i.p.

(a) the titres are defined as described in 3.3.4 (b) assayed on Western blot as described in 3.2.11 i.spleen = intrasplenically, i.p. = introperitoneally i.v. = intravenously, NC = nitrocellulose. DEAE = DEAE Sephacel.

The immunization procedures and the titres obtained. The titres were measured after the second and third injection. (See text 3.3.4).

203 Table 3.3.2

Specific [3H]flunitrazepam Binding to GABA-R bound (cpm + SEM) Stored at -20°C for 12 h 2952+141 diluted 1:10 in assay buffer.

Stored at 4°C for 12 h 2545 + 109 diluted 1:10 in assay buffer.

Stored at 4°C for 12 h 2390 + 167 diluted 1:10 in PBS. Stored at 37°C for 45 min 2316 + 171 diluted 1:10 in PBS. After coating to plates 1461 + 132 for 12 h at 4°C. Binding to plates Plates coated with GABA-R 1162 + 101 for 12 h at 4°C. Plates coated with gelatin. 110 + 39

Ligand binding assay of the GABA-R immobilized on the ELISA plates.

Each assay sample contained 60 ul of GABA-R preparation which is equivalent to the amount incubated in each ELISA well. Values are averages of triplicate assays and are representative of two experiments. (See text 3.3.5.A).

204 Table 3.3.3

Mouse Antigen Fusion cell Wells with +ve Stable inj ected method line growth wells monoclonal (a) (b) 1 native GABA-R 33% PEG SP2 250 8 — i.spleen. 2 native GABA-R 33% PEG SP2 228 6 i.p. 3 native GABA-R 33% PEG SP2 180 4 i.p. 4 native GABA-R 33% PEG SP2 381 5 i.p. 5 native GABA-R 33% PEG NS0 270 2 i.p. + i.spleen. 6 native GABA-R 33% PEG NS0 160 (c) 1 i.p. + i.v. 9 native GABA-R 33% PEG SP2 216 8 i.p. T4 none 50% PEG SP2 790 14 SDS-denatured 50% PEG SP2 808 18 2 (d) GABA-R i.spleen. 17 native GABA-R 50% PEG SP2 812 21 1 i.p. 18 Alum ppt. GABA-R 50% PEG SP2 732 9 1 i.p. 29 Agarose-Ro 7-1986 50% PEG SP2 776 3 i.p. 33 KLH-a3 peptide 50% PEG SP2 610 25 11 (e) i.p.

(a) number of wells of 96-well plates with growing hybridomas. (b) number of positive wells (i.e. O.D. > 3 x background) on the 1st ELISA screening. (c) 24-well plates were used instead of 96-well plates

(d) the antibodies were shown to be directed against soybean trypsin inhibitor (3.3.6)

(e) 10 of the antibodies were shown to recognise KLH and 1 was specific for the a3 peptide. i.p. = intraperitoneally i.spleen. = intrasplenically, i.v. = intraveneously. A summary of the fusions performed. (See text 3.3.6).

205 Table 3.3.4.A

Antigen Additions to the % number of number of inj ected culture medium cells after blast cel 4 days (cells/m ovalbumin none 96% 0.2 x 104 ovalbumin ovalbumin 110% 0.5 x 104 ovalbumin DS, LPS 125% 1.0 x 104 ovalbumin Triton X-100 94% 0.1 x 104 ovalbumin cholate 0% 0 none none 94% 0.1 x 104 none ovalbumin 95% 0.1 X 104 none DS, LPS 121% 1.1 X 104

GABA-R none 93% 0.1 X 104

GABA-R GABA-R 92% 0.1 X 104

GABA-R GABA-R, DS, LPS 123% 1.0 X 104

DS = dextran sulphate. LPS = lipopolysaccharide.

The effect of antigens and mitogens on the growth of spleen cells in tissue culture conditions.

Spleen cells were obtained from immunized and non- immunized mice and were counted before and after 4 days of culture in the presence of the indicated substances. The final number of spleen cells observed is expressed as the percentage of cells present initially. Two separate dishes were counted for each condition. Ovalbumin-injected and

GABA-R-injected mice were mice T1-T3 and mice 11-13 respectively ( Table 3.3.1). (See text 3.3.7)

206 Table 3.3.4.B

Antigen Additions to Panning Total Number of inj ected the culture number of positive into mice medium hybridomas hybridomas

None Ovalbumin No 123 1 DS, LPS

Ovalbumin Ovalbumin No 155 4 DS, LPS

None Ovalbumin Yes 28 0 DS, LPS

Ovalbumin Ovalbumin Yes 43 1 DS, LPS

None GABA-R No 141 0 DS, LPS

GABA-R GABA-R No 158 0 DS, LPS

GABA-R GABA-R Yes 35 0 DS, LPS

None (a) GABA-R No 800 3 (b) DS, LPS, MDP

(a) The spleen cells were from six 2-day old imice (b) The hybridomas were unstable

DS = dextran sulphate, LPS = lipolisaccaride

A summary of fusions carried out with in vitro cultured spleen cells.

Spleen cells were cultured in the presence of the indicated substances and fused with the 33% PEG method (3.2.7.A) except for the 2-day old mice spleen cells which were fused with the 50% PEG method (3.2.7.B). Ovalbumin injected and

GABA-R injected mice were mice T1-T3 and mice 11-13 respectively (see Table 3.3.1). (See text 3.3.7).

207 Table 3.3.4.C

Antibody Dilution % [ 3H]flunitrazepam % [3H]muscimol factor sites precipitated sites precipitated

102D 1:10 51 % 52 %

1:100 51 % 41%

1 :1,000 29 % 27 %

1 :10,000 10 % 14 %

86G 1:10 57 % 45 %

1:100 52 % 43 %

1 :1,000 26 % 11 %

1 :10,000 11 % 12 %

6B6 (a) 1:10 54 % 43 %

1:100 53 % 42 %

1 :1,000 29 % 20 %

1 :10,000 10 % 13 %

(a) anti-acetylcholinesterase monoclonal antibody (cortesy of Dr K.W.K. Tsim)

Immunoprecipitation of purified GABA-R with the ascites fluid of the monoclonal antibodies.

The percentage of ligand binding precipitated was calculated as a fraction of the sum of the radioactivity in the pellet and in the supernatant of each incubation assay.

This total radioactivity varied < 10% between the incubation assays. The above is representative of three experiments. (See text 3.3.11).

208 Table 3.3.5.A

Hybridoma cell line

102D 86G

IgG yield after 1 cycle 1.5 mg 3.5 mg through the protein A Sepharose

IgG yield after 2 cycles 0.7 mg 2.0 mg through the protein A Sepharose

IgG yield after 3 cycles 0.3 mg 0.4 mg through the protein A Sepharose

Reduction in absorbance at 25% 50% 1:500 dilution in the ELISA (a) Titre of purified antibodies 1:500 1:800 in the ELISA

(a) The immunoreactivity of the supernatant was tested in the ELISA assay at 1:500 dilution before and after 3 cycles through the protein A Sepharose column.

Protein A Sepharose chromatography of monoclonal antibodies.

The cloned 102D and 86G hybridoma cell lines were grown in complete DMEM (3.2.4) and purified on a protein A Sepharose affinity column. The above values are representative of 6-8 purification procedures. (See text 3.3.13).

209 Table 3.3.5.B

Method Reduction of Purity of immunoreact iv ity the 1A6 at 1:500 dilution (b) (a)

Ammonium sulphate 51% poor precipitation (30% w/v) Ammonium sulphate 92% poor precipitation (50% w/v)

Boric acid 60% poor precipitation Euglobulin 63% moderate precipitation Ion-exchange 90% moderate purification Gel filtration N.D. moderate purification Euglobulin precipitation 64% best of FCS-free medium

(a) The reduction of immunoreactivity is expressed as percentage of reduction of absorbance in the ELISA assay the sample after the indicated procedure with respect to the initial absorbance of the same sample. (b) The purity of the sample was assessed by SDS-PAGE (see

Figure 3.3.10.A-B)

N. D. = not determined

Purification of the IgM monoclonal 1A6. (See text 3.3.13).

210 Table 3.3.6 Antibody Type of Immunoglobulin Reduction Receptor immunoaffinity coupled (a) of activity purification column (%) (mg/ml gel) (b) (c) 1A6 Pre-activated 86% 1.2 20% (d) — cyanogen bromide Sepharose 4B 0%

Cyanogen bromide 60% 0.8 0% Sepharose 4B freshly prepared

Protein A- 30% (e) 0.5 0% anti-IgM Sepharose 4B "

Affigel 10 77% 1.5 0% —

102D Pre-activated 84% 1.6 0% — cyanogen bromide Sepharose 4B 79% 3.1 0%

Protein A 32% (e) 0.8 0% — Sepharose 4B

Affigel 10 75% 2 .1 0% —

86G Pre-activated 90% 1.0 0% — cyanogen bromide Sepharose 4B 81% 3.5 0%

77% 8.0 0% —

Protein A 40% (e) 1.5 0% — Sepharose 4B

Affigel 10 78 % 6.2 0% — (a) The percentantage of immonoglobulin bound was calculated from the absorbance at A = 280 nm before and after coupling. (b) Expressed as the percentage decrease of [3H]flunitrazepam binding activity after application of the soluble extract to the immunoaffinity column. (c) The purification of the GABA-R was determined by SDS-PAGE

(d) No salt was present in the buffers (3.3.14)

(e) Estimated by the ELISA assay Synthesis of immunopurification columns. (See text 3.3.14). 3.4 Discussion

3.4.1 The production of polyclonal antibodies against the GABA-R

The main objectives in the production of polyclonal anti-GABA-R antibodies were for use in the characterization of the GABA-R both in solution and in Western blots. Additionally they were to be used as a potential probe for the identification of the GABA-R cDNAs. The immunoprecipitation studies of the soluble GABA-R were carried out by Dr Stephenson and the results have been described in Stephenson et al., 1986.

Immunostaining of Western blots of the GABA-R with the same antibodies were carried out in this work and the results showed that only the a subunit was recognised by the antibodies. The immunoprecipitation studies had shown that all the GABA-R ligand binding activities were precipitated in the same proportion by the antibodies.

(Stephenson et al., 1986a). It is however not possible to conclude from the Western blot results and the immunoprecipitation findings that it is sufficient to bind the a subunit in order to immunoprecipitate all the GABA-R binding sites. The antibodies, because they are polyclonal, could theoretically independently recognise

212 separate receptors that contain the various ligand binding sites. Additionally the antibodies could be able to recognise both receptor subunits when presented in the native form.

The minimum amount of GARA-R that could be detected by the antibodies on Western blot was 0.1 ug protein. This experiment was carried out in order to assess whether these antibodies were suitable for the screening of a

Agtll cDNA expression library. It was estimated that each plaque of lysed E.coli transfected with recombinant Agtll may contain 0.01 - 0 .1 ug protein produced by the corresponding cDNA clone (Young and Davies, 1983). Therefore the antibody titre was at the lower limit of that required for screening the library. With hindsight a better test for the quantification of the affinity of the antibodies would have been a dot blot chequer board type of screening assay (3.2.10.D). This is because the efficiency of retention of the receptor by the nitrocellulose filter is higher if applied directly on to it, rather than after SDS-PAGE and Western blot.

Polyclonal antibodies that could also recognise epitopes of the p subunit would have been useful for further screening of the cDNA library. In this study, rabbits were injected with receptor which had been reduced and carboxymethylated in order to prevent the disulphide bonds to reform. The object of treating the receptor in

213 such a way was to partially denature it and to expose epitopes otherwise masked. At the time, it was not known how many disulphide bonds were present in the receptor and what role they played in the receptor structure except for the observation that the receptor showed a similar staining pattern on SDS-PAGE under reduced and non-reduced conditions. However even small changes in the receptor structure could have triggered the desired immune response against the GABA-R p subunit. Alternatively, rabbits could have been injected with SDS-denatured receptor, but this would have introduced the complication that antibodies against SDS might have been raised which would have hampered Western blot immunostaining. It was shown that the antibodies against carboxymethylated receptor also specifically recognised the a subunit in Western blots. This subunit must be antigenically predominant because Vitorica et al. (1988) and Stauber et al.. (1987) also reported polyclonal antibodies mainly directed against the a subunit in immunoblots. The muscle nicotinic acetylcholine receptor has also been shown to have a major immunogenic region which might have pathological implications in the autoimmune disease Myasthenia gravis (Tzartos, 1984) .

Whether the GABA-R also has a major immunogenic region and whether it is implicated in immunological disorders is at present not known.

214 3.4.2 The production of monoclonal antibodies against the GABA-R

Monoclonal antibodies against the purified GABA-R were obtained after nearly all the conditions of the immunization, fusion and cloning methods were varied in successive experiments. During this process some information about the immunogenicity of the GABA-R, and about practical aspects of the fusion techniques, were obtained. The immunization of the spleen-donor animal is the first step in the production of monoclonal antibodies. In this study, mice injected with 200 ug of purified GABA-R per injection generally showed higher titres than those injected with 40 ug. In comparison (see Table 3.4.1) Haring et al., (1985) injected each mouse with 100 ug protein per injection, while Mamalaki et al. (1987) and Vitorica et al. (1988) injected 40 and 50 ug of GABA-R respectively.

It was observed here that titres obtained with injections of native receptor were higher than with injections of heat denatured, or SDS denatured receptor. A possible explanation for this finding is that in the denatured receptor more proteolytic sites are exposed and the antigen may be extensively digested in the injected animal before it can stimulate the immune system.

Freund's adjuvant has been used for the production of

215 all the previously reported monoclonal antibodies. In this work maximum titres of 1 : 1000 were obtained in mice injected with Freund's adjuvant. The highest titre was however obtained from one of the mice injected with alum precipitated GABA-R even though only 50 ug GABA-R per injection were used. This method also proved to be

successful for the production of monoclonal antibodies against the 1,4-dihydropyridine receptor (Norman et al.. 1986). The purified GABA-R was also presented attached to a solid support. Animals injected with DEAE-Sephacel- coupled receptor showed a weak response, while mice which received nitrocellulose implants showed no response. It appears therefore that prolonging the presence of the antigen in the animal does not improve the immunoresponse to the GABA-R. The stimulation of the mouse immune response by intrasplenic injection did not result in the production of monoclonal antibodies against the GABA-R, although two monoclonal antibodies against the soybean trypsin inhibitor were accidentally isolated with this method.

This finding together with the high immune response obtained in mice injected with ovalbumin and KLH (see

Table 3.3.1) shows that proteins can differ considerably in their level of immunogenicity. The economization of antigen and time of the intrasplenic injection method make it extremely useful and worthy of further investigation.

It is possible that in this work the quantity of GABA-R

216 that was injected could have triggered a suppression of the immune response. Monoclonal antibodies obtained with intrasplenic injections include one which recognised an acetylcholinesterase subunit which previously had proven difficult to raise monoclonal antibodies against (Tsim et al., 1988).

The second major step in the production of monoclonal antibodies is the fusion of the spleen cells with the myeloma cells. The SP2 myeloma cell line used in this work, grew efficiently under the conditions described (3.2.4), but many initially positive clones stopped producing antibodies during the screening and cloning steps. Presumably this was caused by the instability of the cell line after fusion. This instability could be due to the fact that SP2 is a hybridoma cell line and thus not ideal for the acceptance of a further set of chromosomes (Galfre and Milstein, 1981). Difficulties were encountered in the maintenance of the NSO cell line before fusion, and it was concluded that culturing of the cells in rotatory bottles would have been advantageous. It might have been worthwhile testing other cell lines, but the SP2 cell line used here was successfuly used in the laboratory for the production of other monoclonal antibodies (Randall et al., 1987? Tsim et al., 1988? Mamalaki et al., 1987).

Important factors for the production of monoclonal antibodies were found to be the PEG used and its time of

217 exposure to the cells. Fusions carried out with 36% PEG

(BDH) for a total of 8 min yielded 200-300 hybridomas, whilst fusions carried out with 50% PEG (BRL) for 1 min yielded 1000-1500 hybridomas (See Table 3.3.3). In addition to the concentration at which they were used, the two PEG differed in that PEG from BRL was purchased in an already sterile and purified form, whilst PEG from BDH had to be autoclaved. It has been shown that autoclaving produces aldehydes and peroxides which are toxic to the cells (Kadish and Wenc, 1983) . Since the type of PEG, its concentration, and the length of exposure time to the cells, were changed simultaneously, it is not possible to determine which factor was responsible for the increase in the number of hybridomas produced, but they probably all play a positive role. The fact that anti-GABA-R monoclonal antibodies were obtained only with the 50% PEG fusion system, is probably due to the production of higher numbers of hybridomas and to the fact that the hybridomas were not exposed to toxic compounds.

The third major step for the production of monoclonal antibodies is the screening of the hybridoma cells produced by the PEG fusion. The ELISA system was used for the screening of all hybridomas produced. The assay was found to be very sensitive and this was highlighted by the accidental isolation of monoclonal antibodies against soybean trypsin inhibitor which was present in the

218 purified GABA-R preparation in quantities undetectable by SDS-PAGE. It was found that the presence of detergent in the purified receptor sample affected the binding of the receptor to the ELISA plates. It was thus necessary to decrease the Triton X-100 concentration in order to obtain a higher signal in the ELISA assay. In the initial ELISA screening of each of the fusions carried out, several positive wells were detected which on later screening were no longer positive. This could be due either to the previously mentioned instability of the newly formed hybridoma or to the fact that the well whose supernatant was assayed may have contained some unfused spleen cells that produced the antibody of interest. Which of the two phenomena actually occurred is difficult to determine but it was observed that if the positive wells were rescreened before expansion of the cells, in the majority of cases they were still positive, indicating that the signal observed was real and not due to a spurious behaviour of the ELISA assay.

A possible shortcoming of the ELISA as an assay system for the screening of monoclonal antibodies that were intended to be used for immunoaffinity purification, is that this system might not detect antibodies that specifically recognise the native soluble receptor. This is because the immobilization of the receptor to the ELISA plates may cause it to denature and it may mask some of the receptor antigenic sites. However, when the receptor

219 bound to the ELISA plates was assayed with [3H]flunitrazepam it was found to have maintained its ligand binding capacity, and has therefore a near-native conformation (see Table 3.3.2).

Alternative screening methods of the hybridoma cells

produced were also tried. The dot blot screening could potentially have been a more sensitive method because it is more effective in immobilizing the receptor. It was however shown that this assay was neither more economical

in terms of purified GABA-R required nor more sensitive for low abundancy antibodies than the ELISA assay (see Figure 3.3.4). The major advantage of the dot blot assay

is that the receptor could be immobilized in 1 h rather than the 36 h required for the ELISA plates.

Screening methods designed for the selection of monoclonal antibodies capable of recognising the receptor

in solution were also tried. One such method was the solid phase radioimmunoassay (3.2.10.C) where a primary antibody, rather than the antigen is immobilized to the microtitre plates. This has proven to be a useful system for screening antibodies against soluble enzymes (Randall et al. . 1987), because reduced amounts of immuno- immobilized enzyme are required to catalyze a reaction whose products are assayed. This method was adapted here for the screening of antibodies that could immobilize the

GABA-R. The wells containing such antibodies would then be

220 identified by the ligand binding assay to the plates. This assay therefore does not benefit from the amplification systems of the enzyme-linked antibody overlaying in the

ELISA assay. It was found that the solid phase radioimmunoassay was not sensitive enough when the monoclonal antibody 1A6 or anti-GABA-R polyclonal antibodies were used in this assay.

Another method for the screening of monoclonal antibodies that recognise protein in solution is to carry out the immunoprecipitation of the receptor with each of the hybridoma supernatants (3.2.21). It must be realized that in the initial screening, the hybridoma cell supernatants contain relatively few antibodies and thus only a proportionally small amount of receptor can be precipitated. Therefore this needs to be assayed with a high specific radioactivity ligand. Monoclonal antibodies against the nicotinic acetylcholine receptor have been isolated with this screening system (Whiting et al., 1985) but [125I]a-bungarotoxin was used as ligand. Vitorica et al. (1988) however used both the ELISA assay and immunoprecipitation assay with [3H]flunitrazepam as their screening system for the isolation of anti-GABA-R monoclonal antibodies. In this work one fusion (mouse 17 Table 3.3.3) was also screened with the immunoprecipitation method but no positive clones were detected while from the same fusion an anti-GABA-R

221 i

monoclonal antibody producing clone was isolated with the ELISA screening method. It is difficult to conclude whether monoclonal antibodies that could recognise the GABA-R in solution were not produced by the animal or the screening systems used were not sensitive enough.

The final step of the monoclonal antibody production is the cloning of the hybridoma cell line producing the desired antibody. The most rapid and efficient method of cloning used here was the "safe" serial dilution described in 3.2.12.B. It was designed to have the safety properties of the limiting dilution cloning system, so that cells susceptible to strong dilution, or present in low abundancy, are not lost because they are also assayed at lower dilution. Additionally it had the rapidity and the economy of antigen used for the serial dilution. Cloning by limiting dilution and by soft agar have the disadvantage of being lengthy procedures and require large amounts of antigen for the screening. Neither of these two cloning systems had any apparent advantages. It was shown that even with limiting dilution, a cell line lost its antibody production capacity always after the same number of cloning steps, which seems to indicate that there are some intrinsic factors of the hybridoma cells that affect their stability.

222 3.4.3 Alternative methods for the production of monoclonal antibodies against the GABA-R

All the above described methods of production of monoclonal antibodies require that an immune response against the antigen of interest is obtained in the spleen- donor animal. The present knowledge of the functioning of the immune system allowed other approaches to be taken namely, in vitro immunization and the production of anti- idiotypic antibodies. The advantages of using an in vitro immunization system for the production of anti GABA-R monoclonal antibodies were that small amounts of antigen are required, the immunization time is only 4-5 days and that Triton X-100, which could have caused a reduction of the immune response in vivo, could be further diluted in vitro. No monoclonal antibodies against the GABA-R were isolated from in vitro immunizations, but this could be partly due to the fact that the less efficient method of 36% PEG fusion was used in the initial experiments and relatively few hybridomas were produced.

In vitro immunization of new-born mice spleen cells was carried out because at this age, the animals are still developing their capacity to recognise "self” from "non self”. Work carried out in this and other laboratories (results to be submitted) have shown that the rat and bovine GABA-R show a high degree of homology and it was

223 assumed that the mouse GABA-R would also be so. The fact that the immune system of the injected mice might have recognised the bovine GABA-R as "self" could have been a reason why low titres were obtained from jLji vivo immunizations. It was therefore postulated that spleen cells from new-born mice would be more responsive to immunization with GABA-R because they would not yet have fully developed their capacity of recognising the GABA-R as "self". When fusions were carried out with spleen cells of new born mice in vitro-immmunized with GABA-R, positive clones were detected in the initial screening. These subsequently stopped producing antibodies and it could be that the fusion product of the young spleen cells with the myeloma cells is particularly unstable.

In vitro immunization was also intended to be used in conjuction with panning. This would have allowed the selection of specific spleen cells before the fusion was carried out, and thus it would have increased the probability of producing the hybridomas of interest. Hybridomas producing antibodies against ovalbumin were isolated with this method (Table 3.3.4.B), but similar results for the isolation of anti-GABA-R antibody- producing hybridomas were not obtained. The ovalbumin- immunized mice used for panning had a higher titre than the GABA-R immunized animals (Table 3.3.1) which probably explains these results. The method for panning that was used was adapted from that of Barald and Wessel (1984) who

224 produced monoclonal antibodies from spleen cells selected on embryonic ganglion neurons. Possibly the use of whole cells as antigen is a more appropriate system for the panning of spleen cells because a wider variety of anigenic sites contribute to the selection of the spleen cells. An alternative system for the selection of spleen cells producing anti-GABA-R antibodies, would have been to couple the receptor with a fluorescent tag and to select those cells to which the receptor had bound with the use of a fluorescence-activated cell sorter. This method has been previously used as a cloning procedure in the production of allotype specific anti-immunoglobulin monoclonal antibodies (Parks et al., 1979). It should however be possible to adapt this method to select the spleen cells before the fusion. An alternative approach for the production of monoclonal antibodies is to raise anti-idiotypic antibodies to one of the ligands of the receptor. In this work the method of directly screening for anti-idiotypic antibodies in mice injected with the ligand was adopted . This method is faster and simpler than the conventional method described in 3.1.4, because it does not require first the production of anti-ligand antibodies and the injection of these in a second animal. Anti-idiotypic antibodies against the nicotinic acetylcholine receptor were obtained with the method used here by injecting mice

225 with bis-qualine (Cleveland and Erlanger, 1986). In this study the ligand Ro 7-1986 was injected immobilized to agarose in an orientation that was known from the affinity purification studies to allow receptor binding. Therefore the ligand's epitope that binds to the receptor was exposed to the immune system of the mice that were injected. The sera from the ligand-injected mice showed antigenicity against the GABA-R when assayed by ELISA. However no antibody affisfity for [3H]flunitrazepam could be measured in the same sera, which means that the presence of the anti-idiotypic antibodies but not of the idiotypic antibodies could be detected. It is possible that the primary antibodies raised against immoblized Ro 7-1986/1 did not cross react with [3H]flunitrazepam. However Fry and Martin (1987) have shown that most anti benzodiazepine antibodies cross react with each other. The presence of anti-GABA-R anti-idiotypic antibodies in the sera was also indicated by the ability of the serum to reduce [3H]flunitrazepam binding to membranes. (3.3.8)

This occurred only if the sera were pre-absorbed with the

immobilized Ro 7-1986/1, which suggests that the anti- idiotypic antibodies have higher affinity for the

idiotypic antibodies than for the receptor itself.

3.4.4 Evaluation of the techniques of hvbridoma

production

The result of all the injections and fusions

226 described was that two monoclonal antibodies were obtained. The first was from a fusion with a mouse which was injected with 200 ug native GABA-R per injection and which had a titre of 1;800 (86G), and the second from a

fusion with a mouse which was injected with alum precipitated receptor and that had a titre of 1:2000

(102D) . In both cases the 50% PEG fusion method was used and the monoclonal antibodies isolated from the 1000-1200 hybridomas were produced. Haring et a_l. (198 5) and Vitorica et al. (1988) have isolated 16 and 7 stable hybridomas cell lines respectively from a single fusion experiment. Mamalaki et al., (1987) instead have reported only 1 stable monoclonal antibody (used in these studies). Table 3.4.1 summarizes the fusion methods used by the various research groups that have produced anti GABA-R monoclonal antibodies. The only major difference that can be seen is the myeloma cell lines that were used. However the most likely explanation for the higher number of monoclonals obtained by Haring et al. (1986) and Vitorica et al. (1988) is probably that the mice that were used had developed a higher immune response against the GABA-R; possibly they were able to select the most suitable animal from a large panel of immunized mice. This is strongly indicated by the fact that Vitorica et al. (1988) obtained

7 monoclonals from only 180 hybridomas. It is unlikely that the screening assay was responsible for the

227 inefficiency in obtaining the desired monoclonal, because, although Vitorica et al. (1988) used in addition to the ELISA screening method the immunoprecipitation screening assay, Haring et al. (1985) used only the ELISA screening assay.

3.4.5 Characterization of the anti-GABA-R monoclonal

antibodies

The antibody 102D recognised on the purified receptor predominantly the a subunit in Western blots. This result is consistent with the finding that the a subunit is immunogenically dominant (3.4.1). However, monoclonal antibodies against the GABA-R p subunit have been raised (Schoch et al., 1985 and Vitorica et al., 1988) and the monoclonal antibody 1A6 recognises both the a and the p subunit (Mamalaki et al., 1987). The immunoblot of total brain membrane proteins with both 1A6 and 102D showed staining of only the a subunit in all the bovine brain regions tested and in the cortex and cerebellum of calf brain (Figure 3.3.9). The intensity of the staining of the membranes is weaker than in the purified receptor and this may be a reason why the staining of the p subunit was not found in immunoblots with 1A6 in membranes. Therefore, the finding that [3H]flunitrazepam photolabels predominantly one subunit in the purified receptor from bovine cortex, but two subunits in crude cortex membrane

228 preparations, is not paralleled by the staining by either monoclonal antibody. Monoclonal 1A6 in fact seems to have opposite properties. This could be because the subunit that is recognised by the antibodies and the one that is photolabelled by [3H]flunitrazepam are not the same, but are instead -i'.r two separate subunits that co-migrate. A two dimensional electrophoresis experiment would be needed to clarify this point.

Immunoblots of brain membranes from a variety of species carried out with both 102D and 1A6 also showed specific staining of the a subunit. This indicates that the two antibodies recognise conserved epitopes. Porcine membranes were however an exception as they were stained by 102D and not by 1A6. This is indirect evidence that the two monoclonal antibodies recognise different epitopes.

One day chick brain also showed only one subunit. This would indicate that the 53 kDa and 54 kDa polypeptide observed in avian brain membranes photolabelled with (3H] flunitrazepam (Hebe, brand et a l .. 1986, 1987) are eguivalent to the a and p subunits of bovine GABA-R as opposed to . . isoforms of the a subunit. Adult avian membranes (dove) were not stained by either antibody. This is probably due to the fact that dove brain membranes contained about one seventh of the [3H]flunitrazepam binding activity of chick brain (see legend Figure

3.3.9) . The monoclonal antibody 86G did not stain Western blots

229 of either purified GABA-R or total cortex membrane proteins which would indicate that the antibody recognises a conformational-dependent epitope. The evidence that 86G specifically recognises the GABA-R comes therefore from the fact that it specifically recognises on the ELISA assay purified GABA-R preparations which were homogeneous when analysed by SDS-PAGE. However, the above described isolation of anti-soybean-trypsin- inhibitor monoclonal antibodies has stressed the sensitivity of the ELISA assay and that minor contaminants can be detected. The production of monoclonal antibodies against the GABA-R was aimed at the construction of an immunoaffinity column. Therefore, it was of interest to establish the immunoprecipitation properties of the monoclonal antibodies that were produced. Both 102D and 86G

immunoprecipitated, over the same dilution range, the purified GABA-R in the presence of 1% BSA. However 102D and 86G did not immunoprecipitate [3H] flunitrazepam or

[3H]muscimol binding activities from crude soluble preparations even when present in the same buffer and detergent as the purified receptor. The same results were obtained with both the cell supernatant culture media,:, and the respective ascites fluids. However, in control experiments where a monoclonal antibody against chick acetylcholinesterase (Tsim et al., 1987) was used, this also precipitated purified GABA-R over the same dilution

230 range observed for 102D and 86G. This would suggest that the observed precipitation of the purified GABA-R was due to a non-specific interaction of the immunoglobulins with the receptor. Mamalaki (1986) reported that 1A6 could precipitate purified receptor but a control with an

irrevelant monoclonal antibody was not performed. The fact that 102D and 86G did not specifically immunoprecipitate the GABA-R could be due to the presence of the detergent. This was indicated by the dot blot experiment where the presence of Triton X-100 inhibited the binding of the antibody to the receptor. Thus the detergent might either interfere with the antibody binding in a non-specific manner or the antibodies could be directed to a hydrophobic region of the receptor which would be masked by the detergent. It should be noted that

in the ELISA assay the microtiter plates were washed with detergent-free buffer before the antibodies were added. Therefore the fact that the monoclonal antibodies that were isolated could recognise only detergent-free GABA-R may be an indirect consequence of the screening system used. Although an immunoprecipitation screening system would have been more appropriate for the isolation of monoclonal antibodies to be used for immunoprecipitation studies, all 16 monoclonal antibodies isolated by Haring et al (1985) with an ELISA screening could precipitate purified GABA-R.

The purification of monoclonal antibodies produced

231 under tissue culture conditions is a necessary step for the construction of an immunoaffinity column. Purification by the protein A column of both 102D and 86G resulted in highly purified immunoglobulin preparations. These

preparations when assayed by ELISA had a lower titre than

expected from the known concentration of these antibodies.

This could be due to either inactivation of the immunoglobulins caused by the low pH elution from the protein A Sepharose column or, to the aggregation of the immunoglobulin molecules. The latter is believed to be more likely as the protein A Sepharose eluates were turbid. Aggregation of the immunoglobulins would

effectively reduce their molar concentration. One of the major disadvantages of monoclonal antibodies of the IgM class such as 1A6, is that they do not bind to protein A. The purification of the IgM monoclonal 1A6 therefore required the use of alternative methods. This task was complicated by the fact that the

culture medium in which the antibody producing cells line were grown, was supplemented with 10-15% FCS which

contains many other proteins. Additionally, IgMs are

secreted in the culture medium at lower concentrations (1 -10 ug/ml) than other immunoglobulins. Conventional chromatographic techniques were not sufficient to obtain a purified immunoglobulin preparation. A more homogeneous

preparation of IgMs was obtained by transferring a large

232 colony of 1A6 cells to a FCS free medium, from which immunoglobulins were precipitated by the euglobulin precipitation method.

Of the antibodies available, 1A6, 102D and 68G, none were ideal candidates for the construction of an immunoaffinity column as they did not immunoprecipitate soluble crude receptor under the assay conditions described (3.2.21). However there was a case for pursuing these antibodies because a relatively high concentration of antibodies can be bound to the matrix which may favour the binding of the soluble receptor when the crude soluble extract is applied to the column. Further, in an immunoaffinity column, one of the reactants i.e. the antibody is immobilized to a solid support which ressembles, although in the reverse fashion, the conditions of the ELISA assay under which the antibodies were selected. Thus many parameters of the construction of immunoaffinity columns and the procedure of immunoaffinity purification were varied and tested (Table 3.3.6). The antibodies were coupled to gel matrices under a variety of conditions for the following reasons: 1) To pre-activated cyanogen bromide Sepharose-4B to maximize the amount of immunoglobulins bound.

2) To freshly prepared cyanogen bromide activated

Sepharose-4B to enable regulation of the amount of

233 a

immunoglobulins bound. It can, in fact, be preferable to reduce the binding capacity of the column to avoid the coupling of each immunoglobulin molecule to the matrix by more than one residue. Such multiple coupling could interfere with the antibody-antigen interaction.

3) To protein A matrix to ensure the correct orientation of the immunoglobulins. Protein A binds to the Fc region of the immunoglobulin and therefore the antigen-binding site will be free.

4) To Affigel-10 to test an alternative coupling to the matrix. Immunomatrices were prepared at different final concentrations of monoclonal antibodies (Table 3.3.6) for the reasons described in point 2) above and to maximise the binding capacity of the immunomatrix.

The construction of an immunoaffinity column with a highly purified immunoglobulin preparation is desirable because the presence of irrelevant proteins decreases the concentration of immunoglobulins bound per unit of gel and also contributes to non-specific absorption of proteins from the crude soluble extract applied to the column.

However, the purification procedure of immunoglobulins, especially the low pH elution from the the protein A

Sepharose, may affect the viability of the antibodies. For this reason, the ascites fluid containing the monoclonal antibodies 102D and 86G were also bound to the matrices prior to purification, but no immunopurification was

234 obtained even under these conditions. The soluble extract was applied to the immunoaffinity columns at room temperature or overnight at 4°C in order to establish the best antigen-antibody binding conditions, and the elutions were carried out at high or low pH or with chaotropic ions. It was found that under all of the above conditions, [3H]flunitrazepam binding activity was

not specifically removed from the soluble extract and

consequently no immunopurification was achieved. It can therefore be concluded that these antibodies are not

suitable for the construction of an affinity column. It should be noted that none of the 16 monoclonal antibodies produced by Haring et al., (1985) have to date been reported to function in an immunoaffinity colum. However,

recently, immunopurification of the GABA-R with one of the monoclonal antibodies raised by Vitorica et al. (1988) has been reported (Park et al., 1987).

3.4.6 The production of antibodies against synthetic peptides of the GABA-R

The production of polyclonal and monoclonal antibodies against the C-terminus of the a3 subunit was part of a larger research programme carried out in the

laboratory, where antibodies to a number of GABA-R peptide sequences are being raised to study the structure of the

GABA-R. The C-terminus of the a3 subunit was chosen

235 because it shoved significant sequence differences from the C-termini of the other receptor subunits, and because the a3 subunit, detected by Northern blots showed a specific unique distribution in the brain (Levitan et al..

1988) . The method used for coupling the peptide via the N-terminal residue maximised the probability that the peptide took a conformation similar to that of the native protein C-terminus. One monoclonal antibody, 75B, that specifically recognised the synthetic peptide was obtained but it did not recognise the GABA-R neither in the ELISA assay nor in Western blots. These results indicate that even though the synthetic peptide was coupled at its N-terminus it must have formed an immunogenic epitope that is not contained in the C-terminus of the native GABA-R a3 subunit.

The sera of rabbits immunized with the peptide showed an immunoreactivity against the GABA-R after the fourth injection. It has been observed in other work carried out in the laboratory, that immune responses against the whole receptor in animals injected with synthetic peptides occurred only after 4 or 5 immunizations and the titre increased further in subsequent injections (M.Duggan and

F.A. Stephenson unpublished results). Further injections of the rabbits with the a3 C-terminal peptide should thus provide a higher titre sera which will be a useful tool for the characterization of this GABA-R subunit.

236 i

Table 3.4.1

Mamalaki Haring Vitorica Casalotti et al. et al. et al. (a) (b) (c) (d) Procedure

Antigen state native native native native/alum (pure GABA-R) amount/inj ection 50 100 50 200/50 (ug protein) site i.p. i.p. i.p. i.p. mouse strain BALB/c BALB/c BALB/c BALB/c PEG 1500 4000 1000 1500 (molecular weight) length of fusion 1 1 1 1 (min) myeloma cell line SP2/0Ag8 PAI P3x63Ag8653 SP2/OAg8 Hybridomas 600-800 (e) NR 180 1200 produced

Screening ELISA ELISA ELISA, ELISA system Immunoppt.

Monoclonal Ab 1 16 7 2 (f) produced

(a) Mamalaki et al., (1987) (b) Haring et al., (1985) (c) Vitorica et al., (1988) (d) Casalotti this work. (e) Personal communication (f) Obtained from two fusion experiments NR = not reported.

A summary of the techniques used for the production of monoclonal antibodies against the GABA-R. (See text 3.4.2).

237 CHAPTER 4

BIOCHEMICAL STUDIES OF THE GABA-R

EXPRESSED IN XENOPOS OOCYTES

238 4.1 introduction

4.1.1 The Xenopus oocvte expression system

The molecular characterization of neuroreceptors has been aided by the application of recombinant DNA techniques. The initial objective is the isolation of the complementary DNAs (cDNA) sequences that encode for the neuroreceptors of interest. It is subsequently necessary to test the functional properties of the protein synthesized from the respective cDNA. However, the expression of neuroreceptors presents particular problems. Firstly, some neuroreceptors such as the GABA-R, have a multi-subunit composition thus all the subunits necessary for the receptor function must be simultaneously expressed and correctly assembled. Secondly, post-translational modifications such as glycosylation must be reproduced and thirdly, the neuroreceptors need to be incorporated into the plasma membrane in order to be tested for functionality e.g. channel ion conductance. The injection of either pure RNA or crude mRNA into Xenopus oocytes has been shown to be able to fulfill the above requisites for the expression of neuroreceptors. (Barnard et al.,

1984). Such results have not been obtained by expression in bacterial hosts. Other practical advantages of the

239 Xenopus oocyte translation system include the fact that the foreign RNA can be injected into the cell without the need of a vector and that the size of the oocytes (1-2 mm diameter) makes them amenable to electrophysiological recording. The Xenopus oocyte expression system has however some limitations. Firstly, the injection of large numbers of oocytes is very laborious and impractical. Secondly, the efficiency of expression of the oocytes can vary greatly seasonally and between batches. Thirdly, the expression of the foreign protein is not continuous and thus new oocytes need to be injected. Finally, the oocyte could already contain the protein of interest, e.g. the muscarinic acetylcholine receptor. The injection of total mRNA preparations or pure RNAs into Xenopus oocytes can be used as a system for the electrophysiological examination of the expression of neuroreceptors. However, the full characterization of an ion channel-gated receptor like the GABA-R, also requires the characterisation of its binding sites. The ideal system for this work would be the transfection of a stable self- proliferating cell line which could be continuously grown to the required quantities and assayed. However the transfection of cells with one or more genes requires vectors which must be specially constructed (Morrison et al., 1984). The oocyte expression system can also be used

240 for the pharmacological characterization of the expressed receptor provided that sufficient protein is expressed and a sensitive assay system is available. The nicotinic acetylcholine receptor (Sumikawa §t al., 1981) and the a2 adrenergic receptor (Kobilka , 1987) have both been expressed and assayed in the oocyte system but a large number of oocytes (ca. 1000) was required for each experiment. The oocyte system can be advantageous for the simultaneous screening of both the pharmacological and electrophysiological properties of site-directed cDNA mutants where it might be impractical to produce cell transfectants for each mutant.

241 4.2 Materials and Methods

4.2.1 Materials

The "SP6 RNA synthesis" kit and rabbit reticulocyte lysate were purchased from Promega. The Xenoous frogs were from Nasco (UK). All other materials were from commercial sources or have been described earlier.

Barth's medium 15 mM Hepes-NaOH pH 7.6, 88 mM NaCl, 1 mM KC1, 2.4 mM NaHC03, 0.3 mM CaN03.4H20, 0.41 mM CaCl2 .6H20, 0.82 mM MgS04.7H20, 10 ug/ml sodium penicillin, 10 ug/ml streptomycin sulphate.

4.2.2 GABA-R al RNA and B RNA synthesis

RNA specific for the al and p subunits was synthesized from linearized pSP65 plasmids (provided by Jeanette

O'Brien) into which the cloned cDNA of the two subunits had been inserted downstream of the SP6 promoter

(Schofield et al., 1987). The Promega Biotec transcription kit was used and the following method carried out. To an autoclaved Eppendorf tube the following were added: 5 x

Promega transcription buffer (20ul), 100 mM DTT (10 ul),

RNasinR ribonuclease inhibitor (1 unit/ul, 4 ul), 2.5 mM

ATP (5 ul), CTP (5ul) , GTP (5 Ul) , UTP (5 ul) , Gppp (9 ul), linearized plasmid DNA (1 ug/ul, 2ul), and riboprobe

242 SP6 RNA polymerase (2 ul) . The volume was brought to 100 ul with H20 that had been filtered through diethylpyrocarbonate (DEPC), and incubated for 2 h at

37°C. R Q 1 ™ DNase was added to a final concentration of 1 unit/ul, and incubated for 15 min at 37°C. Phenol (100 ul) was added, the tubes were vortexed and centrifuged in an

Eppendorf centrifuge at 10,000 g for 2 min. The top aqueous layer was transferred to an Eppendorf tube and to it 1:1 phenol:chloroform (100 ul) was added. The tube was vortexed and centrifuged as above. The top layer was transferred to a tube where chloroform (80 ul) was added. The tube was vortexed and centrifuged as above. The top layer was transferred and incubated for 20 sec at 60°C. The tube was gently shaken and 3M K-acetate, pH 3.5, (7 ul) was added followed by absolute ethanol (2.5 vol, 170 ul) . The tube was incubated in dry ice for 10 min and centrifuged at 10,000 g for 20 min. The supernatant was decanted and 70% (v/v) ethanol (180 ul) was added and the tube centrifuged as above for 10 min. The supernatant was discarded, absolute ethanol (180 ul) was added and the tube was centrifuged as above. All ethanol was removed by inverting the tube on an absorbing tissue and the tube was dried under vacuum for 2 min. DEPC-treated water (20 ul) was added and the tube vortexed for 2 min. The resuspended

RNA was aliquoted and stored at -70°C.

243 4.2.3 Reticulocyte lvsate translation of al RNA and

8 RNA

Both al RNA and 0 RNA (1 ul) (4.2.2) were added separately to 35S-methionine (0.5 ug, 1.5 ul) and rabbit reticulocyte lysate (10.5 ul). The solution was mixed by 4 knocks to the tube, centrifuged for 3 sec, and incubated for 90 min at 30°C. To the tube, SDS-PAGE sample buffer (10 ul) (2.2.7) was added, and SDS-PAGE was carried out in 10% polyacrylamide slab gels (2.2.6.A). The synthesized labelled proteins were detected by gel fluorography (2.2.9.C)

4.2.4 Translation of al RNA and 8 RNA of the GABA-R in Xenoous oocvtes

Oocytes were removed from an anaesthetized Xenopus. dissected individually and incubated overnight at 17°C in

Barths medium (see 4.2.1). Glass microtubes were stretched over a gas flame and further thinned by suspending the microtubes attached to a weight (10 oence coin) in an electrical coil. These glass needles were graduated by aspirating water (1 ul) into the needle and marking 10 equidistant lines. Using a micromanipulator, a RNA and

0 RNA (1:1 ratio, 50 nl) were injected into each oocyte. The oocytes were kept for 3-4 days at 17°C with daily changes of the Barths medium before they were assayed for

GABA-R ligand binding activities.

244 4.2.5 Assay of ligand binding activity to GABA-R expressed in Xenopus oocvtes

Injected and cultured oocytes (40) were homogenized with a glass-teflon homogenizer in buffer A which was 20 mM Tris/HCl pH 7.4, 50 mM NaCl, 10 mM Mg acetate (1 ml), and supplemented with 100 mM NaCl, 1 mM PMSF, 10% (w/v) sucrose. The homogenate was layered on top of a step sucrose gradient of 50% (w/v) sucrose (2 ml) and 20% (w/v) sucrose (2 ml) in buffer A. The tube was centrifuged for 30 min at 15,000 g at 4°C in an HB4 swing rotor. The membrane layer at the interface of the 20%-50% sucrose layers was collected and resuspended in 20 ml of buffer A and centrifuged at 20,000 g for 30 min in a fixed angle rotor at 4°C. The pellet was resuspended in membrane binding assay buffer (see 2.2.5.A), homogenized by several passages through a 23g needle and aliquoted (200 ul) into the assay tubes. The binding assay was performed as described in 2.2.5.A .

245 4.3 Results

4.3.1 Synthesis of the al RNA and the 8 RNA of the GABA-R

The specific RNA coding for the al and the p subunit were prepared as described (4.2.2). Each RNA preparation was tested in a translation experiment by means of the rabbit reticulocyte lysate method (4.2.3). Figure 4.3.1 shows an autoradiogram of the [35S]-labelled products of the RNA translation. The specific products of the translation of a RNA and p RNA were 42 kDa and 43 kDa respectively. A band of molecular weight 35 kDa was also visible, but this was present also in the control samples where no RNA was added.

To further test the functionality of the synthesized

RNAs, these were injected into oocytes which after 3 days were tested electrophysiologically by Dr E. S. Levitan. Following addition of 20 uM GABA onto the impaled oocytes,

Dr Levitan registered a 400 nA inward current, which was comparable to the currents previously recorded in similar experiments carried out by him (Levitan et al.. 1988) .

4.3.2 Liaand binding assays on injected oocvtes

It was found that a total homogenate of 60 non-injected oocytes in a total assay volume of 1.2 ml (i.e. 10 oocytes per binding assay point), when assayed with 10 nM [3H]flunitrazepam by filtration, showed a non-specific binding of 26,000 cpm (see Table 4.3.1) When the membranes of the same number of oocytes were separated from the other cell components by the step sucrose gradient described (4.2.5) only 1200 cpm non-specific binding were measured under the same assay conditions. Therefore this preparation was preferred for further binding assays. It was initially found that the oocytes were not surviving for more than 24 h in culture. This appeared to be a deficiency in the batches of oocytes used. When new Xenopus frogs were purchased, survival rates of 90% after 3 days culture were obtained. However this problem of low survival rates of the oocytes occasionally recurred.

When oocytes which showed high survival rate three days after the injection of al RNA and p RNA, were assayed with

10 nM [3H]flunitrazepam (5 oocytes per binding assay point), no specific binding was detected compared to the same oocyte membrane preparation assayed also in the presence of 10 uM flunitrazepam (Table 4.3.1). Assays of non-injected oocyte membrane preparations were always carried out in parallel and showed no specific binding.

As the presence of functional GABA binding sites in injected oocytes was shown by electrophysiological work carried out in the laboratory, it was decided to test the

247 «

binding of [3H]muscimol in injected oocytes. In two initial experiments where a total of 30 and 98 oocytes were assayed with 20 nM [3H]muscimol, no specific binding was obtained. When a third batch of 85 healthy injected oocytes were assayed with 100 nM [3H]muscimol specific binding was obtained (Table 4.3.1). Non-injected oocytes were shown to have no specific [3H]muscimol binding. Due to the difficulty of injecting large numbers of oocytes, the above results consisted of a single point binding assay therefore, in order to further test the presence of [3H]muscimol binding sites in injected oocytes the assay was carried out in the presence of pentobarbital. In one experiment where 115 oocytes were assayed with 100 nM

[3H] muscimol in the presence and absence of 1 mM pentobabital, it was shown that pentobarbital increased the specific binding of [3H]muscimol to injected oocytes and not to non-injected oocytes (Table 4.3.1). Further experiments with new batches of oocytes should confirm the presence of the above-observed [3H]muscimol binding sites. 4

248 Figure 4.3.1

kOa

68 —

60 —

ABC

Autoradiograph of rabbit reticulolysate in vitro translation of GABA-R RNAs.

Lane A: p RNA translation product. Lane B: a RNA translation product. Lane C: control, no RNA added to the reticulo • • is . lysate. The reticulolysate contained [ S ]methionine and the film was exposed for 3 days. (See text 4.3.1).

2 49 Table 4.3.1

Prep. Oocytes Oocytes Health Assay Specifically bound used per assay ligand (cpm)

A 60 non-inj ected 10 + F 10 nM (26,000) (a) B 60 non-injected 10 + F 10 nM (1028) (a)

B 30 inj ected 5 F 10 nM 0 B 30 non-injected 5 F 10 nM 0

B 30 inj ected 5 + F 10 nM 0 B 30 non-injected 5 + F 10 nM 0

B 45 inj ected 7 F 10 nM 0 B 45 non-injected 7 F 10 nM 0 "

B 45 inj ected 7 + F 10 nM 0 B 45 non-injected 7 + F 10 nM 0

A 60 non-injected 10 + M 20 nM (8,340) (a) B 60 non-injected 10 + M 20 nM (1,320) (a)

B 30 injected 5 M 20 nM 0 B 30 non-injected 5 M 20 nM 0

B 98 injected 12 + M 100 nM 692 ± 234 B 98 non-injected 12 + M 100 nM 316 ± 208

B 118 injected 13 + M 50 nM 120 ± 120 13 + M 50 nM + P 365 ± 132 B 118 non-injected 13 + M 50 nM 0 13 + M 50 nM +P 0

A = Total homogenate B = membrane fraction from step sucrose gradient (4.2.4). (a) total counts F = [3H]flunitrazepam M = [3H]muscimol P = pentobabital (1 mM) Health: + = healthy; - = unhealthy;

Binding assays of oocytes injected with al RNA and 0 RNA.

Each group of data represents a different experiment with a separate batch of injected or non injected oocytes. 4.4 Discussion

4.4.1 Characterization of the GABA-R ligand binding

sites expressed in Xenopus oocvtes

RNA specific for the al and the 0 subunit of the

GABA-R were synthesized in vitro from linearized plasmids containing the cloned cDNA for the respective subunits.

Reticulocyte lysate translation of the RNAs showed the

synthesis of two polypeptides whose molecular weights

indicated that full-length translation of the cDNA and

full-length expression of the RNAs had occurred.

Electrophysiological recording of Xenopus oocytes injected with both the al RNA and the 0 RNA showed that a GABA gated Cl” ion channel had been synthesized in the oocytes.

In this work, the ligand binding properties of the GABA-R expressed in oocytes were analysed.

First of all, it was observed that the rate of oocyte survival in culture and their efficiency of expression of foreign RNA when tested electrophysiologically varied greatly from batch to batch. Furthermore, it was not possible to predict from the external features of the oocytes such as colour, shape and size whether a particular batch would show high efficiency of expression.

Oocytes are known to undergo seasonal variations in their

251 ability to express foreign RNA, possibly reflecting the mating cycles of the Xenopus. (D. Green and I. Smith, personal comunication) The ligand binding studies performed showed that no specific [3]flunitrazepam binding was detected but in two out of four experiments

specific [3H]muscimol was observed, and [3H]muscimol binding was enhanced by pentobarbital. The fact that

[3H]muscimol binding was not detected in the other two

experiments could be attributed both times to low

efficiency of translation in the oocytes.

The oocyte expression system produces quantities of

receptors that are at the limits of the sensitivity of the ligand binding assay used. Previous ligand binding

studies on cloned receptors expressed in oocytes had shown that the a2 adrenergic receptor (Kobilka et al., 1987) and the muscarinic acetylcholine receptor (Kubo et al., 1986)

could be expressed at 1-2 fmol/oocyte and 3-6 fmol/oocytes

respectively. In order to predict the radioactive signal

expected to be bound to oocytes injected with al RNA and p RNA, the following assumptions were made:

1) 1 fmol receptor per oocyte is expressed

2) each GABA-R receptor binds two [3H]muscimol molecules

and one [3H]flunitrazepam molecule (Stephenson, 1988).

3) all the expressed receptors can be fully occupied at the ligand concentration used.

If all the above criteria were satisfied, each oocyte

252 injected with the GABA-R subunit RNAs when assayed with

[3H]flunitrazepam (85 Ci/mmol) would emit 189 dpm which with a 40% counting efficiency would correspond to 76 cpm per oocyte. Similarly when assayed with [3H]muscimol each

oocyte would show 86 dpm or 34 cpm. Therefore, in order to

obtain a detectable signal at least 5-10 oocytes per

incubation assay are required. Thus the observed non

specific binding of the ligands to the oocyte membranes, would give a specific binding signal of 10-30% of the total binding. Higher binding could be obtained if the

GABA-R was expressed more efficiently than 1 fmol/oocyte.

From the electrophysiological studies it was measured that

GABA (20 uM) elicited a Cl” current of 4xl0”7 A. Given that the recordings were carried out at a differential of potential (voltage) of 40 mV, applying the formulae:

1) V = I x R and 2) G = 1 / R thus 3) G = I / V where V = voltage? I = current? R = resistance and G = conductance ? the conductance of the whole oocyte was:

G = 4 X 10"7 A / 0.04 V = lx 10”5 S.

The average conductance of one GABA-R Cl” channel is 20 pS, thus the oocytes apparently contained 5x 105 channels which correspond to 8.3 x 10”19 mol or 0.0001 fmol. This value is thus 4 orders of magnitude lower than the assumed minimum number of sites required for detection. The

253 1

calculation of the number of binding sites in an oocyte based on the total current recorded is however probably an underestimation for several reasons. Firstly, not all the channels will be open simultaneously but they will alternate from a closed to an open state. Secondly, desensitization can occur during the recording and thirdly, some of the receptors expressed may not form functional channels but could nevertheless bind the ligands.

From the results of the ligand binding studies here shown it was evident that the expression of the GABA-R in the oocytes was too low to carry out a detailed biochemical characterization of the expressed receptor. In the experiment where the maximum specific [3H]muscimol binding was observed, the average of a triplicate incubation (20 oocytes/tube) was 692 specific cpm which would correspond to 1 fmol receptor per oocyte.

It remains to be clarified why no [3H]flunitrazepam binding was ever recorded even in batches of injected oocytes that showed GABA-gated Cl“ currents. This could be due to the oocyte translation system being inefficient for the post- translational modification that may be required for the expression of benzodiazepine binding.

Alternatively, an extra subunit to date not yet identified may be required to obtain benzodiazepine binding. Recently a further GABA-R subunit has been cloned

254 (P.S Seeburg and P.R. Schofield unpublished results), but

it has not yet been shown to be the benzodiazepine binding

subunit. Oocytes injected with total rat brain mRNA

(Houamed et al., 1984) have however been shown to exhibit benzodiazepine potentiation of GABA-induced Cl" current.

Further comparison of oocytes injected with total mRNA and pure RNA would have given useful information on the

expression of benzodiazepine binding sites.

255 CHAPTER 5

GENERAL DI8CU88I0N

256 The aim of this project was the study of the molecular

structure of the GABA-R. Because one of the major features

of this receptor is multiplicity of ligand binding sites that are present within its structure, studies were

initially carried out on the distribution of the binding

sites among the GABA-R subunits. The photoaffinity

labelling properties of [3H]muscimol were thus used to

characterise its binding site.

The initial photoaffinity labelling experiments carried

out on cortex membranes were directed mainly at characterizing the time course and specificity of the reaction with the final aim of identifying the labelled proteins by SDS-PAGE. Other aspects of the photolabelling reaction, such as its efficiency and the inactivation of reversible binding sites were also investigated. However the photoaffinity labelling studies with

[3H]flunitrazepam (Mohler, 1982? Gibbs et al.. , 1985?

Herblin, 1985) have shown that detailed analysis of the mechanism and the kinetics of the photoaffinity labelling reactions have provided limited information about the receptor itself. It should be accepted that photoaffinity labelling is a complex mechanism whose details are not fully understood, expecially when it is carried out with molecules such as [3H]muscimol that do not have defined photolabile groups. For the purpose of the study of receptor structure, it is best to concentrate the research efforts on the characterization of the labelled

257 polypeptides rather than on the properties of the photolabelling reaction.

The photoaffinity labelling experiments of the purified GABA-R with [3H]muscimol carried out in this work have shown that [3H]muscimol preferentially photolabels t h e p subunit. The specificity of the reaction was demonstrated by the photoaffinity labelling experiments carried out in the presence of competitive ligands. These proved that the ligand that was irreversibly bound was the one that was present in the active sites at the time of the UV irradiation. It however does not prove whether the residues labelled actually belong to the active site, or that indeed the muscimol binding site is present only on the p subunit. The three-dimensional folding of the protein could be such that the photolabelled residue is,

in the linear sequence of the protein, several amino acids away from the active site.

The labelled peptide that was isolated by HPLC and

SDS-PAGE, can be sequenced and should confirm the localization of the label on the p subunit. This in itself will not be proof of the exact localization of the binding site but it will provide a tool for further investigations. Firstly, it will probably be necessary to reduce the size of the peptide further to identify the amino acid bound to the label. The information obtained could then be used for site-directed mutagenesis studies,

258 and for the synthesis of peptide-specific monoclonal antibodies. These latter two techniques should eventually be able to show if muscimol had bound near, or at, the binding site.

Many difficulties were encountered in the production of monoclonal antibodies against the GABA-R. These stemmed mainly from the fact that the immune response obtained in the injected animals was not sufficiently high. Thus monoclonal antibodies against the GABA-R were obtained only after the fusion procedure was modified in order to yield an increased number of hybridomas. Among the several different conditions in which the purified GABA-R was presented to the animal, the one that yielded the highest titre was the alum precipitation of the antigen. Future attempts for the production of monoclonal antibodies should therefore concentrate on this method and search for a highly responsive animal.

In vitro immunization experiments should be further pursued as some specific hybridomas, although unstable, were produced in the later attempts. The most promising of the non-conventional techniques of monoclonal antibody production pursued here is probably the development of anti-idiotypic antibodies. Animals should be immunized with more injections of immobilized ligand, and the time of appearance of anti-idiotypic antibodies should be better characterized. The potential of this technique is great because it would allow the production of monoclonal

259 antibodies against any receptor without the need to purify

it. Additionally, these antibodies could be used as a tool

for the analysis of the active site.

The role of monoclonal antibodies in the characterization of the GABA-R is changing as advances are being made in this field. Thus at the beginning of this work the emphasis was on the development of antibodies for the construction of an immunoaffinity column with the aim of large scale purification of the GABA-R. Such a column would still be useful for example for the receptor reconstitution studies and for crystallization studies.

However, the recent identification of the amino acid sequence of the receptor has opened the possibility of raising antibodies against specific peptides. Polyclonal antibodies against one such peptide were raised here and further experiments would have led to the isolation of monoclonal antibodies. The use of a panel of peptide specific antibodies will yield useful information on the receptor structure as they have, for example, for the nicotinic acetylcholine receptor (Neumann et al., 1985).

The efficiency of translation of the GABA-R al and p subunit in Xenopus oocytes did not permit a full pharmacological and biochemical characterization of the expressed receptor. The fact that the level of expression was low could be related to features of non-coding regions of the RNAs? for example the expression of the 02“

260 adrenergic receptor was enhanced 10 fold when an upstream region was omitted (Kobilka et a l . , 1987). In the experiments presented here, [3H]muscimol binding to the injected oocytes was measured, while inconclusive data were obtained about [3H]flunitrazepam binding. Considering however that due to its higher specific radioactivity,

[3H]flunitrazepam binding should be more readily detectable than [3H]muscimol binding, the results obtained indicated that the flunitrazepam binding sites were not fully expressed. The absence of [3H]flunitrazepam binding could be due to a deficiency in the oocyte translation system. It is however known that the expression of a functional GABA-R, including all of its modulation properties, is obtained when total brain mRNA is injected into Xenopus oocytes. (Houamed et al., 1984). The results obtained and the latest finding of Levitan et al.,(1988) would therefore suggest that the al RNA and the p RNA together are not sufficient for the full expression of the

GABA-R. The transfection of GABA-R cDNAs in cell lines is however required for a more detailed investigation of the cloned GABA-R.

At the present time there is still controversy on whether heterogeneous GABA-Rs exist in the mammalian brain. Photoaffinity labelling experiments with

[3H]flunitrazepam (Sieghart et al., 1983) have shown that

Type I and Type II benzodiazepine receptors are present on two separate polypeptides. However, polyclonal antibodies

261 4

(Stephenson et al.. , 1986a; Stauber al. , 1987) and monoclonal antibodies (Vitorica st ai- * 1988) raised against the purified receptor, immunoprecipitated a population of GABA-R indistinguishable from the total

GABA-R population. The analysis of the GABA-R at the DNA level, in situ hybridisation and anti-peptide specific antibodies should in the near future indicate whether heterogenous GABA-Rs exist and what role they play.

In summary, in this work, the molecular structure of the GABA-R has been studied with particular reference to one of its binding sites. The methodology for the production of monoclonal antibodies has been investigated and two anti-GABA-R monoclonal antibodies have been isolated. A partial characterization of the cloned receptor subunit expressed in Xenoous oocyte has also been attempted. The goal of the full molecular characterization and understanding of the GABA-R fuctions is still far away, but is one that must be pursued, not only because it will shed light on the functioning of the major inhibitory system of the central nervous system, but also probably help in the understanding of diseases like epilepsy,

Huntington's disease and others (Chugani and Olsen, 1986).

The study of the GABA-R will also help understand the less severe, but much more wide-spread medical problems of benzodiazepine drug dependency. To conclude and to put this thesis into context with ”the real world” I would

262 like to quote from the final paragraphs of J.P.

Change u.x's book The Neuronal Man (1983) :

" .... After destroying our environment are we not now destroying our own brains? A single statistic

indicates the urgency of the problem... seven millions of packets [of benzodiazepines] are sold every month in

France and similar numbers in most of the industrialized nations. One adult in four uses chemical tranquillizers.

Must we put ourselves to sleep in order to endure the environment that we have created? The time has come to consider this problem seriously....” The molecular characterization of the GABA-R will help to understand the problem.

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