Life Sciences, Vol . 25, pp . 783-790 Printed in the U .S .A .

Pergamon Press

SOLUBILIZATION OF HISTAMINE H-1, GABA AID BâNZODIAZEPINE RECEPTORS Moahe Gaviah, Raymond S .L . Chaag and Solomon H. Snyder Departments of Pharmacology and Experimental Therapeutics and Psychiatry and Behavioral Sciences Johns Hopkins University School of Medicine Baltimore, Maryland 21205 (Received in final form July 23, 1979)

Binding sites for histamine H-1, GABA and benzodiazepine receptors have been solubilized from mammalian brain membranes using, as detergents, digitonin for benzodiazepine and H-1 receptors and lysolecithin for GABA receptors . The dissociation constants for 3Fi-mepyramine at histamine H-1 (1 .5 nM), 3H-muscimol at GABA (7 nM, 24 nM) and 3H-fluaitrazepam at benzodiazepine receptors (1 .5 nM) are similar to values in membrane preparations . The relative and absolute potencies of various drugs in competing for 3H-ligand binding to the three receptors are closely similar in the soluble and membrane bound states, indicating that the conformation of the receptor recognition site is maintained during solubillzation . Maximal numbers of binding sites are about 50X of corresponding levels in the membrane bound state. Binding studies with radiolabeled ligande have facilitated an underatandinR of neurotransmitter receptors in the brain (1 ). To clarify molecular mechanisms regulating receptor activity, it would be desirable to solubilize and purify neurotransmitter receptors. However, receptor binding for various neurotransmitters in brain membranes is often readily inactivated by detergents commonly used to solubilize membrane proteins, presumably reflecting an important role for phoapholipids in the recognition function of neurotransmitter receptors . Recently dopamine (2) and muscarinic cholinergic (3,k) receptors from brain membranes have been solubilized under conditions which preserve the binding of ligande in the soluble state . By contrast after solubilization, opiate receptors no longer bind opiates (5) . In the present report we describe the solubilization of histamine H-1, GABA and benzodiazepine receptors with preservation of drug and transmitter binding interactions in the soluble state, Materials and Methods 3H-Fluaitrazepam (84 .8 Ci/mmole), 3H-muscimol (12 .1 Ci/mmole) and 3H~mepyramine (28.5 Ci/mmole) were obtained from New England Nuclear, Boston, Massachusetts . Unlabeled benzodiazepines were obtained from Hofiman-La Roche, Inc . All other compounds were purchased from commercial sources . Solubilization and assay of benzodiazepine receptors : Male Sprague-Dawley rate (175-220 g) were decapitated and the brains minus the brain stem were immediately removed. The tissue was homogenized in 15 volumes of 0.1 M sodium phosphate buffer pH7.4, and 0.32 M sucrose with a glass homogenizes using a Teflon pestle . The homogenate was centrifuged 1000g for 10 min., and the 0024-3205/79/090783-07$02,00/0 Copyright (c) 1979 Pergamon Press Ltd

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supernatant fluid was centrifuged at 49000 g for 15 min, at 4°C, The pellet was resuapended (4 mg protein/ml) in 1X digitonin (Sigma Chemicals) 0 .1 M sodium phosphate buffer pH 7 .4 and 0 .32 M sucrose. The mixture was shaken for 30 min . and the supernatant fluid (100,000 fi for 1 hr) used for binding assays . Binding assay : To 400 ul of solubilized preparation, 50 ul 3H-flunitrazepam were added to a final concentration of 1 .5 nM, unless otherwise indicated, in the presence of 50 ul of buffer (for total binding) or clonazepam (for non specific binding) at a final concentration of 10 uM . The mixture was incubated for 30 min. a t 0 °C whereupon protein was precipitated by the addition of 0.5m1 ice-cold saturated ammonium sulfate, and after 3 min., samples were filtered under vacuum over GF/B filters, washed twice with 5 ml of 50% saturated ammonium sulfate. The filters were placed in vials containing 10 ml of Formula 947 (New England Nuclear Co ., Boston, Mass,) stored overnight, and radioactivity monitored by liquid scintillation spectrometry . Solubilization and assay of histamine H-1 receptors : The preparation of solubilized histamine H-1 receptors was similar to that of benzodiazepine receptors except that Hartley guinea pig brains were employed instead of rat, and the homogenate was centrifuged twice at 49000 g, Binding assay : To 800 ul of solubilized preparation, 100 ul of 3H~mepyramine were added to a final concentration of 2 nM, unless stated otherwise, in the presence of 100 ul of buffer (for total binding) or triprolidine (for non specific binding) at final concentration of 2 uM . The mixtures were incubated for 30 min. at 25°C and then placed on an ice bath for 5 min. To these reaction mixtures, 100 ul of lOX activated charcoal containing 2% bovine serum albumin and 0 .1 M sodium phosphate buffer (pH 7 .4) were added and the mixture rapidly mixed . Two minutes after the addition of charcoal, the samples were centrifuged 5 min, in a Brinkmann microfuge . Eight hundred ul of the supernatant fluid were counted for radioactivity . Solubilization and assay of GABA receptors : Male Sprague-Dawley rats (175220 g) were decapitated and the brains minus the brain stem were immediately removed . The tissue was homogenized in 15 volumes of 50 mM Tris-citrate buffer, pH 7 .1 at 4°C, and 0 .32 M sucrose. The homogenate was centrifuged at 10008 for 10 min. and the supernatant fluid was centrifuged at 490008 for 15 min, at 4°C . The pellet was frozen at least 20 hours . The frozen tissue was homogenized in 50 mM Tris-citrate buffer pH 7 .1 at 4°C, containing 0,05X Triton X-100. The homogenate was incubated 30 min. at 37°C and then washed 5 times with Triscitrate buffer pH 7 .1 at 4°C . The pellet was solubilized by 0 .25% lysolecithin (Sigma Chemicals) in 50 mM Tris-citrate buffer pH 7.1 at 4°C, and 0,32M sucrose and incubated 15 min . at 25 ° C . After incubation the solubilized preparation was diluted to 0 .15X lysolecithin,centrifuged at 100,000 g for 60 min. at 4 ° C and the supernatant fluid used for binding assays . Binding assay: To 400 ul of solubilized preparation 50 ul of 3H-muscimol were added to give a final concentration of 10 nM, unless otherwise indicated, in the presence of 50 ul of buffer (for total binding) or GABA (final concen tration 100 tiaM, for nonspecific binding) . The mixture was incubated for 30min. at 0°C. After the incubation, 100 ul of 10 mg/ml bovine 7-globulin and 200 ul of 30X polyethylene glycol (approx. M,W. 6000, Sigma Chemical) were added. The mixture was filtered on a GF/B filter 5 min. after the addition of polyethylene glycol and washed twice with 8.5% polyethylene glycol . Filters were counted for radioactivity as described previously . Protein was determined by the method of Lawry et al . (6) .

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FIG. 1 Saturation of ligand binding to solubilized receptors. . A. 3H-Mepyramine binding to solubilized preparations as creasing concentrations of 3Ii-mepyramine . B . 3H-Muscimol binding to solubilized preparations as a creasing 3fi-muscimol concentrations . C . 3H-Fluaitrazepam binding to solubilized preparations increasing 3H-flunitrazepam concentrations . Details of preparation of solubilized receptors and the are described under Materials snd Methods.

a function of infunction of inas a function of binding assay

Results Histamine H-1 Receptors : The binding of 3 H-mepyramiae to soluble membrane protein is sat~rable (Fig . 1) . At 2 nM concentration used routinely in binding assays, total H-mepyramine binding is about three times greater than nonspe cific binding assayed in the presence of 2 uM triprolidiae . Specific binding, the difference between total and nonspecific binding, reaches plateau values at about 4 nM with half maximal binding at approximately 1 nM . Scatchard analysis indicates a single population of binding sites with a dissociation constant (ICD) of 1 .3 nM with a maximal number of binding sites (BMAg) of 80 fmoles/mg protein . The (RD) value of 3H-mepyramiae at the soluble receptor sites is similar to the value obtained is brain membranes (7) . The recovery of receptor binding for the solubilization procedure is 40-60% .

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To determine whether the saturable binding of 3H~mepyramine involves H-1 receptors, we examined the potencies of a variety of drugs (Table 1) . As at membrane bound H-1 receptors, the soluble binding sites are influenced in a stereospecific manner by the antihistamine chlorpheniramine, with the pharmacologically active d-isomer being about 100 times more potent than the inactive 1-isomer . The potent antihistamines mepyramine, triprolidine and promethaziae have similar affinities for soluble and membrane bound receptors . The potency of histamine itself is also approximately the same at the soluble and membrane associated receptor . TABLE I Drug Effects on Specific 3H-Mepyramine Binding in Solubilized and Membrane-bound Receptors Compound

d-chlorpheairamine 1-chlorpheniramine Mepyramine Triprolidine Promethazine Histamine

Ki Solubilized 3.7 440 1.2 3.8 2 .8 24,000

(nM) Membrane-bound 1.4 130 0.5 0.6 3.2 27,000

Specific 3H-mepyramine (2 nM) binding to guinea pig brain membrane bound receptors and solubilized receptors, by several kinds of histamine-H1 compounds was assayed in the presence of 5-7 drug concentrations in triplicate to estimate IC50 values (concentration causing 50X inhibition of 3H-mepyramiae binding) . The Ri values were calculated from the equation R1-IC50/(1 + C/KD) where C - 3H-mepyramine concentration; RD = dissociation constaat~ 1 .3 nM, GABA receptors : GABA receptors in membranes can be labeled with 3H-GABA(8) or the potent GABA agonist 3Ii-muscimol (9,10) . The binding of 3fi~muscimol to preparations of brain membranes Solubilized with lysolecithin is saturable (Fig . 1) . At 10 nM 3H-muscimol used in routine experiments total binding is about 10 times higher than nonspecific binding measured in the presence of 0 .1 mM GABA . Specific binding, the difference between total and nonspecific binding, attains plateau values at 50-100 nM and reaches half maximal binding at about 7 nM . Scat chard analysis reveals two populations of binding sites with respective (RD) values of 7 nM and 24 nM and respective (B~) values of 1000 fmoles/mg protein and 770 of fmoles/mg protein . The KD values are similar to those of the membrane bound receptors . The recovery of binding from solubilization is about 50-60X . The binding sites display the drug specificity of GABA receptors . Muscimol itself is about five times more potent thaw GABA (Table 2) .The GABA agoniat imidazoleacetic acid is somewhat less potent than GABA, while 2, 4-diaminobutyric acid, which is inactive at membrane binding sites, is also extremely weak in competing for 3H-muscimol binding to soluble preparations . The GABA aatogoniet bicuculline has about the same potency in soluble as in membrane preparations .

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Beazodiazepine receptors : The binding of 3H-flunitrazepam to membranes solubilized with digitonin also is saturable (Fig .l) . At 2 nM concentration total binding i8 about eight times higher than nonspecific binding assayed in the presence of 10 UM clonazepam . Specific binding plateaus between 5 and Scatchard analysis reveals a single population of binding sites with a lOnM . The RD value is xD value of 1 .5 nM and a BlfA% value of 180 fmolea/mg protein. The binding similar to the value obtained in membrane bound receptors (11) . recovery of solubilization is about 50X. TABLE II Drug Effects on Specific 3H-Muscimol Binding in Solubilized and Membrane-bound Receptors Ri (nM)

Compound

Muscimol GABA (+) -Bicuculline methiodide Imidazoleacetic acid D,L-2,4-Diaminobutyric acid Isoxazole Picroto~d.a

Solubilized 8 28 3000 110 >10000 68 >10000

Membrane-bound 5 16 2000 50 >10000 410 >10000

Specific ~-muscimol (10 nM) binding to rat brain membrane bound receptors and solubilized receptors was assayed in the Ri values presence of 5-7 drug concentrations is triplicate . value for 3fi-muscimol were determined as in Table I using a RD of 7 nM . The drug specificity of the soluble 3H-flunitrazepam binding sites is esaentielly the same as that of membrane bound benzodiazepine receptors (Table 3) . Flunitrazepam and clonazepam are the most potent benzodiazepinea, about ten times as potent as diazepam . Chlordiazepoxide is more thaw two orders o~ magAgents such as RO 5-4964, RO 5-4864 nitude less potent than fluaitrazepam. and RO 5-4933, which are inactive is pharmacological screens and is binding to brain membrane receptors, are also virtually inactive in competing for binding to soluble receptors . Discussion The present study shown that, with appropriate detergents, histamine H-1, GABA and benzodiazepine receptors can be solubilized under conditions which Independently, Tallman _et _al. (13) have succeeded preserve binding of ligaada . in solubiliziag benzodiazepine receptors using a different detergent, Lubrol . Al~o, in a preliminary abstract Greenlee and Olsen reported solubilizing 3ümuacimol binding associated with GABA teceptora using deoaychblate as detergent (14) . Preservation of binding properties in soluble receptors appears to depend For instance, in our laboratory upon the choice of an appropriate detergent . deoxycholate, Triton R-100, and Lubrol have been ineffective in maintaining binding to soluble histamine-H1 receptors .

Selection of as appropriate tech-

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nique for measuring binding to soluble receptors is also critical . Binding assays using Sephadex columns have been relatively unsuccessful in our laboratory, in part because of their restriction to relatively small tissue concentrations as with large columns, 3H-ligand may dissociate . Ammonium sulfate precipitation proved to be efficient for measuring benzodiazepine recéptor binding, but appeared to reduce GAGA eceptor binding. Charcoal binding, which was effective for detecting soluble ~-mepyramine binding could not be used with 31i-muacimol, which fails to bind to charcoal . After solubilization .the B~ value for the three receptors is about 50X of the corresponding value for membrane bound receptors,implying a high recovery of receptors after solubilization : The affinity of 3H-liganda and relative as well as absolute drug potencies are quite similar at the three receptors in the soluble and membrane states suggesting that the recognition site of the receptors changes very little in its conformation during solubilization . TABLE III Drug Effects on Specific 3H-Flunitrazepam Binding at Solubilized and Membrane-bound Receptors Ri

Compound

(nM)

Solubilized Flunitrazepam Clonazepam Diazepam Chlordiazepoxide Bromazepam Medazepam Halazepam Nitrazepam Flurazepam RO 5-4964 RO 5-4864 RO 7-1986/1 RO 5-2904 RO 5-3027 RO 5-3590 RO 5-4528 RO 5-4933 RO 5-6277 *Data from Braestrup and

1 .5 1 .2 14 460 20 9000 320 6 18 >10000 >10000 8 .5 45 2 .5 8 1350 >10000 20 Squires (12) .

Membrane-bound* 2.8 1.9 8.9 574 30 3850 19 16 >10000 14 7 .2 274

Specific 3H-flunitrazepam (1 .5 nM) binding to rat brain membrane bound receptors was assayed in the presence of 5-7 concentrations of each drug in triplicate . Ki values were determined ae in Table I using a value for H-flunitrazepam of 1 .5 nM . ~ Aclcnowledgements Supported by USPHS grants DA-00266, MH-18501, the McKnight and John A. Hartford Foundations and a Damon Runyon fellowship to M,G.

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14 .

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