Gamma-Aminobutyric Acid Binding to Receptor Sites in the Rat Central Nervous System (Neurotransmitter/Bicuculline/Synaptic Membranes/Glycine/Strychnine) STEPHEN R
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Proc. Nat. Acad. Sci. USA Vol. 71, No. 12, pp. 4802-4807, December 1974 Gamma-Aminobutyric Acid Binding to Receptor Sites in the Rat Central Nervous System (neurotransmitter/bicuculline/synaptic membranes/glycine/strychnine) STEPHEN R. ZUKIN, ANNE B. YOUNG, AND SOLOMON H. SNYDER* Departments of Pharmacology and Experimental Therapeutics, and Psychiatry and Behavioral Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 Communicated by David Bodian, September 16, 1974 ABSTRACT 18H]Gamma-aminobutyric acid (GABA) brains were rapidly removed and homogenized in 15 volumes binds to synaptic membrane fractions of rat brain in a M sucrose in a Potter-Elvehjem glass homog- selective fashion representing an interaction with post- of ice-cold 0.32 synaptic GABA receptors. Inhibition of ['H1GABA bind- enizer fitted with a Teflon®D pestle. The homogenate was ing by a variety of amino acids closely parallels their ability centrifuged at 1000 X g for 10 min, the pellet was discarded, to mimic the synaptic inhibitory actions of GABA and and the supernatant fluid was centrifuged at 20,000 X g for 20 does not correlate with their relative affinity for the pre- min. The crude mitochondrial pellet was resuspended in synaptic synaptosomal GABA uptake system. ['HIGABA binding is saturable with an affinity constant of about 0.1 distilled water and dispersed with a Brinkmann Polytron MM. The GABA antagonist bicuculline inhibits ['HIGABA PT-10 sonicator (setting 6) for 30 sec. The suspension was binding with half maximal effects at 5 MM, whereas it re- centrifuged at 8000 X g for 20 min. The supernatant was col- quires a concentration of 0.5 mM to reduce synaptosomal lected and the pellet, a bilayer with a soft, buffy uppercoat, GABA uptake by 50%. In subcellular fractionation experi- was rinsed carefully with the supernatant fluid to collect the ments ['H]GABA binding is most enriched in crude synap- tic membranes. [8IH]GABA binding is greatest in the cere- upper layer. The combined supernatant fraction was then bellum, least in the spinal cord and medulla oblongata- centrifuged at 48,000 X g for 20 min. The final crude synaptic pons, with intermediate values in the thalamus, hippo- membrane pellets were resuspended in H20 and centrifuged campus, hypothalamus, cerebral cortex, midbrain, and at 48,000 X g for 20 min and then stored at -30° for at least corpus striatum. 18 hr. The frozen pellets were resuspended in H20, maintained Gamma-aminobutyric acid (GABA) is a major inhibitory at 250 for 20 min, centrifuged at 48,000 X g for 10 min and the neurotransmitter in the mammalian central nervous system pellets were then suspended in the buffer for GABA binding (1-4). Its inhibitory actions are selectively antagonized by the assay. ['H]GABA binding capacity remained intact for at alkaloid bicuculline, in contrast to the inhibitory actions of least 30 days under these conditions. Storing frozen tissue glycine, which are selectively antagonized by strychnine enhances GABA receptor binding and markedly lowers (5-8). The synaptic activities of GABA may be terminated sodium-dependent GABA binding unrelated to receptor sitest. by a high-affinity, sodium-requiring uptake system into GABA Binding Assay. Aliquots of crude synaptic membranes nerve terminals (9, 10) and possibly glia (11-13). The sodium- (0.3-1.2 mg of protein) were incubated in triplicate at 40 for 5 dependent binding of GABA to membrane preparations of min in 2 ml of 0.05 M Tris-citric acid buffer (pH 7.1 at 40) mammalian brain largely represents binding of GABA to containing approximately 32 nM ['H]-y-aminobutyric acid uptake sites, since the relative affinity of a variety of amino (GABA) (500,000 cpm) alone or in the presence of 1 mM acids and drugs in preventing this binding parallels their GABA, 0.1 mM bicuculline, or other indicated drugs. Time affinity for the GABA uptake system but differs markedly course experiments indicated that specific [3H]GABA binding from their relative abilities to mimic the synaptic inhibitory is at equilibrium within 5 min under these conditions. After actions of GABA (6, 14, 15,t). incubation, the reaction was terminated by centrifugation for Here we report binding of [8H]GABA to crude synaptic 10 min at 48,000 X 9. The supernatant fluid was decanted, membrane fractions of the rat central nervous system which and the pellet was rinsed rapidly and superficially with 5 ml, appears to represent a specific interaction with the post- then 10 ml of ice-cold distilled H20. Bound radioactivity was synaptic GABA receptor. extracted into 1 ml of Protosol (New England Nuclear Corp.), 10 ml of toluene phosphor were added, and radioactivity was MATERIALS AND METHODS assayed by liquid scintillation spectrometry (Packard Tricarb Tissue Preparation. Subcellular fractionation utilized a model 3385 or 3375), at a counting efficiency of 34%. Analysis modification of previously described techniques (16-20). Male of the membrane-bound radioactivity by thin layer chroma- Sprague-Dawley rats (100-200 g) were decapitated and the tography in three solvent systems indicated that all of the bound material was authentic GABA. Specific [8H]GABA binding was obtained by subtracting Abbreviation: GABA, gamma-aminobutyric acid. from the total bound radioactivity the amount not displaced * To whom reprint requests should be sent at the Department of by high concentrations of bicuculline (0.1 mM) or GABA (1 Pharmacology. mM). Except when otherwise indicated, values for nonspecific t S. Enna and S. H. Snyder, in preparation. binding were the same whether bicuculline or GABA was used 4802 Downloaded by guest on September 29, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Gamma-Aminobutyric Acid Receptor 4803 TABLE 1. Subcellular distribution of specific [8H]GABA binding in the rat brain Specific [3H]GABA binding Specific: O 2000 Binding Total binding nonspecific z a (cpm/mg per fraction binding z Fraction of protein) (cpm) ratio m 1600 - Whole homogenate 282 12,708 0.11 Crude nuclear G 1200 - pellet (Pi) 209 2100 0.10 Crude mitochondrial 800 0.44 pellet (P2) 1068 14,858 a. Crude microsomal CD 400 pellet (P8) 0 0 Osmotically shocked P2 subfractions 0 1.0 2.0 3.0 4.0. Mitochondria-myelin mg of MEMBRANE PROTEIN/2ml pellet 210 1829 0.11 FIG. 1. Effect of synaptic membrane concentration on specific Crude synaptic [3HJGABA binding. Various amounts of synaptic membrane membrane pellet 1517 18,177 0.66 suspensions obtained from rat brain were incubated for 5 min at 40 with 34 nM [3HJGABA (500,000 cpm) alone or in the presence Tissues were prepared and subjected to differential centrifuga- of 0.1 mM bicuculline in a total volume of 2 ml. Each point is the tion as described in the text. The crude microsomal pellet was ob- mean of triplicate determinations which varied <10%. The ex- tained by centrifugation of the supernatant fluid remaining after periment has been replicated twice. centrifugation of the crude mitochondrial pellet at 100,000 X g for 1 hr. The various pellets were resuspended in 50 mM Tris-citric pellet probably does not contain large amounts of GABA acid buffer (pH 7.1) (0.7-1.2 mg of membrane protein per 2 ml) binding activity. and assayed as described. The experiment has been replicated When the P2 pellet is subjected to hypotonic shock, nerve twice. endings are lysed and subsequent differential centrifugation can resolve a low-speed pellet containing mitochondria and as a displacer. Protein was measured by the method of Lowry myelin from a high-speed fraction which is enriched in et al. (21). synaptic membranes (16). The crude synaptic membrane fraction contains about 10 times as much GABA binding as Uptake Studies. Uptake of radioactive GABA was assayed the mitochondria-myelin pellet and has a specific activity as previously described (22-25). more than five times that of the original homogenate. The Materials. [3H]GABA (specific activity 10 Ci/mmol) was crude synaptic membrane fraction has been used for routine obtained from New Nuclear GABA binding assays. Specific binding of [3H]G-ABA to England Corp. Picrotoxin, and 2 of whole purchased from Sigma Chemical Corp., and bicuculline, from synaptic membranes is linear between 0.5 mg Pierce Chemical Co., were dissolved immediately prior to use brain membrane protein (Fig. 1). Binding studies are routinely because of their chemical lability. performed within this linear range. During preparation of the crude synaptic membrane frac- RESULTS tion from brain homogenates, the ratio of specific to non- Subeellular localization of [3H]GABA binding specific GABA binding increases progressively. Thus in the in the rat brain whole homogenate specific GABA binding is only 10% of the total. The ratio of specific to nonspecific binding in the crude The distribution of specific [3HIGABA binding in subcellular mitochondrial is four times that of the whole fractions of whole rat brain was measured in fractions prepared pellet homog- enate, while the crude synaptic membrane fraction displays a by a modification of previously described techniques (16-20) six of whole Thus in routine (Table 1). Among the primary fractions obtained by differen- ratio times that the homogenate. assays with the synaptic membrane fraction, about tial the largest amount of binding is recovered 40-50% centrifugation, of [3H]GABA binding is displaced by 0.1 mM bicuculline or 1 in the crude mitochondrial pellet (P2) which contains both mM GABA. pinched-off nerve endings ("synaptosomes") and free mito- chondria. Total specific binding in the P2 pellet is as great or Saturability of I3HJGABA binding to synaptic membranes greater than that of the whole homogenate, while binding in of whole rat brain the crude nuclear pellet is only about 1/6th that of the whole Specific [3H]GABA binding is saturable with increasing con- homogenate, and no specific binding can be detected in the centration, with half-maximal binding occurring at approxi- crude microsomal pellet.