KINETIC AND PHARMACOLOGIC CHARACTERIZATION OF GAMMA-AMINOBUTYRIC ACID RECEPTIVE SITES FROM MAMMALIAN BRAIN

Sodium-dependent (+Na) and sodium-independent (-Na) receptive sites for γ-aminobutyric acid (GABA) have been characterized using synaptic plasma membranes from bovine and rat brain. Synaptic plasma membranes were prepared from either rat cerebellar cortex or calf cerebral cortex by discontinuous sucrose gradient flotation centrifugation of crude mitochondrial pellets, and assayed using equilibrium ligand binding assays to obtain the maximum binding capacity (Bmax) and the thermodynamic constant (KD). Values for KD from equilibrium studies were subsequently confirmed by kinetic analyses of association and dissociation reactions. The KD for +Na GABA binding (5.0 ± 0.2 μM) corresponds to the apparent Michaelis constant for neuronal GABA transport (3.8 ± 0.1 μM)22, while the KD for -Na binding (0.17 ± 0.04 μM) agrees with that determined by Enna and Synder7 for the putative postsynaptic receptor. Maximal binding activities of about 5 and 55 pmole/mg protein were obtained for -Na and +Na binding respectively. The pharmacologic specificities of the two sites were determined using competition binding studies. Nipecotic acid and diaminobutyric acid inhibit both synaptosomal GABA uptake (Ki ≈ 25 μM and 120 μM respectively) and +Na binding of GABA to synaptic plasma membrane (IC50 ≈ 40 μM and 350 μM respectively) but do not inhibit -Na binding. Bicuculline inhibits -Na [3H]GABA binding at low concentrations (IC50 ≈ 15 μM), while affecting the uptake and +Na binding of [3H]GABA only at high concentrations (IC50 ≈ 520 μM and 300 μM respectively). β-Alanine inhibits the -Na binding site (IC50 ≈ 100 μM), but is ineffective at the +Na binding site and does not interfere with synaptosomal uptake of GABA. Finally, chlorpromazine and N-ethylmaleimide inhibit the +Na binding, albeit at high concentrations (IC50 ≈ 600 μM and 5 mM respectively) but are ineffective at the -Na binding site. From these results the -Na binding site is tentatively identified as a postsynaptic receptor and the +Na binding site is identified as the neuronal uptake receptive site. © 1979.