Kinetic and pharmacologic characterization of gamma-aminobutyric acid receptive sites from mammalian brain.

Sodium-dependent (+Na) and sodium-independent (-Na) receptive sites for gamma-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 micron) corresponds to the apparent Michaelis constant for neuronal GABA transport (3.8 +/- 0.1 micron)22, while the KD for -Na binding (0.17 +/- 0.04 micron) agrees with that determined by Enna and Snyder 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 approximately 25 micron and 120 micron respectively) and +Na binding of GABA to synaptic plasma membrane (IC50 approximately 40 micron and 350 micron respectively) but do not inhibit -Na binding. Bicuculline inhibits -Na [3H]GABA binding at low concentrations (IC50 approximately 15 micron), while affecting the uptake and +Na binding of [3H]GABA only at high concentrations (IC50 approximately 520 micron and 300 micron respectively). beta-Alanine inhibits the -Na binding site (IC50 approximately 100 micron), 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 approximately 600 micron 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.