Differential Solubilization of γ‐Aminobutyric Acid Receptive Sites from Membranes of Mammalian Brain

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Abstract
Na-dependent (+Na) and Na-independent (-Na) receptive sites for .gamma.-aminobutyric acid (GABA) residing in or on frozen synaptic plasma membranes (SPM) of bovine cerebral cortex were characterized for binding constants, pharmacologic specificities [nipecotic acid, muscimol] and Na dependence. The SPM fraction was then treated with various concentrations of Triton X-100 resulting in the loss of pharmacologic specificity, binding characteristics and Na dependence associated with +Na GABA receptive sites in SPM. The resulting junctional complex preparation (JC), i.e., a fraction enriched in junctional complexes, possessed only the pharmacologic specificity and binding constants associated with -Na receptive sites whether assayed in the presence or absence of 100 mM-NaCl. This is probably due to the detergent dispersal or solubilization of the +Na GABA receptive site. The binding constants Kd and Bmax for -Na GABA binding in SPM were 170 nM and 4.4 pmol/mg protein, in JC they were 186 nM and 3.7 pmol/mg protein. Under repeated washing the Kd was reduced to 60 .+-. 6.9 nM and the Bmax was reduced to 2.5 .+-. 0.5 pmol/mg protein in JC, probably owing to the removal of endogenous ligand or inhibitor and not to inhibition by residual Triton X-100. Multiple extraction with 0.1 or 0.5% Triton X-100 did not alter the Kd or Bmax values for the binding of [3H]GABA to JC. Na-independent GABA binding was lost from JC membranes with the use of sodium deoxycholate, probably through solubilization.