Differential solubilization of gamma-aminobutyric acid receptive sites from membranes of mammalian brain.
Sodium-dependent (+Na) and sodium-independent (-Na) receptive sites for gamma-aminobutyric acid (GABA) residing in or on frozen synaptic plasma membranes (SPM) of bovine cerebral cortex were characterized as to binding constants, pharmacologic specificities, and sodium dependence. The SPM fraction was then treated with various concentrations of Triton X-100 resulting in the loss of pharmacologic specificity, binding characteristics, and sodium 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, while 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. Sodium-independent GABA binding was lost from JC membranes with the use of sodium deoxycholate, probably through solubilization.
D000818:Animals; D001640:Bicuculline / Q000494:pharmacology; D001665:Binding Sites / Q000187:drug effects; D001667:Binding, Competitive; D002417:Cattle; D002462:Cell Membrane / Q000032:analysis / Q000378:metabolism*; D002540:Cerebral Cortex / Q000378:metabolism* / Q000648:ultrastructure; D007700:Kinetics; D009118:Muscimol / Q000494:pharmacology; D009474:Neurons / Q000378:metabolism; D009557:Nipecotic Acids / Q000494:pharmacology; D011092:Polyethylene Glycols / Q000494:pharmacology; D017981:Receptors, Neurotransmitter / Q000378:metabolism*; D012964:Sodium / Q000494:pharmacology; D012995:Solubility; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D009557:Nipecotic Acids; D017981:Receptors, Neurotransmitter; D009118:Muscimol; D011092:Polyethylene Glycols; D005680:gamma-Aminobutyric Acid; D012964:Sodium; D001640:Bicuculline