Ion and Temperature Effects on the Binding of γ‐Aminobutyrate to Its Receptors and the High‐Affinity Transport System

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Abstract
A set of procedures was developed to study the binding of .gamma.-[3H]aminobutyric acid ([3H]GABA) to GABAA and GABAB receptors, and to the Na+-dependent transport carrier, at 25 and 37.degree. C in the presence of physiological conconcentrations of Na+. The membrane preparation used in these procedures was not subjected to freeze-thawing or treatment with Triton X-100. Isoguvacine, (-)-baclofen, and (-)-nipecotate were used to block selectively the binding to GABAA receptors, GABAB receptors, and the transport site, respectively. Analysis of the binding characteristics of [3H]GABA to the GABAA receptor suggested the existence of high-(KD < 30 nM), middle- (KD = 100-500 nM), and low-affinity KD > 5 .mu.M) binding sites. However, the binding data in the middle-affinity region (100-1,000 nM) were often indicative of cooperativity. The affinity between GABA and the GABAA receptor was reduced modestly by increases in temperature and by the presence of Cl- at physiological concentrations. Binding to the GABAB receptor required Ca2+ and Cl-. Apparent binding to the transport carrier required both Na+ and Cl-. A comparison of Bmax values in three brain regions revealed an inverse relationship between the high-affinity site of the GABAA receptor and the transport binding site.