Functionally relevant gamma-aminobutyric acidA receptors: equivalence between receptor affinity (Kd) and potency...

Many neurotransmitter receptors bind agonists with high affinity (Kd in the nanomolar range), whereas micromolar concentrations of the same agonists are required to elicit a functional effect. We have identified low affinity agonist binding sites for the gamma-amino-butyric acidA (GABAA) receptor-chloride channel under conditions normally used in 36Cl- uptake assays (a measure of receptor function). The GABAA agonist [3H]muscimol bound to a population of receptors with a Kd (2 microM) similar to its EC50 value for 36Cl- uptake. Binding was inhibited by the GABA agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol and by the GABA antagonist bicuculline methiodide. A reduction in the number of [3H]muscimol binding sites (Bmax) by a thiol-modifying reagent produced a corresponding decrease in the Emax for muscimol. The benzodiazepine diazepam enhanced the potency of muscimol in ion flux experiments but did not alter the affinity of [3H]muscimol binding sites. We propose that benzodiazepines enhance GABAergic function by increasing receptor-ion channel coupling, rather than by increasing GABAA receptor affinity. These studies suggest that the study of physiologically relevant (low affinity) binding sites is necessary when examining regulation of receptors by cellular processes, drugs, and disease.