DIRECT EVIDENCE FOR DIAZEPAM MODULATION OF GABA(A) RECEPTOR MICROSCOPIC AFFINITY

Alteration of agonist affinity is a potential mechanism for pharmacolo gical modulation of ligand-gated receptor channel function. The time c ourse for receptor activation and current onset is determined by the c ombined rates for two kinetic transitions that underlie the protein co nformations for binding agonist and channel gating. Using ultrafast li gand exchange techniques, we distinguish between these previously diff icult to separate events and demonstrate their independent pharmacolog ical modulation. Diazepam, which increases apparent affinity of gamma- aminobutyric acid (GABA) to GABA(A) receptors, was used to examine its effects on GABA binding and ion channel gating of expressed alpha(2) beta(1) gamma(2) receptors from excised outside-out patches of acutely transfected HEK 293 cells. Diazepam increased rates of current onset evoked by low concentrations (<1 mM) but not at saturating GABA concen trations. Furthermore, rates of current decay were not affected during brief applications of GABA, and thus, demonstrated a diazepam specifi c effect on ligand binding affinity and not channel gating kinetics. H owever, current decay during and following prolonged GABA applications were altered by diazepam in a fashion similar to that for higher conc entrations of GABA which also increased receptor desensitization. Thes e findings and analysis by computer modeling indicated that diazepam l ikely enhances GABA receptor currents primarily by accelerating GABA a ssociation to its receptor at the first agonist binding site. These re sults provide the first direct physiological evidence for pharmacologi cal modulation of microscopic binding affinity of GABA receptors. Copy right (C) 1996 Elsevier Science Ltd.