DEFINING AFFINITY WITH THE GABA(A) RECEPTOR

At nicotinic and glutamatergic synapses, the duration of the postsynap tic response depends on the affinity of the receptor for transmitter ( Colquhoun et al., 1977; Pan et al., 1993). Affinity is often thought t o be determined by the ligand unbinding rate, whereas the binding rate is assumed to be diffusion-limited. In this view, the receptor select s for those ligands that form a stable complex on binding, but binding is uniformly fast and does not itself affect selectivity. We tested t hese assumptions for the GABA(A) receptor by dissecting the contributi ons of microscopic binding and unbinding kinetics for agonists of equa l efficacy but of widely differing affinities. Agonist pulses applied to outside-out patches of cultured rat hippocampal neurons revealed th at agonist unbinding rates could not account for affinity if diffusion -limited binding was assumed. However, direct measurement of the insta ntaneous competition between agonists and a competitive antagonist rev ealed that binding rates were orders of magnitude slower than expected for free diffusion, being more steeply correlated with affinity than were the unbinding rates. The deviation from diffusion-limited binding indicates that a ligand-specific energy barrier between the unbound a nd bound states determines GABA(A) receptor selectivity. This barrier and our kinetic observations can be quantitatively modeled by requirin g the participation of movable elements within a flexible GABA binding site.