Desensitization mechanism of GABA receptors revealed by single oocyte binding and receptor function

Prolonged exposure of most fast neurotransmitter-operated ion channels to agonist drives the receptors into a nonfunctional, or desensitized, state. Despite extensive investigation, desensitization remains a thoroughly characterized, yet poorly understood, process. Part of the difficulty in elucidating the mechanism of desensitization has been an inability to resolve the kinetics of both agonist binding and functional desensitization in the same set of operable receptors. To overcome this limitation, we applied single oocyte ³H-ligand binding and two-electrode voltage clamp to oocytes expressing recombinant α1β2γ2 GABA receptors. Using this approach, we report several observations fundamental to the mechanism of desensitization. First, we confirm that desensitization reversibly shifts GABA receptors into a high-affinity state. For [³H]GABA binding, the half-maximal binding of the desensitized state was ∼0.040 μM. Second, we show that, upon agonist removal, this high-affinity state disappears with a time constant of 127 ± 12 sec (n = 4), similar to the time constant for functional recovery from desensitization of 124 ± 26 sec (n = 5). [³H]GABA, however, dissociates fourfold faster (τ = 30 ± 2 sec; n = 3) than functional recovery, indicating that desensitized receptors need not be bound by GABA. These data provide direct evidence for a cyclical model of receptor desensitization.