Coupled and uncoupled gating and desensitization effects by pore domain mutations in GABAA receptors

Abstract

GABAA receptors are allosteric ligand-gated ion channels. Agonist-induced gating and desensitization have been proposed to be coupled via pore domain structures. Mutations at two α1 subunit pore-domain (transmembrane domain 2) residues enhance GABA sensitivity, leucine-to-threonine at position 264 (9′), and serine-to-isoleucine at position 270 (15′). We investigated the role of these residues in gating, desensitization, and deactivation of α1β2γ2L GABAA receptors using rapid GABA concentration jumps and patch-clamp electrophysiology. GABA EC50 values for α1(L264T)β2γ2L and α1(S2701)β2γ2L currents were, respectively, ∼80-fold and 13-fold lower than the wild-type EC50. Unlike wild type, both mutant receptors displayed significant picrotoxin-sensitive currents in the absence of GABA, indicating that they enhance gating efficacy. Both mutants displayed current activation rates that matched wild type at 1 μM GABA and above. Desensitization of wild-type and α1(S2701)β2γ2L currents displayed indistinguishable rates and amplitudes, whereas α1(L264T)β2γ2L currents desensitized extremely slowly. Deactivation of wild-type currents displayed two rates and slowed after partial desensitization, whereas currents from both mutants deactivated slowly with single rate constants that were unaffected by desensitization. These results indicate that both α1(L264T) and α1(S2701) mutations increase the gating efficacy of receptors by slowing channel closing, which accounts for nearly all of the similar changes that they produce in macrocurrent dynamics. Because the α1(S2701) mutation uncouples its gating effects from those on rapid desensitization, these two processes are necessarily associated with movements of distinct receptor structures (gates). The effects of the α1(L264T) mutation suggest that the conserved leucines may play a role in gating-desensitization coupling.