Protons modulate gating of recombinant α₁β₂γ₂ GABA_A receptor by affecting desensitization and opening transitions

Protons are potent modulators of GABA_A receptors (GABA_ARs) and α₁Phe64 residue was implicated in their pH sensitivity. Recently, we have demonstrated that this residue is involved in flipping transitions which precede channel opening. We thus re-addressed the mechanism of GABA_AR modulation by protons by considering the gating scheme extended by flipping. The impact of pH changes was examined on currents mediated by wild-type α₁β₂γ₂ receptors or by their α₁Phe64Leu or α₁Phe64Cys mutants and elicited by saturating concentrations of full (GABA) or partial (piperidine-4-sulfonic acid) agonists. To describe the impact of extracellular pH on receptor gating, we combined macroscopic analysis of currents elicited by rapid agonist applications with single-channel studies. Acidification (pH 6.0) increased current amplitudes (in the case of leucine mutants effect was stronger when P4S was used) and decreased the rate and the extent of desensitization whereas alkalization (pH 8.0) had the opposite but weaker effect. Deactivation kinetics for wild-type receptors was slowed down by acidification while in the case of mutants this effect was observed upon alkalization. Moreover, α₁Phe64 mutations enhanced GABA_AR sensitivity to alkaline pH. Single-channel analysis revealed that acidification prolonged burst durations and affected shut but not open time distributions. Model simulations for macroscopic and single-channel activity indicated a novel mechanism in which protons primarily affected opening and desensitization rates but not flipping/unflipping. This evidence for the impact of protons on the receptor gating together with previously demonstrated effect on the agonist binding, point to a complex effect of extracellular pH on GABA_AR macromolecule.