Mutation of the 9' leucine in the GABA(A) receptor gamma2L subunit produces an apparent decrease in desensitization by stabilizing open states without altering desensitized states

Abstract

A conserved leucine near the middle (9' position) of the second transmembrane domain of ligand-gated ion channels has been implicated in both gating and desensitization. Specifically, L9'S and L9'T mutations decreased agonist EC50, decreased apparent desensitization and prolonged deactivation in members of the LGIC superfamily, suggesting that this residue may regulate channel properties including desensitization. GABA(A) receptors desensitize in three phases, but in previous 9' leucine studies, only slow phases of desensitization were resolved. We used excised patches containing alpha1beta3gamma2L or alpha1beta3gamma2L(L9'S) GABA(A) receptors and combined single channel recording and concentration jump techniques to reevaluate the effects of this mutation on desensitization. Although desensitization extent was decreased in mutated channels, desensitization still occurred in three phases, suggesting that desensitized states may be intact. Interestingly, deactivation rate was slowed by the mutation, opposite to that expected if desensitization was attenuated. alpha1beta3gamma2L(L9'S) receptor single channels had increased open durations. Simulations revealed that stabilizing the open state (by decreasing the channel closing rate) could account for multiple macroscopic findings: left-shifted GABA EC50, smaller extent of desensitization, slower desensitization rate, and longer deactivation. We concluded that changes in efficacy can alter macroscopic desensitization without affecting desensitized states per se.