Allosteric Activation Mechanism of the α1β2γ2 γ-Aminobutyric Acid Type A Receptor Revealed by Mutation of the Conserved M2 Leucine

摘要: A conserved leucine residue in the midpoint of second transmembrane domain (M2) ligand-activated ion channel family has been proposed to play an important role receptor activation. In this study, we assessed importance activation rat alpha 1 beta 2 gamma GABA receptors expressed Xenopus laevis oocytes by site-directed mutagenesis and two-electrode voltage clamp. The hydrophobic M2 leucines alpha1(L263), beta2(L259), 2(L274) subunits were mutated hydrophilic amino acid serine coexpressed all possible combinations with their wild-type and/or mutant counterparts. mutation any one subunit decreased EC(50) created spontaneous openings that blocked picrotoxin and, surprisingly, competitive antagonist bicuculline. magnitudes shifts IC(50) as well degree correlated number carrying mutation. Simultaneous binding site (beta 2Y157S; increased EC(50)) 2L259S; produced predicted intermediate agonist sensitivity, indicating two mutations affect gating independently. results are discussed light a allosteric mechanism.