Allosteric activation mechanism of the α1β2γ2 γ-aminobutyric acid type A receptor revealed by mutation of the conserved M2 leucine

A conserved leucine residue in the midpoint of the second transmembrane domain (M2) of the ligand-activated ion channel family has been proposed to play an important role in receptor activation. In this study, we assessed the importance of this leucine in the activation of rat α1β2γ2 GABA receptors expressed in Xenopus laevis oocytes by site-directed mutagenesis and two- electrode voltage clamp. The hydrophobic conserved M2 leucines in α1(L263), β2(L259), and γ2(L274) subunits were mutated to the hydrophilic amino acid residue serine and coexpressed in all possible combinations with their wild- type and/or mutant counterparts. The mutation in any one subunit decreased the EC₅₀ and created spontaneous openings that were blocked by picrotoxin and, surprisingly, by the competitive antagonist bicuculline. The magnitudes of the shifts in GABA EC₅₀ and picrotoxin IC₅₀ as well as the degree of spontaneous openings were all correlated with the number of subunits carrying the leucine mutation. Simultaneous mutation of the GABA binding site (β2Y157S; increased the EC₅₀) and the conserved M2 leucine (β2L259S; decreased the EC₅₀) produced receptors with the predicted intermediate agonist sensitivity, indicating the two mutations affect binding and gating independently. The results are discussed in light of a proposed allosteric activation mechanism.