Yc. Chang et Ds. Weiss, Allosteric activation mechanism of the alpha 1 beta 2 gamma 2 gamma-aminobutyric acid type A receptor revealed by mutation of the conserved M2 leucine, BIOPHYS J, 77(5), 1999, pp. 2542-2551
A conserved leucine residue in the midpoint of the second transmembrane dom
ain (M2) of the ligand-activated ion channel family has been proposed to pl
ay an important role in receptor activation. In this study, we assessed the
importance of this leucine in the activation of rat alpha 1 beta 2 gamma 2
GABA receptors expressed in Xenopus laevis oocytes by site-directed mutage
nesis and two-electrode voltage clamp. The hydrophobic conserved M2 leucine
s in alpha 1(L263), beta 2(L259), and gamma 2(L274) subunits were mutated t
o 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 tha
t were blocked by picrotoxin and, surprisingly, by the competitive antagoni
st bicuculline. The magnitudes of the shifts in GABA EC50 and picrotoxin IC
50 as well as the degree of spontaneous openings were all correlated with t
he number of subunits carrying the leucine mutation. Simultaneous mutation
of the GABA binding site (beta 2Y157S; increased the EC50) and the conserve
d M2 leucine (beta 2L259S; decreased the EC50) produced receptors with the
predicted intermediate agonist sensitivity, indicating the two mutations af
fect binding and gating independently. The results are discussed in light o
f a proposed allosteric activation mechanism.