Biophys J, November 1999, p. 2542-2551, Vol. 77, No. 5

Allosteric Activation Mechanism of the 122 -Aminobutyric Acid Type A Receptor Revealed by Mutation of the Conserved M2 Leucine

Departments of Neurobiology and Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35294-0021 USA

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 122 GABA receptors expressed in Xenopus laevis oocytes by site-directed mutagenesis and two-electrode voltage clamp. The hydrophobic conserved M2 leucines in 1(L263), 2(L259), and 2(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 (2Y157S; increased the EC₅₀) and the conserved M2 leucine (2L259S; 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.

Biophys J, November 1999, p. 2542-2551, Vol. 77, No. 5
© 1999 by the Biophysical Society   0006-3495/99/11/2542/10  $2.00

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