Mutation of glutamate 155 of the GABAA receptor beta2 subunit produces a spontaneously open channel: a trigger for channel activation.

Protein movements underlying ligand-gated ion channel activation are poorly understood. The binding of agonist initiates a series of conformational movements that ultimately lead to the opening of the ion channel pore. Although little is known about local movements within the GABA-binding site, a recent structural model of the GABA(A) receptor (GABA(A)R) ligand-binding domain predicts that beta2Glu155 is a key residue for direct interactions with the neurotransmitter (Cromer et al., 2002). To elucidate the role of the beta2Ile154-Asp163 region in GABA(A)R activation, each residue was individually mutated to cysteine and coexpressed with wild-type alpha1 subunits in Xenopus laevis oocytes. Seven mutations increased the GABA EC50 value (8- to 3400-fold), whereas three mutations (E155C, S156C, and G158C) also significantly increased the 2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl) pyridazinium (SR-95531) K(I) value. GABA, SR-95531, and pentobarbital slowed N-biotinylaminoethyl methanethiosulfonate modification of T160C and D163C, indicating that beta2Thr160 and beta2Asp163 are located in or near the GABA-binding site and that this region undergoes structural rearrangements during channel gating. Cysteine substitution of beta2Glu155 resulted in spontaneously open GABA(A)Rs and differentially decreased the GABA, piperidine-4-sulfonic acid (partial agonist), and SR-95531 sensitivities, indicating that the mutation perturbs ligand binding as well as channel gating. Tethering thiol-reactive groups onto beta2E155C closed the spontaneously open channels, suggesting that beta2Glu155 is a control element involved in coupling ligand binding to channel gating. Structural modeling suggests that the beta2 Ile154-Asp163 region is a protein hinge that forms a network of interconnections that couples binding site movements to the cascade of events leading to channel opening.