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.
Newell
J Glen
JG
Department of Physiology, University of Wisconsin, Madison, Wisconsin 53706, USA.
McDevitt
Ross A
RA
Czajkowski
Cynthia
C
D000818:Animals; D001665:Binding Sites; D001710:Biotin / Q000031:analogs & derivatives*; D003545:Cysteine; D058785:GABA-A Receptor Agonists; D018698:Glutamic Acid; D015640:Ion Channel Gating / Q000235:genetics / Q000502:physiology*; D007700:Kinetics; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D018408:Patch-Clamp Techniques; D010424:Pentobarbital / Q000494:pharmacology; D011487:Protein Conformation; D051381:Rats; D011963:Receptors, GABA-A / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins; D014982:Xenopus laevis
D058785:GABA-A Receptor Agonists; C502185:Gabrb2 protein, rat; D008024:Ligands; C419543:N-biotinylaminoethyl methanethiosulfonate; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; D018698:Glutamic Acid; D001710:Biotin; D010424:Pentobarbital; D003545:Cysteine