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The Journal of Neuroscience, December 15, 2004, 24(50):11226-11235; doi:10.1523/JNEUROSCI.3746-04.2004

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Cellular/Molecular
Mutation of Glutamate 155 of the GABA_A Receptor ₂ Subunit Produces a Spontaneously Open Channel: A Trigger for Channel Activation

J. Glen Newell, Ross A. McDevitt, and Cynthia Czajkowski

Department of Physiology, University of Wisconsin, Madison, Wisconsin 53706

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_AR) ligand-binding domain predicts that ₂Glu¹⁵⁵ is a key residue for direct interactions with the neurotransmitter (Cromer et al., 2002). To elucidate the role of the ₂Ile¹⁵⁴-Asp¹⁶³ region in GABA_AR activation, each residue was individually mutated to cysteine and coexpressed with wild-type ₁ 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 ₂Thr¹⁶⁰ and ₂Asp¹⁶³ are located in or near the GABA-binding site and that this region undergoes structural rearrangements during channel gating. Cysteine substitution of ₂Glu¹⁵⁵ resulted in spontaneously open GABA_ARs 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 ₂E155C closed the spontaneously open channels, suggesting that ₂Glu¹⁵⁵ is a control element involved in coupling ligand binding to channel gating. Structural modeling suggests that the ₂ 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.

Key words: GABA; SR-95531; gabazine; picrotoxin; piperidine-4-sulfonic acid; mutagenesis; substituted cysteine accessibility method; pentobarbital; Xenopus laevis oocytes; methanethiosulfonate; two-electrode voltage clamp

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Received Sep 9, 2004; revised November 1, 2004; accepted November 1, 2004.

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