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The Journal of Neuroscience, April 26, 2006, 26(17):4492-4499; doi:10.1523/JNEUROSCI.0224-06.2006

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Cellular/Molecular
State-Dependent Cross-Linking of the M2 and M3 Segments: Functional Basis for the Alignment of GABA_A and Acetylcholine Receptor M3 Segments

Michaela Jansen and Myles H. Akabas

Departments of Physiology and Biophysics and of Neuroscience, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461

Correspondence should be addressed to Dr. Myles H. Akabas, Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461. Email: makabas{at}aecom.yu.edu

Construction of a GABA_A receptor homology model based on the acetylcholine (ACh) receptor structure is complicated by the low sequence similarity between GABA_A and ACh M3 transmembrane segments that creates significant uncertainty in their alignment. We determined the orientation of the GABA_A M2 and M3 transmembrane segments using disulfide cross-linking. The M2 residues 1M266 (11') and 1T267 (12') were mutated to cysteine in either wild type or single M3 cysteine mutant (1V297C, 1A300C to 1A305C) backgrounds. We assayed spontaneous and induced disulfide bond formation. Reduction with DTT significantly potentiated GABA-induced currents in 1T267C-L301C and 1T267C-F304C. Copper phenanthroline-induced oxidation inhibited GABA-induced currents in these mutants and in 1T267C-A305C. Intrasubunit disulfide bonds formed between these Cys pairs, implying that the -carbon separation was at most 5.6 Å. The reactive 1M3 residues (L301, F304, A305) lie on the same face of an -helix. The unresponsive ones (A300, I302, E303) lie on the opposite face. In the resting state, the reactive side of 1M3 faces M2-1T267. In conjunction with the ACh structure, our data indicate that alignment of GABA_A and ACh M3 requires a single gap in the GABA_A M2–M3 loop. In the presence of GABA, oxidation of 1T267C-L301C and 1T267C-F304C had no effect, but oxidation of 1T267C-A305C caused a significant increase in spontaneous channel opening. We infer that, as the channel opens, the distance and/or orientation between M2-1T267 and M3-1A305 changes such that the disulfide bond stabilizes the open state. This begins to define the conformational motion that M2 undergoes during channel opening.

Key words: GABA_A receptor; ion channel; acetylcholine receptor; disulfide cross-linking; glycine; serotonin

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Received Jan. 17, 2006; revised March 20, 2006; accepted March 20, 2006.

Correspondence should be addressed to Dr. Myles H. Akabas, Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461. Email: makabas{at}aecom.yu.edu

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