State-dependent cross-linking of the M2 and M3 segments: functional basis for the alignment of GABAA and acetylcholine receptor M3 segments.

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
Michaela Jansen, Myles H Akabas

Abstract

Construction of a GABAA receptor homology model based on the acetylcholine (ACh) receptor structure is complicated by the low sequence similarity between GABAA and ACh M3 transmembrane segments that creates significant uncertainty in their alignment. We determined the orientation of the GABAA M2 and M3 transmembrane segments using disulfide cross-linking. The M2 residues alpha1M266 (11') and alpha1T267 (12') were mutated to cysteine in either wild type or single M3 cysteine mutant (alpha1V297C, alpha1A300C to alpha1A305C) backgrounds. We assayed spontaneous and induced disulfide bond formation. Reduction with DTT significantly potentiated GABA-induced currents in alpha1T267C-L301C and alpha1T267C-F304C. Copper phenanthroline-induced oxidation inhibited GABA-induced currents in these mutants and in alpha1T267C-A305C. Intrasubunit disulfide bonds formed between these Cys pairs, implying that the alpha-carbon separation was at most 5.6 A. The reactive alpha1M3 residues (L301, F304, A305) lie on the same face of an alpha-helix. The unresponsive ones (A300, I302, E303) lie on the opposite face. In the resting state, the reactive side of alpha1M3 faces M2-alpha1T267. In conjunction with the ACh structure, our data indicate that align...Continue Reading

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