期刊
JOURNAL OF NEUROSCIENCE
卷 26, 期 17, 页码 4492-4499出版社
SOC NEUROSCIENCE
DOI: 10.1523/JNEUROSCI.0224-06.2006
关键词
GABA(A) receptor; ion channel; acetylcholine receptor; disulfide cross-linking; glycine; serotonin
资金
- NIGMS NIH HHS [GM77660] Funding Source: Medline
- NINDS NIH HHS [NS30808, R01 NS030808] Funding Source: Medline
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 alpha 1M266 (11') and alpha 1T267 (12') were mutated to cysteine in either wild type or single M3 cysteine mutant (alpha 1V297C, alpha 1A300C to alpha 1A305C) backgrounds. We assayed spontaneous and induced disulfide bond formation. Reduction with DTT significantly potentiated GABA-induced currents in alpha 1T267C- L301C and alpha 1T267C- F304C. Copper phenanthroline- induced oxidation inhibited GABA-induced currents in these mutants and in alpha 1T267C-A305C. Intrasubunit disulfide bonds formed between these Cys pairs, implying that the alpha-carbon separation was at most 5.6 angstrom. The reactive alpha 1M3 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 alpha 1M3 faces M2-alpha 1T267. In conjunction with the ACh structure, our data indicate that alignment of GABA(A) and ACh M3 requires a single gap in the GABAA M2-M3 loop. In the presence of GABA(A) oxidation of alpha 1T267C-L301C and alpha 1T267C- F304C had no effect, but oxidation of alpha 1T267C-A305C caused a significant increase in spontaneous channel opening. Weinfer that, as the channel opens, the distance and/ or orientation between M2-alpha 1T267 and M3-alpha 1A305 changes such that the disulfide bond stabilizes the open state. This begins to define the conformational motion that M2 undergoes during channel opening.
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