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Cellular/Molecular State-Dependent Cross-Linking of the M2 and M3 Segments

Summary: Cellular/Molecular
State-Dependent Cross-Linking of the M2 and M3 Segments:
Functional Basis for the Alignment of GABAA 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
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 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 . 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 GABAA and ACh M3 requires a single gap in the GABAA M2M3 loop. In the presence of
GABA,oxidationof 1T267C-L301Cand 1T267C-F304Chadnoeffect,butoxidationof 1T267C-A305Ccausedasignificantincreasein
spontaneouschannelopening.Weinferthat,asthechannelopens,thedistanceand/ororientationbetweenM2- 1T267andM3- 1A305
channel opening.


Source: Akabas, Myles - Department of Physiology and Biophysics, Albert Einstein College of Medicine, Yeshiva University


Collections: Biology and Medicine