Journal
JOURNAL OF NEUROSCIENCE
Volume 29, Issue 10, Pages 3083-3092Publisher
SOC NEUROSCIENCE
DOI: 10.1523/JNEUROSCI.6090-08.2009
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Funding
- National Institutes of Health [NS030808, GM077660, K99NS059841]
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The molecular basis of general anesthetic interactions with GABA(A) receptors is uncertain. An accurate homology model would facilitate studies of anesthetic action. Construction of a GABAA model based on the 4 angstrom resolution acetylcholine receptor structure is complicated by alignment uncertainty between the acetylcholine and GABAA receptor M3 and M4 transmembrane segments. Using disulfide crosslinking we previously established the orientation of M2 and M3 within a single GABA(A) subunit. The resultant model predicts that the beta M3 residue beta 2M286, implicated in anesthetic binding, faces the adjacent alpha 1-M1 segment and not into the beta 2 subunit interior as some models have suggested. To assess the proximity of beta 2M286 to the alpha 1-M1 segment we expressed beta 2M286C and gamma 2 with 10 consecutive alpha 1-M1 cysteine (Cys) mutants, alpha 1I223C to alpha 1L232C, in and flanking the extracellular end of alpha 1-M1. In activated states, beta 2M286C formed disulfide bonds with alpha 1Y225C and alpha 1Q229C based on electrophysiological assays and dimers on Western blots, but not with other alpha 1-M1 mutants. beta 2F289, one helical turn below beta 2M286, formed disulfide bonds with alpha 1I228C, alpha 1Q229C and alpha 1L232C in activated states. The intervening residues, beta 2G287C and beta 2C288, did not form disulfide bonds with alpha 1-M1 Cys mutants. We conclude that the beta 2-M3 residues beta 2M286 and beta 2F289 face the intersubunit interface in close proximity to alpha 1-M1 and that channel gating induces a structural rearrangement in the transmembrane subunit interface that reduces the beta M3 to alpha M1 separation by similar to 7 angstrom. This supports the hypothesis that some intravenous anesthetics bind in the beta M3-alpha M1 subunit interface consistent with azi-etomidate photoaffinity labeling.
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