GABA-induced intersubunit conformational movement in the GABA A receptor α1M1-β2M3 transmembrane subunit interface: Experimental basis for homology modeling of an intravenous anesthetic binding site

Moez Bali, Michaela Jansen, Myles H. Akabas

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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 GABA A model based on the 4 Å resolution acetylcholine receptor structure is complicated by alignment uncertainty between the acetylcholine and GABA A 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 βM3 residue β2M286, implicated in anesthetic binding, faces the adjacent α1-M1 segment and not into the β2 subunit interior as some models have suggested. To assess the proximity of β2M286 to the α1-M1 segment we expressed β2M286C and γl with 10 consecutive α1-M1 cysteine (Cys) mutants, α1I223C to α1L232C, in and flanking the extracellular end of α1-M1. In activated states, β2M286C formed disulfide bonds with α1Y225C and α1Q229C based on electrophysiological assays and dimers on Western blots, but not with other α1-M1 mutants. β2F289, one helical turn below β2M286, formed disulfide bonds with α1I228C, α1Q229C and α1L232C in activated states. The intervening residues, β2G287C and β2C288, did not form disulfide bonds with α1-M1 Cys mutants. We conclude that the β2-M3 residues β2M286 and β2F289 face the intersubunit interface in close proximity to α1-M1 and that channel gating induces a structural rearrangement in the transmembrane subunit interface that reduces the βM3 to αM1 separation by ∼7 Å. This supports the hypothesis that some intravenous anesthetics bind in the βM3-αM1 subunit interface consistent with azi-etomidate photoaffinity labeling.

Original languageEnglish (US)
Pages (from-to)3083-3092
Number of pages10
JournalJournal of Neuroscience
Issue number10
Publication statusPublished - Mar 11 2009


ASJC Scopus subject areas

  • Neuroscience(all)

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