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The Journal of Neuroscience, March 11, 2009, 29(10):3083-3092; doi:10.1523/JNEUROSCI.6090-08.2009

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Cellular/Molecular
GABA-Induced Intersubunit Conformational Movement in the GABA_A Receptor 1M1-β2M3 Transmembrane Subunit Interface: Experimental Basis for Homology Modeling of an Intravenous Anesthetic Binding Site

Moez Bali,¹ Michaela Jansen,¹ and Myles H. Akabas^1,2,3

Departments of ¹Physiology and Biophysics, ²Neuroscience, and ¹Medicine, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461

Correspondence should be addressed to Dr. Myles H. Akabas, Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. Email: makabas{at}aecom.yu.edu

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 1-M1 segment and not into the β2 subunit interior as some models have suggested. To assess the proximity of β2M286 to the 1-M1 segment we expressed β2M286C and 2 with 10 consecutive 1-M1 cysteine (Cys) mutants, 1I223C to 1L232C, in and flanking the extracellular end of 1-M1. In activated states, β2M286C formed disulfide bonds with 1Y225C and 1Q229C based on electrophysiological assays and dimers on Western blots, but not with other 1-M1 mutants. β2F289, one helical turn below β2M286, formed disulfide bonds with 1I228C, 1Q229C and 1L232C in activated states. The intervening residues, β2G287C and β2C288, did not form disulfide bonds with 1-M1 Cys mutants. We conclude that the β2-M3 residues β2M286 and β2F289 face the intersubunit interface in close proximity to 1-M1 and that channel gating induces a structural rearrangement in the transmembrane subunit interface that reduces the βM3 to M1 separation by 7 Å. This supports the hypothesis that some intravenous anesthetics bind in the βM3-M1 subunit interface consistent with azi-etomidate photoaffinity labeling.

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Received Dec. 22, 2008; accepted Jan. 27, 2009.

Correspondence should be addressed to Dr. Myles H. Akabas, Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. Email: makabas{at}aecom.yu.edu

This article has been cited by other articles:

				G.-D. Li, D. C. Chiara, J. B. Cohen, and R. W. Olsen Neurosteroids Allosterically Modulate Binding of the Anesthetic Etomidate to {gamma}-Aminobutyric Acid Type A Receptors J. Biol. Chem., May 1, 2009; 284(18): 11771 - 11775. [Abstract] [Full Text] [PDF]

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