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Protein mobility and GABA-induced conformational changes in GABAA receptor pore-lining M2 segment

Nature Neuroscience volume 4, pages 477–485 (2001)Cite this article

Abstract

Protein movements underlying ligand-gated ion channel activation are poorly understood. Here we used disulfide bond trapping to examine the proximity and mobility of cysteines substituted for aligned GABAA receptor α1 and β1 M2 segment channel-lining residues in resting and activated receptors. With or without GABA, disulfide bonds formed at α1N275C/β1E270C (20′) and α1S272C/β1H267C (17′), near the extracellular end, suggesting that this end is more mobile and/or flexible than the rest of the segment. Near the middle of M2, at α1T261C/β1T256C (6′), a disulfide bond formed only in the presence of GABA and locked the channels open. Channel activation must involve an asymmetric rotation of two adjacent subunits toward each other. This would move aligned engineered cysteines on different subunits into proximity and allow disulfide bond formation without blocking conduction. Asymmetric rotation of M2 segments is probably a common gating mechanism in other ligand-gated ion channels.

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Figure 1: Effects of oxidation and reduction on wild-type α1β1 GABAA receptors (a) Aligned channel-lining residues in the α1 and β1 M2 membrane-spanning segments.
Figure 2: Disulfide bonds form in α1N20′Cβ1E20′C receptors.
Figure 3: Disulfide bonds form in α1S17′Cβ1H17′C and in α1β1H17′C receptors.
Figure 4: In α1T6′Cβ1T6′C, co-application of Cu:phen and GABA induces formation of a disulfide bond that locks the channels in an open state.
Figure 5: Asymmetric rotation of adjacent β subunits upon channel opening.

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Acknowledgements

This work was supported in part by grants from the National Institutes of Health NS30808 (M.H.A.), GM61925 (M.H.A.) and NS34727 (C.C.), and a Burroughs Wellcome Fund New Investigator Award (C.C.). We thank A. Finkelstein for comments on this manuscript.

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Authors and Affiliations

  1. Department of Physiology and Biophysics and Department of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, 10461, New York, USA

    Jeffrey Horenstein & Myles H. Akabas

  2. Department of Physiology, Columbia University, 630 W 168th Street, New York, 10032, New York, USA

    Jeffrey Horenstein

  3. Department of Physiology, University of Wisconsin-Madison, 1300 University Avenue, Madison, 53706, Wisconsin, USA

    David A. Wagner & Cynthia Czajkowski

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  1. Jeffrey Horenstein
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Correspondence to Myles H. Akabas.

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Horenstein, J., Wagner, D., Czajkowski, C. et al. Protein mobility and GABA-induced conformational changes in GABAA receptor pore-lining M2 segment. Nat Neurosci 4, 477–485 (2001). https://doi.org/10.1038/87425

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