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A discrete alcohol pocket involved in GIRK channel activation

Nature Neuroscience volume 12, pages 988–995 (2009)Cite this article

Abstract

Ethanol modifies neural activity in the brain by modulating ion channels. Ethanol activates G protein–gated inwardly rectifying K+ channels, but the molecular mechanism is not well understood. Here, we used a crystal structure of a mouse inward rectifier containing a bound alcohol and structure-based mutagenesis to probe a putative alcohol-binding pocket located in the cytoplasmic domains of GIRK channels. Substitutions with bulkier side-chains in the alcohol-binding pocket reduced or eliminated activation by alcohols. By contrast, alcohols inhibited constitutively open channels, such as IRK1 or GIRK2 engineered to strongly bind PIP2. Mutations in the hydrophobic alcohol-binding pocket of these channels had no effect on alcohol-dependent inhibition, suggesting an alternate site is involved in inhibition. Comparison of high-resolution structures of inwardly rectifying K+ channels suggests a model for activation of GIRK channels using this hydrophobic alcohol-binding pocket. These results provide a tool for developing therapeutic compounds that could mitigate the effects of alcohol.

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Figure 1: A conserved alcohol-binding pocket in IRK1 and GIRK2 channels.
Figure 2: MPD activates GIRK2 in a manner similar to other alcohols.
Figure 3: Alanine/tryptophan scan of the hydrophobic alcohol-binding pocket in GIRK2.
Figure 4: Comprehensive mutagenesis of GIRK2-L257 in the βD-βE ribbon of hydrophobic alcohol-binding pocket reveals changes in alcohol- and Gβγ-activated currents.
Figure 5: Reduced alcohol activation with increasing bulkiness of amino acid substitutions at GIRK2-L257.
Figure 6: Mutations in the hydrophobic alcohol-binding pocket of GIRK4* alter alcohol-activated currents.
Figure 7: Mutations in the hydrophobic alcohol-binding pocket of IRK1 have no effect on alcohol-dependent inhibition.
Figure 8: Model for alcohol-dependent activation of GIRK channels.

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Acknowledgements

We would like to thank Y. Kurachi for GIRK2 coordinates, M. Lazdunsky for GIRK2 cDNA, D. Clapham for GIRK4 cDNA, N. Dascal for m-Phos cDNA, S. Pegan for initial discussions on structure of IRK1-MPD and members of the Slesinger laboratory for helpful comments. This work was funded, in part, by a pre-doctoral National Research Service Award from the National Institute on Alcohol Abuse and Alcoholism (F31AA017042, P.A.), by the National Institute on General Medical Sciences (R01GM056653, S.C.), and by the H.N. & Frances C. Berger Foundation and the Salk Institute for Biological Studies (P.A.S.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute on Alcohol Abuse and Alcoholism or the National Institute on General Medical Sciences.

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Authors and Affiliations

  1. Peptide Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California, USA

    Prafulla Aryal & Paul A Slesinger

  2. Division of Biology, Graduate Program in Biology, University of California, San Diego, La Jolla, California, USA

    Prafulla Aryal, Senyon Choe & Paul A Slesinger

  3. Structural Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California, USA

    Hay Dvir & Senyon Choe

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  1. Prafulla Aryal
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  2. Hay Dvir
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Contributions

P.A.S. and P.A. designed the experiments and analyzed the data. P.A. conducted the molecular cloning, electrophysiology and imaging experiments. H.D. and P.A. collaborated on structural analysis and figure production. H.D. conducted modeling experiments. P.A., H.D. and P.A.S co-wrote and revised the manuscript. P.A.S and S.C. supervised the project.

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Correspondence to Senyon Choe or Paul A Slesinger.

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Aryal, P., Dvir, H., Choe, S. et al. A discrete alcohol pocket involved in GIRK channel activation. Nat Neurosci 12, 988–995 (2009). https://doi.org/10.1038/nn.2358

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