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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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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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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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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DOI: https://doi.org/10.1038/nn.2358
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