Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

A discrete alcohol pocket involved in GIRK channel activation

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.

This is a preview of subscription content

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

Accession codes

Accessions

Protein Data Bank

References

  1. 1

    Mihic, S.J. et al. Sites of alcohol and volatile anaesthetic action on GABAA and glycine receptors. Nature 389, 385–389 (1997).

    CAS  Article  Google Scholar 

  2. 2

    Lovinger, D.M., White, G. & Weight, F.F. Ethanol inhibits NMDA-activated ion current in hippocampal neurons. Science 243, 1721–1724 (1989).

    CAS  Article  Google Scholar 

  3. 3

    Cardoso, R.A. et al. Effects of ethanol on recombinant human neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes. J. Pharmacol. Exp. Ther. 289, 774–780 (1999).

    CAS  PubMed  Google Scholar 

  4. 4

    Zhou, Q. & Lovinger, D.M. Pharmacologic characteristics of potentiation of 5-HT3 receptors by alcohols and diethyl ether in NCB-20 neuroblastoma cells. J. Pharmacol. Exp. Ther. 278, 732–740 (1996).

    CAS  PubMed  Google Scholar 

  5. 5

    Harris, R.A., Trudell, J.R. & Mihic, S.J. Ethanol's molecular targets. Sci. Signal. 1, re7 (2008).

    Article  Google Scholar 

  6. 6

    Wick, M.J. et al. Mutations of gamma-aminobutyric acid and glycine receptors change alcohol cutoff: evidence for an alcohol receptor? Proc. Natl. Acad. Sci. USA 95, 6504–6509 (1998).

    CAS  Article  Google Scholar 

  7. 7

    Lewohl, J.M. et al. G protein–coupled inwardly rectifying potassium channels are targets of alcohol action. Nat. Neurosci. 2, 1084–1090 (1999).

    CAS  Article  Google Scholar 

  8. 8

    Kobayashi, T. et al. Ethanol opens G protein–activated inwardly rectifying K+ channels. Nat. Neurosci. 2, 1091–1097 (1999).

    CAS  Article  Google Scholar 

  9. 9

    Covarrubias, M., Vyas, T.B., Escobar, L. & Wei, A. Alcohols inhibit a cloned potassium channel at a discrete saturable site. Insights into the molecular basis of general anesthesia. J. Biol. Chem. 270, 19408–19416 (1995).

    CAS  Article  Google Scholar 

  10. 10

    Blednov, Y.A., Stoffel, M., Alva, H. & Harris, R.A. A pervasive mechanism for analgesia: activation of GIRK2 channels. Proc. Natl. Acad. Sci. USA 100, 277–282 (2003).

    CAS  Article  Google Scholar 

  11. 11

    Blednov, Y.A., Stoffel, M., Chang, S.R. & Harris, R.A. Potassium channels as targets for ethanol: studies of G protein–coupled inwardly rectifying potassium channel 2 (GIRK2) null mutant mice. J. Pharmacol. Exp. Ther. 298, 521–530 (2001).

    CAS  PubMed  Google Scholar 

  12. 12

    Reuveny, E. et al. Activation of the cloned muscarinic potassium channel by G protein βγ-subunits. Nature 370, 143–146 (1994).

    CAS  Article  Google Scholar 

  13. 13

    Wickman, K.D. et al. Recombinant G protein βγ-subunits activate the muscarinic-gated atrial potassium channel. Nature 368, 255–257 (1994).

    CAS  Article  Google Scholar 

  14. 14

    Huang, C.L., Slesinger, P.A., Casey, P.J., Jan, Y.N. & Jan, L.Y. Evidence that direct binding of G βγ to the GIRK1 G protein–gated inwardly rectifying K+ channel is important for channel activation. Neuron 15, 1133–1143 (1995).

    CAS  Article  Google Scholar 

  15. 15

    Kunkel, M.T. & Peralta, E.G. Identification of domains conferring G protein regulation on inward rectifier potassium channels. Cell 83, 443–449 (1995).

    CAS  Article  Google Scholar 

  16. 16

    Krapivinsky, G. βγ binding to GIRK4 subunit is critical for G protein–gated K+ channel activation. J. Biol. Chem. 273, 16946–16952 (1998).

    CAS  Article  Google Scholar 

  17. 17

    He, C., Zhang, H., Mirshahi, T. & Logothetis, D.E. Identification of a potassium channel site that interacts with G protein βγ subunits to mediate agonist-induced signaling. J. Biol. Chem. 274, 12517–12524 (1999).

    CAS  Article  Google Scholar 

  18. 18

    Ivanina, T. et al. Mapping the Gβγ-binding sites in GIRK1 and GIRK2 subunits of the G protein–activated K+ channel. J. Biol. Chem. 278, 29174–29183 (2003).

    CAS  Article  Google Scholar 

  19. 19

    Finley, M., Arrabit, C., Fowler, C., Suen, K.F. & Slesinger, P.A. βL-βM loop in the C-terminal domain of G protein–activated inwardly rectifying K+ channels is important for G(βγ) subunit activation. J. Physiol. (Lond.) 555, 643–657 (2004).

    CAS  Article  Google Scholar 

  20. 20

    Hara, K., Lewohl, J.M., Yamakura, T. & Harris, R.A. Mutational analysis of ethanol interactions with G protein–coupled inwardly rectifying potassium channels. Alcohol 24, 5–8 (2001).

    CAS  Article  Google Scholar 

  21. 21

    Pegan, S., Arrabit, C., Slesinger, P.A. & Choe, S. Andersen's syndrome mutation effects on the structure and assembly of the cytoplasmic domains of Kir2.1. Biochemistry 45, 8599–8606 (2006).

    CAS  Article  Google Scholar 

  22. 22

    Kruse, S.W., Zhao, R., Smith, D.P. & Jones, D.N. Structure of a specific alcohol-binding site defined by the odorant binding protein LUSH from Drosophila melanogaster. Nat. Struct. Biol. 10, 694–700 (2003).

    CAS  Article  Google Scholar 

  23. 23

    Nishida, M. & MacKinnon, R. Structural basis of inward rectification: cytoplasmic pore of the G protein–gated inward rectifier GIRK1 at 1.8 Å resolution. Cell 111, 957–965 (2002).

    CAS  Article  Google Scholar 

  24. 24

    Pegan, S. et al. Cytoplasmic domain structures of Kir2.1 and Kir3.1 show sites for modulating gating and rectification. Nat. Neurosci. 8, 279–287 (2005).

    CAS  Article  Google Scholar 

  25. 25

    Inanobe, A., Matsuura, T., Nakagawa, A. & Kurachi, Y. Structural diversity in the cytoplasmic region of G protein–gated inward rectifier K+ channels. Channels (Austin) 1, 39–45 (2007).

    Article  Google Scholar 

  26. 26

    Rishal, I., Porozov, Y., Yakubovich, D., Varon, D. & Dascal, N. Gβγ-dependent and Gβγ -independent basal activity of G protein–activated K+ channels. J. Biol. Chem. 280, 16685–16694 (2005).

    CAS  Article  Google Scholar 

  27. 27

    Vivaudou, M. et al. Probing the G-protein regulation of GIRK1 and GIRK4, the two subunits of the KACh channel, using functional homomeric mutants. J. Biol. Chem. 272, 31553–31560 (1997).

    CAS  Article  Google Scholar 

  28. 28

    Zhang, H., He, C., Yan, X., Mirshahi, T. & Logothetis, D.E. Activation of inwardly rectifying K+ channels by distinct PtdIns(4,5)P2 interactions. Nat. Cell Biol. 1, 183–188 (1999).

    CAS  Article  Google Scholar 

  29. 29

    Zhou, W., Arrabit, C., Choe, S. & Slesinger, P.A. Mechanism underlying bupivacaine inhibition of G protein–gated inwardly rectifying K+ channels. Proc. Natl. Acad. Sci. USA 98, 6482–6487 (2001).

    CAS  Article  Google Scholar 

  30. 30

    Jenkins, A. et al. Evidence for a common binding cavity for three general anesthetics within the GABAA receptor. J. Neurosci. 21, RC136 (2001).

    CAS  Article  Google Scholar 

  31. 31

    Huang, C.L., Feng, S. & Hilgemann, D.W. Direct activation of inward rectifier potassium channels by PIP2 and its stabilization by Gβγ. Nature 391, 803–806 (1998).

    CAS  Article  Google Scholar 

  32. 32

    Peoples, R.W. & Ren, H. Inhibition of N-methyl-d-aspartate receptors by straight-chain diols: implications for the mechanism of the alcohol cutoff effect. Mol. Pharmacol. 61, 169–176 (2002).

    CAS  Article  Google Scholar 

  33. 33

    Dildy-Mayfield, J.E., Mihic, S.J., Liu, Y., Deitrich, R.A. & Harris, R.A. Actions of long chain alcohols on GABAA and glutamate receptors: relation to in vivo effects. Br. J. Pharmacol. 118, 378–384 (1996).

    CAS  Article  Google Scholar 

  34. 34

    Ramaswamy, S., Eklund, H. & Plapp, B.V. Structures of horse liver alcohol dehydrogenase complexed with NAD+ and substituted benzyl alcohols. Biochemistry 33, 5230–5237 (1994).

    CAS  Article  Google Scholar 

  35. 35

    Svensson, S., Hoog, J.O., Schneider, G. & Sandalova, T. Crystal structures of mouse class II alcohol dehydrogenase reveal determinants of substrate specificity and catalytic efficiency. J. Mol. Biol. 302, 441–453 (2000).

    CAS  Article  Google Scholar 

  36. 36

    Weinhold, E.G. & Benner, S.A. Engineering yeast alcohol dehydrogenase. Replacing Trp54 by Leu broadens substrate specificity. Protein Eng. 8, 457–461 (1995).

    CAS  Article  Google Scholar 

  37. 37

    Thode, A.B., Kruse, S.W., Nix, J.C. & Jones, D.N. The role of multiple hydrogen-bonding groups in specific alcohol binding sites in proteins: insights from structural studies of LUSH. J. Mol. Biol. 376, 1360–1376 (2008).

    CAS  Article  Google Scholar 

  38. 38

    Lu, T. et al. Probing ion permeation and gating in a K+ channel with backbone mutations in the selectivity filter. Nat. Neurosci. 4, 239–246 (2001).

    CAS  Article  Google Scholar 

  39. 39

    Nishida, M., Cadene, M., Chait, B.T. & MacKinnon, R. Crystal structure of a Kir3.1-prokaryotic Kir channel chimera. EMBO J. 26, 4005–4015 (2007).

    CAS  Article  Google Scholar 

  40. 40

    Yi, B.A., Lin, Y.F., Jan, Y.N. & Jan, L.Y. Yeast screen for constitutively active mutant G protein–activated potassium channels. Neuron 29, 657–667 (2001).

    CAS  Article  Google Scholar 

  41. 41

    Sadja, R., Smadja, K., Alagem, N. & Reuveny, E. Coupling Gβγ -dependent activation to channel opening via pore elements in inwardly rectifying potassium channels. Neuron 29, 669–680 (2001).

    CAS  Article  Google Scholar 

  42. 42

    Jin, T. et al. The βγ subunits of G proteins gate a K+ channel by pivoted bending of a transmembrane segment. Mol. Cell 10, 469–481 (2002).

    CAS  Article  Google Scholar 

  43. 43

    Sarac, R. et al. Mutation of critical GIRK subunit residues disrupts N- and C-termini association and channel function. J. Neurosci. 25, 1836–1846 (2005).

    CAS  Article  Google Scholar 

  44. 44

    Riven, I., Kalmanzon, E., Segev, L. & Reuveny, E. Conformational rearrangements associated with the gating of the G protein–coupled potassium channel revealed by FRET microscopy. Neuron 38, 225–235 (2003).

    CAS  Article  Google Scholar 

  45. 45

    Ford, C.E. et al. Molecular basis for interactions of G protein βγ subunits with effectors. Science 280, 1271–1274 (1998).

    CAS  Article  Google Scholar 

  46. 46

    Clancy, S.M. et al. Pertussis toxin–sensitive Galpha subunits selectively bind to C-terminal domain of neuronal GIRK channels: evidence for a heterotrimeric G protein–channel complex. Mol. Cell. Neurosci. 28, 375–389 (2005).

    CAS  Article  Google Scholar 

  47. 47

    Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K. & Pease, L.R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51–59 (1989).

    CAS  Article  Google Scholar 

  48. 48

    Dundas, J. et al. CASTp: computed atlas of surface topography of proteins with structural and topographical mapping of functionally annotated residues. Nucleic Acids Res. 34, W116–118 (2006).

    CAS  Article  Google Scholar 

  49. 49

    Harpaz, Y., Gerstein, M. & Chothia, C. Volume changes on protein folding. Structure 2, 641–649 (1994).

    CAS  Article  Google Scholar 

Download references

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.

Author information

Affiliations

Authors

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.

Corresponding authors

Correspondence to Senyon Choe or Paul A Slesinger.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 (PDF 245 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing