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 prokaryotic proton-gated ion channel from the nicotinic acetylcholine receptor family

Abstract

Ligand-gated ion channels (LGICs) mediate excitatory and inhibitory transmission in the nervous system. Among them, the pentameric or ‘Cys-loop’ receptors (pLGICs) compose a family that until recently was found in only eukaryotes. Yet a recent genome search identified putative homologues of these proteins in several bacterial species1. Here we report the cloning, expression and functional identification of one of these putative homologues from the cyanobacterium Gloeobacter violaceus. It was expressed as a homo-oligomer in HEK 293 cells and Xenopus oocytes, generating a transmembrane cationic channel that is opened by extracellular protons and shows slow kinetics of activation, no desensitization and a single channel conductance of 8 pS. Electron microscopy and cross-linking experiments of the protein fused to the maltose-binding protein and expressed in Escherichia coli are consistent with a homo-pentameric organization. Sequence comparison shows that it possesses a compact structure, with the absence of the amino-terminal helix, the canonical disulphide bridge and the large cytoplasmic domain found in eukaryotic pLGICs. Therefore it embodies a minimal structure required for signal transduction. These data establish the prokaryotic origin of the family. Because Gloeobacter violaceus carries out photosynthesis and proton transport at the cytoplasmic membrane2, this new proton-gated ion channel might contribute to adaptation to pH change.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: The Glvi protein is related to eukaryotic pLGICs.
Figure 2: Expression and oligomerization of the Glvi protein.
Figure 3: The Glvi protein functions as a proton-gated ion channel.
Figure 4: Single-channel currents through Glvi protein in HEK cells.

References

  1. Tasneem, A., Iyer, L. M., Jakobsson, E. & Aravind, L. Identification of the prokaryotic ligand-gated ion channels and their implications for the mechanisms and origins of animal Cys-loop ion channels. Genome Biol. 6, R4 (2005)

    Article  Google Scholar 

  2. Rippka, R., Waterbury, J. & Cohen-Bazire, G. A cyanobacterium which lacks thylakoids. Arch. Microbiol. 100, 419–436 (1974)

    Article  CAS  Google Scholar 

  3. Gunthorpe, M. J., Smith, G. D., Davis, J. B. & Randall, A. D. Characterisation of a human acid-sensing ion channel (hASIC1a) endogenously expressed in HEK293 cells. Pflügers Arch. 442, 668–674 (2001)

    Article  CAS  Google Scholar 

  4. Hansen, S. B. et al. Tryptophan fluorescence reveals conformational changes in the acetylcholine binding protein. J. Biol. Chem. 277, 41299–41302 (2002)

    Article  CAS  Google Scholar 

  5. Grutter, T. et al. A chimera encoding the fusion of an acetylcholine-binding protein to an ion channel is stabilized in a state close to the desensitized form of ligand-gated ion channels. C. R. Biol. 328, 223–234 (2005)

    Article  CAS  Google Scholar 

  6. Cartaud, J., Benedetti, E. L., Sobel, A. & Changeux, J. P. A morphological study of the cholinergic receptor protein from Torpedo marmorata in its membrane environment and in its detergent-extracted purified form. J. Cell Sci. 29, 313–337 (1978)

    CAS  PubMed  Google Scholar 

  7. Taly, A. et al. Normal mode analysis suggests a quaternary twist model for the nicotinic receptor gating mechanism. Biophys. J. 88, 3954–3965 (2005)

    Article  ADS  CAS  Google Scholar 

  8. Brejc, K. et al. Crystal structure of an ACh-binding protein reveals the ligand-binding domain of nicotinic receptors. Nature 411, 269–276 (2001)

    Article  ADS  CAS  Google Scholar 

  9. Miyazawa, A., Fujiyoshi, Y. & Unwin, N. Structure and gating mechanism of the acetylcholine receptor pore. Nature 423, 949–955 (2003)

    Article  ADS  CAS  Google Scholar 

  10. Unwin, N. Refined structure of the nicotinic acetylcholine receptor at 4 Å resolution. J. Mol. Biol. 346, 967–989 (2005)

    Article  CAS  Google Scholar 

  11. Giraudat, J., Dennis, M., Heidmann, T., Chang, J. Y. & Changeux, J. P. Structure of the high-affinity binding site for noncompetitive blockers of the acetylcholine receptor: serine-262 of the δ subunit is labeled by [3H]chlorpromazine. Proc. Natl Acad. Sci. USA 83, 2719–2723 (1986)

    Article  ADS  CAS  Google Scholar 

  12. Corringer, P. J. et al. Mutational analysis of the charge selectivity filter of the α7 nicotinic acetylcholine receptor. Neuron 22, 831–843 (1999)

    Article  CAS  Google Scholar 

  13. Wilson, G. G. & Karlin, A. The location of the gate in the acetylcholine receptor channel. Neuron 20, 1269–1281 (1998)

    Article  CAS  Google Scholar 

  14. Schofield, C. M., Trudell, J. R. & Harrison, N. L. Alanine-scanning mutagenesis in the signature disulfide loop of the glycine receptor α1 subunit: critical residues for activation and modulation. Biochemistry 43, 10058–10063 (2004)

    Article  CAS  Google Scholar 

  15. Grutter, T. et al. Molecular tuning of fast gating in pentameric ligand-gated ion channels. Proc. Natl Acad. Sci. USA 102, 18207–18212 (2005)

    Article  ADS  CAS  Google Scholar 

  16. Lee, W. Y. & Sine, S. M. Principal pathway coupling agonist binding to channel gating in nicotinic receptors. Nature 438, 243–247 (2005)

    Article  ADS  CAS  Google Scholar 

  17. England, P. M., Zhang, Y., Dougherty, D. A. & Lester, H. A. Backbone mutations in transmembrane domains of a ligand-gated ion channel: implications for the mechanism of gating. Cell 96, 89–98 (1999)

    Article  CAS  Google Scholar 

  18. Revah, F. et al. Mutations in the channel domain alter desensitization of a neuronal nicotinic receptor. Nature 353, 846–849 (1991)

    Article  ADS  CAS  Google Scholar 

  19. Chang, Y. & Weiss, D. S, Substitutions of the highly conserved M2 leucine create spontaneously opening ρ1 γ-aminobutyric acid receptors. Mol. Pharmacol. 53, 511–523 (1998)

    Article  CAS  Google Scholar 

  20. Yakel, J. L., Lagrutta, A., Adelman, J. P. & North, R. A. Single amino acid substitution affects desensitization of the 5-hydroxytryptamine type 3 receptor expressed in Xenopus oocytes. Proc. Natl Acad. Sci. USA 90, 5030–5033 (1993)

    Article  ADS  CAS  Google Scholar 

  21. Wilkins, M. E., Hosie, A. M. & Smart, T. G. Proton modulation of recombinant GABA(A) receptors: influence of GABA concentration and the β subunit TM2–TM3 domain. J. Physiol. (Lond.) 567, 365–377 (2005)

    Article  CAS  Google Scholar 

  22. Schnizler, K. et al. A novel chloride channel in Drosophila melanogaster is inhibited by protons. J. Biol. Chem. 280, 16254–16262 (2005)

    Article  CAS  Google Scholar 

  23. Hopfield, J. F., Tank, D. W., Greengard, P. & Huganir, R. L. Functional modulation of the nicotinic acetylcholine receptor by tyrosine phosphorylation. Nature 336, 677–680 (1988)

    Article  ADS  CAS  Google Scholar 

  24. Kelley, S. P., Dunlop, J. I., Kirkness, E. F., Lambert, J. J. & Peters, J. A. A cytoplasmic region determines single-channel conductance in 5-HT3 receptors. Nature 424, 321–324 (2003)

    Article  ADS  CAS  Google Scholar 

  25. Sola, M. et al. Structural basis of dynamic glycine receptor clustering by gephyrin. EMBO J. 23, 2510–2519 (2004)

    Article  CAS  Google Scholar 

  26. Chen, G. Q., Cui, C., Mayer, M. L. & Gouaux, E. Functional characterization of a potassium-selective prokaryotic glutamate receptor. Nature 402, 817–821 (1999)

    Article  ADS  CAS  Google Scholar 

  27. Belkin, S., Mehlhorn, R. J. & Packer, L. Proton gradients in intact cyanobacteria. Plant Physiol. 84, 25–30 (1987)

    Article  CAS  Google Scholar 

  28. Sallette, J. et al. Nicotine upregulates its own receptors through enhanced intracellular maturation. Neuron 46, 595–607 (2005)

    Article  CAS  Google Scholar 

  29. Paoletti, P., Ascher, P. & Neyton, J. High-affinity zinc inhibition of NMDA NR1–NR2A receptors. J. Neurosci. 17, 5711–5725 (1997)

    Article  CAS  Google Scholar 

  30. Fischer, M., Corringer, P. J., Schott, K., Bacher, A. & Changeux, J. P. A new method for soluble overexpression of the α7 nAChR extracellular domain. Proc. Natl Acad. Sci. USA 98, 3567–3570 (2001)

    Article  ADS  CAS  Google Scholar 

Download references

Acknowledgements

We wish to thank N. Le Novère, J. L. Popot, P. Delepelaire and C. Beloin for useful assistance, and S. Edelstein for critical reading. This work was supported by the Région Ile de France, the Association Française contre les Myopathies, the Collège de France, the Commission of the European Communities (CEC) and the Association pour la Recherche sur le Cancer.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Pierre-Jean Corringer.

Ethics declarations

Competing interests

Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Methods, and two Supplementary Figures analysing the ionic permeability of the Glvi ion channel, by whole-cell patch clamp recording of Glvi-transfected HEK cells. The Supplementary Figures present the reversal potential of pH5-elicited currents in different external NaCl and proton concentrations. (PDF 268 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Bocquet, N., Prado de Carvalho, L., Cartaud, J. et al. A prokaryotic proton-gated ion channel from the nicotinic acetylcholine receptor family. Nature 445, 116–119 (2007). https://doi.org/10.1038/nature05371

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature05371

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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