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Probing ion-channel pores one proton at a time

Nature volume 438, pages 975–980 (2005)Cite this article

Abstract

Although membrane proteins often rely on ionizable residues for structure and function, their ionization states under physiological conditions largely elude experimental estimation. To gain insight into the effect of the local microenvironment on the proton affinity of ionizable residues, we have engineered individual lysines, histidines and arginines along the α-helical lining of the transmembrane pore of the nicotinic acetylcholine receptor. We can detect individual proton binding–unbinding reactions electrophysiologically at the level of a single proton on a single side chain as brief blocking–unblocking events of the passing cation current. Kinetic analysis of these fluctuations yields the position-dependent rates of proton transfer, from which the corresponding pKa values and shifts in pKa can be calculated. Here we present a self-consistent, residue-by-residue description of the microenvironment around the pore-lining transmembrane α-helices (M2) in the open-channel conformation, in terms of the excess free energy that is required to keep the engineered basic side chains protonated relative to bulk water. A comparison with closed-channel data leads us to propose that the rotation of M2, which is frequently invoked as a hallmark of the gating mechanism of Cys-loop receptors, is minimal, if any.

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Figure 1: Protonation of lysines causes partial channel block.
Figure 2: Experimental estimates of p K a values.
Figure 3: pH dependence of proton-transfer reactions.

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References

  1. Perutz, M. F. Electrostatic effects in proteins. Science 201, 1187–1191 (1978)

    Article  ADS  CAS  Google Scholar 

  2. Warshel, A. Electrostatic basis of structure–function correlation in proteins. Acc. Chem. Res. 14, 284–290 (1981)

    Article  CAS  Google Scholar 

  3. Davis, M. E. & McCammon, J. A. Electrostatics in biomolecular structure and dynamics. Chem. Rev. 90, 509–521 (1990)

    Article  CAS  Google Scholar 

  4. Honig, B. & Nicholls, A. Classical electrostatics in biology and chemistry. Science 268, 1144–1149 (1995)

    Article  ADS  CAS  Google Scholar 

  5. Imoto, K. et al. Rings of negatively charged amino acids determine the acetylcholine receptor channel conductance. Nature 335, 645–648 (1988)

    Article  ADS  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  7. Heinemann, S. H., Terlau, H., Stühmer, W., Imoto, K. & Numa, S. Calcium channel characteristics conferred on the sodium channel by single mutations. Nature 356, 441–443 (1992)

    Article  ADS  CAS  Google Scholar 

  8. Yang, J., Ellinor, P. T., Sather, W. A., Zhang, J. F. & Tsien, R. W. Molecular determinants of Ca2+ selectivity and ion permeation in L-type Ca2+ channels. Nature 366, 158–161 (1993)

    Article  ADS  CAS  Google Scholar 

  9. Keramidas, A., Moorhouse, A. J., Pierce, K. D., Schofield, P. R. & Barry, P. H. Cation-selective mutations in the M2 domain of the inhibitory glycine receptor channel reveal determinants of ion-charge selectivity. J. Gen. Physiol. 119, 393–410 (2002)

    Article  CAS  Google Scholar 

  10. Lu, Z. & MacKinnon, R. Electrostatic tuning of Mg2+ affinity in an inward-rectifier K+ channel. Nature 371, 243–246 (1994)

    Article  ADS  CAS  Google Scholar 

  11. Khan, A., Romantseva, L., Lam, A., Lipkind, G. & Fozzard, H. A. Role of outer ring carboxylates of the rat skeletal muscle sodium channel pore in proton block. J. Physiol. Lond. 543, 71–84 (2002)

    Article  CAS  Google Scholar 

  12. Schulte, U. & Fakler, B. Gating of inward-rectifier K+ channels by intracellular pH. Eur. J. Biochem. 267, 5837–5841 (2000)

    Article  CAS  Google Scholar 

  13. Aggarwal, S. K. & MacKinnon, R. Contribution of the S4 segment to gating charge in the Shaker K+ channel. Neuron 16, 1169–1177 (1996)

    Article  CAS  Google Scholar 

  14. Seoh, S.-A., Sigg, D., Papazian, D. M. & Bezanilla, F. Voltage-sensing residues in the S2 and S4 segments of the Shaker K+ channel. Neuron 16, 1159–1167 (1996)

    Article  CAS  Google Scholar 

  15. Schutz, C. N. & Warshel, A. What are the dielectric ‘constants’ of proteins and how to validate electrostatic models? Proteins 44, 400–417 (2001)

    Article  CAS  Google Scholar 

  16. Mehler, E. L., Fuxreiter, M., Simon, I. & García-Moreno, E. B. The role of hydrophobic microenvironments in modulating pKa shifts in proteins. Proteins 48, 283–292 (2002)

    Article  CAS  Google Scholar 

  17. Parzefall, F., Wilhelm, R., Heckmann, M. & Dudel, J. Single channel currents at six microsecond resolution elicited by acetylcholine in mouse myoballs. J. Physiol. Lond. 512, 181–188 (1998)

    Article  CAS  Google Scholar 

  18. Dao-pin, S., Anderson, D. E., Baase, W. A., Dahlquist, F. W. & Matthews, B. W. Structural and thermodynamic consequences of burying a charged residue within the hydrophobic core of T4 lysozyme. Biochemistry 30, 11521–11529 (1991)

    Article  CAS  Google Scholar 

  19. Prod'hom, B., Pietrobon, D. & Hess, P. Direct measurement of proton transfer rates to a group controlling the dihydropyridine-sensitive Ca2+ channel. Nature 329, 243–246 (1987)

    Article  ADS  CAS  Google Scholar 

  20. Root, M. J. & MacKinnon, R. Two identical noninteracting sites in an ion channel revealed by proton transfer. Science 265, 1852–1856 (1994)

    Article  ADS  CAS  Google Scholar 

  21. Lester, H. The permeation pathway of neurotransmitter-gated ion channels. Annu. Rev. Biophys. Biomol. Struct. 21, 267–292 (1992)

    Article  CAS  Google Scholar 

  22. Unwin, N. Acetylcholine receptor channel imaged in the open state. Nature 373, 37–43 (1995)

    Article  ADS  CAS  Google Scholar 

  23. 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 

  24. Horenstein, J., Wagner, D. A., Czajkowski, C. & Akabas, M. H. Protein mobility and GABA-induced conformational changes in GABAA receptor pore-lining M2 segment. Nature Neurosci. 4, 477–485 (2001)

    Article  CAS  Google Scholar 

  25. 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 

  26. Adcock, C., Smith, G. R. & Sansom, M. S. P. Electrostatics and the ion selectivity of ligand-gated channels. Biophys. J. 75, 1211–1222 (1998)

    Article  CAS  Google Scholar 

  27. Stopar, D., Sprujit, R. B., Wolfs, C. J. & Hemminga, M. A. Local dynamics of the M13 major coat protein in different membrane-mimicking systems. Biochemistry 35, 15467–15473 (1996)

    Article  CAS  Google Scholar 

  28. Grosman, C., Zhou, M. & Auerbach, A. Mapping the conformational wave of acetylcholine receptor channel gating. Nature 403, 773–776 (2000)

    Article  ADS  CAS  Google Scholar 

  29. Cymes, G. D., Grosman, C. & Auerbach, A. Structure of the transition state of gating in the acetylcholine receptor channel pore: a φ-value analysis. Biochemistry 41, 5548–5555 (2002)

    Article  CAS  Google Scholar 

  30. Grosman, C. Free-energy landscapes of ion-channel gating are malleable: changes in the number of bound ligands are accompanied by changes in the location of the transition state in acetylcholine-receptor channels. Biochemistry 42, 14977–14987 (2003)

    Article  CAS  Google Scholar 

  31. Goren, E. N., Reeves, D. C. & Akabas, M. H. Loose protein packing around the extracellular half of the GABAA receptor β1 subunit M2 channel-lining segment. J. Biol. Chem. 279, 11198–11205 (2004)

    Article  CAS  Google Scholar 

  32. Kim, S., Chamberlain, A. K. & Bowie, J. U. A model of the closed form of the nicotinic acetylcholine receptor M2 channel pore. Biophys. J. 87, 792–799 (2004)

    Article  CAS  Google Scholar 

  33. Dahan, D. et al. A fluorophore attached to nicotinic acetylcholine receptor βM2 detects productive binding of agonist to the αδ site. Proc. Natl Acad. Sci. USA 101, 10195–10200 (2004)

    Article  ADS  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  35. White, B. H. & Cohen, J. B. Agonist-induced changes in the structure of the acetylcholine receptor M2 regions revealed by photoincorporation of an uncharged nicotinic noncompetitive antagonist. J. Biol. Chem. 267, 15770–15783 (1992)

    CAS  PubMed  Google Scholar 

  36. Arévalo, E., Chiara, D. C., Forman, S. A., Cohen, J. B. & Miller, K. W. Gating-enhanced accessibility of hydrophobic sites within the transmembrane region of the nicotinic acetylcholine receptor's δ subunit. A time-resolved photolabeling study. J. Biol. Chem. 280, 13631–13640 (2005)

    Article  Google Scholar 

  37. O'Mara, M., Barry, P. H. & Chung, S. H. A model of the glycine receptor deduced from Brownian dynamics. Proc. Natl Acad. Sci. USA 100, 4310–4315 (2003)

    Article  ADS  CAS  Google Scholar 

  38. Corry, B., Vora, T. & Chung, S. H. Electrostatic basis of valence selectivity in cationic channels. Biochim. Biophys. Acta 1711, 72–86 (2005)

    Article  CAS  Google Scholar 

  39. Akabas, M. H., Kaufmann, C., Archdeacon, P. & Karlin, A. Identification of acetylcholine receptor channel-lining residues in the entire M2 segment of the α subunit. Neuron 13, 919–927 (1994)

    Article  CAS  Google Scholar 

  40. Qin, F. Restoration of single-channel currents using the segmental k-means method based on hidden Markov modeling. Biophys. J. 86, 1488–1501 (2004)

    Article  ADS  CAS  Google Scholar 

  41. Qin, F., Auerbach, A. & Sachs, F. Estimating single-channel kinetic parameters from idealized patch-clamp data containing missed events. Biophys. J. 70, 264–280 (1996)

    Article  CAS  Google Scholar 

  42. Ohno, K. et al. Congenital myasthenic syndrome caused by prolonged acetylcholine receptor channel openings due to a mutation in the M2 domain of the epsilon subunit. Proc. Natl Acad. Sci. USA 92, 758–762 (1995)

    Article  ADS  CAS  Google Scholar 

  43. Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Graphics 14, 33–38 (1996)

    Article  CAS  Google Scholar 

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Acknowledgements

We thank S. Sine for wild-type muscle AChR subunit cDNA; J. Jasielec and J. Gasser for technical assistance; S. Varma and B. García-Moreno E. for discussions; and E. Tajkhorshid for introducing us to the VMD imaging program. This work was supported by a grant from the NIH (to C.G.).

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Authors and Affiliations

  1. Department of Molecular and Integrative Physiology, Center for Biophysics and Computational Biology, and Neuroscience Program, University of Illinois at Urbana-Champaign, Illinois, 61801, Urbana, USA

    Gisela D. Cymes, Ying Ni & Claudio Grosman

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  1. Gisela D. Cymes
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  3. Claudio Grosman
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Correspondence to Claudio Grosman.

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Cymes, G., Ni, Y. & Grosman, C. Probing ion-channel pores one proton at a time. Nature 438, 975–980 (2005). https://doi.org/10.1038/nature04293

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