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Pore-opening mechanism of the nicotinic acetylcholine receptor evinced by proton transfer

Nature Structural & Molecular Biology volume 15, pages 389–396 (2008)Cite this article

Abstract

The conformational changes underlying cysteine-loop receptor channel gating remain elusive and controversial. We previously developed a single-channel electrophysiological method that allows structural inferences about the transient open-channel conformation to be made from the effect and properties of introduced charges on systematically engineered ionizable amino acids. Here we have applied this methodology to the entire M1 and M3 segments of the muscle nicotinic acetylcholine receptor, two transmembrane α-helices that pack against the pore-lining M2 α-helix. Together with our previous results on M2, these data suggest that the pore dilation that underlies channel opening involves only a subtle rearrangement of these three transmembrane helices. Such a limited conformational change seems optimal to allow rapid closed-open interconversion rates, and hence a fast postsynaptic response upon neurotransmitter binding. Thus, this receptor-channel seems to have evolved to take full advantage of the steep dependence of ion- and water-conduction rates on pore diameter that is characteristic of model hydrophobic nanopores.

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Figure 1: General properties of the AChR.
Figure 2: Geometrical relationships between M1, M2, M3 and the pore in the closed state.
Figure 3: pH- and position-dependence of proton-transfer events.
Figure 4: Extent of channel block.
Figure 5: Two engineered lysines create two proton binding sites.
Figure 6: Experimental estimation of pKa values using electrophysiological recordings.
Figure 7: pKa-shifts at protein-protein interfaces.

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References

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

    Article  CAS  Google Scholar 

  2. Dilger, J.P. & Liu, Y. Desensitization of acetylcholine receptors in BC3H–1 cells. Pflugers Arch. 420, 479–485 (1992).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. 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 

  5. Sansom, M.S.P. Twist to open. Curr. Biol. 5, 373–375 (1995).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  7. Law, R.J., Henchman, R.H. & McCammon, J.A. A gating mechanism proposed from a simulation of a human α7 nicotinic acetylcholine receptor. Proc. Natl. Acad. Sci. USA 102, 6813–6818 (2005).

    Article  CAS  Google Scholar 

  8. Paas, Y. et al. Pore conformations and gating mechanism of a Cys-loop receptor. Proc. Natl. Acad. Sci. USA 102, 15877–15882 (2005).

    Article  CAS  Google Scholar 

  9. Cheng, X., Ivanov, I., Wang, H., Sine, S.M. & McCammon, J.A. Nanosecond time scale conformational dynamics of the human α7 nicotinic acetylcholine receptor. Biophys. J. 93, 2622–2634 (2007).

    Article  CAS  Google Scholar 

  10. Cheng, X., Wang, H., Grant, B., Sine, S.M. & McCammon, J.A. Targeted molecular dynamics study of C-loop closure and channel gating in nicotinic receptors. PLOS Comput. Biol. 2, e134 (2006).

    Article  Google Scholar 

  11. Cymes, G.D., Ni, Y. & Grosman, C. Probing ion-channel pores one proton at a time. Nature 438, 975–980 (2005).

    Article  CAS  Google Scholar 

  12. Warshel, A. Calculations of enzymatic reactions: calculations of pKa, proton transfer reactions, and general catalysis reactions in enzymes. Biochemistry 20, 3167–3177 (1981).

    Article  CAS  Google Scholar 

  13. 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 

  14. 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 

  15. Fitch, C.A. et al. pKa values of buried residues: analysis with continuum methods and role of water penetration. Biophys. J. 82, 3289–3304 (2002).

    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. Guillén Schlippe, Y.V. & Hedstrom, L. A twisted base? The role of arginine in enzyme-catalyzed proton abstractions. Arch. Biochem. Biophys. 433, 266–278 (2005).

    Article  Google Scholar 

  18. Kim, J., Mao, J. & Gunner, M.R. Are acidic and basic groups in buried proteins predicted to be ionized? J. Mol. Biol. 348, 1283–1298 (2005).

    Article  CAS  Google Scholar 

  19. Niemeyer, M.I. et al. Neutralization of a single arginine residue gates open a two-pore domain, alkali-activated K+ channel. Proc. Natl. Acad. Sci. USA 104, 666–671 (2007).

    Article  CAS  Google Scholar 

  20. Jansen, M. & Akabas, M.H. State-dependent cross-linking of the M2 and M3 segments: functional basis for the alignment of GABAA and acetylcholine receptor M3 segments. J. Neurosci. 26, 4492–4499 (2006).

    Article  CAS  Google Scholar 

  21. Norberg, J., Foloppe, N. & Nilsson, L. Intrinsic relative stabilities of the neutral tautomers of arginine side-chain models. J. Chem. Theory Comput. 1, 986–993 (2005).

    Article  CAS  Google Scholar 

  22. Blanton, M.P. & Cohen, J.B. Identifying the lipid-protein interface of the Torpedo nicotinic acetylcholine receptor: secondary structure implications. Biochemistry 33, 2859–2872 (1994).

    Article  CAS  Google Scholar 

  23. Hamouda, A.K., Chiara, D.C., Sauls, D., Cohen, J.B. & Blanton, M.P. Cholesterol interacts with transmembrane α-helices M1, M3, and M4 of the Torpedo nicotinic acetylcholine receptor: photolabeling studies using [3H]azicholesterol. Biochemistry 45, 976–986 (2006).

    Article  CAS  Google Scholar 

  24. Tamamizu, S. et al. Functional effects of periodic tryptophan substitutions in the α M4 transmembrane domain of the Torpedo californica nicotinic acetylcholine receptor. Biochemistry 39, 4666–4673 (2000).

    Article  CAS  Google Scholar 

  25. Dwyer, T.M., Adams, D.J. & Hille, B. The permeability of the endplate channel to organic cations in frog muscle. J. Gen. Physiol. 75, 469–492 (1980).

    Article  CAS  Google Scholar 

  26. Cohen, B.N., Labarca, C., Davidson, N. & Lester, H.A. Mutations in M2 alter the selectivity of the mouse nicotinic acetylcholine receptor for organic and alkali metal cations. J. Gen. Physiol. 100, 373–400 (1992).

    Article  CAS  Google Scholar 

  27. Smart, O.S., Neduvelil, J.G., Wang, X., Wallace, B.A. & Sansom, M.S.P. HOLE: a program for the analysis of the pore dimensions of ion channel structural models. J. Mol. Graph. 14, 354–360 (1996).

    Article  CAS  Google Scholar 

  28. Varma, S., Chiu, S.W. & Jakobsson, E. The influence of amino acid protonation states on molecular dynamics simulations of the bacterial porin OmpF. Biophys. J. 90, 112–123 (2006).

    Article  CAS  Google Scholar 

  29. Beckstein, O. & Sansom, M.S.P. A hydrophobic gate in an ion channel: the closed state of the nicotinic acetylcholine receptor. Phys. Biol. 3, 147–159 (2006).

    Article  CAS  Google Scholar 

  30. Corry, B. An energy-efficient gating mechanism in the acetylcholine receptor channel suggested by molecular and Brownian dynamics. Biophys. J. 90, 799–810 (2006).

    Article  CAS  Google Scholar 

  31. 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 

  32. Beckstein, O., Biggin, P.C. & Sansom, M.S.P. A hydrophobic gating mechanism for nanopores. J. Phys. Chem. B 105, 12902–12905 (2001).

    Article  CAS  Google Scholar 

  33. Lynden-Bell, R.M. & Rasaiah, J.C. Mobility and solvation of ions in channels. J. Chem. Phys. 105, 9266–9280 (1996).

    Article  CAS  Google Scholar 

  34. Beckstein, O. & Sansom, M.S.P. The influence of geometry, surface character, and flexibility on the permeation of ions and water through biological pores. Phys. Biol. 1, 42–52 (2004).

    Article  CAS  Google Scholar 

  35. Peter, C. & Hummer, G. Ion transport through membrane-spanning nanopores studied by molecular dynamics simulations and continuum electrostatics calculations. Biophys. J. 89, 2222–2234 (2005).

    Article  CAS  Google Scholar 

  36. Sotomayor, M., van der Straaten, T.A., Ravaioli, U. & Schulten, K. Electrostatic properties of the mechanosensitive channel of small conductance MscS. Biophys. J. 90, 3496–3510 (2006).

    Article  CAS  Google Scholar 

  37. Vora, T., Corry, B. & Chung, S.H. Brownian dynamics investigation into the conductance state of the MscS channel crystal structure. Biochim. Biophys. Acta 1758, 730–737 (2006).

    Article  CAS  Google Scholar 

  38. Pathak, M.M. et al. Closing in on the resting state of the Shaker K+ channel. Neuron 56, 124–140 (2007).

    Article  CAS  Google Scholar 

  39. 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  CAS  Google Scholar 

  40. Schwarz, G. Estimating the dimension of a model. Ann. Statist. 6, 461–464 (1978).

    Article  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. Grosman, C., Salamone, F.N., Sine, S.M. & Auerbach, A. The extracellular linker of muscle acetylcholine receptor channels is a gating control element. J. Gen. Physiol. 116, 327–339 (2000).

    Article  CAS  Google Scholar 

  43. Engel, A.G. et al. New mutations in acetylcholine receptor subunit genes reveal heterogeneity in the slow-channel congenital myasthenic syndrome. Hum. Mol. Genet. 5, 1217–1227 (1996).

    Article  CAS  Google Scholar 

  44. Grosman, C. & Auerbach, A. Asymmetric and independent contribution of the second transmembrane segment 12′ residues to diliganded gating of acetylcholine receptor channels. A single-channel study with choline as the agonist. J. Gen. Physiol. 115, 637–651 (2000).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  47. Frishman, D. & Argos, P. Knowledge-based protein secondary structure assignment. Proteins 23, 566–579 (1995).

    Article  CAS  Google Scholar 

  48. Blanton, M.P., McCardy, E.A., Huggins, A. & Parikh, D. Probing the structure of the nicotinic acetylcholine receptor with the hydrophobic photoreactive probes [125I]TID-BE and [125I]TIDPC/16. Biochemistry 37, 14545–14555 (1998).

    Article  CAS  Google Scholar 

  49. Tanokura, M. 1H-NMR study on the tautomerism of the imidazole ring of histidine residues. I. Microscopic pK values and molar ratios of tautomers in histidine-containing peptides. Biochim. Biophys. Acta 742, 576–585 (1983).

    Article  CAS  Google Scholar 

  50. Thurlkill, R.L., Grimsley, G.R., Scholtz, J.M. & Pace, C.N. pK values of the ionizable groups of proteins. Protein Sci. 15, 1214–1218 (2006).

    Article  CAS  Google Scholar 

  51. Warwicker, J. Improved pKa calculations through flexibility based sampling of a water-dominated interaction scheme. Protein Sci. 13, 2793–2805 (2004).

    Article  CAS  Google Scholar 

  52. Hilf, R.J.C. & Dutzler, R. X-ray structure of a prokaryotic pentameric ligand-gated ion channel. Nature advance online publication, doi:10.1038/nature06717 (5 March 2008).

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Acknowledgements

We thank S. Sine (Mayo Clinic College of Medicine, Rochester, Minnesota, USA) for the wild-type muscle AChR subunit cDNA; G. Westfield and J. Gasser for technical assistance; and E. Tajkhorshid, M. Sotomayor, S. Varma and F.D. González-Nilo for advice and discussions. This work was supported by a grant from the US National Institutes of Health (R01-NS042169 to C.G.).

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  1. Department of Molecular and Integrative Physiology, Center for Biophysics and Computational Biology and Neuroscience Program, University of Illinois at Urbana-Champaign, 407 South Goodwin Avenue, 524 Burrill Hall, Urbana, 61801, Illinois, USA

    Gisela D Cymes & Claudio Grosman

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  1. Gisela D Cymes
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G.D.C. designed and performed the experiments, and analyzed data; C.G. designed experiments, analyzed data and wrote the manuscript.

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Correspondence to Claudio Grosman.

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Cymes, G., Grosman, C. Pore-opening mechanism of the nicotinic acetylcholine receptor evinced by proton transfer. Nat Struct Mol Biol 15, 389–396 (2008). https://doi.org/10.1038/nsmb.1407

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