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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$189.00 per year
only $15.75 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Unwin, N. Refined structure of the nicotinic acetylcholine receptor at 4 Å resolution. J. Mol. Biol. 346, 967–989 (2005).
Dilger, J.P. & Liu, Y. Desensitization of acetylcholine receptors in BC3H–1 cells. Pflugers Arch. 420, 479–485 (1992).
Unwin, N. Acetylcholine receptor channel imaged in the open state. Nature 373, 37–43 (1995).
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).
Sansom, M.S.P. Twist to open. Curr. Biol. 5, 373–375 (1995).
Miyazawa, A., Fujiyoshi, Y. & Unwin, N. Structure and gating mechanism of the acetylcholine receptor pore. Nature 423, 949–955 (2003).
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).
Paas, Y. et al. Pore conformations and gating mechanism of a Cys-loop receptor. Proc. Natl. Acad. Sci. USA 102, 15877–15882 (2005).
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).
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).
Cymes, G.D., Ni, Y. & Grosman, C. Probing ion-channel pores one proton at a time. Nature 438, 975–980 (2005).
Warshel, A. Calculations of enzymatic reactions: calculations of pKa, proton transfer reactions, and general catalysis reactions in enzymes. Biochemistry 20, 3167–3177 (1981).
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).
Schutz, C.N. & Warshel, A. What are the dielectric “constants” of proteins and how to validate electrostatic models? Proteins 44, 400–417 (2001).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
Corry, B. An energy-efficient gating mechanism in the acetylcholine receptor channel suggested by molecular and Brownian dynamics. Biophys. J. 90, 799–810 (2006).
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).
Beckstein, O., Biggin, P.C. & Sansom, M.S.P. A hydrophobic gating mechanism for nanopores. J. Phys. Chem. B 105, 12902–12905 (2001).
Lynden-Bell, R.M. & Rasaiah, J.C. Mobility and solvation of ions in channels. J. Chem. Phys. 105, 9266–9280 (1996).
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).
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).
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).
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).
Pathak, M.M. et al. Closing in on the resting state of the Shaker K+ channel. Neuron 56, 124–140 (2007).
Qin, F. Restoration of single-channel currents using the segmental k-means method based on hidden Markov modeling. Biophys. J. 86, 1488–1501 (2004).
Schwarz, G. Estimating the dimension of a model. Ann. Statist. 6, 461–464 (1978).
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).
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).
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).
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).
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).
Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Graph. 14, 33–38 (1996).
Frishman, D. & Argos, P. Knowledge-based protein secondary structure assignment. Proteins 23, 566–579 (1995).
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).
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).
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).
Warwicker, J. Improved pKa calculations through flexibility based sampling of a water-dominated interaction scheme. Protein Sci. 13, 2793–2805 (2004).
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).
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.).
Author information
Authors and Affiliations
Contributions
G.D.C. designed and performed the experiments, and analyzed data; C.G. designed experiments, analyzed data and wrote the manuscript.
Corresponding author
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1 and 2 and Supplementary Table 1 (PDF 302 kb)
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nsmb.1407
This article is cited by
-
A distinct mechanism for activating uncoupled nicotinic acetylcholine receptors
Nature Chemical Biology (2013)
-
The Structural Mechanism of the Cys-Loop Receptor Desensitization
Molecular Neurobiology (2013)
-
RETRACTED ARTICLE: Deprotonation of Arginines in S4 is Involved in NaChBac Gating
The Journal of Membrane Biology (2012)
-
Emerging approaches to probing ion channel structure and function
Neuroscience Bulletin (2012)
-
Tunable pKa values and the basis of opposite charge selectivities in nicotinic-type receptors
Nature (2011)