Abstract
Nicotinic acetylcholine receptors are heteropentameric ion channels that open upon activation to a single conducting state. The second transmembrane segments of each subunit were identified as channel-forming elements, but their respective contribution in the gating process remains unclear. Moreover, the detailed impact of variations of the membrane potential, such as occurring during an action potential, on the transmembrane domains, is unknown. Residues at the 12′ position, close to the center of each second transmembrane segment, play a key role in channel gating. We examined their functional symmetry by substituting a lysine to that position of each subunit and measuring the electrical activity of single channels. For 12′ lysines in the α, γ and δ subunits rapid transitions between an intermediate and large conductance appeared, which are interpreted as single lysine protonation events. From the kinetics of these transitions we calculated the pK a values of respective lysines and showed that they vary differently with membrane hyperpolarization. Respective mutations in β or ε subunits gave receptors with openings of either intermediate or large conductance, suggesting extreme pK a values in two open state conformations. The results demonstrate that these parts of the highly homologous transmembrane domains, as probed by the 12′ lysines, sense unequal microenvironments and are differently affected by physiologically relevant voltage changes. Moreover, observation of various gating events for mutants of α subunits suggests that the open channel pore exists in multiple conformations, which in turn supports the notion of functional asymmetry of the channel.
Similar content being viewed by others
Abbreviations
- nAChR:
-
Nicotinic acetylcholine receptor
- TM1/TM2:
-
First/second transmembrane segment
- GFP:
-
Green fluorescent protein
References
Absalom NL, Lewis TM, Schofield PR (2004) Mechanisms of channel gating of the ligand-gated ion channel superfamily inferred from protein structure. Exp Physiol 89:145–153
Akabas MH, Kaufmann C, Archdeacon P, Karlin A (1994) Identification of acetylcholine receptor channel-lining residues in the entire M2 segment of the alpha subunit. Neuron 13:919–927
Auerbach A, Sigurdson W, Chen J, Akk G (1996) Voltage dependence of mouse acetylcholine receptor gating: different charge movements in di-, mono- and unliganded receptors. J Physiol 494(Pt 1):155–170
Camacho P, Liu Y, Mandel G, Brehm P (1993) The epsilon subunit confers fast channel gating on multiple classes of acetylcholine receptors. J Neurosci 13:605–613
Cymes GD, Ni Y, Grosman C (2005) Probing ion-channel pores one proton at a time. Nature 438:975–980
Dahan DS, Dibas MI, Petersson EJ, Auyeung VC, Chanda B, Bezanilla F, Dougherty DA, Lester HA (2004) A fluorophore attached to nicotinic acetylcholine receptor beta M2 detects productive binding of agonist to the alpha delta site. Proc Natl Acad Sci USA 101:10195–10200
Grosman C, Auerbach A (2000) 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
Grutter T, Changeux JP (2001) Nicotinic receptors in wonderland. Trends Biochem Sci 26:459–463
Hucho F, Oberthur W, Lottspeich F (1986) The ion channel of the nicotinic acetylcholine receptor is formed by the homologous helices M II of the receptor subunits. FEBS Lett 205:137–142
Maconochie DJ, Fletcher GH, Steinbach JH (1995) The conductance of the muscle nicotinic receptor channel changes rapidly upon gating. Biophys J 68:483–490
Miyazawa A, Fujiyoshi Y, Unwin N (2003) Structure and gating mechanism of the acetylcholine receptor pore. Nature 423:949–955
Kim S, Aaron K. Chamberlain, James U. Bowie (2004). A model of the closed form of the nicotinic acetylcholine receptor m2 channel pore. Biophys J 87:792–799
Newland CF, Beeson D, Vincent A, Newsom-Davis J (1995) Functional and non-functional isoforms of the human muscle acetylcholine receptor. J Physiol 489(Pt 3):767–778
Qin F, Auerbach A, Sachs F (1996) Estimating single-channel kinetic parameters from idealized patch-clamp data containing missed events. Biophys J 70:264–280
Sigworth FJ (1985) Open channel noise I. Noise in acetylcholine receptor currents suggests conformational fluctuations. Biophys J 47:709–720
Unwin N (2005) Refined structure of the nicotinic acetylcholine receptor at 4A resolution. J Mol Biol 346:967–989
Acknowledgments
We thank Karen Martinez for critical manuscript reading and Pierre-Jean Corringer and David Beeson for providing cDNA of wild-type nAChR subunits. This work was supported by the Swiss National Science Foundation (31–57023.99 and 31–00A0–102062/1) and the European Commission via contract LSHG-CT-2004–504601 (E-MeP).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Grandl, J., Danelon, C., Hovius, R. et al. Functional asymmetry of transmembrane segments in nicotinic acetylcholine receptors. Eur Biophys J 35, 685–693 (2006). https://doi.org/10.1007/s00249-006-0078-2
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00249-006-0078-2