Abstract
The muscle acetylcholine receptor channel (AChR) is a large (Mr ≅290K) transmembrane protein that mediates synaptic transmission. Theactivation of this ion channel can be understood in the framework of athermodynamic cycle with spontaneous gating (i.e., the closed ⇌ open reaction) and ligand-binding events as the elementary steps. Becauseagonists bind more tightly to the open than to the closed state, gating ofliganded receptors is more favorable than that of unliganded receptors.Accordingly, channel opening must involve two major conformationalchanges: the ACh-binding sites switch from a low-affinity to a high-affinityform, and the pore (located ∼ 45 Å away from the binding sites)switches from an ion-impermeable to an ion-permeable conformation. Togain insight into the reaction mechanism of fully-liganded gating, wecharacterized the corresponding transition state in the context of the `linearfree-energy relationships' of physical organic chemistry (Φ-valueanalysis). Gating of fully-liganded AChRs was studied by recordingsingle-channel currents using the patch-clamp technique. Perturbations tothe wild-type receptor were either series of different mutations at individualpositions or series of different agonists. Based on the obtained `snapshot'of the gating reaction at the transition state, and aware of the lack ofinformation about the rest of the energy profile, the most parsimoniousmechanism seems to be one where opening proceeds asynchronously, withthe low-to-high affinity change at the binding sites preceding the completeopening of the distant pore.
Keywords: Brønsted plots, ion channels, nicotinic receptors, transition state
Full Text
The Full Text of this article is available as a PDF (141.9 KB).
References
- 1.Monod J., Wyman J., Changeux .-P. On the Nature of Allosteric Transitions: A Plausible Model. J. Mol. Biol. 1965;12:88–118. doi: 10.1016/s0022-2836(65)80285-6. [DOI] [PubMed] [Google Scholar]
- 2.Karlin A. On the Application of 'A Plausible Model' of Allosteric Proteins to the Receptor for Acetylcholine. J. Theor. Biol. 1967;16:306–320. doi: 10.1016/0022-5193(67)90011-2. [DOI] [PubMed] [Google Scholar]
- 3.Jackson M.B. Spontaneous Openings of the Acetylcholine Receptor Channel. Proc. Natl. Acad. Sci. USA. 1984;81:3901–3904. doi: 10.1073/pnas.81.12.3901. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Edelstein S.J., Changeux J.-P. Allosteric Transitions of the Acetylcholine Receptor. Adv. Prot. Chem. 1998;51:121–184. doi: 10.1016/s0065-3233(08)60652-x. [DOI] [PubMed] [Google Scholar]
- 5.Grosman C., Auerbach A. Kinetic, Mechanistic, and Structural Aspects of Unliganded Gating of Acetylcholine Receptor Channels. A Single-Channel Study of M2 12' Mutants. J. Gen. Physiol. 2000;115:621–635. doi: 10.1085/jgp.115.5.621. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Valenzuela C.F., Weign P., Yguerabide J., Johnson D.A. Transverse Distance between the Membrane and the Agonist Binding Sites on the Tor pedo Acetylcholine Receptor: A Fluorescence Study. Biophys. J. 1994;66:674–682. doi: 10.1016/s0006-3495(94)80841-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Leffler J.E., Grunwald E. Rates and Equilibria of Organic Reactions. New York: John Wiley & Sons; 1963. [Google Scholar]
- 8.Hammett L.P. Linear Free Energy Relationships in Rate and Equilibrium Phenomena. Trans. Faraday Soc. 1938;34:156–165. [Google Scholar]
- 9.Fersht A.R., Leatherbarrow R.J., Wells T.N.C. Quantitative Analysis of Structure-Activity Relationships in Engineered Proteins by Linear Free-Energy Relationships. Nature. 1986;322:284–286. doi: 10.1021/bi00393a013. [DOI] [PubMed] [Google Scholar]
- 10.Itzhaki L.S., Otzen D.E., Fersht A.R. The Structure of the Transition State for Fold-ing of Chymotrypsin Inhibitor 2 Analyzed by Protein Engineering Methods: Evidence for a Nucleation-Condensation Mechanism for Protein Folding. J. Mol. Biol. 1995;254:260–288. doi: 10.1006/jmbi.1995.0616. [DOI] [PubMed] [Google Scholar]
- 11.Grosman C., Zhou M., Auerbach A. Mapping the Conformational Wave of Acetylcholine Receptor Channel Gating. Nature. 2000;403:773–776. doi: 10.1038/35001586. [DOI] [PubMed] [Google Scholar]
- 12.Leffler J.E. Parameters for the Description of Transition States. Science. 1953;117:340–341. doi: 10.1126/science.117.3039.340. [DOI] [PubMed] [Google Scholar]
- 13.Grosman C., Auerbach A. Asymmetric and Independent Contribution of the Second Transmembrane Segment 12' Residues to Diliganded Gating of Acetylcholine Receptor Chan-nels: A Single-Channel Study with Choline as the Agonist. J. Gen. Physiol. 2000;115:637–651. doi: 10.1085/jgp.115.5.637. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Qin F., Auerbach A., Sachs F. Estimating Single-Channel Kinetic Parameters from Idealized Patch-Clamp Data Containing Missed Events. Biophys. J. 1996;70:264–280. doi: 10.1016/S0006-3495(96)79568-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Jaffé H.H. A Reexamination of the Hammett Equation. Chem. Rev. 1953;53:191–261. [Google Scholar]