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Acetylcholine Receptor Gating at Extracellular Transmembrane Domain Interface: the “Pre-M1” Linker

Acetylcholine Receptor Gating at Extracellular Transmembrane Domain Interface: the “Pre-M1” Linker Charged residues in the β10–M1 linker region (“pre-M1”) are important in the expression and function of neuromuscular acetylcholine receptors (AChRs). The perturbation of a salt bridge between pre-M1 residue R209 and loop 2 residue E45 has been proposed as being a principle event in the AChR gating conformational “wave.” We examined the effects of mutations to all five residues in pre-M1 (positions M207–P211) plus E45 in loop 2 in the mouse α 1 -subunit. M207, Q208, and P211 mutants caused small (approximately threefold) changes in the gating equilibrium constant (K eq ), but the changes for R209, L210, and E45 were larger. Of 19 different side chain substitutions at R209 on the wild-type background, only Q, K, and H generated functional channels, with the largest change in K eq (67-fold) from R209Q. Various R209 mutants were functional on different E45 backgrounds: H, Q, and K (E45A), H, A, N, and Q (E45R), and K, A, and N (E45L). Φ values for R209 (on the E45A background), L210, and E45 were 0.74, 0.35, and 0.80, respectively. Φ values for R209 on the wt and three other backgrounds could not be estimated because of scatter. The average coupling energy between 209/45 side chains (six different pairs) was only −0.33 kcal/mol (for both α subunits, combined). Pre-M1 residues are important for expression of functional channels and participate in gating, but the relatively modest changes in closed- vs. open-state energy caused mutations, the weak coupling energy between these residues and the functional activity of several unmatched-charge pairs are not consistent with the perturbation of a salt bridge between R209 and E45 playing the principle role in gating. Footnotes Abbreviations used in this paper: AChR, acetylcholine receptor; ECD, extracellular domain; REFER, rate-equilibrium free energy relationship; TMD, transmembrane domain. Submitted: 17 July 2007 Accepted: 8 November 2007 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of General Physiology Rockefeller University Press

Acetylcholine Receptor Gating at Extracellular Transmembrane Domain Interface: the “Pre-M1” Linker

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References (32)

Publisher
Rockefeller University Press
Copyright
Copyright © 2007, by The Rockefeller University Press
ISSN
0022-1295
eISSN
1540-7748
DOI
10.1085/jgp.200709857
pmid
18040058
Publisher site
See Article on Publisher Site

Abstract

Charged residues in the β10–M1 linker region (“pre-M1”) are important in the expression and function of neuromuscular acetylcholine receptors (AChRs). The perturbation of a salt bridge between pre-M1 residue R209 and loop 2 residue E45 has been proposed as being a principle event in the AChR gating conformational “wave.” We examined the effects of mutations to all five residues in pre-M1 (positions M207–P211) plus E45 in loop 2 in the mouse α 1 -subunit. M207, Q208, and P211 mutants caused small (approximately threefold) changes in the gating equilibrium constant (K eq ), but the changes for R209, L210, and E45 were larger. Of 19 different side chain substitutions at R209 on the wild-type background, only Q, K, and H generated functional channels, with the largest change in K eq (67-fold) from R209Q. Various R209 mutants were functional on different E45 backgrounds: H, Q, and K (E45A), H, A, N, and Q (E45R), and K, A, and N (E45L). Φ values for R209 (on the E45A background), L210, and E45 were 0.74, 0.35, and 0.80, respectively. Φ values for R209 on the wt and three other backgrounds could not be estimated because of scatter. The average coupling energy between 209/45 side chains (six different pairs) was only −0.33 kcal/mol (for both α subunits, combined). Pre-M1 residues are important for expression of functional channels and participate in gating, but the relatively modest changes in closed- vs. open-state energy caused mutations, the weak coupling energy between these residues and the functional activity of several unmatched-charge pairs are not consistent with the perturbation of a salt bridge between R209 and E45 playing the principle role in gating. Footnotes Abbreviations used in this paper: AChR, acetylcholine receptor; ECD, extracellular domain; REFER, rate-equilibrium free energy relationship; TMD, transmembrane domain. Submitted: 17 July 2007 Accepted: 8 November 2007

Journal

The Journal of General PhysiologyRockefeller University Press

Published: Dec 1, 2007

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