1. Acetylcholine receptor gating at extracellular transmembrane domain interface: the 'Pre-M1' linker
- Author
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Purohit, Prasad and Auerbach, Anthony
- Subjects
Gating system (Founding) -- Methods ,Gating system (Founding) -- Analysis ,Mutation (Biology) -- Risk factors ,Mutation (Biology) -- Analysis ,Acetylcholine -- Receptors ,Acetylcholine -- Health aspects ,Acetylcholine -- Analysis ,Biological sciences ,Health - Abstract
Charged residues in the [beta]10-M1 linker region ('pre-M1') are important in the expression and traction 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 [[alpha].sub.1]-subunit. M207, Q208, and P211 mutants caused small (approximately threefold) changes in the gating equilibrium constant ([K.sub.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.sub.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). [PHI] values for R209 (on the E45A background), L210, and E45 were 0.74, 0.35, and 0.80, respectively, [PHI] 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 [alpha] 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.
- Published
- 2007