Backbone mutations in transmembrane domains of a ligand-gated ion channel: implications for the mechanism of gating.

An approach to identify backbone conformational changes underlying nicotinic acetylcholine receptor (nAChR) gating was developed. Specific backbone peptide bonds were replaced with an ester, which disrupts backbone hydrogen bonds at the site of mutation. At a conserved proline residue (alphaPro221) in the first transmembrane (M1) domain, the amide-to-ester mutation provides receptors with near-normal sensitivity, although the natural amino acids tested other than Pro produce receptors that gate with a much larger EC50 than normal. Therefore, a backbone hydrogen bond at this site may interfere with normal gating. In the alphaM2 domain, the amide-to-ester mutation yielded functional receptors at 15 positions, 3 of which provided receptors with >10-fold lower EC50 than wild type. These results support a model for gating that includes significant changes of backbone conformation within the M2 domain.