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 nicotini c acetylcholine receptor (nAChR) gating was developed. Specific backbone pe ptide bonds were replaced with an ester, which disrupts backbone hydrogen b onds at the site of mutation. At a conserved proline residue (alpha Pro221) in the first transmembrane (M1) domain, the amide-to-ester mutation provid es 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 alpha M2 domain, the amide-to-ester mutation yi elded functional receptors at 15 positions, 3 of which provided receptors w ith >10-fold lower EC50 than wild type. These results support a model for g ating that includes significant changes of backbone conformation within the M2 domain.