Ligand-gated ion channels contain a conserved leucine at position 9' (L9') in the M2 transmembrane domain. We used multiple substitutions at this position in the gamma subunit of the mouse acetylcholine receptor (AChR) (gammaL9') to examine the role of residue polarity at this position in the gating process at both the macroscopic and single-channel levels. The midpoint of the macroscopic dose-response relationship (EC(50)) and the channel closing rate constant, alpha, decreased as the polarity of the residue at that position increased, suggesting a stabilization of the open state of the channel. Both parameters showed similar dependencies on the polarity of the substituted residue. These data support the notion that during AChR gating, the amino acid at the 9' position moves into a polar environment, and that interactions between this residue and the polar environment determine the stability of the open state. Since this residue is conserved in all other members of the ligand-gated ion channel family, we suggest that a similar mechanism applies to the other members of the family.