Acetylcholine receptor (AChR) channels with proline (P) mutations in the putative pore-forming domain (at the 12′ position of the M2 segment) were examined at the single-channel level. For all subunits (α, β, ϵ, and δ), a 12′P mutation increased the open channel lifetime >5-fold. To facilitate the estimation of binding and gating rate constants, subunits with 12′P mutations were co-expressed with α subunits having a binding site mutation that slows channel opening (αD200N). In these AChRs, a 12′P mutation in ϵ or β slowed the closing rate constant ∼6-fold but had no effect on either the channel opening rate constant or the equilibrium dissociation constant for ACh (Kd). In contrast, a 12′P mutation in δ slowed the channel closing rate constant only ∼2-fold and significantly increased both the channel opening rate constant and the Kd. Pairwise expression of 12′P subunits indicates that mutations in ϵ and β act nearly independently, but one in δ reduces the effect of a homologous mutation in ϵ or β. The results suggest that a 12′P mutation in ϵ and β has mainly local effects, whereas one in δ has both local and distributed effects that influence both agonist binding and channel gating.
