Nicotinic receptor interloop proline anchors β1-β2 and Cys loops in coupling agonist binding to channel gating

Nicotinic acetylcholine receptors (AChRs) mediate rapid excitatory synaptic transmission throughout the peripheral and central nervous systems. They transduce binding of nerve-released ACh into opening of an intrinsic channel, yet the structural basis underlying transduction is not fully understood. Previous studies revealed a principal transduction pathway in which alpha Arg 209 of the pre-M1 domain and alpha Glu 45 of the beta 1-beta 2 loop functionally link the two regions, positioning alpha Val 46 of the beta 1-beta 2 loop in a cavity formed by alpha Pro 272 through alpha Ser 269 of the M2-M3 loop. Here we investigate contributions of residues within and proximal to this pathway using single-channel kinetic analysis, site-directed mutagenesis, and thermodynamic mutant cycle analysis. We find that in contributing to channel gating, alpha Val 46 and alpha Val 132 of the signature Cys loop couple energetically to alpha Pro 272. Furthermore, these residues are optimized in both their size and hydrophobicity to mediate rapid and efficient channel gating, suggesting naturally occurring substitutions at these positions enable a diverse range of gating rate constants among the Cys-loop receptor superfamily. The overall results indicate that alpha Pro 272 functionally couples to flanking Val residues extending from the beta 1-beta 2 and Cys loops within the ACh binding to channel opening transduction pathway.