Binding to gating transduction in nicotinic receptors: Cys-loop energetically couples to pre-M1 and M2-M3 regions

The nicotinic acetylcholine receptor (AChR) transduces binding of nerve-released ACh into opening of an intrinsic ion channel, yet the intraprotein interactions behind transduction remain to be fully elucidated. Attention has focused on the region of the AChR in which the beta1-beta2 and Cys-loops from the extracellular domain project into a cavity framed by residues preceding the first transmembrane domain (pre-M1) and the linker spanning transmembrane domains M2 and M3. Previous studies identified a principal transduction pathway in which the pre-M1 domain is coupled to the M2-M3 linker through the beta1-beta2 loop. Here we identify a parallel pathway in which the pre-M1 domain is coupled to the M2-M3 linker through the Cys-loop. Mutagenesis, single-channel kinetic analyses and thermodynamic mutant cycle analyses reveal energetic coupling among alphaLeu 210 from the pre-M1 domain, alphaPhe 135 and alphaPhe 137 from the Cys-loop, and alphaLeu 273 from the M2-M3 linker. Residues at equivalent positions of non-alpha-subunits show negligible coupling, indicating these interresidue couplings are specific to residues in the alpha-subunit. Thus, the extracellular beta1-beta2 and Cys-loops bridge the pre-M1 domain and M2-M3 linker to transduce agonist binding into channel gating.