αM2M3–TBS coupling. (A) Examples of MCA. Single-channel currents are C↔O clusters (see Fig. 3 B); open is down. Arrows, gating free energy change caused by the mutation or mutations. αS268 is in αM2M3, and αW149/αY93 are at the TBS; αY93 shows no interaction (pair = sum), but αW149 is coupled significantly (sum > pair by 0.9 kcal/mol). (B) Map of αM2M3–TBS coupling. Connecting lines mark interacting amino acids: thick-dashed, strong (>1.2 kcal/mol); and thin-solid, significant (0.6–1.2 kcal/mol). TBS structure is AChBP (PDB accession code 3WIP), and M2M3 structure is α4β2 AChR (PDB accession code 5KXI); spheres are αC (TBS: green, aromatic; pink, proline; blue, glycine). (C) MCA coupling values. Dashed lines are at ±0.6 kcal/mol. Main, TBS aromatics; inset, TBS prolines and glycines. TBS mutations were to Ala except where indicated; the M2M3 mutations are shown except for *, Pro and **, Gly. All pairs are adult-type AChRs except for αY190F and αY198A (f, for fetal; 1 and 2 indicate modes; see Fig. 3 B).