The nicotinic acetylcholine receptor (AChR) is a pentameric ligand-gated ion channel that can switch between ion-impermeable (closed) and ion-permeable (open) conformations. The transition state structures that link the closed and open states are largely unresolved. In this study, I used single-molecule kinetic analysis and rate-equilibrium linear free energy relationships (REFERs) to obtain information about the transition state of the gating reaction and elucidate the sequence of molecular events between the closed and open conformational states. REFER analyses of 87 mutants in the M4...