Gating Nanodomains of the Acetylcholine Receptor: Single-channel Transition State Analysis of the Transmembrane Segments

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 transmembrane segments indicate that the ¿M4 segment moves as single domain and in the middle of the gating reaction, with both ¿¿subunits moving synchronously. Between the subunits, the sequence of M4 motions is ¿¿¿¿¿. In the pore-lining M2 segments, I show that the two ¿-subunits experience distinct energy barriers to gating at the equator and that the subunit motions in this region are asymmetric with regard to their reaction progress in the AChR gating conformational 'wave'. Future experiments involving a more extensive REFER analysis will provide finer details of this ion channel nanomachine.