Linear free-energy relationships and the dynamics of gating in the acetylcholine receptor channel: A φ-value analysis of an allosteric transition at the single-molecule level

The muscle acetylcholine receptor channel (AChR) is a large (M_r ≅ 290K) transmembrane protein that mediates synaptic transmission. The activation of this ion channel can be understood in the framework of a thermodynamic cycle with spontaneous gating (i.e., the closed ⇋ open reaction) and ligand-binding events as the elementary steps. Because agonists bind more tightly to the open than to the closed state, gating of liganded receptors is more favorable than that of unliganded receptors. Accordingly, channel opening must involve two major conformational changes: the ACh-binding sites switch from a low-affinity to a high-affinity form, and the pore (located ∼ 45 Å away from the binding sites) switches from an ion-impermeable to an ion-permeable conformation. To gain insight into the reaction mechanism of fully-liganded gating, we characterized the corresponding transition state in the context of the 'linear free-energy relationships' of physical organic chemistry (Φ-value analysis). Gating of fully-liganded AChRs was studied by recording single-channel currents using the patch-clamp technique. Perturbations to the wild-type receptor were either series of different mutations at individual positions or series of different agonists. Based on the obtained 'snapshot' of the gating reaction at the transition state, and aware of the lack of information about the rest of the energy profile, the most parsimonious mechanism seems to be one where opening proceeds asynchronously, with the low-to-high affinity change at the binding sites preceding the complete opening of the distant pore.