Acetylcholine Receptor Gating: Movement in the α-Subunit Extracellular Domain

Abstract

Acetylcholine receptor channel gating is a brownian conformational cascade in which nanometer-sized domains (“Φ blocks”) move in staggering sequence to link an affinity change at the transmitter binding sites with a conductance change in the pore. In the α-subunit, the first Φ-block to move during channel opening is comprised of residues near the transmitter binding site and the second is comprised of residues near the base of the extracellular domain. We used the rate constants estimated from single-channel currents to infer the gating dynamics of Y127 and K145, in the inner and outer sheet of the β-core of the α-subunit. Y127 is at the boundary between the first and second Φ blocks, at a subunit interface. αY127 mutations cause large changes in the gating equilibrium constant and with a characteristic Φ-value (Φ = 0.77) that places this residue in the second Φ-block. We also examined the effect on gating of mutations in neighboring residues δI43 (Φ = 0.86), εN39 (complex kinetics), αI49 (no effect) and in residues that are homologous to αY127 on the ε, β, and δ subunits (no effect). The extent to which αY127 gating motions are coupled to its neighbors was estimated by measuring the kinetic and equilibrium constants of constructs having mutations in αY127 (in both α subunits) plus residues αD97 or δI43. The magnitude of the coupling between αD97 and αY127 depended on the αY127 side chain and was small for both H (0.53 kcal/mol) and C (−0.37 kcal/mol) substitutions. The coupling across the single α–δ subunit boundary was larger (0.84 kcal/mol). The Φ-value for K145 (0.96) indicates that its gating motion is correlated temporally with the motions of residues in the first Φ-block and is not synchronous with those of αY127. This suggests that the inner and outer sheets of the α-subunit β-core do not rotate as a rigid body.