Initial Coupling of Binding to Gating Mediated by Conserved Residues in the Muscle Nicotinic Receptor

Abstract

We examined functional consequences of intrasubunit contacts in the nicotinic receptor α subunit using single channel kinetic analysis, site-directed mutagenesis, and structural modeling. At the periphery of the ACh binding site, our structural model shows that side chains of the conserved residues αK145, αD200, and αY190 converge to form putative electrostatic interactions. Structurally conservative mutations of each residue profoundly impair gating of the receptor channel, primarily by slowing the rate of channel opening. The combined mutations αD200N and αK145Q impair channel gating to the same extent as either single mutation, while αK145E counteracts the impaired gating due to αD200K, further suggesting electrostatic interaction between these residues. Interpreted in light of the crystal structure of acetylcholine binding protein (AChBP) with bound carbamylcholine (CCh), the results suggest in the absence of ACh, αK145 and αD200 form a salt bridge associated with the closed state of the channel. When ACh binds, αY190 moves toward the center of the binding cleft to stabilize the agonist, and its aromatic hydroxyl group approaches αK145, which in turn loosens its contact with αD200. The positional changes of αK145 and αD200 are proposed to initiate the cascade of perturbations that opens the receptor channel: the first perturbation is of β-strand 7, which harbors αK145 and is part of the signature Cys-loop, and the second is of β-strand 10, which harbors αD200 and connects to the M1 domain. Thus, interplay between these three conserved residues relays the initial conformational change from the ACh binding site toward the ion channel.