Initial coupling of binding to gating mediated by conserved residues in the muscle nicotinic receptor

We examined functional consequences of intrasubunit contacts in the nicotinic receptor alpha 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 alpha K145, alpha D200, and alpha Y190 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 alpha D200N and alpha K145Q impair channel gating to the same extent as either single mutation, while alpha K145E counteracts the impaired gating due to alpha D200K, 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, alpha K145 and alpha D200 form a salt bridge associated with the closed state of the channel. When ACh binds, alpha Y190 moves toward the center of the binding cleft to stabilize the agonist, and its aromatic hydroxyl group approaches alpha K145, which in turn loosens its contact with alpha D200. The positional changes of alpha K145 and alpha D200 are proposed to initiate the cascade of perturbations that opens the receptor channel: the first perturbation is of beta-strand 7, which harbors alpha K145 and is part of the signature Cys-loop, and the second is of beta-strand 10, which harbors alpha D200 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.