title: 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 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 alphaK145, alphaD200, and alphaY190 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 alphaD200N and alphaK145Q impair channel gating to the same extent as either single mutation, while alphaK145E counteracts the impaired gating due to alphaD200K, 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, alphaK145 and alphaD200 form a salt bridge associated with the closed state of the channel. When ACh binds, alphaY190 moves toward the center of the binding cleft to stabilize the agonist, and its aromatic hydroxyl group approaches alphaK145, which in turn loosens its contact with alphaD200. The positional changes of alphaK145 and alphaD200 are proposed to initiate the cascade of perturbations that opens the receptor channel: the first perturbation is of beta-strand 7, which harbors alphaK145 and is part of the signature Cys-loop, and the second is of beta-strand 10, which harbors alphaD200 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.
authors.affiliation: Receptor Biology Laboratory, Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
authors.forename: Nuriya
authors.initials: N
authors.lastname: Mukhtasimova
authors.forename: Chris
authors.initials: C
authors.lastname: Free
authors.forename: Steven M
authors.initials: SM
authors.lastname: Sine
mesh_terms: D000109:Acetylcholine / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000378:metabolism; D001665:Binding Sites; D002460:Cell Line; D017124:Conserved Sequence; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D007668:Kidney / Q000378:metabolism*; D008564:Membrane Potentials / Q000502:physiology*; D018482:Muscle, Skeletal / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D000596:Amino Acids; D011978:Receptors, Nicotinic; D000109:Acetylcholine