title: Invariant aspartic Acid in muscle nicotinic receptor contributes selectively to the kinetics of agonist binding.
abstract: We examined functional contributions of interdomain contacts within the nicotinic receptor ligand binding site using single channel kinetic analyses, site-directed mutagenesis, and a homology model of the major extracellular region. At the principal face of the binding site, the invariant alphaD89 forms a highly conserved interdomain contact near alphaT148, alphaW149, and alphaT150. Patch-clamp recordings show that the mutation alphaD89N markedly slows acetylcholine (ACh) binding to receptors in the resting closed state, but does not affect rates of channel opening and closing. Neither alphaT148L, alphaT150A, nor mutations at both positions substantially affects the kinetics of receptor activation, showing that hydroxyl side chains at these positions are not hydrogen bond donors for the strong acceptor alphaD89. However substituting a negative charge at alphaT148, but not at alphaT150, counteracts the effect of alphaD89N, demonstrating that a negative charge in the region of interdomain contact confers rapid association of ACh. Interpreted within the structural framework of ACh binding protein and a homology model of the receptor ligand binding site, these results implicate main chain amide groups in the domain harboring alphaW149 as principal hydrogen bond donors for alphaD89. The specific effect of alphaD89N on ACh association suggests that interdomain hydrogen bonding positions alphaW149 for optimal interaction with ACh.
authors.affiliation: Department of Physiology and Biophysics, Mayo Clinic College of Medicine, 200 First St., SW, MSB 1-35, Rochester, MN 55905, USA.
authors.forename: Won Yong
authors.initials: WY
authors.lastname: Lee
authors.forename: Steven M
authors.initials: SM
authors.lastname: Sine
mesh_terms: D000109:Acetylcholine / Q000378:metabolism*; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002460:Cell Line; D006801:Humans; D007668:Kidney / Q000502:physiology*; D007700:Kinetics; D008024:Ligands; D008564:Membrane Potentials / Q000502:physiology*; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012680:Sensitivity and Specificity; D013329:Structure-Activity Relationship
chemical_list: D008024:Ligands; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins; D001224:Aspartic Acid; D000109:Acetylcholine