title: Mutation causing severe myasthenia reveals functional asymmetry of AChR signature cystine loops in agonist binding and gating.
abstract: We describe a highly disabling congenital myasthenic syndrome (CMS) associated with rapidly decaying, low-amplitude synaptic currents, and trace its cause to a valine to leucine mutation in the signature cystine loop (cys-loop) of the AChR alpha subunit. The recently solved crystal structure of an ACh-binding protein places the cys-loop at the junction between the extracellular ligand-binding and transmembrane domains where it may couple agonist binding to channel gating. We therefore analyzed the kinetics of ACh-induced single-channel currents to identify elementary steps in the receptor activation mechanism altered by the alphaV132L mutation. The analysis reveals that alphaV132L markedly impairs ACh binding to receptors in the resting closed state, decreasing binding affinity for the second binding step 30-fold, but attenuates gating efficiency only about twofold. By contrast, mutation of the equivalent valine residue in the delta subunit impairs channel gating approximately fourfold with little effect on ACh binding, while corresponding mutations in the beta and epsilon subunits are without effect. The unique functional contribution of the alpha subunit cys-loop likely owes to its direct connection via a beta strand to alphaW149 at the center of the ligand-binding domain. The overall findings reveal functional asymmetry between cys-loops of the different AChR subunits in contributing to ACh binding and channel gating.
authors.affiliation: Department of Neurology and Neuromuscular Research Laboratory, Mayo Clinic, Rochester, Minnesota 55905, USA.
authors.forename: Xin-Ming
authors.initials: XM
authors.lastname: Shen
authors.forename: Kinji
authors.initials: K
authors.lastname: Ohno
authors.forename: Akira
authors.initials: A
authors.lastname: Tsujino
authors.forename: Joan M
authors.initials: JM
authors.lastname: Brengman
authors.forename: Monique
authors.initials: M
authors.lastname: Gingold
authors.forename: Steven M
authors.initials: SM
authors.lastname: Sine
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Engel
mesh_terms: D000109:Acetylcholine / Q000378:metabolism; D000595:Amino Acid Sequence; D016022:Case-Control Studies; D002460:Cell Line; D002675:Child, Preschool; D003545:Cysteine / Q000737:chemistry; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D015640:Ion Channel Gating; D007700:Kinetics; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020294:Myasthenic Syndromes, Congenital / Q000235:genetics* / Q000378:metabolism*; D010641:Phenotype; D017354:Point Mutation; D021122:Protein Subunits; D011950:Receptors, Cholinergic / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D021122:Protein Subunits; D011950:Receptors, Cholinergic; D011994:Recombinant Proteins; D003545:Cysteine; D000109:Acetylcholine