title: Investigation of Congenital Myasthenia Reveals Functional Asymmetry of Invariant Acetylcholine Receptor (AChR) Cys-loop Aspartates.
abstract: We identify two heteroallelic mutations in the acetylcholine receptor δ-subunit from a patient with severe myasthenic symptoms since birth: a novel δD140N mutation in the signature Cys-loop and a mutation in intron 7 of the δ-subunit gene that disrupts splicing of exon 8. The mutated Asp residue, which determines the disease phenotype, is conserved in all eukaryotic members of the Cys-loop receptor superfamily. Studies of the mutant acetylcholine receptor expressed in HEK 293 cells reveal that δD140N attenuates cell surface expression and apparent channel gating, predicting a reduced magnitude and an accelerated decay of the synaptic response, thus reducing the safety margin for neuromuscular transmission. Substituting Asn for Asp at equivalent positions in the α-, β-, and ϵ-subunits also suppresses apparent channel gating, but the suppression is much greater in the α-subunit. Mutant cycle analysis applied to single and pairwise mutations reveals that αAsp-138 is energetically coupled to αArg-209 in the neighboring pre-M1 domain. Our findings suggest that the conserved αAsp-138 and αArg-209 contribute to a principal pathway that functionally links the ligand binding and pore domains.
authors.affiliation: From the Department of Neurology and Neuromuscular Research Laboratory, Mayo Clinic, Rochester, Minnesota 55905, shen.xinming@mayo.edu.
authors.forename: Xin-Ming
authors.initials: XM
authors.lastname: Shen
authors.affiliation: From the Department of Neurology and Neuromuscular Research Laboratory, Mayo Clinic, Rochester, Minnesota 55905.
authors.forename: Joan
authors.initials: J
authors.lastname: Brengman
authors.affiliation: the Department of Pediatric Neurology, University Children's Hospital, Ljubljana 1525, Slovenia, and.
authors.forename: David
authors.initials: D
authors.lastname: Neubauer
authors.affiliation: From the Department of Neurology and Neuromuscular Research Laboratory, Mayo Clinic, Rochester, Minnesota 55905, the Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota 55905.
authors.forename: Steven M
authors.initials: SM
authors.lastname: Sine
authors.affiliation: From the Department of Neurology and Neuromuscular Research Laboratory, Mayo Clinic, Rochester, Minnesota 55905.
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Engel
mesh_terms: D000109:Acetylcholine / Q000737:chemistry / Q000378:metabolism*; D019943:Amino Acid Substitution; D002038:Bungarotoxins / Q000494:pharmacology; D002648:Child; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D007438:Introns; D008024:Ligands; D008958:Models, Molecular; D018908:Muscle Weakness / Q000209:etiology; D009154:Mutation; D020294:Myasthenic Syndromes, Congenital / Q000235:genetics* / Q000378:metabolism / Q000503:physiopathology; D018722:Nicotinic Agonists / Q000494:pharmacology; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D012326:RNA Splicing; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012720:Severity of Illness Index
chemical_list: D002038:Bungarotoxins; D008024:Ligands; D018722:Nicotinic Agonists; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins; D000109:Acetylcholine
keywords: Cys-loop receptor superfamily; congenital myasthenic syndrome; invariant Cys-loop aspartate; invariant pre-M1 arginine; ion channel; mutant cycle analysis; nicotinic acetylcholine receptors (nAChR); patch clamp; receptor structure-function; structure-function