Abstract
ObjectiveTo characterize the molecular and phenotypic basis of a severe slow‐channel congenital myasthenic syndrome (SCCMS).MethodsIntracellular and single‐channel recordings from patient endplates; alpha‐bungarotoxin binding studies; direct sequencing of AChR genes; microsatellite analysis; kinetic analysis of AChR activation; homology modeling of adult human AChR structure.ResultsAmong 24 variants reported to cause SCCMS only two appear in the AChR δ‐subunit. We here report a 16‐year‐old patient harboring a novel δL273F mutation (δL294F in HGVS nomenclature) in the second transmembrane domain (M2) of the AChR δ subunit. Kinetic analyses with ACh and the weak agonist choline indicate that δL273F prolongs the channel opening bursts 9.4‐fold due to a 75‐fold increase in channel gating efficiency, whereas a previously identified εL269F mutation (εL289F in HGVS nomenclature) at an equivalent location in the AChR ε‐subunit prolongs channel opening bursts 4.4‐fold due to a 30‐fold increase in gating efficiency. Structural modeling of AChR predicts that inter‐helical hydrophobic interactions between the mutant residue in the δ and ε subunit and nearby M2 domain residues in neighboring α subunits contribute to structural stability of the open relative to the closed channel states.InterpretationThe greater increase in gating efficiency by δL273F than by εL269F explains why δL273F has more severe clinical effects. Both δL273F and εL269F impair channel gating by disrupting hydrophobic interactions with neighboring α‐subunits. Differences in the extent of impairment of channel gating in δ and ε mutant receptors suggest unequal contributions of ε/α and δ/α subunit pairs to gating efficiency.
Highlights
The congenital myasthenic syndromes (CMS) are heterogeneous disorders associated with fatigable muscle weakness due to a compromised safety margin of neuromuscular transmission
The clinical, electrophysiologic, ultrastructural, and molecular genetics data allowed us to trace the cause of a severe slow-channel congenital myasthenic syndrome (SCCMS) to a novel dominant missense mutation, dL237F, in the M2 of the acetylcholine receptor (AChR) d subunit
To the best of our knowledge, this is the third report of SCCMS caused by a d subunit mutation at a novel site.[7,8,9]
Summary
The congenital myasthenic syndromes (CMS) are heterogeneous disorders associated with fatigable muscle weakness due to a compromised safety margin of neuromuscular transmission. Prolonged EPPs allow excessive calcium influx into the postsynaptic region, which initiates focal degeneration of the junctional folds, loss of AChR and apoptosis of subjunctional nuclei, collectively referred to as an endplate myopathy.[5] AChRs from SCCMS patients are prone to desensitization, which reduces the number of AChRs available for activation.[6] To date, 24 slow-channel mutations have been reported in different domains of AChR subunits,[7] but only two mutations involving the same residue were identified in the d subunit.[8,9] Most slow-channel mutations appear in transmembrane domains of AChR subunits, but detailed analysis of their kinetic consequences has been hindered by the inability of the recording instruments to capture the fastest gating steps with ACh as agonist. We compare the kinetic consequences of dL273F to those of a previously reported eL269F mutation[12] located at a position equivalent to that of dL273F
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