Shen, Xin‐Ming

AbstractObjectiveTo 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.

We identify 2 homozygous mutations in the ε-subunit of the muscle acetylcholine receptor (AChR) in 3 patients with severe congenital myasthenia: εR218W in the pre-M1 region in 2 patients and εE184K in the β8-β9 linker in 1 patient. Arg218 is conserved in all eukaryotic members of the Cys-loop receptor superfamily, while Glu184 is conserved in the α-, δ-, and ε-subunits of AChRs from all species. εR218W reduces channel gating efficiency 338-fold and AChR expression on the cell surface 5-fold, whereas εE184K reduces channel gating efficiency 11-fold but does not alter AChR cell surface expression. Determinations of the effective channel gating rate constants, combined with mutant cycle analyses, demonstrate strong energetic coupling between εR218 and εE184, and between εR218 and εE45 from the β1-β2 linker, as also observed for equivalent residues in the principal coupling pathway of the α-subunit. Thus, efficient and rapid gating of the AChR channel is achieved not only by coupling between conserved residues within the principal coupling pathway of the α-subunit, but also between corresponding residues in the ε-subunit.

AbstractCongenital myasthenic syndromes (CMS) are heterogeneous disorders in which the safety margin of neuromuscular transmission is impaired by one or more specific mechanisms. Since the advent of next‐generation sequencing methods, the discovery of novel CMS targets and phenotypes has proceeded at an accelerated rate. Here, we review the current classification of CMS and describe our findings in five of these targets identified and investigated in our laboratory in the past 5 years. Defects in LRP4 hinder synaptic development and maintenance; the defects in PREPL are predicted to diminish filling of the synaptic vesicle with acetylcholine; and defects in SNAP25, Munc13‐1, and synaptotbrevin‐1 impede synaptic vesicle exocytosis.

AbstractObjectiveTo identify the molecular basis and elucidate the pathogenesis of a fatal congenital myasthenic syndrome.MethodsWe performed clinical electrophysiology studies, exome and Sanger sequencing, and analyzed functional consequences of the identified mutation.ResultsClinical electrophysiology studies of the patient revealed several‐fold potentiation of the evoked muscle action potential by high frequency nerve stimulation pointing to a presynaptic defect. Exome sequencing identified a homozygous c.340delA frameshift mutation in synaptobrevin 1 (SYB1), one of the three SNARE proteins essential for synaptic vesicle exocytosis. Analysis of both human spinal cord gray matter and normal human muscle revealed expression of the SYB1A and SYB1D isoforms, predicting expression of one or both isoforms in the motor nerve terminal. The identified mutation elongates the intravesicular C‐terminus of the A isoform from 5 to 71, and of the D isoform from 4 to 31 residues. Transfection of either mutant isoform into bovine chromaffin cells markedly reduces depolarization‐evoked exocytosis, and transfection of either mutant isoform into HEK cells significantly decreases expression of either mutant compared to wild type.InterpretationThe mutation is pathogenic because elongation of the intravesicular C‐terminus of the A and D isoforms increases the energy required to move their C‐terminus into the synaptic vesicle membrane, a key step for fusion of the synaptic vesicle with the presynaptic membrane, and because it is predicted to reduce expression of either isoform in the nerve terminal.