Abstract:ObjectiveTo 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 f… Show more
“…In contrast, the C-terminal half is splayed from the TMD of the cognate SNARE protein syntaxin 49 and cholesterol, known to promote fusion pore opening 50 , has a further effect on the tilt 25 . Addition of charged residues at the C-terminal end restricts VAMP2 TMD mobility and reduces or delays fusion pore opening 24, 51, 52 which was interpreted as a consequence of reduced vertical “pull” of the TMD into the membrane 53 . However, such a C-terminal modification will also restrict lateral mobility of the transmembrane domain and potentially prevents the tilt of angle accompanying the transition from an α helix to a β sheet conformation 21 .Figure 8Proposed model of conformation of VAMP2 transmembrane domain during exocytosis.…”
Exocytosis depends on cytosolic domains of SNARE proteins but the function of the transmembrane domains (TMDs) in membrane fusion remains controversial. The TMD of the SNARE protein synaptobrevin2/VAMP2 contains two highly conserved small amino acids, G100 and C103, in its central portion. Substituting G100 and/or C103 with the β-branched amino acid valine impairs the structural flexibility of the TMD in terms of α-helix/β-sheet transitions in model membranes (measured by infrared reflection-absorption or evanescent wave spectroscopy) during increase in protein/lipid ratios, a parameter expected to be altered by recruitment of SNAREs at fusion sites. This structural change is accompanied by reduced membrane fluidity (measured by infrared ellipsometry). The G100V/C103V mutation nearly abolishes depolarization-evoked exocytosis (measured by membrane capacitance) and hormone secretion (measured biochemically). Single-vesicle optical (by TIRF microscopy) and biophysical measurements of ATP release indicate that G100V/C103V retards initial fusion-pore opening, hinders its expansion and leads to premature closure in most instances. We conclude that the TMD of VAMP2 plays a critical role in membrane fusion and that the structural mobility provided by the central small amino acids is crucial for exocytosis by influencing the molecular re-arrangements of the lipid membrane that are necessary for fusion pore opening and expansion.
“…In contrast, the C-terminal half is splayed from the TMD of the cognate SNARE protein syntaxin 49 and cholesterol, known to promote fusion pore opening 50 , has a further effect on the tilt 25 . Addition of charged residues at the C-terminal end restricts VAMP2 TMD mobility and reduces or delays fusion pore opening 24, 51, 52 which was interpreted as a consequence of reduced vertical “pull” of the TMD into the membrane 53 . However, such a C-terminal modification will also restrict lateral mobility of the transmembrane domain and potentially prevents the tilt of angle accompanying the transition from an α helix to a β sheet conformation 21 .Figure 8Proposed model of conformation of VAMP2 transmembrane domain during exocytosis.…”
Exocytosis depends on cytosolic domains of SNARE proteins but the function of the transmembrane domains (TMDs) in membrane fusion remains controversial. The TMD of the SNARE protein synaptobrevin2/VAMP2 contains two highly conserved small amino acids, G100 and C103, in its central portion. Substituting G100 and/or C103 with the β-branched amino acid valine impairs the structural flexibility of the TMD in terms of α-helix/β-sheet transitions in model membranes (measured by infrared reflection-absorption or evanescent wave spectroscopy) during increase in protein/lipid ratios, a parameter expected to be altered by recruitment of SNAREs at fusion sites. This structural change is accompanied by reduced membrane fluidity (measured by infrared ellipsometry). The G100V/C103V mutation nearly abolishes depolarization-evoked exocytosis (measured by membrane capacitance) and hormone secretion (measured biochemically). Single-vesicle optical (by TIRF microscopy) and biophysical measurements of ATP release indicate that G100V/C103V retards initial fusion-pore opening, hinders its expansion and leads to premature closure in most instances. We conclude that the TMD of VAMP2 plays a critical role in membrane fusion and that the structural mobility provided by the central small amino acids is crucial for exocytosis by influencing the molecular re-arrangements of the lipid membrane that are necessary for fusion pore opening and expansion.
“…Synaptobrevin-1 CMS was investigated in collaboration with Scola and her coworkers. 28 A Brazilian girl born to first-cousin unaffected parents had severe myasthenic symptoms since birth, but her eye movements were only mildly restricted. At 6 months of age, she lacked head control and could not sit without support.…”
Congenital 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.
“…For instance, mutations in DPAGT1 may cause a severe CDG with impaired cognitive and motor development or they may cause CMS; overexpression of DPAGT1 is thought to have a role in oral cancers, and DPAGT1 is the target of several antibiotics . Similarly, it is clear that mutations in GMPPB can underlie several different phenotypic pictures, and, for many mutations uncovered in presynaptic proteins, myasthenic symptoms are only one component of a much more extensive and severe phenotype . Whereas treatment of the myasthenic symptoms is often remarkably effective, medication for these multicomponent disorders will require the development of new therapeutic strategies.…”
The congenital myasthenic syndromes (CMS) are hereditary disorders of neuromuscular transmission. The number of cases recognized, at around 1:100,000 in the United Kingdom, is increasing with improved diagnosis. The advent of next-generation sequencing has facilitated the discovery of many genes that harbor CMS-associated mutations. An emerging group of CMS, characterized by a limb-girdle pattern of muscle weakness, is caused by mutations in genes that encode proteins involved in the initial steps of the N-linked glycosylation pathway, which is surprising, since this pathway is found in all mammalian cells. However, mutations in these genes may also give rise to multisystem disorders (congenital disorders of glycosylation) or muscle disorders where the myasthenic symptoms constitute only one component within a wider phenotypic spectrum. We also report a CMS due to mutations in COL13A1, which encodes an extracellular matrix protein that is concentrated at the neuromuscular junction and highlights a role for these extracellular matrix proteins in maintaining synaptic stability that is independent of the AGRN/MuSK clustering pathway. Knowledge about the neuromuscular synapse and the different proteins involved in maintaining its structure as well as function enables us to tailor treatments to the underlying pathogenic mechanisms.
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