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Vesicular acetylcholine transporter defect underlies devastating congenital myasthenia syndrome.

ABSTRACT: To identify the genetic basis of a recessive congenital neurologic syndrome characterized by severe hypotonia, arthrogryposis, and respiratory failure.Identification of the responsible gene by exome sequencing and assessment of the effect of the mutation on protein stability in transfected rat neuronal-like PC12A123.7 cells.Two brothers from a nonconsanguineous Yemeni Jewish family manifested at birth with severe hypotonia and arthrogryposis. The older brother died of respiratory failure at 5 days of age. The proband, now 4.5 years old, has been mechanically ventilated since birth with virtually no milestones achievement. Whole exome sequencing revealed homozygosity of SLC18A3 c.1078G>C, p.Gly360Arg in the affected brothers but not in other family members. SLC18A3 p.Gly360Arg is not reported in world populations but is present at a carrier frequency of 1:30 in healthy Yemeni Jews. SLC18A3 encodes the vesicular acetylcholine transporter (VAChT), which loads newly synthesized acetylcholine from the neuronal cytoplasm into synaptic vesicles. Mice that are VAChT-null have been shown to die at birth of respiratory failure. In human VAChT, residue 360 is located in a conserved region and substitution of arginine for glycine is predicted to disrupt proper protein folding and membrane embedding. Stable transfection of wild-type and mutant human VAChT into neuronal-like PC12A123.7 cells revealed similar mRNA levels, but undetectable levels of the mutant protein, suggesting post-translational degradation of mutant VAChT.Loss of function of VAChT underlies severe arthrogryposis and respiratory failure. While most congenital myasthenic syndromes are caused by defects in postsynaptic proteins, VAChT deficiency is a presynaptic myasthenic syndrome.

SUBMITTER: Aran A

PROVIDER: S-EPMC5384838 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Vesicular acetylcholine transporter defect underlies devastating congenital myasthenia syndrome.

Aran Adi A Segel Reeval R Kaneshige Kota K Gulsuner Suleyman S Renbaum Paul P Oliphant Scott S Meirson Tomer T Weinberg-Shukron Ariella A Hershkovitz Yair Y Zeligson Sharon S Lee Ming K MK Samson Abraham O AO Parsons Stanley M SM King Mary-Claire MC Levy-Lahad Ephrat E Walsh Tom T

Neurology 20170210 11

<h4>Objective</h4>To identify the genetic basis of a recessive congenital neurologic syndrome characterized by severe hypotonia, arthrogryposis, and respiratory failure.<h4>Methods</h4>Identification of the responsible gene by exome sequencing and assessment of the effect of the mutation on protein stability in transfected rat neuronal-like PC12<sup>A123.7</sup> cells.<h4>Results</h4>Two brothers from a nonconsanguineous Yemeni Jewish family manifested at birth with severe hypotonia and arthrogr ...[more]

PMID: 28188302

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Project description:Invariant E309 is in contact with critical and invariant D398 in a three-dimensional homology model of vesicular acetylcholine transporter (VAChT, TC 2.A.1.2.13) [Vardy, E., et al. (2004) Protein Sci. 13, 1832-1840]. In the work reported here, E309 and D398 in human VAChT were mutated singly and together to test their functions, assign pK values to them, and determine whether the residues are close to each other in three-dimensional space. Mutants were stably expressed in the PC12(A123.7) cell line, and transport and binding properties were characterized at different pH values using radiolabeled ligands and filtration assays. Contrary to a prior conclusion, the results demonstrate that most D398 mutants do not bind the allosteric inhibitor vesamicol even weakly. Earlier work showed that most D398 mutants do not transport ACh. D398 therefore probably is the residue that must deprotonate with a pK of 6.5 for binding of vesamicol and with a pK of approximately 5.9 for transport of ACh. Because E309Q has no effect on VAChT functions at physiological pH, E309 has no apparent critical role. However, radical mutations in E309 cause decreases in ACh and vesamicol affinities and total loss of ACh transport. Unlike wild-type VAChT, which exhibits a peak of [(3)H]vesamicol binding centered at pH 7.4, mutants E309Q, E309D, E309A, and E309K all exhibit peaks of binding centered at pH >or=9. The combination of high pH and mutated E309 apparently produces a relaxed (in contrast to tense) conformation of VAChT that binds vesamicol exceptionally tightly. No compensatory interactions between E309 and D398 in double mutants were discovered. Proof of a close spatial relationship between E309 and D398 was not found. Nevertheless, the data are more consistent with the homology model than an alternative hydropathy model of VAChT that likely locates E309 far from D398 and the ACh binding site in three-dimensional space. Also, a probable network of interactions involving E309 and an unknown residue having a pK of 10 has been revealed.

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Vesicular acetylcholine transporter defect underlies devastating congenital myasthenia syndrome.

Publications

Vesicular acetylcholine transporter defect underlies devastating congenital myasthenia syndrome.

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