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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 ?-subunit from a patient with severe myasthenic symptoms since birth: a novel ?D140N mutation in the signature Cys-loop and a mutation in intron 7 of the ?-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 ?D140N 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 ?-, ?-, and ?-subunits also suppresses apparent channel gating, but the suppression is much greater in the ?-subunit. Mutant cycle analysis applied to single and pairwise mutations reveals that ?Asp-138 is energetically coupled to ?Arg-209 in the neighboring pre-M1 domain. Our findings suggest that the conserved ?Asp-138 and ?Arg-209 contribute to a principal pathway that functionally links the ligand binding and pore domains.

SUBMITTER: Shen XM

PROVIDER: S-EPMC4751375 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Investigation of Congenital Myasthenia Reveals Functional Asymmetry of Invariant Acetylcholine Receptor (AChR) Cys-loop Aspartates.

Shen Xin-Ming XM Brengman Joan J Neubauer David D Sine Steven M SM Engel Andrew G AG

The Journal of biological chemistry 20151223 7

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 δ ...[more]

PMID: 26698174

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Project description:Neuromuscular transmission failure in myasthenia gravis (MG) is most commonly elicited by autoantibodies (ab) to the acetylcholine receptor or the muscle-specific kinase, constituting AChR-MG and MuSK-MG. It is controversial whether these MG subtypes arise through different T helper (Th) 1, Th2 or Th17 polarized immune reactions and how these reactions are blunted by immunosuppression. To address these questions, plasma levels of cytokines related to various Th subtypes were determined in patients with AChR-MG, MuSK-MG and healthy controls (CON). Peripheral blood mononuclear cells (PBMC) were activated in vitro by anti-CD3, and cytokines were quantified in supernatants. In purified blood CD4+ T cells, RNA of various cytokines, Th subtype specific transcription factors and the co-stimulatory molecule, CD40L, were quantified by qRT-PCR. Plasma levels of Th1, Th2 and Th17 related cytokines were overall not significantly different between MG subtypes and CON. By contrast, in vitro stimulated PBMC from MuSK-MG but not AChR-MG patients showed significantly increased secretion of the Th1, Th17 and T follicular helper cell related cytokines, IFN-γ, IL-17A and IL-21. Stimulated expression of IL-4, IL-6, IL-10 and IL-13 was not significantly different. At the RNA level, expression of CD40L by CD4+ T cells was reduced in both AChR-MG and MuSK-MG patients while expression of Th subset related cytokines and transcription factors were normal. Immunosuppression treatment had two effects: First, it reduced levels of IL12p40 in the plasma of AChR-MG and MuSK-MG patients, leaving other cytokine levels unchanged; second, it reduced spontaneous secretion of IFN-γ and increased secretion of IL-6 and IL-10 by cultured PBMC from AChR-MG, but not MuSK-MG patients. We conclude that Th1 and Th17 immune reactions play a role in MuSK-MG. Immunosuppression attenuates the Th1 response in AChR-MG and MuSK-MG, but otherwise modulates immune responses in AChR-MG and MuSK-MG patients differentially.

Dataset Information

Investigation of Congenital Myasthenia Reveals Functional Asymmetry of Invariant Acetylcholine Receptor (AChR) Cys-loop Aspartates.

Publications

Investigation of Congenital Myasthenia Reveals Functional Asymmetry of Invariant Acetylcholine Receptor (AChR) Cys-loop Aspartates.

Similar Datasets

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