Dataset Information

Disease-linked mutations alter the stoichiometries of HCN-KCNE2 complexes.

ABSTRACT: The four hyperpolarization-activated cylic-nucleotide gated (HCN) channel isoforms and their auxiliary subunit KCNE2 are important in the regulation of peripheral and central neuronal firing and the heartbeat. Disruption of their normal function has been implicated in cardiac arrhythmias, peripheral pain, and epilepsy. However, molecular details of the HCN-KCNE2 complexes are unknown. Using single-molecule subunit counting, we determined that the number of KCNE2 subunits in complex with the pore-forming subunits of human HCN channels differs with each HCN isoform and is dynamic with respect to concentration. These interactions can be altered by KCNE2 gene-variants with functional implications. The results provide an additional consideration necessary to understand heart rhythm, pain, and epileptic disorders.

SUBMITTER: Lussier Y

PROVIDER: S-EPMC6591248 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Disease-linked mutations alter the stoichiometries of HCN-KCNE2 complexes.

Lussier Yoann Y Fürst Oliver O Fortea Eva E Leclerc Marc M Priolo Dimitri D Moeller Lena L Bichet Daniel G DG Blunck Rikard R D'Avanzo Nazzareno N

Scientific reports 20190624 1

The four hyperpolarization-activated cylic-nucleotide gated (HCN) channel isoforms and their auxiliary subunit KCNE2 are important in the regulation of peripheral and central neuronal firing and the heartbeat. Disruption of their normal function has been implicated in cardiac arrhythmias, peripheral pain, and epilepsy. However, molecular details of the HCN-KCNE2 complexes are unknown. Using single-molecule subunit counting, we determined that the number of KCNE2 subunits in complex with the pore ...[more]

PMID: 31235733

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Project description:Amelogenin plays a crucial role in tooth enamel formation, and mutations on X-chromosomal amelogenin cause X-linked amelogenesis imperfecta (AI). Amelogenin pre-messenger RNA (mRNA) is highly alternatively spliced, and during alternative splicing, exon4 is mostly skipped, leading to the formation of a microRNA (miR-exon4) that has been suggested to function in enamel and bone formation. While delivering the functional variation of amelogenin proteins, alternative splicing of exon4 is the decisive first step to producing miR-exon4. However, the factors that regulate the splicing of exon4 are not well understood. This study aimed to investigate the association between known mutations in exon4 and exon5 of X chromosome amelogenin that causes X-linked AI, the splicing of exon4, and miR-exon4 formation. Our results showed mutations in exon4 and exon5 of the amelogenin gene, including c.120T>C, c.152C>T, c.155C>G, and c.155delC, significantly affected the splicing of exon4 and subsequent miR-exon4 production. Using an amelogenin minigene transfected in HEK-293 cells, we observed increased inclusion of exon4 in amelogenin mRNA and reduced miR-exon4 production with these mutations. In silico analysis predicted that Ser/Arg-rich RNA splicing factor (SRSF) 2 and SRSF5 were the regulatory factors for exon4 and exon5 splicing, respectively. Electrophoretic mobility shift assay confirmed that SRSF2 binds to exon4 and SRSF5 binds to exon5, and mutations in each exon can alter SRSF binding. Transfection of the amelogenin minigene to LS8 ameloblastic cells suppressed expression of the known miR-exon4 direct targets, Nfia and Prkch, related to multiple pathways. Given the mutations on the minigene, the expression of Prkch has been significantly upregulated with c.155C>G and c.155delC mutations. Together, we confirmed that exon4 splicing is critical for miR-exon4 production, and mutations causing X-linked AI in exon4 and exon5 significantly affect exon4 splicing and following miR-exon4 production. The change in miR-exon4 would be an additional etiology of enamel defects seen in some X-linked AI.

Dataset Information

Disease-linked mutations alter the stoichiometries of HCN-KCNE2 complexes.

Publications

Disease-linked mutations alter the stoichiometries of HCN-KCNE2 complexes.

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