Dataset Information

Alternative splicing of the cardiac sodium channel creates multiple variants of mutant T1620K channels.

ABSTRACT: Alternative splicing creates several Na(v)1.5 transcripts in the mammalian myocardium and in various other tissues including brain, dorsal root ganglia, breast cancer cells as well as neuronal stem cell lines. In total nine Na(v)1.5 splice variants have been discovered. Four of them, namely Na(v)1.5a, Na(v)1.5c, Na(v)1.5d, and Na(v)1.5e, generate functional channels in heterologous expression systems. The significance of alternatively spliced transcripts for cardiac excitation, in particular their role in SCN5A channelopathies, is less well understood. In the present study, we systematically investigated electrophysiological properties of mutant T1620K channels in the background of all known functional Na(v)1.5 splice variants in HEK293 cells. This mutation has been previously associated with two distinct cardiac excitation disorders: with long QT syndrome type 3 (LQT3) and isolated cardiac conduction disease (CCD). When investigating the effect of the T1620K mutation, we noticed similar channel defects in the background of hNa(v)1.5, hNa(v)1.5a, and hNa(v)1.5c. In contrast, the hNa(v)1.5d background produced differential effects: In the mutant channel, some gain-of-function features did not emerge, whereas loss-of-function became more pronounced. In case of hNa(v)1.5e, the neonatal variant of hNa(v)1.5, both the splice variant itself as well as the corresponding mutant channel showed electrophysiological properties that were distinct from the wild-type and mutant reference channels, hNa(v)1.5 and T1620K, respectively. In conclusion, our data show that alternative splicing is a mechanism capable of generating a variety of functionally distinct wild-type and mutant hNa(v)1.5 channels. Thus, the cellular splicing machinery is a potential player affecting genotype-phenotype correlations in SCN5A channelopathies.

SUBMITTER: Walzik S

PROVIDER: S-EPMC3084281 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Alternative splicing of the cardiac sodium channel creates multiple variants of mutant T1620K channels.

Walzik Stefan S Schroeter Annett A Benndorf Klaus K Zimmer Thomas T

PloS one 20110428 4

Alternative splicing creates several Na(v)1.5 transcripts in the mammalian myocardium and in various other tissues including brain, dorsal root ganglia, breast cancer cells as well as neuronal stem cell lines. In total nine Na(v)1.5 splice variants have been discovered. Four of them, namely Na(v)1.5a, Na(v)1.5c, Na(v)1.5d, and Na(v)1.5e, generate functional channels in heterologous expression systems. The significance of alternatively spliced transcripts for cardiac excitation, in particular the ...[more]

PMID: 21552533

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Project description:Voltage-gated sodium channels are the target sites of both DDT and pyrethroid insecticides. The importance of alternative splicing as a key mechanism governing the structural and functional diversity of sodium channels and the resulting development of insecticide and acaricide resistance is widely recognized, as shown by the extensive research on characterizing alternative splicing and variants of sodium channels in medically and agriculturally important insect species. Here we present the first comparative study of multiple variants of the sodium channel transcripts in the mosquito Culex quinquefasciatus. The variants were classified into two categories, CxNa-L and CxNa-S based on their distinguishing sequence sizes of ~6.5 kb and ~4.0 kb, respectively, and generated via major extensive alternative splicing with minor small deletions/ insertions in susceptible S-Lab, low resistant HAmCq(G0), and highly resistant HAmCq(G8)Culex strains. Four alternative Cx-Na-L splice variants were identified, including three full length variants with three optional exons (2, 5, and 21i) and one with in-frame-stop codons. Large, multi-exon-alternative splices were identified in the CxNa-S category. All CxNa-S splicing variants in the S-Lab and HAmCq(G0) strains contained in-frame stop codons, suggesting that any resulting proteins would be truncated. The ~1000 to ~3000-fold lower expression of these splice variants with stop codons compared with the CxNa-L splicing variants may support the lower importance of these variants in S-Lab and HAmCq(G0). Interestingly, two alternative splicing variants of CxNa-S in HAmCq(G8) included entire ORFs but lacked exons 5 to18 and these two variants had much higher expression levels in HAmCq(G8) than in S-Lab and HAmCq(G0). These results provide a functional basis for further characterizing how alternative splicing of a voltage-gated sodium channel contributes to diversity in neuronal signaling in mosquitoes in response to pyrethroids, and possibly indicates the role of these variants in the development of pyrethroid resistance.

Project description:Over 1,500 missense variants of sodium channel hNav1.5, which are reported in the ClinVar database, are associated with cardiac diseases. For most of the variants, the clinical significance is uncertain (VUS), not provided (NP), or has conflicting interpretations of pathogenicity (CIP). Reclassifying these variants as pathogenic/likely pathogenic (P/LP) variants is important for diagnosing genotyped patients. In our earlier work, several bioinformatics tools and paralogue annotation method consensually predicted that 74 VUS/NP/CIP variants of 54 wild type residues (set w54) are potentially damaging variants (PDVs). Atomic mechanisms underlying dysfunction of the PDVs are unknown. Here we employed a recent cryo-EM structure of the hNav1.5 channel with likely inactivated pore domain (PD) and activated voltage-sensing domains (VSDs), and ad hoc models of the closed and open PD and resting VSDs to explore intersegment contacts of w54 residues. We found that 44 residues from set w54 contact 84 residues with 118 disease missense variants. These include 104 VUS/NP/CIP variants, most of which are associated with the loss-of-function Brugada syndrome (BrS1) or gain-of-function long QT syndrome (LQT3). Matrix representation of the PDVs and their contact variants facilitated recognition of coupled mutations associated with the same disease. In particular, BrS1-associated coupled mutations, which disturb the P-loops region with the selectivity filter slow inactivation gate, would cause the channel dysfunction. Other likely causes of the channel dysfunction include coupled BrS1-associated variants within VSDs that would destabilize their activated states and coupled LQT3-associated variants, which would stabilize the open PD or activated VSDs. Our study proposes mechanisms of channel dysfunction for scores of BrS1- and LQT3-associated variants, confirms status for 82% of PDVs, and suggests damaging status for their contact variants, which are currently categorized as VUS/NP/CIP variants.

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Alternative splicing of the cardiac sodium channel creates multiple variants of mutant T1620K channels.

Publications

Alternative splicing of the cardiac sodium channel creates multiple variants of mutant T1620K channels.

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