Dominant-negative effect of SCN5A N-terminal mutations through the interaction of Na(v)1.5 α-subunits.
OBJECTIVE
Brugada syndrome (BrS) is an autosomal-inherited cardiac arrhythmia characterized by an ST-segment elevation in the right precordial leads of the electrocardiogram and an increased risk of syncope and sudden death. SCN5A, encoding the cardiac sodium channel Na(v)1.5, is the main gene involved in BrS. Despite the fact that several mutations have been reported in the N-terminus of Na(v)1.5, the functional role of this region remains unknown. We aimed to characterize two BrS N-terminal mutations, R104W and R121W, a construct where this region was deleted, ΔNter, and a construct where only this region was present, Nter.


RESULTS
Patch-clamp recordings in HEK293 cells demonstrated that R104W, R121W, and ΔNter abolished the sodium current I(Na). Moreover, R104W and R121W mutations exerted a strong dominant-negative effect on wild-type (WT) channels. Immunocytochemistry of rat neonatal cardiomyocytes revealed that both mutants were mostly retained in the endoplasmic reticulum and that their co-expression with WT channels led to WT channel retention. Furthermore, co-immunoprecipitation experiments showed that Na(v)1.5-subunits were interacting with each other, even when mutated, deciphering the mutation dominant-negative effect. Both mutants were mostly degraded by the ubiquitin-proteasome system, while ΔNter was addressed to the membrane, and Nter expression induced a two-fold increase in I(Na). In addition, the co-expression of N-terminal mutants with the gating-defective but trafficking-competent R878C-Na(v)1.5 mutant gave rise to a small I(Na).


CONCLUSIONS
This study reports for the first time the critical role of the Na(v)1.5 N-terminal region in channel function and the dominant-negative effect of trafficking-defective channels occurring through α-subunit interaction.
INSERM, UMR_S 956, IFR14, Paris,  France.
Jérôme
J
Clatot
Azza
A
Ziyadeh-Isleem
Svetlana
S
Maugenre
Isabelle
I
Denjoy
Haiyan
H
Liu
Gilles
G
Dilanian
Stéphane N
SN
Hatem
Isabelle
I
Deschênes
Alain
A
Coulombe
Pascale
P
Guicheney
Nathalie
N
Neyroud
D000328:Adult; D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D053840:Brugada Syndrome / Q000235:genetics*; D005816:Genetic Complementation Test; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D032383:Myocytes, Cardiac / Q000378:metabolism; D062554:NAV1.5 Voltage-Gated Sodium Channel / Q000235:genetics* / Q000378:metabolism; D010375:Pedigree; D051381:Rats
D062554:NAV1.5 Voltage-Gated Sodium Channel; C568320:SCN5A protein, human