Ubiquitin-activating Enzymes UBE1 And UBA6 Regulate Ubiquitination And Expression Of Cardiac Sodium Channel Na_v1.5

The human SCN5A gene encoded the?subunit of the main cardiac sodium channel Na_v1.5 is located on chromosome 3p21.This channel predominates inward sodium current and plays a critical role in the initiation and conduction of cardiac action potentials.SCN5A mutation or abnormal Na_v1.5 is associated with long QT syndrome(LQTS),Brugada syndrome(Br S),progressive cardiac conduction defect disease(PCCD),family atrial fibrillation(AF)and dilated cardiomyopathy(DCM).Previous genetic,electrophysiological,and molecular studies have identified the arrhythmic and cardiac structural characteristics induced by abnormal Na_v1.5.However,due to the variation of disease manifestations and genetic background,impact of environmental factors,as well as the presence of mixed phenotypes,the detailed and individualized physiological mechanisms in various Na_v1.5-related syndromes are not fully elucidated.Understanding the molecular mechanisms of Na_v1.5 expression is very useful in developing new therapeutic approaches.Ubiquitin(Ub)is an evolutionarily conserved protein that post-translationally marks proteins for degradation.Ub is covalently coupled to lysine residues on target proteins by a cascade of enzymatic reactions carried out by activating(E1),conjugating(E2)and ligating(E3)enzymes.Ub is first activated by E1 and is then transferred onto E2 conjugating enzyme.Subsequently,E3 Ub ligases interact at the same time with a Ub-loaded E2 and the substrate protein and mediate isopeptide bond formation between the C terminus of Ub and a substrate lysine.Eventually,the substrate is ubiquitinated and degradation through the cascade of enzymatic reaction.Ubiquitination is now known to be important for protein localization,trafficking,and recognition by signaling or regulatory complexes,thus affecting all aspects of cellular signaling and homeostasis.Ubiquitination of ion channels modulate their internalization and endoplasmic reticulum-associated degradation,thereby directly regulating their physiological functions.Therefore,in-depth understanding of the ubiquitination mechanism of Na_v1.5 provides a good therapeutic approach for prevention and treatment of arrhythmia or heart failure.The human genome contains>600 annotated Ub E3 ligases and>40 E2 ligases.Our lad recently reported that UBC9,a SUMOylation E2enzyme,serves as an E2 regulating Na_v1.5 ubiquitination.In addition,our and others reported Nedd4-2 serves as an E3 ligase involve in ubiquitination of Na_v1.5.Because a complete ubiquitination pathway for Na_v1.5 has not been reported,this study aimed to identify the E1 enzyme(s).Here are two E1s in the human genome,including UBE1 and UBA6.In both the HEK293/Na_v1.5 stable line and neonatal cardiacmyocytes,overexpression of either UBE1 or UBA6 significantly reduces Na_v1.5 expression and sodium current density,whereas knockdown of UBE1 or UBA6 expression significantly increases Na_v1.5 expression and sodium current density.Overexpression of either UBE1 or UBA6 increases ubiquitination of Na_v1.5.The effects of UBE1 or UBA6 overexpression on the ubiquitination and expression of Na_v1.5 are abolished by knockdown UBC9 expression.These data suggest that both UBE1 and UBA6 are required for regulating ubiquitination of Na_v1.5 through UBC9.Then,we investigated the relationship of UBE1 and UBA6 on Na_v1.5.The effect of the knockdown of both UBE1 and UBA6 on the expression of Na_v1.5 was more than the combined additive effects of knockdown of UBE1 or UBA6 alone,suggesting that UBE1 and UBA6 had a synergistic effect.However,the synergistic effect was not obvious in the case of overexpression of both UBE1 and UBA6.Interestingly,in the case of overexpression of both UBE1 and UBA6,the effect of UBE1 or UBA6 on Na_v1.5 is dose-dependent.As the amount of either UBE1 or UBA6 expression plasmid DNA in transfection was increased,Western blot analysis showed that the expression level of Na_v1.5 was decreased in a dose-dependent manner.Finally,bioinformatic analysis predicted two ubiquitination sites at K590 and K591,and the mutation of either site to alanine abolishes the effects of UBE1 or UBA6 on Na_v1.5 expression and sodium current density.To summarize,our study identifies the E1 enzymes,include UBE1 and UBA6,involved in Na_v1.5 ubiquitination through UBC9.Both UBE1 and UBA6 have dose-dependent and synergistic effect on regulating Na_v1.5 expression.Moreover,we find out the ubiquitination sites of UBE1 and UBA6 resulting in Na_v1.5,which located on lysine at position 590 and 591 of Na_v1.5.Our study is also the first to reveal the regulatory role of either UBE1 or UBA6 in ion channels,which may play a role in better understanding the molecular of Na_v1.5 expression.