Ubiquitination-activating enzymes UBE1 and UBA6 regulate ubiquitination and expression of cardiac sodium channel Nav1.5

Cardiac sodium channel Na(v)1.5 is associated with cardiac arrhythmias and heart failure. Protein ubiquitination is catalyzed by an E1-E2-E3 cascade of enzymes. However, the E1 enzyme catalyzing Na(v)1.5 ubiquitination is unknown. Here, we show that UBE1 and UBA6 are two E1 enzymes regulating Na(v)1.5 ubiquitination and expression. Western blot analysis and patch-clamping recordings showed that overexpression of UBE1 or UBA6 increased the ubiquitination of Na(v)1.5 and significantly reduced Na(v)1.5 expression and sodium current density, and knockdown of UBE1 or UBA6 expression significantly increased Na(v)1.5 expression and sodium current density in HEK293/Na(v)1.5 cells. Similar results were obtained in neonatal cardiomyocytes. Bioinformatic analysis predicted two ubiquitination sites at K590 and K591. Mutations of K590 and K591 to K590A and K591A abolished the effects of overexpression or knockdown of UBE1 or UBA6 on Na(v)1.5 expression and sodium current density. Western blot analysis showed that the effects of UBE1 or UBA6 overexpression on the ubiquitination and expression of Na(v)1.5 were abolished by knockdown of UBC9, a putative E2 enzyme reported for Na(v)1.5 ubiquitination by us. Interestingly, real-time RT-PCR analysis showed that the expression level of UBE1, but not UBA6, was significantly up-regulated in ventricular tissues from heart failure patients. These data establish UBE1 and UBA6 as the E1 enzymes involved in Na(v)1.5 ubiquitination, and suggest that UBE1 and UBA6 regulate ubiquitination of Na(v)1.5 through UBC9. Our study is the first to reveal the regulatory role of the UBE1 or UBA6 E1 enzyme in the ubiquitination of an ion channel and links UBE1 up-regulation to heart failure.