UBC9 regulates cardiac sodium channel Na_v1.5 ubiquitination, degradation and sodium current density

Voltage-gated sodium channel Na_v1.5 is critical for generation and conduction of cardiac action potentials. Mutations and expression level changes of Na_v1.5 are associated with cardiac arrhythmias and sudden death. The ubiquitin (Ub) conjugation machinery utilizes three enzyme activities, E1, E2, and E3, to regulate protein degradation. Previous studies from us and others showed that Nedd4-2 acts as an E3 ubiquitin-protein ligase involved in ubiquitination and degradation of Na_v1.5, however, more key regulators remain to be identified. In this study, we show that UBC9, a SUMO-conjugating enzyme, regulates ubiquitination and degradation of Na_v1.5. Overexpression of UBC9 significantly decreased Na_v1.5 expression and reduced sodium current densities, whereas knockdown of UBC9 expression significantly enhanced Na_v1.5 expression and increased sodium current densities, in both HEK293 cells and primary neonatal cardiomyocytes. Overexpression of UBC9 increased ubiquitination of Na_v1.5, and proteasome inhibitor MG132 blocked the effect of UBC9 overexpression on Na_v1.5 degradation. Co-immunoprecipitation showed that UBC9 interacts with Nedd4-2. UBC9 with mutation C93S, which suppresses SUMO-conjugating activity of UBC9, was as active as wild type UBC9 in regulating Na_v1.5 levels, suggesting that UBC9 regulates Na_v1.5 expression levels in a SUMOylation-independent manner. Our findings thus identify a key structural element of the ubiquitin-conjugation machinery for Na_v1.5 and provide important insights into the regulatory mechanism for ubiquitination and turnover of Na_v1.5.