Brugada syndrome (BrS) is a genetically determined disease which predisposes affected individuals to fatal arrhythmias and sudden cardiac death (1). Although mutations in SCN5A leading to a loss of function of the cardiac sodium channel (Nav1.5) are the most common genotype found among BrS patients, the precise mechanisms are not well understood (2). E3 ubiquitin ligase Nedd4-2 binds to the PPxY motif at the C-terminus of Nav1.5 with its WW domain and downregulates the protein expression and functional current of Nav1.5 channel (3). Sequence alignment showed that BrS-associated L1239P mutant of SCN5A produces a new binding site (from LLEY to LPEY) of Nedd4-2 (4). We hypothesize that this additional Nedd4-2-binding site may enhance Nav1.5 ubiquitination and decrease mutant channel function. To test this, site directed mutagenesis was used to generate mutant DNA, Western blot analysis was used to quantify the protein expression and whole-cell patch clamp was used to record sodium current. Our data revealed that L1239P-SCN5A mutation resulted in loss-of-function properties with decreased protein expression and no detectable sodium current. Treatment of HEK293 cells transiently expressing L1239P-SCN5A with proteasome inhibitor MG-132 robustly increases the expression of the L1239P, which almost equal to wild type (WT), implying that increased degradation of Nav1.5 maybe the possible mechanism. To further investigate whether Nedd4-2 takes part in this process, WT-SCN5A or L1239P-SCN5A together with Nedd4-2 were transfected into HEK293 cells and co-immunoprecipitaion was used to detect Nav1.5-Nedd4-2 interaction. Our results demonstrated that the interaction between L1239P and Nedd4-2 is stronger than that between WT and Nedd4-2. Furthermore, a double mutation L1239P/Y1977A was generated which disrupt the basal biding site at Y1977 for Nedd4-2. Western blot result showed that the expression of L1239P/Y1977A increases almost two folds compared with that of L1239P. Co-immunoprecipitaion result showed the interaction between L1239P and Nedd4-2 is stronger than that between L1239P/Y1977A and Nedd4-2, which indicate that L1239P is an extra biding site besides Y1977. In order to investigate the importance of L1239P in the degradation of Nav1.5L-1239 was also mutated to R and H. Our data showed that protein expression and sodium current in L1239R-SCN5A and L1239H-SCN5A are almost equal to that of WT-SCN5A, thus supporting the role of L1239P as a significant biding site for Nedd4-2. In summary, these data demonstrated a novel mechanism of BrS-associated L1239P-SCN5A mutation, that additional Nedd4-2-binding site increases the Nedd4-2-Nav1.5 interaction and enhances the ubiquitination and degradation of Nav1.5 channel, which underlying the pathophysiology of BrS.