Proteomic and functional mapping of cardiac NaV1.5 channel phosphorylation sites

Abstract

Phosphorylation of the voltage-gated Na+ (NaV) channel NaV1.5 regulates cardiac excitability, yet the phosphorylation sites regulating its function and the underlying mechanisms remain largely unknown. Using a systematic, quantitative phosphoproteomic approach, we analyzed NaV1.5 channel complexes purified from nonfailing and failing mouse left ventricles, and we identified 42 phosphorylation sites on NaV1.5. Most sites are clustered, and three of these clusters are highly phosphorylated. Analyses of phosphosilent and phosphomimetic NaV1.5 mutants revealed the roles of three phosphosites in regulating NaV1.5 channel expression and gating. The phosphorylated serines S664 and S667 regulate the voltage dependence of channel activation in a cumulative manner, whereas the nearby S671, the phosphorylation of which is increased in failing hearts, regulates cell surface NaV1.5 expression and peak Na+ current. No additional roles could be assigned to the other clusters of phosphosites. Taken together, our results demonstrate that ventricular NaV1.5 is highly phosphorylated and that the phosphorylation-dependent regulation of NaV1.5 channels is highly complex, site specific, and dynamic.

Functional cross-talk between phosphorylation and disease-causing mutations in the cardiac sodium channel Na v 1.5

“…To date, most confirmed Na v 1.5 phosphorylation sites are situated within the DI-DII linker. Although multiple lines of evidence suggest that all intracellular linkers can be phosphorylated ( 18 , 54 , 55 ), mass spectrometry–based work on cardiac and neuronal Na v channels from native tissues has yet to find evidence for phosphorylation in the DIII-DIV linker ( 20 , 56 , 57 ). This is most likely due to the close association of the DIII-DIV linker with the transmembrane helices ( 11 ) and its highly basic sequence content (12/53 amino acids are basic), both of which reduce mass spectrometry sequence coverage.…”

Genetic Characteristics and Transcriptional Regulation of Sodium Channel Related Genes in Chinese Patients With Brugada Syndrome

“…Some mutations lead to a decrease in current density, others do not lead to a decrease in I Na , while some location-specific SCN5A mutations resulted in poorer outcomes during follow-up (44). As different mutation (45)(46)(47)(48) including SUMOylation (49), ubiquitination (50), acetylation (51), etc. In our previous study, we revealed that miR-192-5p bound to the 3 ′ -UTR of human SCN5A to negatively regulate the expression of Nav1.5 and reduce I Na density.…”

FHF2 phosphorylation and regulation of native myocardial NaV1.5 channels

“…The tryptic peptides from mouse left ventricular Na V channel complexes were generated and labeled as described previously (Lorenzini et al, 2021). Briefly, the IP eluates were thawed on ice, reduced, and denatured by heating for 10 min at 95ºC.…”

“…Immnunoprecipitation (IP) of Na V channel complexes from mouse left ventricles was performed as described previously (Lorenzini et al, 2021). Briefly, flash-frozen left ventricles from 13-weeks-old male C57/BL6J wild-type (WT) mice were homogenized individually in ice-cold lysis buffer containing 20 mM HEPES (pH 7.4), 150 mM NaCl, 0.5% amidosulfobetaine, 1X complete protease inhibitor cocktail tablet, 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.7 μg/ml pepstatin A (Thermo Fisher Scientific, Waltham, MA) and 1X Halt phosphatase inhibitor cocktail (Thermo Fisher Scientific).…”

“…Cultured cardiomyocytes were lysed and western blot analyses of cardiomyocyte lysates were completed as described previously (Lorenzini et al, 2021). Briefly, cells were washed twice in ice-cold PBS (pH 7.4) and lysed in ice-cold lysis buffer containing 20 mM HEPES (pH 7.4), 150 mM NaCl, 0.5% amidosulfobetaine, 1X complete protease inhibitor cocktail tablet, 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.7 μg/ml pepstatin A (Thermo Fisher Scientific) and 1X Halt phosphatase inhibitor cocktail (Thermo Fisher Scientific).…”