Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII) Regulates Cardiac Sodium Channel NaV1.5 Gating by Multiple Phosphorylation Sites

• Published in: The Journal of biological chemistry Vol. 287; no. 24; pp. 19856 - 19869
• Main Authors: Ashpole, Nicole M., Herren, Anthony W., Ginsburg, Kenneth S., Brogan, Joseph D., Johnson, Derrick E., Cummins, Theodore R., Bers, Donald M., Hudmon, Andy
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 08.06.2012; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Arrhythmia; Brugada Syndrome - genetics; Brugada Syndrome - metabolism; Brugada Syndrome - pathology; Ca2+/Calmodulin-dependent Protein Kinase; Calcium Signaling; Calcium-Calmodulin-Dependent Protein Kinase Type 2 - genetics; Calcium-Calmodulin-Dependent Protein Kinase Type 2 - metabolism; CaMKII; Heart Failure - genetics; Heart Failure - metabolism; Heart Failure - pathology; HEK293 Cells; Humans; Ion Channel Gating - genetics; Ion Channels; Long QT Syndrome - genetics; Long QT Syndrome - metabolism; Long QT Syndrome - pathology; Membrane Potentials - genetics; Mice; Molecular Biophysics; Muscle Proteins - genetics; Muscle Proteins - metabolism; Myocardium - metabolism; Myocardium - pathology; Myocytes, Cardiac - metabolism; Myocytes, Cardiac - pathology; Na+ Current; NAV1.5 Voltage-Gated Sodium Channel; Phosphorylation - genetics; Protein Phosphorylation; Protein Structure, Tertiary; Sodium Channels; Sodium Channels - genetics; Sodium Channels - metabolism; CaMKII; Ion Channels; Arrhythmia; Ca2+/Calmodulin-dependent Protein Kinase; Sodium Channels; Protein Phosphorylation; Na+ Current; Calcium Signaling
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M111.322537

The cardiac Na+ channel NaV1.5 current (INa) is critical to cardiac excitability, and altered INa gating has been implicated in genetic and acquired arrhythmias. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is up-regulated in heart failure and has been shown to cause INa gating changes that mimic those induced by a point mutation in humans that is associated with combined long QT and Brugada syndromes. We sought to identify the site(s) on NaV1.5 that mediate(s) the CaMKII-induced alterations in INa gating. We analyzed both CaMKII binding and CaMKII-dependent phosphorylation of the intracellularly accessible regions of NaV1.5 using a series of GST fusion constructs, immobilized peptide arrays, and soluble peptides. A stable interaction between δC-CaMKII and the intracellular loop between domains 1 and 2 of NaV1.5 was observed. This region was also phosphorylated by δC-CaMKII, specifically at the Ser-516 and Thr-594 sites. Wild-type (WT) and phosphomutant hNaV1.5 were co-expressed with GFP-δC-CaMKII in HEK293 cells, and INa was recorded. As observed in myocytes, CaMKII shifted WT INa availability to a more negative membrane potential and enhanced accumulation of INa into an intermediate inactivated state, but these effects were abolished by mutating either of these sites to non-phosphorylatable Ala residues. Mutation of these sites to phosphomimetic Glu residues negatively shifted INa availability without the need for CaMKII. CaMKII-dependent phosphorylation of NaV1.5 at multiple sites (including Thr-594 and Ser-516) appears to be required to evoke loss-of-function changes in gating that could contribute to acquired Brugada syndrome-like effects in heart failure. CaMKII is up-regulated in heart failure and modulates Na+ current (INa), yet the mechanism is unclear. CaMKII phosphorylates several sites in the first intracellular loop of NaV1.5, thereby altering INa gating properties. This multisite phosphorylation may contribute to acquired arrhythmogenesis. Identification of these regulatory sites is critical for potential therapeutic targeting of CaMKII and NaV1.5 in failing hearts.