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

The cardiac Na+ channel Na(V)1.5 current (I-Na) is critical to cardiac excitability, and altered I-Na 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 I-Na 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 Na(V)1.5 that mediate(s) the CaMKII-induced alterations in I-Na gating. We analyzed both CaMKII binding and CaMKII-dependent phosphorylation of the intracellularly accessible regions of Na(V)1.5 using a series of GST fusion constructs, immobilized peptide arrays, and soluble peptides. A stable interaction between delta(C)-CaMKII and the intracellular loop between domains 1 and 2 of Na(V)1.5 was observed. This region was also phosphorylated by delta(C)-CaMKII, specifically at the Ser-516 and Thr-594 sites. Wild-type (WT) and phospho-mutant hNa(V)1.5 were co-expressed with GFP-delta(C)-CaMKII in HEK293 cells, and I-Na was recorded. As observed in myocytes, CaMKII shifted WT I-Na availability to a more negative membrane potential and enhanced accumulation of I-Na 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 I-Na availability without the need for CaMKII. CaMKII-dependent phosphorylation of Na(V)1.5 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.