Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates cardiac sodium channel NaV1.5 gating by multiple phosphorylation sites

Nicole M. Ashpole, Anthony W. Herren, Kenneth S. Ginsburg, Joseph D. Brogan, Derrick E. Johnson, Theodore R. Cummins, Donald M. Bers, Andy Hudmon

Research output: Contribution to journalArticle

104 Scopus citations

Abstract

The cardiac Na+ channel NaV1.5 current (I Na) is critical to cardiac excitability, and altered INa gating has been implicated in genetic and acquired arrhythmias. Ca 2+/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 I Na gating.Weanalyzed 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 hNa V1.5 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.

Original languageEnglish (US)
Pages (from-to)19856-19869
Number of pages14
JournalJournal of Biological Chemistry
Volume287
Issue number24
DOIs
StatePublished - Jun 8 2012

    Fingerprint

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this