title: Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates cardiac sodium channel NaV1.5 gating by multiple phosphorylation sites.
abstract: 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. Ca(2+)/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 δ(C)-CaMKII and the intracellular loop between domains 1 and 2 of Na(V)1.5 was observed. This region was also phosphorylated by δ(C)-CaMKII, specifically at the Ser-516 and Thr-594 sites. Wild-type (WT) and phosphomutant hNa(V)1.5 were co-expressed with GFP-δ(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.
authors.affiliation: Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
authors.forename: Nicole M
authors.initials: NM
authors.lastname: Ashpole
authors.forename: Anthony W
authors.initials: AW
authors.lastname: Herren
authors.forename: Kenneth S
authors.initials: KS
authors.lastname: Ginsburg
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Brogan
authors.forename: Derrick E
authors.initials: DE
authors.lastname: Johnson
authors.forename: Theodore R
authors.initials: TR
authors.lastname: Cummins
authors.forename: Donald M
authors.initials: DM
authors.lastname: Bers
authors.forename: Andy
authors.initials: A
authors.lastname: Hudmon
mesh_terms: D000818:Animals; D053840:Brugada Syndrome / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006333:Heart Failure / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D015640:Ion Channel Gating / Q000235:genetics; D008133:Long QT Syndrome / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008564:Membrane Potentials / Q000235:genetics; D051379:Mice; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism* / Q000473:pathology; D032383:Myocytes, Cardiac / Q000378:metabolism / Q000473:pathology; D062554:NAV1.5 Voltage-Gated Sodium Channel; D010766:Phosphorylation / Q000235:genetics; D017434:Protein Structure, Tertiary; D015222:Sodium Channels / Q000235:genetics / Q000378:metabolism*
chemical_list: D009124:Muscle Proteins; D062554:NAV1.5 Voltage-Gated Sodium Channel; C568320:SCN5A protein, human; C568321:Scn5a protein, mouse; D015222:Sodium Channels; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2