Ca²⁺/calmodulin-dependent protein kinase II regulates cardiac Na⁺ channels
J. Clin. Invest. Stefan Wagner, et al. 116:3127 doi:10.1172/JCI26620 [Go to this article.]

Figure 7
CaMKII-dependent association with and phosphorylation of Na⁺ channels. (A and B) Equal amounts of CaMKII were IP from mouse hearts (A, Tg versus WT) and rabbit myocytes (B, CaMKIIδ_c versus nontransfected cells) and subjected to immunoblotting. CaMKII IP showed CaMKII association with Na⁺ channels in WT and Tg hearts (n = 3 and n = 3, respectively), while IP with polyclonal rabbit anti-Ca_v1.2a did not. Similar results were seen in rabbit myocytes (CaMKIIδ_C and nontransfected cells, n = 4 and n = 2, respectively). (C) Colocalization of CaMKII and Na⁺ channel in rabbit myocytes with immunocytological staining. (D and E) Autoradiograms measuring ³²P incorporation into Na⁺ channels. Equal amounts of Na⁺ channels were immunoprecipitated from WT mouse hearts (n = 3) and directly phosphorylated with or without CaMKII, KN93 (50 μM), AIP (5 μM), or PKA/PKC inhibitor cocktail (PKA/C-I; 1 μM). (F) Endogenous CaMKII-dependent Na⁺ channel phosphorylation was activated in permeabilized rabbit myocytes by preincubating for 5 minutes in internal solutions of 500 nM [Ca²⁺] plus 2 μM CaM (versus 50 nM [Ca²⁺]). Sites not already phosphorylated were subsequently back-phosphorylated in Na⁺ channel immunoprecipitates (equal amounts) by exogenous preactivated CaMKII and ATP-γ-³²P. The intensity of ³²P is inversely related to the CaMKII-dependent phosphorylation during preincubation. (G and H) Western blots (anti-Na_v Pan) show upregulation of Na⁺ channel expression in CaMKIIδ_C-Tg versus WT mice (relative to calsequestrin [CSQ]) but unaltered expression in CaMKIIδ_C rabbit myocytes (data pooled for MOI 10 and 100).