α 1 -Adrenoceptor stimulation potentiates L-type Ca 2+ current through Ca 2+ /calmodulin-dependent PK II (CaMKII) activation in rat ventricular myocytes

Abstract

α₁-Adrenoceptor stimulation (α₁ARS) modulates cardiac muscle contraction under physiological conditions by means of changes in Ca²⁺ current through L-type channels (I_Ca,L) and Ca²⁺ ensitivity of the myofilaments. However, the cellular mechanisms of α₁ARS are not fully clarified. In this study, we investigated the role of Ca²⁺/calmodulin-dependent PK II (CaMKII) in the regulation of I_Ca,L during α₁ARS in isolated adult rat ventricular myocytes by using the perforated patch–clamp technique. CaMKII inhibition with 0.5 μM KN-93 abolished the potentiation in I_Ca,L observed during α₁ARS by 10 μM phenylephrine. In the presence of PKC inhibitor (10 μM chelerythrine), the potentiation of I_Ca,L by phenylephrine also disappeared. In Western immunoblotting analysis, phenylephrine (≥1 μM) increased the amount of autophosphorylated CaMKII (active CaMKII) significantly, and this increase was abolished by CaMKII inhibition or PKC inhibition. Also, we investigated changes in the subcellular localization of active CaMKII by using immunofluorescence microscopy and immunoelectron microscopy. Before α₁ARS, active CaMKII was exclusively located just beneath the plasmalemma. However, after α₁ARS, active CaMKII was localized close to transverse tubules, where most of L-type Ca²⁺ channels are located. From these results, we propose that CaMKII, which exists near transverse tubules, is activated and phosphorylated by α₁ARS and that CaMKII activation directly potentiates I_Ca,L in rat ventricular myocytes.