Interaction of calcium/calmodulin-dependent protein kinase IIδ C with sorcin indirectly modulates ryanodine receptor function in cardiac myocytes

Abstract

Calcium/calmodulin dependent protein kinase II delta C (CaMKIIδ C) and the EF-hand Ca 2+-binding protein, sorcin have both been shown to regulate the excitation-contraction coupling process. This study explores the possibility that these two proteins interact directly and, as a result of this interaction, modulate cardiac calcium handling. Two independent methods (surface plasmon resonance (SPR) and overlay assays) were used to determine whether CaMKIIδ C and sorcin interacted in a direct manner. The nature of this interaction was explored by (i) examining the effects of sorcin on CaMKIIδ C activity using a selective kinase assay and (ii) studying whether sorcin was a substrate for CaMKIIδ C using autoradiography. Ryanodine binding assays on mouse ventricular cardiomyocytes were used to determine specific functional effects of this interaction. SPR studies suggested that sorcin interacts with CaMKIIδ C in a concentration-dependent manner. This interaction occurs in the presence of Ca 2+ and in the presence or absence of calmodulin (CaM). Overlay assays confirmed the existence of this interaction. Further experiments suggested that this interaction is reciprocal. Firstly, sorcin significantly inhibited both recombinant and native CaMKIIδ C activity to similar extents. Secondly, sorcin was phosphorylated by CaMKIIδ C. Thirdly, sorcin inhibition of CaMKII activity occurred under conditions where sorcin remained dephosphorylated. Functionally, CaMKIIδ C-mediated phosphorylation of sorcin served to abolish the inhibitory effect of sorcin on ryanodine receptor (RyR 2) open probability (Po). Since both proteins are capable of directly modulating RyR 2 activity, this interaction may serve as an additional or alternative indirect route by which both proteins can regulate RyR 2 opening status in cardiac myocytes.