Calcium/calmodulin-dependent protein kinase IIdelta associates with the ryanodine receptor complex and regulates channel function in rabbit heart

Abstract

Cardiac ryanodine receptors (RyR2s) play a critical role in excitation-contraction coupling by providing a pathway for the release of Ca(2+) from the sarcoplasmic reticulum into the cytosol. RyR2s exist as macromolecular complexes that are regulated via binding of Ca(2+) and protein phosphorylation/dephosphorylation. The present study examined the association of endogenous CaMKII (calcium/calmodulin-dependent protein kinase II) with the RyR2 complex and whether this enzyme could modulate RyR2 function in isolated rabbit ventricular myocardium. Endogenous phosphorylation of RyR2 was verified using phosphorylation site-specific antibodies. Co-immunoprecipitation studies established that RyR2 was physically associated with CaMKIIdelta. Quantitative assessment of RyR2 protein was performed by [(3)H]ryanodine binding to RyR2 immunoprecipitates. Parallel kinase assays allowed the endogenous CaMKII activity associated with these immunoprecipitates to be expressed relative to the amount of RyR2. The activity of RyR2 in isolated cardiac myocytes was measured in two ways: (i) RyR2-mediated Ca(2+) release (Ca(2+) sparks) using confocal microscopy and (ii) Ca(2+)-sensitive [(3)H]ryanodine binding. These studies were performed in the presence and absence of AIP (autocamtide-2-related inhibitory peptide), a highly specific inhibitor of CaMKII. At 1 microM AIP Ca(2+) spark duration, frequency and width were decreased significantly. Similarly, 1 microM AIP decreased [(3)H]ryanodine binding. At 5 microM AIP, a more profound inhibition of Ca(2+) sparks and a decrease in [(3)H]ryanodine binding was observed. Separate measurements showed that AIP (1-5 microM) did not affect sarcoplasmic reticulum Ca(2+)-ATPase-mediated Ca(2+) uptake. These results suggest the existence of an endogenous CaMKIIdelta that associates directly with RyR2 and specifically modulates RyR2 activity.

CaMKII inhibition targeted to the sarcoplasmic reticulum inhibits frequency-dependent acceleration of relaxation and Ca2+ current facilitation

“…This leads to increased RyR channel opening in bilayers and to an increased Ca 2+ spark frequency in myocytes at a given SR Ca 2+ content [3,19,34,[48][49][50]. Controversy remains about which RyR amino acid is the critical CaMKII target (Ser2809 vs. Ser2815) [19,48,49].…”

“…CaMKII is known to associate with ryanodine receptors (RyRs) [3,4], L-type Ca 2+ channels [5][6][7] and possibly phospholamban (PLB) [8]. This basal localization near targets may be important for efficient and specific regulation of these CaMKII effector proteins.…”

Calcium/Calmodulin-dependent Protein Kinase II Phosphorylation Drives Synapse-associated Protein 97 into Spines

“…Because it is known that CaMKII can be associated to RyRs (16,17), we tested whether CaMKII-dependent modulation of SAP97 trafficking was correlated to activation of RyRs by examining the distribution pattern of SAP97 in neurons exposed to caffeine (Fig. 4).…”

Dynamic Alterations in the CaV1.2/CaM/CaMKII Signaling Pathway in the Left Ventricular Myocardium of Ischemic Rat Hearts

“…In this study, our findings revealed that there was a significant reduction of CaMKII levels in the acute phase of ischemia, which might decrease the activation of LTCC and RYR2 and intracellular Ca 2 + concentration. Phospho-CaMKII may promote activation of RYR2 of the SR, thereby increasing RyR2 Ca 2 + sensitivity and spontaneous release of Ca 2 + from internal storage (Currie et al, 2004). Further, phospho-CaMKII could cause increased PLB phosphorylation at T 17 , which might help preserve SR Ca 2 + , and disabled PLB's ability to inhibit SERCA2a, thereby increasing Ca 2 + reuptake into intracellular stores (Schwoerer et al, 2008).…”

“…Ryanodine receptor 2 (RYR2) was a CaMKII-regulated protein that was involved in the regulation of the intranuclear Ca 2 + concentration. Currie et al (2004) have reported that CaMKII may promote activation of RYR2 of the sarcoplasmic reticulum (SR), thereby increasing RyR2 Ca 2 + sensitivity and spontaneous release of Ca 2 + from internal stores. The PLB was also defined as a CaMKII-regulated phosphoprotein (Sag et al, 2007).…”