Functional Consequence of Protein Kinase A-dependent Phosphorylation of the Cardiac Ryanodine Receptor SENSITIZATION OF STORE OVERLOAD-INDUCED Ca2+ RELEASE

Abstract The phosphorylation of the cardiac Ca2+-release channel (ryanodine receptor, RyR2) by protein kinase A (PKA) has been extensively characterized, but its functional consequence remains poorly defined and controversial. We have previously shown that RyR2 is phosphorylated by PKA at two major sites, serine 2030 and serine 2808, of which Ser-2030 is the major PKA site responding to β-adrenergic stimulation. Here we investigated the effect of the phosphorylation of RyR2 by PKA on the properties of single channels and on spontaneous Ca2+ release during sarcoplasmic reticulum Ca2+ overload, a process we have referred to as store overload-induced Ca2+ release (SOICR). We found that PKA activated single RyR2 channels in the presence, but not in the absence, of luminal Ca2+. On the other hand, PKA had no marked effect on the sensitivity of the RyR2 channel to activation by cytosolic Ca2+. Importantly, the S2030A mutation, but not mutations of Ser-2808, diminished the effect of PKA on RyR2. Furthermore, a phosphomimetic mutation, S2030D, potentiated the response of RyR2 to luminal Ca2+ and enhanced the propensity for SOICR in HEK293 cells. In intact rat ventricular myocytes, the activation of PKA by isoproterenol reduced the amplitude and increased the frequency of SOICR. Confocal line-scanning fluorescence microscopy further revealed that the activation of PKA by isoproterenol increased the rate of Ca2+ release and the propagation velocity of spontaneous Ca2+ waves, despite reduced wave amplitude and resting cytosolic Ca2+. Collectively, our data indicate that PKA-dependent phosphorylation enhances the response of RyR2 to luminal Ca2+ and reduces the threshold for SOICR and that this effect of PKA is largely mediated by phosphorylation at Ser-2030.