Functional consequence of protein kinase A-dependent phosphorylation of the cardiac ryanodine receptor: sensitization of store overload-induced Ca2+ release.
The phosphorylation of the cardiac Ca(2+)-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 2,030 and serine 2,808, of which Ser-2,030 is the major PKA site responding to beta-adrenergic stimulation. Here we investigated the effect of the phosphorylation of RyR2 by PKA on the properties of single channels and on spontaneous Ca(2+) release during sarcoplasmic reticulum Ca(2+) overload, a process we have referred to as store overload-induced Ca(2+) release (SOICR). We found that PKA activated single RyR2 channels in the presence, but not in the absence, of luminal Ca(2+). On the other hand, PKA had no marked effect on the sensitivity of the RyR2 channel to activation by cytosolic Ca(2+). Importantly, the S2030A mutation, but not mutations of Ser-2,808, diminished the effect of PKA on RyR2. Furthermore, a phosphomimetic mutation, S2030D, potentiated the response of RyR2 to luminal Ca(2+) 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 Ca(2+) release and the propagation velocity of spontaneous Ca(2+) waves, despite reduced wave amplitude and resting cytosolic Ca(2+). Collectively, our data indicate that PKA-dependent phosphorylation enhances the response of RyR2 to luminal Ca(2+) and reduces the threshold for SOICR and that this effect of PKA is largely mediated by phosphorylation at Ser-2,030.
Xiao
Bailong
B
Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
Tian
Xixi
X
Xie
Wenjun
W
Jones
Peter P
PP
Cai
Shitian
S
Wang
Xianhua
X
Jiang
Dawei
D
Kong
Huihui
H
Zhang
Lin
L
Chen
Keyun
K
Walsh
Michael P
MP
Cheng
Heping
H
Chen
S R Wayne
SR
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D006352:Heart Ventricles / Q000473:pathology; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D008954:Models, Biological; D009154:Mutation; D009206:Myocardium / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*; D012694:Serine / Q000737:chemistry
D019837:Ryanodine Receptor Calcium Release Channel; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases; D007545:Isoproterenol; D002118:Calcium