The effect of PKA-mediated phosphorylation of ryanodine receptor on SR Ca2+ leak in ventricular myocytes Journal Article

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Authors: Bovo, E; Huke, S.; Blatter, L. A.; Zima, A. V.
Article Title: The effect of PKA-mediated phosphorylation of ryanodine receptor on SR Ca2+ leak in ventricular myocytes
Abstract: Functional impact of cardiac ryanodine receptor (type 2 RyR or RyR2) phosphorylation by protein kinase A (PKA) remains highly controversial. In this study, we characterized a functional link between PKA-mediated RyR2 phosphorylation level and sarcoplasmic reticulum (SR) Ca2+ release and leak in permeabilized rabbit ventricular myocytes. Changes in cytosolic [Ca2+] and intra-SR [Ca2+]SR were measured with Fluo-4 and Fluo-5N, respectively. Changes in RyR2 phosphorylation at two PKA sites, serine-2031 and -2809, were measured with phospho-specific antibodies. cAMP (10muM) increased Ca2+ spark frequency approximately two-fold. This effect was associated with an increase in SR Ca2+ load from 0.84 to 1.24mM. PKA inhibitory peptide (PKI; 10muM) abolished the cAMP-dependent increase of SR Ca2+ load and spark frequency. When SERCA was completely blocked by thapsigargin, cAMP did not affect RyR2-mediated Ca2+ leak. The lack of a cAMP effect on RyR2 function can be explained by almost maximal phosphorylation of RyR2 at serine-2809 after sarcolemma permeabilization. This high RyR2 phosphorylation level is likely the consequence of a balance shift between protein kinase and phosphatase activity after permeabilization. When RyR2 phosphorylation at serine-2809 was reduced to its "basal" level (i.e. RyR2 phosphorylation level in intact myocytes) using kinase inhibitor staurosporine, SR Ca2+ leak was significantly reduced. Surprisingly, further dephosphorylation of RyR2 with protein phosphatase 1 (PP1) markedly increased SR Ca2+ leak. At the same time, phosphorylation of RyR2 at serine 2031 did not significantly change under identical experimental conditions. These results suggest that RyR2 phosphorylation by PKA has a complex effect on SR Ca2+ leak in ventricular myocytes. At an intermediate level of RyR2 phosphorylation SR Ca2+ leak is minimal. However, complete dephosphorylation and maximal phosphorylation of RyR2 increases SR Ca2+ leak.
Journal Title: Journal of Molecular and Cellular Cardiology
Volume: 104
ISSN: 1095-8584; 0022-2828
Publisher: Unknown  
Journal Place: England
Date Published: 2017
Start Page: 9
End Page: 16
Language: eng
DOI/URL:

S0022-2828(17)30027-5
Notes: LR: 20170307; CI: Copyright (c) 2017; GR: P01 HL080101/HL/NHLBI NIH HHS/United States; GR: R01 HL130231/HL/NHLBI NIH HHS/United States; GR: R01 HL101235/HL/NHLBI NIH HHS/United States; GR: R01 HL128044/HL/NHLBI NIH HHS/United States; GR: R01 HL062231/HL/NHLBI NIH HHS/United States; JID: 0262322; NIHMS848183; OTO: NOTNLM; PMCR: 2018/03/01; 2016/09/13 [received]; 2016/11/22 [revised]; 2017/01/24 [accepted]; ppublish