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+]SRwere 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 (10μM) 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; 10μM) 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.
authors.affiliation: Department of Cell and Molecular Physiology, Loyola University Chicago, 2160 South First Avenue, Maywood, IL 60153, United States.
authors.forename: Elisa
authors.initials: E
authors.lastname: Bovo
authors.affiliation: Medicine-Division of Cardiovascular Disease, University of Alabama at Birmingham, 901 19th Street South, Birmingham, AL 35294, United States.
authors.forename: Sabine
authors.initials: S
authors.lastname: Huke
authors.affiliation: Department of Molecular Biophysics and Physiology, Rush University Medical Center, 1750 West Harrison Street, Chicago, IL 60612, United States.
authors.forename: Lothar A
authors.initials: LA
authors.lastname: Blatter
authors.affiliation: Department of Cell and Molecular Physiology, Loyola University Chicago, 2160 South First Avenue, Maywood, IL 60153, United States. Electronic address: azima@luc.edu.
authors.forename: Aleksey V
authors.initials: AV
authors.lastname: Zima
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D006352:Heart Ventricles / Q000378:metabolism*; D015640:Ion Channel Gating; D009206:Myocardium / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000378:metabolism*; D010766:Phosphorylation; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
chemical_list: D019837:Ryanodine Receptor Calcium Release Channel; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D002118:Calcium
keywords: Ca(2+) spark; Cardiomyocyte; Protein kinase A; Protein phosphatases; Ryanodine receptor; Sarcoplasmic reticulum Ca(2+) leak