title: Protein kinase A phosphorylation of the ryanodine receptor does not affect calcium sparks in mouse ventricular myocytes.
abstract: Ryanodine receptor (RyR) phosphorylation by protein kinase A (PKA) may be important in modulating resting sarcoplasmic reticulum (SR) Ca2+ release, especially in heart failure. However, clear cellular data on PKA-dependent modulation of cardiac RyRs is limited because of difficulty in distinguishing between PKA effects on RyR, phospholamban (PLB), and Ca2+ current. To clarify this, we measured resting Ca2+ sparks in streptolysin-O permeabilized ventricular myocytes from wild-type (WT) and PLB knockout (PLB-KO) mice and transgenic mice expressing only double-mutant PLB (PLB-DM) that lacks the regulatory phosphorylation sites (S16A/T17A). In WT myocytes, cAMP dramatically increased Ca2+ spark frequency (CaSpF) by 2- and 3-fold when [Ca2+] was clamped at 50 and 10 nmol/L (and the SR Ca2+ content also rose by 40% and 50%). However, in PLB-KO and PLB-DM, neither CaSpF nor SR Ca2+ load was changed by the addition of 10 micromol/L cAMP (even with phosphatase inhibition). PKA activation also increased Ca2+ spark amplitude, duration, and width in WT, but not in PLB-KO or PLB-DM. RyR phosphorylation was confirmed by measurements of 32P incorporation on immunoprecipitated RyR. In intact resting myocytes, PKA activation increased CaSpF 2.8-fold in WT, but not in PLB-KO, confirming results in permeabilized myocytes. We conclude that the PKA-dependent increase in myocyte CaSpF and size is entirely attributable to PLB phosphorylation and consequent enhanced SR Ca2+ load. PKA does not seem to have any appreciable effect on resting RyR function in these ventricular myocytes. Moreover, the data provide compelling evidence that elevated intra-SR [Ca2+] increases RyR gating independent of cytosolic [Ca2+] (which was clamped).
authors.affiliation: Department of Physiology and Cardiovascular Institute, Loyola University Chicago, Stritch School of Medicine, Maywood, Ill 60153, USA.
authors.forename: Yanxia
authors.initials: Y
authors.lastname: Li
authors.forename: Evangelia G
authors.initials: EG
authors.lastname: Kranias
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Mignery
authors.forename: Donald M
authors.initials: DM
authors.lastname: Bers
mesh_terms: D000818:Animals; D001426:Bacterial Proteins; D001665:Binding Sites / Q000235:genetics; D002110:Caffeine / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000172:deficiency / Q000235:genetics; D002463:Cell Membrane Permeability / Q000187:drug effects; D002469:Cell Separation; D000242:Cyclic AMP / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D006352:Heart Ventricles / Q000166:cytology / Q000378:metabolism*; D066298:In Vitro Techniques; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D013301:Streptolysins / Q000494:pharmacology; D000099211:Phospholamban
chemical_list: D001426:Bacterial Proteins; D002110:Caffeine; D002135:Calcium-Binding Proteins; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D019837:Ryanodine Receptor Calcium Release Channel; D013301:Streptolysins; D000099211:Phospholamban; C025964:streptolysin O