Functional Consequence of Protein Kinase A-dependent Phosphorylation of the Cardiac Ryanodine Receptor

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. 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. Ventricular tachycardia (VT) 4The abbreviations used are: VT, ventricular tachycardia; HF, heart failure; SR, sarcoplasmic reticulum; RyR2, ryanodine receptor 2; SOICR, store overload-induced Ca2+ release; AR, β-adrenergic receptor; PKA, protein kinase A; PLB, phospholamban; FKBP, FK506-binding protein; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Iso, isoproterenol; wt, wild type; SERCA, sarcoplasmic-endoplasmic reticulum calcium ATPase. is the leading cause of sudden death, particularly in patients with heart failure (HF), but the molecular mechanisms underlying the high incidence of VT in HF are not completely understood (1Pogwizd S.M. Bers D.M. Trends Cardiovasc. Med. 2004; 14: 61-66Crossref PubMed Scopus (292) Google Scholar). A major cause of VT is believed to be delayed afterdepolarizations, which are produced by spontaneous Ca2+ release from the sarcoplasmic reticulum (SR) via the cardiac ryanodine receptor (RyR2) during SR Ca2+ overload (2Kass R.S. Tsien R.W. Biophys. J. 1982; 38: 259-269Abstract Full Text PDF PubMed Scopus (131) Google Scholar, 3Orchard C. Eisner D. Allen D. Nature. 1983; 304: 735-738Crossref PubMed Scopus (172) Google Scholar, 4Stern M. Kort A. Bhatnagar G. Lakatta E. J. Gen. Physiol. 1983; 82: 119-153Crossref PubMed Scopus (82) Google Scholar, 5Wier W. Kort A. Stern M. Lakatta E. Marban E. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 7367-7371Crossref PubMed Scopus (111) Google Scholar), a process we referred to as store overload-induced Ca2+ release (SOICR) (6Jiang D. Xiao B. Yang D. Wang R. Choi P. Zhang L. Cheng H. Chen S.R.W. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13062-13067Crossref PubMed Scopus (361) Google Scholar, 7Jiang D. Wang R. Xiao B. Kong H. Hunt D.J. Choi P. Zhang L. Chen S.R.W. Circ. Res. 2005; 97: 1173-1181Crossref PubMed Scopus (299) Google Scholar). Physical or emotional stresses, which activate the β-adrenergic receptor (AR)/protein kinase A (PKA) signaling pathway, are common triggers for SOICR. The activation of PKA leads to the phosphorylation of several key Ca2+ handling proteins, including the L-type Ca2+ channel, phospholamban (PLB), and RyR2. The outcome of this PKA activation is an increase in the SR Ca2+ load, SR Ca2+ release, and consequently cardiac output (8Bers D.M. Nature. 2002; 415: 198-205Crossref PubMed Scopus (3397) Google Scholar). Although the impact of PKA phosphorylation of the L-type Ca2+ channel and PLB on Ca2+ influx and SR Ca2+ uptake is well understood, the functional consequence and physiological significance of the phosphorylation of RyR2 by PKA remains unclear and controversial (9Bers D.M. Eisner D.A. Valdivia H.H. Circ. Res. 2003; 93: 487-490Crossref PubMed Scopus (250) Google Scholar). Marks' group has shown that RyR2 is phosphorylated by PKA at a single residue, Ser-2808 (10Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1686) Google Scholar, 11Wehrens X.H. Lehnart S.E. Reiken S. Vest J.A. Wronska A. Marks A.R. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 511-518Crossref PubMed Scopus (279) Google Scholar), which was originally identified as a unique Ca2+- and calmodulin-dependent protein kinase II phosphorylation site (12Witcher D.R. Kovacs R.J. Schulman H. Cefali D.C. Jones L.R. J. Biol. Chem. 1991; 266: 11144-11152Abstract Full Text PDF PubMed Google Scholar, 13Witcher D.R. Strifler B.A. Jones L.R. J. Biol. Chem. 1992; 267: 4963-4967Abstract Full Text PDF PubMed Google Scholar). They propose that in HF this residue is hyperphosphorylated by PKA, resulting in the dissociation of a 12.6-kDa FK506-binding protein (FKBP12.6) from RyR2 and consequently increasing the sensitivity of the channel to activation by cytosolic Ca2+ and enhancing SR Ca2+ leak. However, we have recently shown that RyR2 is phosphorylated by PKA at two major sites, Ser-2030 and Ser-2808, of which Ser-2030 is the major site responding to PKA activation upon β-adrenergic stimulation, and that there is no hyperphosphorylation of RyR2 by PKA in canine HF (14Xiao B. Jiang M.T. Zhao M. Yang D. Sutherland C. Lai F.A. Walsh M.P. Warltier D.C. Cheng H. Chen S.R.W. Circ. Res. 2005; 96: 847-855Crossref PubMed Scopus (169) Google Scholar). Jiang et al. (15Jiang M.T. Lokuta A.J. Farrell E.F. Wolff M.R. Haworth R.A. Valdivia H.H. Circ. Res. 2002; 91: 1015-1022Crossref PubMed Scopus (212) Google Scholar) have also demonstrated that there are no measurable differences in the phosphorylation level of RyR2 by PKA between failing and non-failing canine hearts. Moreover, stoichiometric phosphorylation of both native and recombinant RyR2 at Ser-2808 and mutations of Ser-2808 fail to dissociate FKBP12.6 from RyR2 (16Stange M. Xu L. Balshaw D. Yamaguchi N. Meissner G. J. Biol. Chem. 2003; 278: 51693-51702Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 17Xiao B. Sutherland C. Walsh M.P. Chen S.R.W. Circ. Res. 2004; 94: 487-495Crossref PubMed Scopus (142) Google Scholar). Furthermore, neither mutations of Ser-2808, nor the dissociation of FKBP12.6 affected the properties of RyR2 channels (16Stange M. Xu L. Balshaw D. Yamaguchi N. Meissner G. J. Biol. Chem. 2003; 278: 51693-51702Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 18Timerman A.P. Onoue H. Xin H.B. Barg S. Copello J. Wiederrecht G. Fleischer S. J. Biol. Chem. 1996; 271: 20385-20391Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). Hence the mechanism by which PKA modulates RyR2 requires further investigation. The results of most in vitro studies using isolated RyR2 and SR membrane vesicles indicate that the phosphorylation of RyR2 by PKA increases its open probability or its responsiveness to fast Ca2+ transients (10Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1686) Google Scholar, 19Hain J. Onoue H. Mayrleitner M. Fleischer S. Schindler H. J. Biol. Chem. 1995; 270: 2074-2081Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 20Valdivia H.H. Kaplan J.H. Ellis-Davies G.C. Lederer W.J. Science. 1995; 267: 1997-2000Crossref PubMed Scopus (319) Google Scholar, 21Uehara A. Yasukochi M. Mejia-Alvarez R. Fill M. Imanaga I. Pflügers Arch. 2002; 444: 202-212Crossref PubMed Scopus (32) Google Scholar, 22Carter S. Colyer

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