Stoichiometric Phosphorylation of Cardiac Ryanodine Receptor on Serine 2809 by Calmodulin-dependent Kinase II and Protein Kinase A

Abstract

The ryanodine receptor of cardiac muscle performs a central role in excitation-contraction coupling. Phosphorylation of the channel on serine 2809 (in rabbit or the corresponding serine 2808 in man) alters function in vitro, although the impact of this in vivo has not been established. We have produced a pair of antisera to the serine 2809 phosphorylation site to aid description of the incidence and consequence of phosphorylation of this receptor. One of these antisera is specific for the serine 2809 phosphorylated form of the cardiac ryanodine receptor; the other antiserum is specific for the serine 2809 dephosphorylated receptor. These antibodies have been used to demonstrate that both protein kinase A and calmodulin-dependent kinase II are capable of phosphorylating serine 2809 in vitro. Both kinases phosphorylate serine 2809 to full stoichiometry, but this is accompanied by the incorporation of more (radioactive) phosphate into the receptor by calmodulin-dependent kinase II than by protein kinase A. These data suggest that calmodulin-dependent kinase II phosphorylates at least four sites in addition to serine 2809 in vitro.The ryanodine receptor (RYR2) of cardiac muscle plays a central role in the coupling of electrical excitation of the muscle to mechanical contraction. It is a Ca 2ϩ channel, which resides primarily in the sarcoplasmic reticulum (SR) 1 at junctions between this organelle and the t-tubular system (a specialized invaginated domain of the plasma membrane). Upon depolarization of the plasma membrane, Ca 2ϩ enters the cell across the t-tubule membrane and interacts with the RYR2. Ca 2ϩ binding to RYR2 opens the channel, and Ca 2ϩ stored in the SR moves through the channel into the cytosol to initiate contraction (1).A variety of strategies are used by the cell to regulate RYR2 channel activity. It is anticipated that these regulatory strategies facilitate the fine control of E-C coupling, although evidence for this in live cells is rather limited. In vitro studies have shown that the binding of Ca 2ϩ (2), Mg 2ϩ (3), ATP (4), cADP ribose (5), calmodulin (6), and FKBP12.6 (7) affect channel activity, as does the binding of pharmacological agents such as the plant alkaloid ryanodine (which was originally used to identify the channel protein; Ref. 8). In addition the channel is phosphorylated on at least a single residue, and this phosphorylation alters channel behavior in vitro (7,9).In an effort to understand the regulatory role of RYR2 phosphorylation, research has focused on three aspects of the process. First, the identity of sites of phosphorylation in the receptor and kinases capable of using these sites; second, the functional consequence of site-specific phosphorylation in vitro; and third, the incidence and functional consequence of sitespecific phosphorylation of the receptor in living cells. To date Ser-2809 (in the rabbit sequence (10) or the corresponding Ser-2808 in man (11)) has been identified as a site of phosphorylation on RYR2, which is used in vitro (7,9). It has a counterpart in...

Cardiac ryanodine receptor phosphorylation: target sites and functional consequences

“…It seems likely that the only appreciable PKA-dependent phosphorylation occurs at Ser-2030 and Ser-2808, since mutating both serine sites to alanine in RyR2 abolishes PKA-dependent phosphate incorporation [17]. However, Rodriguez et al [16] showed that the stoichiometry of RyR2 phosphorylation by CaMKII was 4-fold higher than for PKA, and this is consistent with greater amounts of 32 P incorporation via CaMKII than PKA [18,19]. Thus CaMKII can surely phosphorylate RyR2 at Ser-2814, and probably also at Ser-2808, and may also have as yet unidentified sites.…”

“…As attractive as this working hypothesis is, the attention it has received and its extensive testing by this group, it remains controversial. This is because numerous laboratory groups have found results that do not support different aspects of this hypothesis [1,[13][14][15][16][17]. These include findings that RyR2 is not hyperphosphorylated by PKA in HF, that PKA-dependent phosphorylation does not cause FKBP12.6 release, that PKA activation does not increase basal RyR2 activation, that there are additional PKA sites on RyR2 and that Ser-2809 can also be phosphorylated by CaMKII or PKG (protein kinase G).…”

Ryanodine Receptors: Structure, Expression, Molecular Details, and Function in Calcium Release

“…RyRs have several potential phosphorylation sites in their cytoplasmic domains. PKA, CaMKII, and cGMP-dependent kinase (PKG) have all been shown to phosphorylate RyR isoforms (Rodriguez et al 2003;Wehrens et al 2004;Xiao et al 2006;Huke and Bers 2007).…”

CaMKII inhibition targeted to the sarcoplasmic reticulum inhibits frequency-dependent acceleration of relaxation and Ca2+ current facilitation

“…Reduced SR Ca 2+ leak explains unaltered SR Ca 2+ load despite decreased SERCA activity CaMKII associates with and directly phosphorylates the cardiac RyR [19,34,48,49]. This leads to increased RyR channel opening in bilayers and to an increased Ca 2+ spark frequency in myocytes at a given SR Ca 2+ content [3,19,34,[48][49][50].…”

“…This leads to increased RyR channel opening in bilayers and to an increased Ca 2+ spark frequency in myocytes at a given SR Ca 2+ content [3,19,34,[48][49][50]. Controversy remains about which RyR amino acid is the critical CaMKII target (Ser2809 vs. Ser2815) [19,48,49].…”