Biol Res 37: 521-525, 2004

Phosphorylation of Ryanodine Receptors

CRISTINA I. DANILA and SUSAN L. HAMILTON

Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030, USA

Dirección para Correspondencia

ABSTRACT

Both cardiac and skeletal muscle ryanodine receptors (RyRs) are parts of large complexes that include a number of kinases and phosphatases. These RyRs have several potential phosphorylation sites in their cytoplasmic domains, but the functional consequences of phosphorylation and the identity of the enzymes responsible have been subjects of considerable controversy. Hyperphosphorylation of Ser-2809 in RyR2 (cardiac isoform) and Ser-2843 in RyR1 (skeletal isoform) has been suggested to cause the dissociation of the FK506-binding protein (FKBP) from RyRs, producing "leaky channels," but some laboratories find no relationship between phosphorylation and FKBP binding. Also debated is the identity of the kinases that phosphorylate these serines: cAMP-dependent protein kinase (PKA) versus calmodulin kinase II (CaMKII). Phosphorylation of other targets of these kinases could also alter calcium homeostasis. For example, PKA also phosphorylates phospholamban (PLB), altering the Sarco-endoplasmic reticulum Ca²⁺ ATPase (SERCA) activity. This review summarizes the major findings and controversies associated with phosphorylation of RyRs.

Key words: ryanodine receptor, phosphorylation site, protein kinase, Ser-2809/Ser-2843, FK506-binding protein

Excitation-contraction (E-C) coupling is the process in which the depolarization of muscle fiber membrane elicited by neuromuscular transmission triggers the release of Ca²⁺ from the sarcoplasmic reticulum (SR). The rise in cytosolic Ca²⁺ activates a series of events that culminate in muscle contraction. Resting Ca²⁺ levels are restored primarily by the SERCA action in the longitudinal SR, allowing the muscle to relax.

Two ion channels, an L-type Ca²⁺ channel (dihydropyridine receptor, DHPR) and a calcium release channel (ryanodine receptor, RyR), are involved in the release of Ca²⁺ from SR internal stores. In the skeletal muscle, E-C coupling is mediated by a direct physical interaction between DHPR, positioned in transverse tubular infoldings (t-tubules), and the skeletal muscle RyR1 in the sarcoplasmic reticulum (see Franzini-Armstrong, this issue). This process is defined as voltage activated calcium-release (VAC). In the cardiac muscle, E-C coupling requires the influx of Ca²⁺ ions through the DHPR channels, and the entering Ca²⁺ triggers Ca²⁺ release via RyR2. This type of release is term calcium-induced calcium release (CICR).

RyRs are members of a family of Ca²⁺ release channels found in intracellular Ca²⁺ storage/release organelles (i.e.: sarco/endoplasmic reticulum) of a variety of cells. The 3 mammalian RyRs, RyR1, RyR2, and RyR3, share over 60% amino acid sequence identity and are encoded by separate genes. Isoform 1 (RyR1) is highly enriched in skeletal muscle, isoform 2 (RyR2) is enriched in the cardiac muscle, and isoform 3 (RyR3), along with RyR1 and RyR2, is found at lower levels in a variety of tissues. These channels are the largest known ion channels. All isoforms have a large cytoplasmic domain (~ 4/5 of the molecule) and a smaller hydrophobic membrane-spanning region (~1/5 of the molecule) and are homotetramers with each subunit having a molecular mass of around 565 kDa.

Several modulatory molecules bind directly to RyRs, including, on the cytoplasmic side of the sarcoplasmic reticulum (SR) membrane, Ca²⁺, Mg²⁺, ATP, calmodulin and FK506-binding proteins (FKBP). The FKBPs belong to the family of immunophilins (ie.: proteins that bind the immunosuppressive drugs, FK506 and rapamycin) and have cis-trans prolyl isomerase activity (PPIase or rotamase). Their role appears to not only stabilize RyR conformation (Brillantes et al., 1994) but also synchronize the gating of neighboring RyRs, coupled gating (Marx et al., 1998, 2001). Recent studies have shown that FKBP12 and FKBP12.6 bind to both RyR1 and RyR2 (Jeyakumar et al., 2001). Although RyR1 has a higher affinity for FKBP12.6 than for FKBP12, the higher tissue concentrations of FKBP12 result in FKBP12 being the predominant form bound to RyR1. In cardiac tissue the concentration of FKBP12 is considerably higher than FKBP12.6, but the preferential interaction of FKBP12.6 with RyR2 is driven by the higher affinity of RyR2 for FKBP12.6 compared to FKBP12 (Timerman et al., 1996).

RyRs in skeletal and cardiac tissue are part of large macromolecular complexes that include cAMP-dependent protein kinase (PKA) and its muscle protein kinase A-anchoring protein (m-AKAP), protein phosphatases (PP1 in skeletal and cardiac and PP2A, only in cardiac muscle) and their anchoring proteins (spinophilin and PR130), and calcineurin (PP2B). Marks et al., (2002) found that the targeting of PKA, PP1, and PP2A to RyR2 is dependent upon the binding of targeting proteins to the channel via highly conserved leucine/isoleucine zippers (LIZs) motifs.

Protein phosphorylation is a fundamental mechanism in which the interplay of kinases and phosphatases regulate a wide variety of biological processes in living cells. Modulation of skeletal and cardiac E-C coupling results, in part, from changes in the phosphorylation state of numerous SR proteins involved in calcium homeostasis. Changes in the levels of phosphorylation might contribute to an impaired contractile function of the striated muscle.

In its cytoplasmic region RyR has many conserved, potential phosphorylation sites. Several studies have suggested that phosphorylation of RyR2 Ser-2809 in rabbit sequence (Otsu et al., 1990) or Ser-2808 in human sequence (Tunwell et al., 1996) is the site of in vitro phosphorylation (Marx et al., 2000; Witcher et al., 1991). In the skeletal RyR1 the corresponding Ser-2843 is also phosphorylated in vitro (Suko et al., 1993). CaMKII (CaM-dependent protein kinase) was originally identified as the kinase responsible for the phosphorylation of these serines. More recently, PKA (cAMP-dependent protein kinase) has been shown to also phosphorylate these sites. CaMKII in vitro phosphorylates four sites in addition to Ser-2809 of RyR2 (Rodriguez et al., 2003). Other kinases, such as protein kinase C and protein kinase G, can phosphorylate RyR1 in vitro, but the physiological relevance of these phosphorylation events remains to be established (Suko et. al. 1993). Phosphorylation of RyRs at Ser-2809 in RyR2 and Ser-2843 in RyR1 has been shown to cause the release of FKBPs from the RyRs and to increased channel activity. Marx and coworkers (2000) suggested that the stimulation of the sympathetic nervous system (SNS) results in PKA phosphorylation of the RyR and modulation of the channel. The "fight or flight" response is a classic stress pathway that involves activation of the SNS, leading to b-adrenergic stimulation of the muscle. During SNS stimulation, cathecolamines bind to b-adrenergic receptors, activate adenylate cyclase via G-proteins, and increase the intracellular levels of the second messenger cAMP, thus activating PKA.

Marks and coworkers (Marx et al., 2000, Reiken et al., 2003) found that PKA hyperphosphorylation of RyR2 and RyR1 at Ser-2809/Ser-2843 in animal models of heart failure (HF) and human failing hearts shifted the sensitivity of RyRs to CICR, resulting in "leaky" channels that produce diastolic Ca²⁺ release, and a reduced SR Ca²⁺ load. These findings suggest that specific serine phosphorylation may underlie the generalized EC coupling defects found in both types of striated muscles (cardiac and skeletal) in heart failure. The proposed "culprit" in this process was the increased dissociation of FKBP from RYRs, which allowed channels to open more readily. This would suggest that the physiologic role of FKBP is to stabilize the closed state of the channel. Marks and colleagues identified the PKA phosphorylation site using site-directed mutagenesis. The mutation of this serine to an alanine mimicked the dephosphorylated state while mutation to an aspartic acid mimicked a constitutively phosphorylated state of RyR. The functional consequences of these mutations were analyzed following transient transfection in HEK293 cells. Single channel recordings showed that the mutant RyR2 (S2809A) that mimics the dephosphorylated RyR2 had properties similar to native RyR2. The mutants with the S2809D mutation that mimic phosphorylation had an increased open-probability compared with the wild type RyR2. In FKBP12.6 binding assays it was found that the S2809A mutant bound to FKBP12.6 was similar to wildtype (wt) RYR2, but S2809D displayed a significant reduction in the amount of bound FKBP12.6.

RyR1 from normal skeletal muscle is also phosphorylated in vitro by PKA. Using coimmunoprecipitation and immunoblotting, Reiken et al. (2003), found that in animal models of HF there was a significant reduction in amount of FKBP12 bound to RYR1 compared to normal muscle. An examination of Ca²⁺ sparks showed slow, reduced amplitude, and longer-lasting SR Ca²⁺ release events. These workers proposed that a chronic hyperadrenergic state during HF alters RyRs phosphorylation and represents the underlying mechanism for the E-C myopathy. This implies that a possible therapeutic target for HF could be one of the components involved in PKA phosphorylation and dephosphorylation of RyRs. This novel hypothesis has, however, been challenged by a number of other laboratories. Using a similar approach, Stange et al., (2003) prepared four recombinant RyRs that carry the Ser mutations: RyR1-S2843D, RyR2-S2809D, RyR1-S2843A, and RyR2-S2809A. These constructs were transiently transfected into HEK293 and the functional and biochemical effects of these RyR mutants were evaluated. These workers also coimmunoprecipitated RyRs and FKBPs. The phosphorylated mutants displayed no significant alterations in their FKBP/RyR binding ratio. The single channel measurements using the planar lipid bilayer method showed no differences in either the open probability or the gating of the mutant channels compared to wtRyR. [³H]-ryanodine, widely used because of its preferential binding to the open channel state, was also unaffected by the mutations. In addition to Ser-2809, Stange et al., (2003) suggested the possibility of additional phosphorylation sites in RyR2.

The PKA hyperphosphorylation /FKBP dissociation hypothesis has also been challenged by Xiao et al., (2004). These workers assessed the ability of FKBP12.6 to bind to Ser-2808 phosphorylated RyR2 and S2808D mutant. They also evaluated the ability of PKA phosphorylation at Ser-2808 to dissociate FKBP12.6 from RyR2. Sequence-specific antibodies, which recognize the phosphorylated and nonphosphorylated Ser-2808 site, were used in these studies. A significant proportion of the RyR2 channels appeared to be phosphorylated at Ser-2808 even prior to the addition of exogenous kinases. FKBP12.6 was found to bind to both S2808D and S2808A mutants of RYR2 to a similar extent as to wildtype RYR2. The binding of FKBP12.6 was abolished in the presence of rapamycin. In addition, phosphorylation of the expressed RyR2s by exogenous PKA did not dissociate FKBP12.6.

The discrepancies between studies could arise from the different experimental conditions used among different laboratories. Another possibility is that PKA phosphorylation of RyR2 at Ser-2808 is, in itself, insufficient to dissociate FKBP12.6 from RyR2. Li et al., (2002) suggested that clear cellular data on PKA-dependent modulation of cardiac RyR are limited because of the difficulty in distinguishing between PKA effects on RyR, phospholamban (PLB), and Ca²⁺ currents. They measured resting Ca²⁺ sparks in streptolysin-O permeabilized ventricular myocytes from wild type and PLB knockout (PLB-KO) and transgenic mice expressing only double-mutant PLB (PLB-DM) that lacks the regulatory phosphorylation sites (S16A/T17A) and concluded that PKA-dependent RyR phosphorylation does not affect resting SR Ca²⁺ leak in these myocytes. They suggested that the increased Ca²⁺ spark frequency seen in wild-type mice is entirely dependent on PLB phosphorylation and subsequent increases in SR Ca²⁺ content. In support of this interpretation, Jiang et al., (2002) found that the phosphorylation levels and basal activity of the RyR2 in a canine model of HF and human failing hearts were indistinguishable from those of RyR2 from normal hearts. In their study RyRs remain largely unaffected by the pathophysiological mechanisms that occur in HF, and that an abnormal Ca²⁺ uptake is more likely to contribute to the depressed and slowed Ca²⁺ transient characteristic of HF. Decreased SERCA 2a activity may lead to depressed SR Ca²⁺ load and, indirectly, to reduced Ca²⁺ release.

Terentyev et al., (2003) found that dephosphorylation of RyR2 rather than phosphorylation enhances the channel activity. They investigated the effects of protein phosphatases PP1 and PP2A on spontaneous Ca²⁺ sparks and SR Ca²⁺ load in myocytes permeabilized with saponin. PP1 or PP2A caused a dramatic increase in frequency of Ca²⁺ sparks followed by a nearly complete disappearance of events. These effects were accompanied by depletion of the SR Ca²⁺ stores, as determined by application of caffeine. These changes in Ca²⁺ release and SR Ca²⁺ load could be prevented by the inhibitors of PP1 and PP2A phosphatase activities. At the single channel level, PP1 increased the open probability of RyRs incorporated into lipid bilayers. PP1-mediated RyR dephosphorylation was confirmed biochemically by quantitative immunoblotting using a phospho-specific anti-RyR antibody.

Recently, a new piece to the puzzle was added by Wehrens et al., (2004). Using site-directed mutagenesis technique, they found a specific Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) phosphorylation site on recombinant RyR2 distinct from the site for protein kinase A (PKA) phosphorylation that mediated the "fight-or-flight" stress response. The CaM kinase II phosphorylation site is Ser-2815. CaMKII activated by increased heart rates phosphorylates RyR2 and enhances Ca²⁺-induced Ca²⁺ release. Moreover, rate-dependent CaMKII phosphorylation of RyR2 was defective in heart failure. CaMKII-mediated phosphorylation of RyR2 may contribute to the enhanced contractility observed at higher heart rates.

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Corresponding author: Susan L. Hamilton, Department of Molecular Physiology and Biophysics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. Phone: (713) 798-3894, Fax: (713) 798-5441, E-mail: susanh@bcm.tmc.edu

Received: May 24, 2004. Accepted: July 2, 2004.