Ser-2030, but not Ser-2808, is the major phosphorylation site in cardiac ryanodine receptors responding to protein kinase A activation upon beta-adrenergic stimulation in normal and failing hearts

We have recently shown that RyR2 (cardiac ryanodine receptor) is phosphorylated by PKA (protein kinase A/cAMP-dependent protein kinase) at two major sites, Ser-2030 and Ser-2808. In the present study, we examined the properties and physiological relevance of phosphorylation of these two sites. Using site- and phospho-specific antibodies, we demonstrated that Ser-2030 of both recombinant and native RyR2 from a number of species was phosphorylated by PKA, indicating that Ser-2030 is a highly conserved PKA site. Furthermore, we found that the phosphorylation of Ser-2030 responded to isoproterenol (isoprenaline) stimulation in rat cardiac myocytes in a concentration- and time-dependent manner, whereas Ser-2808 was already substantially phosphorylated before beta-adrenergic stimulation, and the extent of the increase in Ser-2808 phosphorylation after beta-adrenergic stimulation was much less than that for Ser-2030. Interestingly, the isoproterenol-induced phosphorylation of Ser-2030, but not of Ser-2808, was markedly inhibited by PKI, a specific inhibitor of PKA. The basal phosphorylation of Ser-2808 was also insensitive to PKA inhibition. Moreover, Ser-2808, but not Ser-2030, was stoichiometrically phosphorylated by PKG (protein kinase G). In addition, we found no significant phosphorylation of RyR2 at the Ser-2030 PKA site in failing rat hearts. Importantly, isoproterenol stimulation markedly increased the phosphorylation of Ser-2030, but not of Ser-2808, in failing rat hearts. Taken together, these observations indicate that Ser-2030, but not Ser-2808, is the major PKA phosphorylation site in RyR2 responding to PKA activation upon beta-adrenergic stimulation in both normal and failing hearts, and that RyR2 is not hyperphosphorylated by PKA in heart failure. Our results also suggest that phosphorylation of RyR2 at Ser-2030 may be an important event associated with altered Ca2+ handling and cardiac arrhythmia that is commonly observed in heart failure upon beta-adrenergic stimulation.     

Three-dimensional localization of serine 2808, a phosphorylation site in cardiac ryanodine receptor

Type 2 ryanodine receptor (RyR2) is the major calcium release channel in cardiac muscle. Phosphorylation of RyR2 by cAMP-dependent protein kinase A and by calmodulin-dependent protein kinase II modulates channel activity. Hyperphosphorylation at a single amino acid residue, Ser-2808, has been proposed to directly disrupt the binding of a 12.6-kDa FK506-binding protein (FKBP12.6) to RyR2, causing a RyR2 malfunction that triggers cardiac arrhythmias in human heart failure. To determine the structural basis of the interaction between Ser-2808 and FKBP12.6, we have employed two independent approaches to map this phosphorylation site in RyR2 by three-dimensional cryo-electron microscopy. In one approach, we inserted a green fluorescent protein (GFP) after amino acid Tyr-2801, and mapped the GFP three-dimensional location in the RyR2 structure. In another approach, the binding site of monoclonal antibody 34C was mapped in the three-dimensional structure of skeletal muscle RyR1. The epitope of antibody 34C has been mapped to amino acid residues 2,756 through 2,803 of the RyR1 sequence, corresponding to residues 2,722 through 2,769 of the RyR2 sequence. These locations of GFP insertion and antibody binding are adjacent to one another in domain 6 of the cytoplasmic clamp region. Importantly, the three-dimensional location of the Ser-2808 phosphorylation site is 105-120 A distance from the FKBP12.6 binding site mapped previously, indicating that Ser-2808 is unlikely to be directly involved in the binding of FKBP12.6 to RyR2, as had been proposed previously.     

Three amino acid residues determine selective binding of FK506-binding protein 12.6 to the cardiac ryanodine receptor.

FK506-binding protein (FKBP12) has been found to be associated with the skeletal muscle ryanodine receptor (RyR1) (calcium release channel), whereas FKBP12.6, a novel isoform of FKBP, is selectively associated with the cardiac ryanodine receptor (RyR2). For both RyRs, the stoichiometry is 4 FKBP/RyR. Although FKBP12.6 differs from FKBP12 by only 18 of 108 amino acids, FKBP12.6 selectively binds to RyR2 and exchanges with bound FKBP12.6 of RyR2, whereas both FKBP isoforms bind to RyR1 and exchange with bound FKBP12 of RyR1. To assess the amino acid residues of FKBP12.6 that are critical for selective binding to RyR2, the residues of FKBP12.6 that differ with FKBP12 were mutated to the respective residues of FKBP12. RyR2 of cardiac sarcoplasmic reticulum, prelabeled by exchange with [35S]FKBP12.6, was used as assay system for binding/exchange with the mutants. The triple mutant (Q31E/N32D/F59W) of FKBP12.6 was found to lack selective binding to the cardiac RyR2, comparable with that of FKBP12.0. In complementary studies, mutations of FKBP12 to the three critical amino acids of FKBP12.6, conferred selective binding to RyR2. Each of the FKBP12.6 and FKBP12 mutants retained binding to the skeletal muscle RyR1. We conclude that three amino acid residues (Gln31, Asn32, and Phe59) of human FKBP12.6 account for the selective binding to cardiac RyR2.     

PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts

The ryanodine receptor (RyR)/calcium release channel on the sarcoplasmic reticulum (SR) is the major source of calcium (Ca2+) required for cardiac muscle excitation-contraction (EC) coupling. The channel is a tetramer comprised of four type 2 RyR polypeptides (RyR2) and four FK506 binding proteins (FKBP12.6). We show that protein kinase A (PKA) phosphorylation of RyR2 dissociates FKBP12.6 and regulates the channel open probability (Po). Using cosedimentation and coimmunoprecipitation we have defined a macromolecular complex comprised of RyR2, FKBP12.6, PKA, the protein phosphatases PP1 and PP2A, and an anchoring protein, mAKAP. In failing human hearts, RyR2 is PKA hyperphosphorylated, resulting in defective channel function due to increased sensitivity to Ca2+-induced activation.     

Phosphorylation-dependent regulation of ryanodine receptors: a novel role for leucine/isoleucine zippers

Ryanodine receptors (RyRs), intracellular calcium release channels required for cardiac and skeletal muscle contraction, are macromolecular complexes that include kinases and phosphatases. Phosphorylation/dephosphorylation plays a key role in regulating the function of many ion channels, including RyRs. However, the mechanism by which kinases and phosphatases are targeted to ion channels is not well understood. We have identified a novel mechanism involved in the formation of ion channel macromolecular complexes: kinase and phosphatase targeting proteins binding to ion channels via leucine/isoleucine zipper (LZ) motifs. Activation of kinases and phosphatases bound to RyR2 via LZs regulates phosphorylation of the channel, and disruption of kinase binding via LZ motifs prevents phosphorylation of RyR2. Elucidation of this new role for LZs in ion channel macromolecular complexes now permits: (a) rapid mapping of kinase and phosphatase targeting protein binding sites on ion channels; (b) predicting which kinases and phosphatases are likely to regulate a given ion channel; (c) rapid identification of novel kinase and phosphatase targeting proteins; and (d) tools for dissecting the role of kinases and phosphatases as modulators of ion channel function.     

Functional consequence of protein kinase A-dependent phosphorylation of the cardiac ryanodine receptor: sensitization of store overload-induced Ca2+ release

The phosphorylation of the cardiac Ca(2+)-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 2,030 and serine 2,808, of which Ser-2,030 is the major PKA site responding to beta-adrenergic stimulation. Here we investigated the effect of the phosphorylation of RyR2 by PKA on the properties of single channels and on spontaneous Ca(2+) release during sarcoplasmic reticulum Ca(2+) overload, a process we have referred to as store overload-induced Ca(2+) release (SOICR). We found that PKA activated single RyR2 channels in the presence, but not in the absence, of luminal Ca(2+). On the other hand, PKA had no marked effect on the sensitivity of the RyR2 channel to activation by cytosolic Ca(2+). Importantly, the S2030A mutation, but not mutations of Ser-2,808, diminished the effect of PKA on RyR2. Furthermore, a phosphomimetic mutation, S2030D, potentiated the response of RyR2 to luminal Ca(2+) 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 Ca(2+) release and the propagation velocity of spontaneous Ca(2+) waves, despite reduced wave amplitude and resting cytosolic Ca(2+). Collectively, our data indicate that PKA-dependent phosphorylation enhances the response of RyR2 to luminal Ca(2+) and reduces the threshold for SOICR and that this effect of PKA is largely mediated by phosphorylation at Ser-2,030.     

PKA phosphorylation activates the calcium release channel (ryanodine receptor) in skeletal muscle: defective regulation in heart failure

The type 1 ryanodine receptor (RyR1) on the sarcoplasmic reticulum (SR) is the major calcium (Ca2+) release channel required for skeletal muscle excitation-contraction (EC) coupling. RyR1 function is modulated by proteins that bind to its large cytoplasmic scaffold domain, including the FK506 binding protein (FKBP12) and PKA. PKA is activated during sympathetic nervous system (SNS) stimulation. We show that PKA phosphorylation of RyR1 at Ser2843 activates the channel by releasing FKBP12. When FKB12 is bound to RyR1, it inhibits the channel by stabilizing its closed state. RyR1 in skeletal muscle from animals with heart failure (HF), a chronic hyperadrenergic state, were PKA hyperphosphorylated, depleted of FKBP12, and exhibited increased activity, suggesting that the channels are "leaky." RyR1 PKA hyperphosphorylation correlated with impaired SR Ca2+ release and early fatigue in HF skeletal muscle. These findings identify a novel mechanism that regulates RyR1 function via PKA phosphorylation in response to SNS stimulation. PKA hyperphosphorylation of RyR1 may contribute to impaired skeletal muscle function in HF, suggesting that a generalized EC coupling myopathy may play a role in HF.     

FKBP12.6 deficiency and defective calcium release channel (ryanodine receptor) function linked to exercise-induced sudden cardiac death

Arrhythmias, a common cause of sudden cardiac death, can occur in structurally normal hearts, although the mechanism is not known. In cardiac muscle, the ryanodine receptor (RyR2) on the sarcoplasmic reticulum releases the calcium required for muscle contraction. The FK506 binding protein (FKBP12.6) stabilizes RyR2, preventing aberrant activation of the channel during the resting phase of the cardiac cycle. We show that during exercise, RyR2 phosphorylation by cAMP-dependent protein kinase A (PKA) partially dissociates FKBP12.6 from the channel, increasing intracellular Ca(2+) release and cardiac contractility. FKBP12.6(-/-) mice consistently exhibited exercise-induced cardiac ventricular arrhythmias that cause sudden cardiac death. Mutations in RyR2 linked to exercise-induced arrhythmias (in patients with catecholaminergic polymorphic ventricular tachycardia [CPVT]) reduced the affinity of FKBP12.6 for RyR2 and increased single-channel activity under conditions that simulate exercise. These data suggest that "leaky" RyR2 channels can trigger fatal cardiac arrhythmias, providing a possible explanation for CPVT.     

Three-dimensional visualization of FKBP12.6 binding to an open conformation of cardiac ryanodine receptor

The cardiac isoform of the ryanodine receptor (RyR2) from dog binds predominantly a 12.6-kDa isoform of the FK506-binding protein (FKBP12.6), whereas RyR2 from other species binds both FKBP12.6 and the closely related isoform FKBP12. The role played by FKBP12.6 in modulating calcium release by RyR2 is unclear at present. We have used cryoelectron microscopy and three-dimensional (3D) reconstruction techniques to determine the binding position of FKBP12.6 on the surface of canine RyR2. Buffer conditions that should favor the "open" state of RyR2 were used. Quantitative comparison of 3D reconstructions of RyR2 in the presence and absence of FKBP12.6 reveals that FKBP12.6 binds along the sides of the square-shaped cytoplasmic region of the receptor, adjacent to domain 9, which forms part of the four clamp (corner-forming) structures. The location of the FKBP12.6 binding site on "open" RyR2 appears similar, but slightly displaced (by 1-2 nm) from that found previously for FKBP12 binding to the skeletal muscle ryanodine receptor that was in the buffer that favors the "closed" state. The conformation of RyR2 containing bound FKBP12.6 differs considerably from that depleted of FKBP12.6, particularly in the transmembrane region and in the clamp structures. The x-ray structure of FKBP12.6 was docked into the region of the 3D reconstruction that is attributable to bound FKBP12.6, to show the relative orientations of amino acid residues (Gln-31, Asn-32, Phe-59) that have been implicated as being critical in interactions with RyR2. A thorough understanding of the structural basis of RyR2-FKBP12.6 interaction should aid in understanding the roles that have been proposed for FKBP12.6 in heart failure and in certain forms of sudden cardiac death.     

FKBP12.6 and cADPR regulation of Ca2+ release in smooth muscle cells

Intracellular Ca²⁺ release through ryanodine receptors (RyRs) plays important roles in smooth muscle excitation-contraction coupling, but the underlying regulatory mechanisms are poorly understood. Here we show that FK506 binding protein of 12.6 kDa (FKBP12.6) associates with and regulates type 2 RyRs (RyR2) in tracheal smooth muscle. FKBP12.6 binds to RyR2 but not other RyR or inositol 1,4,5-trisphosphate receptors, and FKBP12, known to bind to and modulate skeletal RyRs, does not associate with RyR2. When dialyzed into tracheal myocytes, cyclic ADP-ribose (cADPR) alters spontaneous Ca²⁺ release at lower concentrations and produces macroscopic Ca²⁺ release at higher concentrations; neurotransmitter-evoked Ca²⁺ release is also augmented by cADPR. These actions are mediated through FKBP12.6 because they are inhibited by molar excess of recombinant FKBP12.6 and are not observed in myocytes from FKBP12.6-knockout mice. We also report that force development in FKBP12.6-null mice, observed as a decrease in the concentration/tension relationship of isolated trachealis segments, is impaired. Taken together, these findings point to an important role of the FKBP12.6/RyR2 complex in stochastic (spontaneous) and receptor-mediated Ca²⁺ release in smooth muscle. FK506 binding protein 12.6; ryanodine receptor type 2; calcium sparks; calcium-activated chloride currents     

Targeting of protein kinase A by muscle A kinase-anchoring protein (mAKAP) regulates phosphorylation and function of the skeletal muscle ryanodine receptor

Protein kinase A anchoring proteins (AKAPs) tether cAMP-dependent protein kinase (PKA) to specific subcellular locations. The muscle AKAP, mAKAP, co-localizes with the sarcoplasmic reticulum Ca2+ release channel or ryanodine receptor (RyR). The purpose of this study was to determine whether anchoring of PKA by mAKAP regulates RyR function. Either mAKAP or mAKAP-P, which is unable to anchor PKA, was expressed in CHO cells stably expressing the skeletal muscle isoform of RyR (CHO-RyR1). Immunoelectron microscopy showed that mAKAP co-localized with RyR1 in disrupted skeletal muscle. Following the addition of 10 microm forskolin to activate adenylyl cyclase, RyR1 phosphorylation in CHO-RyR1 cells expressing mAKAP increased by 42.4 +/- 6.6% (n = 4) compared with cells expressing mAKAP-P. Forskolin treatment alone did not increase the amplitude of the cytosolic Ca2+ transient in CHO-RyR1 cells expressing mAKAP or mAKAP-P; however, forskolin plus 10 mm caffeine elicited a cytosolic Ca2+ transient, the amplitude of which increased by 22% (p < 0.05) in RyR1/mAKAP-expressing cells compared with RyR1/mAKAP-P-expressing cells. Therefore, localization of PKA by mAKAP at RyR1 increases both PKA-dependent RyR phosphorylation as well as efflux of Ca2+ through the RyR. Therefore, RyR1 function is regulated by mAKAP targeting of PKA, implying an important functional role for PKA phosphorylation of RyR in skeletal muscle.     

Localization of the dantrolene-binding sequence near the FK506-binding protein-binding site in the three-dimensional structure of the ryanodine receptor

Dantrolene is believed to stabilize interdomain interactions between the NH2-terminal and central regions of ryanodine receptors by binding to the NH2-terminal residues 590-609 in skeletal ryanodine receptor (RyR1) and residues 601-620 in cardiac ryanodine receptor (RyR2). To gain further insight into the structural basis of dantrolene action, we have attempted to localize the dantrolene-binding sequence in RyR1/RyR2 by using GFP as a structural marker and three-dimensional cryo-EM. We inserted GFP into RyR2 after residues Arg-626 and Tyr-846 to generate GFP-RyR2 fusion proteins, RyR2Arg-626-GFP and RyR2Tyr-846-GFP. Insertion of GFP after residue Arg-626 abolished the binding of a bulky GST- or cyan fluorescent protein-tagged FKBP12.6 but not the binding of a smaller, nontagged FKBP12.6, suggesting that residue Arg-626 and the dantrolene-binding sequence are located near the FKBP12.6-binding site. Using cryo-EM, we have mapped the three-dimensional location of Tyr-846-GFP to domain 9, which is also adjacent to the FKBP12.6-binding site. To further map the three-dimensional location of the dantrolene-binding sequence, we generated 10 FRET pairs based on four known three-dimensional locations (FKBP12.6, Ser-437-GFP, Tyr-846-GFP, and Ser-2367-GFP). Based on the FRET efficiencies of these FRET pairs and the corresponding distance relationships, we mapped the three-dimensional location of Arg-626-GFP or -cyan fluorescent protein, hence the dantrolene-binding sequence, to domain 9 near the FKBP12.6-binding site but distant to the central region around residue Ser-2367. An allosteric mechanism by which dantrolene stabilizes interdomain interactions between the NH2-terminal and central regions is proposed.     

Involvement of the cardiac ryanodine receptor/calcium release channel in catecholaminergic polymorphic ventricular tachycardia.

The cardiac ryanodine receptor (RyR2), the major calcium release channel on the sarcoplasmic reticulum (SR) in cardiomyocytes, has recently been shown to be involved in at least two forms of sudden cardiac death (SCD): (1) Catecholaminergic polymorphic ventricular tachycardia (CPVT) or familial polymorphic VT (FPVT); and (2) Arrhythmogenic right ventricular dysplasia type 2 (ARVD2). Eleven RyR2 missense mutations have been linked to these diseases. All eleven RyR2 mutations cluster into 3 regions of RyR2 that are homologous to the three malignant hyperthermia (MH)/central core disease (CCD) mutation regions of the skeletal muscle ryanodine receptor/calcium release channel RyR1. MH/CCD RyR1 mutations have been shown to alter calcium-induced calcium release. Sympathetic nervous system stimulation leads to phosphorylation of RyR2 by protein kinase A (PKA). PKA phosphorylation of RyR2 activates the channel. In conditions associated with high rates of SCD such as heart failure RyR2 is PKA hyperphosphorylated resulting in "leaky" channels. SR calcium leak during diastole can generate "delayed after depolarizations" that can trigger fatal cardiac arrhythmias (e.g., VT). We propose that RyR2 mutations linked to genetic forms of catecholaminergic-induced SCD may alter the regulation of the channel resulting in increased SR calcium leak during sympathetic stimulation.     

Central domain of the human cardiac muscle ryanodine receptor does not mediate interaction with FKBP12.6

The immunophilin, FK506-binding protein (FKBP12), is an essential component of the ryanodine receptor channel complex of skeletal muscle (RyR1) and modulates intracellular calcium signaling from the nedoplasmic reticulum. The cardiac muscle RyR isoform (RyR2) specifically associates with a distinct FKBP isoform, FKBP12.6. Previous studies have led to the proposal that the central domain of RyR1 exclusively mediates the interaction with FKBP12. To characterize the topography of the FKBP 12.6 binding site on the human cardiac RyR2, we have applied complementary protein-protein interaction methods using both in vivo yeast two-hybrid analysis and in vitro immunoprecipitation experiments. Our results indicate an absence of interaction of FKBP12/12.6 with fragments containin the central domain of either RyR1, RyR2, or RyR3. Furthermore, no interaction was detected between FKBP12.6 with a series of overlapping fragments encompassing the entire RyR2, either individually or in multiple combination. We also found that a distinct, alternatively spliced variant of FKBP12.6 was unable to interact with RyR. In contrast, we successfully demonstrated a robust association between the cytoplasmic domain of transforming growth factor-β receptor type I and both FKBP12 and FKBP12.6 in parallel positive control experiments, as well as between native RyR2 and FKBP12.6. These results suggest that the specific interaction of FKBP12.6 with RyR2, and generally of FKBPs with any RyR isoform, is not readily reconstituted by peptide fragments corresponding to central RyR domains. Further structural analysis will be necessary to unravel this intricate signaling system and the current model of FKBP-12-RyR interaction via a single, central RyR, epitope may therefore require revision.     

Interaction of FKBP12.6 with the cardiac ryanodine receptor C-terminal domain

The ryanodine receptor-calcium release channel complex (RyR) plays a pivotal role in excitation-contraction coupling in skeletal and cardiac muscle. RyR channel activity is modulated by interaction with FK506-binding protein (FKBP), and disruption of the RyR-FKBP association has been implicated in cardiomyopathy, cardiac hypertrophy, and heart failure. Evidence for an interaction between RyR and FKBP is well documented, both in skeletal muscle (RyR1-FKBP12) and in cardiac muscle (RyR2-FKBP12.6), however definition of the FKBP-binding site remains elusive. Early reports proposed interaction of a short RyR central domain with FKBP12/12.6, however this site has been questioned, and recently an alternative FKBP12.6 interaction site has been identified within the N-terminal half of RyR2. In this study, we report evidence for the human RyR2 C-terminal domain as a novel FKBP12.6-binding site. Using competition binding assays, we find that short C-terminal RyR2 fragments can displace bound FKBP12.6 from the native RyR2, although they are unable to exclusively support interaction with FKBP12.6. However, expression of a large RyR2 C-terminal construct in mammalian cells encompassing the pore-forming transmembrane domains exhibits rapamycin-sensitive binding specifically to FKBP12.6 but not to FKBP12. We also obtained some evidence for involvement of the RyR2 N-terminal, but not the central domain, in FKBP12.6 interaction. Our studies suggest that a novel interaction site for FKBP12.6 may be present at the RyR2 C terminus, proximal to the channel pore, a sterically appropriate location that would enable this protein to play a central role in the modulation of this critical ion channel.     

The Ca 2+ leak paradox and rogue ryanodine receptors: SR Ca 2+ efflux theory and practice

Ca²⁺ efflux from the sarcoplasmic reticulum (SR) is routed primarily through SR Ca²⁺ release channels (ryanodine receptors, RyRs). When clusters of RyRs are activated by trigger Ca²⁺ influx through L-type Ca²⁺ channels (dihydropyridine receptors, DHPR), Ca²⁺ sparks are observed. Close spatial coupling between DHPRs and RyR clusters and the relative insensitivity of RyRs to be triggered by Ca²⁺ together ensure the stability of this positive-feedback system of Ca²⁺ amplification. Despite evidence from single channel RyR gating experiments that phosphorylation of RyRs by protein kinase A (PKA) or calcium-calmodulin dependent protein kinase II (CAMK II) causes an increase in the sensitivity of the RyR to be triggered by [Ca²⁺]_i there is little clear evidence to date showing an increase in Ca²⁺ spark rate. Indeed, there is some evidence that the SR Ca²⁺ content may be decreased in hyperadrenergic disease states. The question is whether or not these observations are compatible with each other and with the development of arrhythmogenic extrasystoles that can occur under these conditions. Furthermore, the appearance of an increase in the SR Ca²⁺ “leak” under these conditions is perplexing. These and related complexities are analyzed and discussed in this report. Using simple mathematical modeling discussed in the context of recent experimental findings, a possible resolution to this paradox is proposed. The resolution depends upon two features of SR function that have not been confirmed directly but are broadly consistent with several lines of indirect evidence: (1) the existence of unclustered or “rogue” RyRs that may respond differently to local [Ca²⁺]_i in diastole and during the [Ca²⁺]_i transient; and (2) a decrease in cooperative or coupled gating between clustered RyRs in response to physiologic phosphorylation or hyper-phosphorylation of RyRs in disease states such as heart failure. Taken together, these two features may provide a framework that allows for an improved understanding of cardiac Ca²⁺ signaling.     

Ryanodine receptors in muscarinic receptor-mediated bronchoconstriction

Ryanodine receptors (RyRs), intracellular calcium release channels essential for skeletal and cardiac muscle contraction, are also expressed in various types of smooth muscle cells. In particular, recent studies have suggested that in airway smooth muscle cells (ASMCs) provoked by spasmogens, stored calcium release by the cardiac isoform of RyR (RyR2) contributes to the calcium response that leads to airway constriction (bronchoconstriction). Here we report that mouse ASMCs also express the skeletal muscle and brain isoforms of RyRs (RyR1 and RyR3, respectively). In these cells, RyR1 is localized to the periphery near the cell membrane, whereas RyR3 is more centrally localized. Moreover, RyR1 and/or RyR3 in mouse airway smooth muscle also appear to mediate bronchoconstriction caused by the muscarinic receptor agonist carbachol. Inhibiting all RyR isoforms with > or = 200 microM ryanodine attenuated the graded carbachol-induced contractile responses of mouse bronchial rings and calcium responses of ASMCs throughout the range of carbachol used (50 nM to > or = 3 microM). In contrast, inhibiting only RyR1 and RyR3 with 25 microM dantrolene attenuated these responses caused by high (>500 nM) but not by low concentrations of carbachol. These data suggest that, as the stimulation of muscarinic receptor in the airway smooth muscle increases, RyR1 and/or RyR3 also mediate the calcium response and thus bronchoconstriction. Our findings provide new insights into the complex calcium signaling in ASMCs and suggest that RyRs are potential therapeutic targets in bronchospastic disorders such as asthma.     

Novel in vitro and in vivo phosphorylation sites on protein phosphatase 1 inhibitor CPI-17

CPI-17 is a protein phosphatase 1 (PP1) inhibitor that has been shown to act on the myosin light chain phosphatase. CPI-17 is phosphorylated on Thr-38 in vivo, thus enhancing its ability to inhibit PP1. Thr-38 has been shown to be the target of several protein kinases in vitro. Originally, the expression of CPI-17 was proposed to be smooth muscle specific. However, it has recently been found in platelets and we show in this report that it is endogenously phosphorylated in brain on Ser-128 in a domain unique to CPI-17. Ser-128 is within a consensus phosphorylation site for protein kinase A (PKA) and calcium calmodulin kinase II. However, these two kinases do not phosphorylate Ser-128 in vitro but phosphorylate Ser-130 and Thr-38, respectively. The kinase responsible for Ser-128 phosphorylation remains to be identified. CPI-17 has strong sequence similarity with PHI-1 (which is also a phosphatase inhibitor) and LimK-2 kinase. The novel in vivo and in vitro phosphorylation sites (serines 128 and 130) are in a region/domain unique to CPI-17, suggesting a specific interaction domain that is regulated by phosphorylation.     

Molecular regulation of cardiac ryanodine receptor ion channel

The release of Ca(2+) ions from intracellular stores is a key step in a wide variety of cellular functions. In striated muscle, the release of Ca(2+) from the sarcoplasmic reticulum (SR) leads to muscle contraction. Ca(2+) release occurs through large, high-conductance Ca(2+) release channels, also known as ryanodine receptors (RyRs) because they bind the plant alkaloid ryanodine with high affinity and specificity. The RyRs are isolated as 30S protein complexes comprised of four 560 kDa RyR2 subunits and four 12 kDa FK506 binding protein (FKBP12) subunits. Multiple endogenous effector molecules and posttranslational modifications regulate the RyRs. This review focuses on current research toward understanding the control of the isolated cardiac Ca(2+) release channel/ryanodine receptor (RyR2) by Ca(2+), calmodulin, thiol oxidation/reduction and nitrosylation, and protein phosphorylation.     

Functional properties of ryanodine receptors from rat dorsal root ganglia

The properties of ryanodine receptors (RyRs) from rat dorsal root ganglia (DRGs) have been studied. The density of RyRs (Bmax) determined by [3H]ryanodine binding was 63 fmol/mg protein with a dissociation constant (Kd) of 1.5 nM. [3H]Ryanodine binding increased with caffeine, decreased with ruthenium red and tetracaine, and was insensitive to millimolar concentrations of Mg2+ or Ca2+. DRG RyRs reconstituted in planar lipid bilayers were Ca2+-dependent and displayed the classical long-lived subconductance state in response to ryanodine; however, unlike cardiac and skeletal RyRs, they lacked Ca2+-dependent inactivation. Antibodies against RyR3, but not against RyR1 or RyR2, detected DRG RyRs. Thus, DRG RyRs are immunologically related to RyR3, but their lack of divalent cation inhibition is unique among RyR subtypes.