Search

The phosphorylation of the cardiac calcium release channel/ryanodine receptor (RyR2) by protein kinase A (PKA) has been extensively investigated, but its functional consequences remain poorly understood and controversial. We have previously shown that S2030 is a major PKA site in RyR2 responding to beta-adrenergic stimulation, and that phosphomimetic mutation of this PKA site (S2030D) enhances luminal calcium activation of single RyR2 channels and increases the propensity for spontaneous calcium release in HEK293 cells during store calcium overload. To further investigate the physiological and pathophysiological significance of PKA phosphorylation of S2030, we generated a knock-in mouse model harboring the phosphomimetic S2030D mutation in RyR2. We found that S2030D mutant mice displayed higher isoproterenol-induced heart rate increase than wt mice. ECG recordings revealed that S2030D mutant mice were more susceptible to bidirectional ventricular tachycardia induced by the injection of epinephrine plus caffeine as compared to wt mice. To determine the impact of the S2030D mutation on sarcoplasmic reticulum (SR) calcium handling, ventricular myocytes isolated from wt and mutant mice were loaded with fluo-4 and field-stimulated. Calcium imaging showed that S2030D mutant cells exhibited enhanced spontaneous calcium release activity in the presence of isoproterenol. These observations indicate that the S2030D mutation in RyR2 enhances the beta-adrenergic response, and suggest that excessive PKA phosphorylation of S2030 in RyR2 may increase the propensity for stress-induced spontaneous SR calcium release and ventricular arrhythmias (supported by CIHR).

Global conformational changes in the three-dimensional (3D) structure of the calcium release channel/ryanodine receptor (RyR) occur upon ligand activation. Of several ligands that activate RyR, Ca2+ is the primary activator. Although RyR Ca2+ activation is well characterized functionally, little is known about the conformational changes in RyR induced by Ca2+. Here we generated three fluorescence resonance energy transfer (FRET)-based conformational probes. Each of these probes was constructed by inserting a CFP into one domain and a YFP into a neighboring domain in the cardiac RyR (RyR2) to yield a CFP- and YFP-dual labeled RyR2 (CFP/YFP FRET pair). These CFP/YFP FRET pairs were located in the “clamp” region (RyR2S2367-CFP/Y2801-YFP), the calmodulin binding region (RyR2R3595-CFP/K4269-YFP), and the “bridge” region (RyR2S437-YFP/S2367-CFP), respectively. We monitored the conformational changes in these regions by recording the FRET signals, and the extent of Ca2+ release by measuring store Ca2+ depletion in HEK293 cells expressing each of the CFP/YFP FRET pairs upon activation by Ca2+, caffeine, and ATP. Surprisingly, we found that different ligands induced different conformational changes in different regions of RyR2. For instance, we detected conformational changes in the clamp region for caffeine and ATP, but not for Ca2+, although they all induced Ca2+ release. Considering Ca2+ as the primary activator of RyR2, we determined the impact of cytosolic Ca2+ sensing mutation E3987A on conformational changes. Interestingly, this single mutation abolishes caffeine-induced conformational changes, but not caffeine-induced Ca2+ release. These observations demonstrate that conformational changes in RyR2 are ligand-dependent, and that E3987, which is critical for cytosolic Ca2+ sensing, is essential for ligand-induced conformational changes (Supported by CFI, CIHR, HSFC, NIH, and LCIA).

Calmodulin (CaM), a 16 kDa ubiquitous calcium-sensing protein, is known to bind tightly to the cardiac calcium release channel/ryanodine receptor (RyR2) at low and high Ca2+ concentrations, and modulate the function of the channel. CaM binding studies using RyR fragments or synthetic peptides have revealed that multiple regions in the RyR's primary sequence may be involved in CaM binding. However, the locations of these potential CaM binding regions in the three dimensional structure of RyRs have yet to be determined. In the present study, we inserted GFP or GST into these proposed CaM binding sequences and mapped some of them onto the three-dimensional structure of intact RyR2 by cryo-electron microscopy and single particle image analysis. Surprisingly, we found that some of these potential CaM binding regions, e.g. Arg-3595 and Lys-4269, are located in close proximity and are adjacent to the CaM binding sites that were mapped previously by 3D cryo-EM. These observations suggest that multiple regions in the RyR2 sequence may form a binding pocket for CaM. (Supported by NIH and CIHR).

Malignant Hyperthermia (MH) and Central Core Disease (CCD) are skeletal muscle disorders linked to mutations in the skeletal muscle ryanodine receptor (RyR1). Based on their phenotypes, disease-causing RyR1 mutations can be separated into three major groups: MH-only, both MH and CCD (MH/CCD), and CCD-only. The molecular basis for these different genotype-phenotype relationships is largely undefined. We have recently demonstrated that the porcine MH mutation, R615C, increases the sensitivity of the RyR1 channel to luminal Ca2+ activation and reduces the threshold for spontaneous Ca2+ release during store Ca2+ overload, also known as store-overload-induced Ca2+ release (SOICR). To investigate whether human MH and CCD mutations also alter the luminal Ca2+ activation of RyR1 and SOICR, we have generated a number of MH-only, MH/CCD, and CCD-only mutations located in the NH2 terminal, central, and COOH-terminal regions, and established stable, inducible HEK293 cell lines expressing these mutants. Using single cell Ca2+ imaging, we found that MH-only and MH/CCD mutations enhance the propensity for SOICR. On the other hand, some CCD-only mutations suppress or abolish SOICR, but retain caffeine-induced Ca2+ release, while other CCD-only mutants display little or no ryanodine- or caffeine-sensitive channel activity. Single channel studies reveal that, like the R615C MH mutation, MH/CCD mutations markedly sensitize the RyR1 channel to activation by luminal Ca2+. To assess their impact in the context of muscle cells, we are currently establishing stable, inducible mouse skeletal muscle C2C12 cell lines expressing MH and CCD RyR1 mutants. Further studies will address the question of whether altered luminal Ca2+ activation of RyR1 underlies a common defect of human MH and CCD mutations.

It has long been known that Ca2+ release from the sarcoplasmic reticulum (SR) can occur spontaneously when the SR Ca2+ content reaches a critical threshold, and that this spontaneous SR Ca2+ release can lead to lethal cardiac arrhythmias. Although it is clear that spontaneous SR Ca2+ release results from the activation of the cardiac ryanodine receptor (RyR2) by luminal Ca2+, the molecular basis of luminal Ca2+ activation of RyR2 remains undefined and controversial. An increasing body of evidence indicates that the RyR2 channel contains a luminal Ca2+ sensor distinct from its cytosolic Ca2+ sensor. To localize this luminal Ca2+ sensor, we have systematically mutated each of the negatively charged residues in the predicted pore-forming region of RyR2 that is likely to be accessible to luminal Ca2+. We found that alanine-mutations within and near the COOH-terminal end of the TM10 transmembrane helix (the pore inner helix) abolish the luminal, but not the cytosolic, Ca2+ activation of single RyR2 channels. HEK293 cells expressing these alanine-mutants display caffeine-induced Ca2+ release, but show no store-overload-induced Ca2+ release. Interestingly, introducing histidines into this region creates a high affinity metal binding site. The histidine-mutant channels are blocked by Ni2+ (

Highly ordered arrays of ryanodine receptor type 2 (RyR2) are believed to be critical for synchronous Ca release and effective, stable excitation-contraction coupling in adult cardiomyocytes. Altered RyR2 distribution and intracellular architectures have been implicated in the genesis of dyssynchronous Ca release often observed in disease hearts. To gain insights into the expression and distribution of RyR2 and their correlation with function in situ in adult cardiomyocytes, we generated a knock-in mouse model in which the green fluorescence protein (GFP) has been inserted into RyR2 after residue T1366. The GFP-tagged RyR2 mice show no gross structural and functional abnormalities. Confocal laser scanning microscopy of isolated cardiomyocytes from the GFP-tagged RyR2 mice revealed discrete clusters of GFP-RyR2 located along Z-lines. Confocal Ca imaging analysis of GFP-tagged RyR2 cardiomyocytes loaded with Rhod-2 AM showed that Ca sparks originate from GFP-RyR2 clusters and rarely occur in non-Z-line region. These observations suggest that the production of Ca sparks may require clustering of RyR2. To further define the distribution of GFP-RyR2 clusters, we employed a camelid single-domain antibody against GFP (GFP-nanobody) conjugated with the Alexa Fluor (AF) 647 fluorescent dye. The distribution of the GFP-nanobody staining in GFP-tagged RyR2 cardiomyocytes was found to be identical to that of GFP-RyR2 clusters. Further super-resolution imaging using the AF647-labelled GFP-nanobody should provide new and detailed insights into the nano-distribution of RyR2 in cardiomyocytes (Supported by CFI, CIHR, and LCIA).