Ryanodine Binding Research Articles

Extensive Ca2+ leak through K4750Q cardiac ryanodine receptors caused by cytosolic and luminal Ca2+ hypersensitivity.

Various ryanodine receptor 2 (RyR2) point mutations cause catecholamine-induced polymorphic ventricular tachycardia (CPVT), a life-threatening arrhythmia evoked by diastolic intracellular Ca2+ release dysfunction. These mutations occur in essential regions of RyR2 that regulate Ca2+ release. The molecular dysfunction caused by CPVT-associated RyR2 mutations as well as the functional consequences remain unresolved. Here, we study the most severe CPVT-associated RyR2 mutation (K4750Q) known to date. We define the molecular and cellular dysfunction generated by this mutation and detail how it alters RyR2 function, using Ca2+ imaging, ryanodine binding, and single-channel recordings. HEK293 cells and cardiac HL-1 cells expressing RyR2-K4750Q show greatly enhanced spontaneous Ca2+ oscillations. An endoplasmic reticulum-targeted Ca2+ sensor, R-CEPIA1er, revealed that RyR2-K4750Q mediates excessive diastolic Ca2+ leak, which dramatically reduces luminal [Ca2+]. We further show that the K4750Q mutation causes three RyR2 defects: hypersensitization to activation by cytosolic Ca2+, loss of cytosolic Ca2+/Mg2+-mediated inactivation, and hypersensitization to luminal Ca2+ activation. These defects combine to kinetically stabilize RyR2-K4750Q openings, thus explaining the extensive diastolic Ca2+ leak from the sarcoplasmic reticulum, frequent Ca2+ waves, and severe CPVT phenotype. As the multiple concurrent defects are induced by a single point mutation, the K4750 residue likely resides at a critical structural point at which cytosolic and luminal RyR2 control input converge.

Enhanced Cytosolic Ca2+ Activation Underlies a Common Defect of Central Domain Cardiac Ryanodine Receptor Mutations Linked to Arrhythmias

Recent three-dimensional structural studies reveal that the central domain of ryanodine receptor (RyR) serves as a transducer that converts long-range conformational changes into the gating of the channel pore. Interestingly, the central domain encompasses one of the mutation hotspots (corresponding to amino acid residues 3778–4201) that contains a number of cardiac RyR (RyR2) mutations associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) and atrial fibrillation (AF). However, the functional consequences of these central domain RyR2 mutations are not well understood. To gain insights into the impact of the mutation and the role of the central domain in channel function, we generated and characterized eight disease-associated RyR2 mutations in the central domain. We found that all eight central domain RyR2 mutations enhanced the Ca2+-dependent activation of [3H]ryanodine binding, increased cytosolic Ca2+-induced fractional Ca2+ release, and reduced the activation and termination thresholds for spontaneous Ca2+ release in HEK293 cells. We also showed that racemic carvedilol and the non-beta-blocking carvedilol enantiomer, (R)-carvedilol, suppressed spontaneous Ca2+ oscillations in HEK293 cells expressing the central domain RyR2 mutations associated with CPVT and AF. These data indicate that the central domain is an important determinant of cytosolic Ca2+ activation of RyR2. These results also suggest that altered cytosolic Ca2+ activation of RyR2 represents a common defect of RyR2 mutations associated with CPVT and AF, which could potentially be suppressed by carvedilol or (R)-carvedilol.

A type 2 ryanodine receptor variant associated with reduced Ca2+ release and short-coupled torsades de pointes ventricular arrhythmia

Ventricular fibrillation may be caused by premature ventricular contractions (PVCs) whose coupling intervals are <300 ms, a characteristic of the short-coupled variant of torsades de pointes (scTdP). The purpose of this study was to analyze the underlying cardiac ryanodine receptor (RyR2) variants in patients with scTdP. Seven patients with scTdP (mean age 34 ± 12 years; 4 men and 3 women) were enrolled in this study. The RyR2 gene was screened by targeted gene sequencing methods; variant minor allele frequency was confirmed in 3 databases; and the pathogenicity was investigated in silico analysis using multiple tools. The activity of wild-type and mutant RyR2 channels was evaluated by monitoring Ca2+ signals of HEK293 cells with a [3H]ryanodine binding assay. The mean coupling interval of PVCs was 282 ± 13 ms. The 12-lead electrocardiogram had no specific findings except PVCs with an extremely short-coupling interval. Genetic analysis revealed 3 novel RyR2 variants and 1 polymorphism, all located in the cytoplasmic region. p.Ser4938Phe was not detected in 3 databases, and in silico analysis indicated its pathogenicity. In functional analysis, p.Ser4938Phe demonstrated loss of function and impaired RyR2 channel Ca2+ release, while 2 other variants, p.Val1024Ile and p.Ala2673Val, had mild gain-of-function effects but were similar to the polymorphism p.Asn1551Ser. We identified an RyR2 variant associated with reduced Ca2+ release and short-coupled torsades de pointes ventricular arrhythmia. The mechanisms of arrhythmogenesis remain unclear.

Genotype-Dependent and -Independent Calcium Signaling Dysregulation in Human Hypertrophic Cardiomyopathy.

Aberrant calcium signaling may contribute to arrhythmias and adverse remodeling in hypertrophic cardiomyopathy (HCM). Mutations in sarcomere genes may distinctly alter calcium handling pathways. We analyzed gene expression, protein levels, and functional assays for calcium regulatory pathways in human HCM surgical samples with (n=25) and without (n=10) sarcomere mutations compared with control hearts (n=8). Gene expression and protein levels for calsequestrin, L-type calcium channel, sodium-calcium exchanger, phospholamban, calcineurin, and calcium/calmodulin-dependent protein kinase type II (CaMKII) were similar in HCM samples compared with controls. CaMKII protein abundance was increased only in sarcomere-mutation HCM (P<0.001). The CaMKII target pT17-phospholamban was 5.5-fold increased only in sarcomere-mutation HCM (P=0.01), as was autophosphorylated CaMKII (P<0.01), suggestive of constitutive activation. Calcineurin (PPP3CB) mRNA was not increased, nor was RCAN1 mRNA level, indicating a lack of calcineurin activation. Furthermore, myocyte enhancer factor 2 and nuclear factor of activated T cell transcription factor activity was not increased in HCM, suggesting that calcineurin pathway activation is not an upstream cause of increased CAMKII protein abundance or activation. SERCA2A mRNA transcript levels were reduced in HCM regardless of genotype, as was sarcoplasmic endoplasmic reticular calcium ATPase 2/phospholamban protein ratio (45% reduced; P=0.03). 45Ca sarcoplasmic endoplasmic reticular calcium ATPaseuptake assay showed reduced uptake velocity in HCM regardless of genotype (P=0.01). The cardiac ryanodine receptor was not altered in transcript, protein, or phosphorylated (pS2808, pS2814) protein abundance, and [3H]ryanodine binding was not different in HCM, consistent with no major modification of the ryanodine receptor. Human HCM demonstrates calcium mishandling through both genotype-specific and common pathways. Posttranslational activation of the CaMKII pathway is specific to sarcomere mutation-positive HCM, whereas sarcoplasmic endoplasmic reticular calcium ATPase 2 abundance and sarcoplasmic reticulum Ca uptake are depressed in both sarcomere mutation-positive and -negative HCM.

An Extended Structure-Activity Relationship of Nondioxin-Like PCBs Evaluates and Supports Modeling Predictions and Identifies Picomolar Potency of PCB 202 Towards Ryanodine Receptors.

Nondioxin-like polychlorinated biphenyls (NDL PCBs) activate ryanodine-sensitive Ca2+ channels (RyRs) and this activation has been associated with neurotoxicity in exposed animals. RyR-active congeners follow a distinct structure-activity relationship and a quantitative structure-activity relationship (QSAR) predicts that a large number of PCBs likely activate the receptor, which requires validation. Additionally, previous structural based conclusions have been established using receptor ligand binding assays but the impact of varying PCB structures on ion channel gating behavior is not understood. We used [3H]Ryanodine ([3H]Ry) binding to assess the RyR-activity of 14 previously untested PCB congeners evaluating the predictability of the QSAR. Congeners determined to display widely varying potency were then assayed with single channel voltage clamp analysis to assess direct influences on channel gating kinetics. The RyR-activity of individual PCBs assessed in in vitro assays followed the general pattern predicted by the QSAR but binding and lipid bilayer experiments demonstrated higher potency than predicted. Of the 49 congeners tested to date, tetra-ortho PCB 202 was found to be the most potent RyR-active congener increasing channel open probability at 200 pM. Shifting meta-substitutions to the para-position resulted in a > 100-fold reduction in potency as seen with PCB 197. Non-ortho PCB 11 was found to lack activity at the receptor supporting a minimum mono-ortho substitution for PCB RyR activity. These findings expand and support previous SAR assessments; where out of the 49 congeners tested to date 42 activate the receptor demonstrating that the RyR is a sensitive and common target of PCBs.

Genotype-Phenotype Correlations of Malignant Hyperthermia and Central Core Disease Mutations in the Central Region of the RYR1 Channel.

Type 1 ryanodine receptor (RYR1) is a Ca2+ release channel in the sarcoplasmic reticulum of skeletal muscle and is mutated in some muscle diseases, including malignant hyperthermia (MH) and central core disease (CCD). Over 200 mutations associated with these diseases have been identified, and most mutations accelerate Ca2+ -induced Ca2+ release (CICR), resulting in abnormal Ca2+ homeostasis in skeletal muscle. However, it remains largely unknown how specific mutations cause different phenotypes. In this study, we investigated the CICR activity of 14 mutations at 10 different positions in the central region of RYR1 (10 MH and four MH/CCD mutations) using a heterologous expression system in HEK293 cells. In live-cell Ca2+ imaging, the mutant channels exhibited an enhanced sensitivity to caffeine, a reduced endoplasmic reticulum Ca2+ content, and an increased resting cytoplasmic Ca2+ level. The three parameters for CICR (Ca2+ sensitivity for activation, Ca2+ sensitivity for inactivation, and attainable maximum activity, i.e., gain) were obtained by [3 H]ryanodine binding and fitting analysis. The mutant channels showed increased gain and Ca2+ sensitivity for activation in a site-specific manner. Genotype-phenotype correlations were explained well by the near-atomic structure of RYR1. Our data suggest that divergent CICR activity may cause various disease phenotypes by specific mutations.

Malignant hyperthermia-associated mutations in the S2-S3 cytoplasmic loop of type 1 ryanodine receptor calcium channel impair calcium-dependent inactivation.

Channel activities of skeletal muscle ryanodine receptor (RyR1) are activated by micromolar Ca2+ and inactivated by higher (∼1 mM) Ca2+ To gain insight into a mechanism underlying Ca2+-dependent inactivation of RyR1 and its relationship with skeletal muscle diseases, we constructed nine recombinant RyR1 mutants carrying malignant hyperthermia or centronuclear myopathy-associated mutations and determined RyR1 channel activities by [3H]ryanodine binding assay. These mutations are localized in or near the RyR1 domains which are responsible for Ca2+-dependent inactivation of RyR1. Four RyR1 mutations (F4732D, G4733E, R4736W, and R4736Q) in the cytoplasmic loop between the S2 and S3 transmembrane segments (S2-S3 loop) greatly reduced Ca2+-dependent channel inactivation. Activities of these mutant channels were suppressed at 10-100 μM Ca2+, and the suppressions were relieved by 1 mM Mg2+ The Ca2+- and Mg2+-dependent regulation of S2-S3 loop RyR1 mutants are similar to those of the cardiac isoform of RyR (RyR2) rather than wild-type RyR1. Two mutations (T4825I and H4832Y) in the S4-S5 cytoplasmic loop increased Ca2+ affinities for channel activation and decreased Ca2+ affinities for inactivation, but impairment of Ca2+-dependent inactivation was not as prominent as those of S2-S3 loop mutants. Three mutations (T4082M, S4113L, and N4120Y) in the EF-hand domain showed essentially the same Ca2+-dependent channel regulation as that of wild-type RyR1. The results suggest that nine RyR1 mutants associated with skeletal muscle diseases were differently regulated by Ca2+ and Mg2+ Four malignant hyperthermia-associated RyR1 mutations in the S2-S3 loop conferred RyR2-type Ca2+- and Mg2+-dependent channel regulation.

Structure-function relationships of peptides forming the calcin family of ryanodine receptor ligands.

Calcins are a novel family of scorpion peptides that bind with high affinity to ryanodine receptors (RyRs) and increase their activity by inducing subconductance states. Here, we provide a comprehensive analysis of the structure-function relationships of the eight calcins known to date, based on their primary sequence, three-dimensional modeling, and functional effects on skeletal RyRs (RyR1). Primary sequence alignment and evolutionary analysis show high similarity among all calcins (≥78.8% identity). Other common characteristics include an inhibitor cysteine knot (ICK) motif stabilized by three pairs of disulfide bridges and a dipole moment (DM) formed by positively charged residues clustering on one side of the molecule and neutral and negatively charged residues segregating on the opposite side. [(3)H]Ryanodine binding assays, used as an index of the open probability of RyRs, reveal that all eight calcins activate RyR1 dose-dependently with Kd values spanning approximately three orders of magnitude and in the following rank order: opicalcin1 > opicalcin2 > vejocalcin > hemicalcin > imperacalcin > hadrucalcin > maurocalcin >> urocalcin. All calcins significantly augment the bell-shaped [Ca(2+)]-[(3)H]ryanodine binding curve with variable effects on the affinity constants for Ca(2+) activation and inactivation. In single channel recordings, calcins induce the appearance of a subconductance state in RyR1 that has a unique fractional value (∼20% to ∼60% of the full conductance state) but bears no relationship to binding affinity, DM, or capacity to stimulate Ca(2+) release. Except for urocalcin, all calcins at 100 nM concentration stimulate Ca(2+) release and deplete Ca(2+) load from skeletal sarcoplasmic reticulum. The natural variation within the calcin family of peptides offers a diversified set of high-affinity ligands with the capacity to modulate RyRs with high dynamic range and potency.

Conformation of ryanodine receptor-2 gates store-operated calcium entry in rat pulmonary arterial myocytes.

Store-operated Ca(2+) entry (SOCE) contributes to a multitude of physiological and pathophysiological functions in pulmonary vasculatures. SOCE attributable to inositol 1,4,5-trisphosphate receptor (InsP3R)-gated Ca(2+) store has been studied extensively, but the role of ryanodine receptor (RyR)-gated store in SOCE remains unclear. The present study aims to delineate the relationship between RyR-gated Ca(2+) stores and SOCE, and characterize the properties of RyR-gated Ca(2+) entry in pulmonary artery smooth muscle cells (PASMCs). PASMCs were isolated from intralobar pulmonary arteries of male Wister rats. Application of the RyR1/2 agonist 4-chloro-m-cresol (4-CmC) activated robust Ca(2+) entry in PASMCs. It was blocked by Gd(3+) and the RyR2 modulator K201 but was unaffected by the RyR1/3 antagonist dantrolene and the InsP3R inhibitor xestospongin C, suggesting RyR2 is mainly involved in the process. siRNA knockdown of STIM1, TRPC1, and Orai1, or interruption of STIM1 translocation with ML-9 significantly attenuated the 4-CmC-induced SOCE, similar to SOCE induced by thapsigargin. However, depletion of RyR-gated store with caffeine failed to activate Ca(2+) entry. Inclusion of ryanodine, which itself did not cause Ca(2+) entry, uncovered caffeine-induced SOCE in a concentration-dependent manner, suggesting binding of ryanodine to RyR is permissive for the process. This Ca(2+) entry had the same molecular and pharmacological properties of 4-CmC-induced SOCE, and it persisted once activated even after caffeine washout. Measurement of Ca(2+) in sarcoplasmic reticulum (SR) showed that 4-CmC and caffeine application with or without ryanodine reduced SR Ca(2+) to similar extent, suggesting store-depletion was not the cause of the discrepancy. Moreover, caffeine/ryanodine and 4-CmC failed to initiate SOCE in cells transfected with the ryanodine-binding deficient mutant RyR2-I4827T. RyR2-gated Ca(2+) store contributes to SOCE in PASMCs; however, store-depletion alone is insufficient but requires a specific RyR conformation modifiable by ryanodine binding to activate Ca(2+) entry.

Modulation by CGP-37157 (CGP) Analogs of the Sarcoplasmic Reticulum Calcium ATPase SERCA)

We have reported that CGP, a 4-1 benzothiazepine (BZT) associated with cardioprotection in ischemia, is a SERCA inhibitor. CGP blocking action differs from that of thapsigargin because is Ca2+ dependent; i.e., CGP blocking potency increases when Ca2+ levels decrease. We utilized purified sarcoplasmic reticulum microsomal membranes (SRM; from rabbit skeletal muscle and pig ventricle) to test the inhibitory actions of various novel CGP analogs on ATPase function (estimated by measurements of Ca2+ loading and ATPase activity). The 4,1-benzothiazepin-2-one structural arrangement seems to be important for SERCA blocking action since function is lost within compounds where: the Sulfur is substituted by Nitrogen (as seen in benzodiazepines); the Oxygen is replaced by an alkyl group; or the arrangement between Nitrogen, Oxygen, and Sulfur in the BZT ring is reorganized (as seen in Diltiazem). Removal of Chlorine from position 7 in the BZT ring did not affect SERCA blocking potency. A combination of substitutions: 3H (benzylaminomethyl-carboxymethyl), alkylation of the Nitrogen (neopentyl) and replacement of the position 1 of benzyl group (naphtyl), significantly increased potency 10-fold (IC50 ∼ 1.5 μM, in presence of 2 μM free Ca2+). We determined (using SRM Ca2+ leak assay, [3H] ryanodine binding and channel studies in bilayers) CGP analogs can also activate ryanodine receptors (RyRs), albeit with low potency. In summary, we have identified structural features that may confer Ca2+-dependent SERCA blocking in 4,1 BZT by utilizing CGP-like molecules. We also identified ph000995, a potent blocker, which may have potential for assessing SERCA roles in cells as well as being a potential drug-template for therapeutic intervention in diseases of abnormal Ca2+ homeostasis, such as ischemia. (Supported by American Heart Association, Eskridge Foundation and APS Frontiers in Physiology Research Program).

S4-S5 Linker Regulates RyR2 Channel Gating through Multiple Interactions

Type 2 ryanodine receptor (RyR2) is a Ca2+ release channel in the sarcoplasmic reticulum of cardiac muscle and plays an important role in excitation-contraction coupling. Although recent cryo-EM studies provided near-atomic structures of type 1 ryanodine receptor (RyR1), molecular mechanism of the channel opening remains largely unknown. The S4-S5 linker (S45L) is an α-helical structure connecting the S4 and S5 transmembrane segments and mediates signal transmission in a wide variety of channels. To address the role of S45L in the RyR2 channel gating, we systematically mutated residues in S45L (Thr4751-Asn4762) and neighboring transmembrane segments (S5 and S6). The mutant RyR2 was stably expressed in HEK293 cells, and the channel activity was investigated by ER luminal Ca2+ measurements and [3H]ryanodine binding. Using high sequence identity between RyR1 and RyR2 (65% in the whole molecule and 93% in the core channel domain after S4), we constructed structural model of RyR2 based on the structures of RyR1, and the phenotypes of mutants were interpreted by the model. Plausible interactions between amino acid residues in S45L and neighboring domains that may regulate channel gating will be discussed.

Ablation of two Major Phosphorylation Sites in RyR2 Alter Sarcoplamic Reticulum Calcium Handling and Increases the Propensity to Atrial Fibrillation

Rationale: Compelling data indicate that excessive Ca2+ leak from the sarcoplasmic reticulum (SR) contribute to atrial fibrillation (AF) pathogenesis. However, the role of RyR2 phosphorylation in Ca2+ leak in particular and cardiac diseases in general remains controversial. Aim: To investigate the effect of genetic inhibition of two major RyR2 phosphorylation sites on the channel function and atrial arrhythmogenesis. Methods and Results: We performed programmed electrical stimulation in anesthetized wild type (WT) mice and in mice harboring two RyR2 mutations that prevent PKA and CaMKII phosphorylation (S2808A and S2814A, respectively). Surprisingly, the double knock-in (DKI) mice exhibited increased incidence of AF episodes (5.2±3% WT vs 45±5% DKI, p<0.05). Confocal Ca2+ imaging showed that atrial myocytes from the DKI mouse have 30% increases in fractional Ca2+ release. Patch clamping in voltage mode coupled to confocal imaging revealed increased EC coupling gain in DKI myocytes (1.69±0.14 WT vs 2.53±0.28 DKI, at +10 mV). In I-current mode, DKI myocytes showed increased incidence of DADs after 2.0 Hz of pacing (5.94±0.10 min-1 WT vs 7.9±0.30 DKI min-1; p<0.05). Importantly, whereas the response of SR Ca2+ release to adrenergic stimulation was intact in DKI myocytes, the RyR2-mediated leak was higher (spark mass x spark frequency: 38.22±5.72 WT vs 115.1 ± 26.4 DKI; p<0.05). RyR2 restitution assays showed a faster recovery from refractoriness (τ = 170±12 WT vs 120±11 ms DKI, p<0.05). Moreover, [3H]ryanodine binding assays showed 2-fold increase of RyR2 activity in atrial homogenates from DKI at equivalent systolic and diastolic ]Ca2+]. Conclusion: Ablation of two major RyR2 phosphorylation sites increased the activity of the channel and was associated with increased susceptibility to AF.

Dampened Activity of Single Ryanodine Receptor Channels in Mice Devoid of TRIC-A

TRIC-A and TRIC-B are proteins localized to endo/sarcoplasmic reticulum (ER/SR). Their physiological roles are not established although there is evidence that they can function as monovalent cation channels (Yazawa et al., 2007). Mice lacking both TRIC-A and TRIC-B die in embryonic heart failure displaying serious defects in SR Ca2+-release (Yazawa et al., 2007). TRIC-A knockout (KO) mice survive but exhibit hypertension and abnormalities in cardiac and skeletal excitation-contraction coupling. We incorporated SR vesicles derived from the skeletal muscle of wild type (WT) and TRIC-A KO mice into planar lipid bilayers under voltage-clamp conditions to investigate if the single-channel properties of ryanodine receptors (RyR) and SR K+ channels are altered.We found no significant difference in single channel conductance, open probability (Po) and voltage-dependence of SR K+ channels from wild type (WT) or TRIC-A KO mice. [3H]ryanodine binding studies indicate that the Ca2+ sensitivity of RyR derived from TRIC-A KO or WT mice is similar (WT EC50 =4.14 µM; TRIC-A KO EC50 =3.65 µM; n=6). This was confirmed by the similar Po of RyR from WT (0.0023±0.0011, SEM, n=14) and TRIC-A KO (0.0017±0.0006, SEM, n=21) mice with 10 µM cytosolic Ca2+ as the sole activator. However, in the presence of 1 mM free Mg2+, 3 mM ATP was significantly less effective at activating RyR from TRIC-A KO mice (Po=0.0464±0.0133; SEM, n=21) than from WT mice (Po =0.1149±0.0290; n=14, SEM; p<0.05). Our results indicate that TRIC-A indirectly modulates RyR channel function either by reducing the inhibitory effect of Mg2+ or by increasing sensitivity to the effects of ATP and may, at least in part, explain why SR Ca2+-release is impaired in TRIC-A KO mice.Funded by the BHF.

Structural Dynamics of Calmodulin in Regulation of Cardiac Calcium Release in Health and Disease

Regulation of the cardiac calcium release channel, ryanodine receptor (RyR), by calmodulin (CaM) is disrupted by oxidation and disease-causing mutations. However, the structural basis for this regulatory change and the role CaM plays in the development of heart failure and arrhythmias is not well understood. Several studies suggest that the modulatory role of CaM is closely tied to its conformation when bound to RyR, but the association between structure and function is largely unknown. Testing the hypothesis that the modulatory action of CaM on RyR is caused by structural changes in the CaM-RyR complex, and establishment of these structure-function connections will be crucial in the analysis and treatment of heart disease. To address this clear deficit of knowledge, we use site-directed spectroscopic techniques to characterize structural changes that contribute to calcium deregulation in the heart. The approach is to prepare CaM mutants that contain a single Cys on each of the two lobes, then attach probes to those sites and measure intramolecular distance distributions using time-resolved spectroscopy. Previous studies from this lab employed EPR (DEER) spectroscopy, but the present study focuses on time-resolved FRET, in order to obtain sufficient sensitivity to study CaM bound to RyR in sarcoplasmic reticulum membranes. We will compare these measurements with those of CaM bound to a peptide corresponding to the CaM binding domain of RyR (RyR1 residues: 3614-3643), a comparison that has not been previously investigated. Ryanodine binding measurements were performed to ensure functional integrity of labeled CaM constructs.

Characterization of Ca2+-Induced Ca2+ Release via RyR2 Carrying Arrhythmogenic Mutations

The type 2 ryanodine receptor (RyR2) is the Ca2+ release channel on cardiac sarcoplasmic reticulum and plays a crucial role in cardiac E-C coupling. The mutations in RyR2 have been implicated in a number of arrhythmogenic disorders including catecholaminergic polymorphic ventricular tachycardia (CPVT), arrhythmogenic right ventricular cardiomyopathy (ARVC), idiopathic ventricular fibrillation (I-VF) and atrial fibrillation (AF). In this study, we aimed to characterize Ca2+ release properties of RyR2s carrying the above 4 types of disease mutations. Wild type and mutant RyR2 channels were expressed in HEK293 cells and spontaneous Ca2+ oscillations were monitored by live-cell Ca2+ imaging using Ca2+ indicators for cytoplasm (fluo-4, GECOs) and ER (CEPIAs). In addition, the Ca2+-induced Ca2+ release (CICR) activity were determined with [3H]ryanodine binding assay. There were good correlations between CICR activity, Ca2+ oscillation frequencies and ER Ca2+ levels among all of mutants examined. While all CPVT mutants showed enhanced CICR activity, I-VF mutants showed divergent CICR profiles. Our results suggest that further analyses of experimental results and clinical data are needed to understand mechanisms of arrhythmogenic diseases due to RyR2 mutations.

Simvastatin Activates Single Skeletal RyR1 Channels but Exerts More Complex Regulation of the Cardiac Isoform, RyR2

Statins are widely prescribed for the treatment of hypercholesterolemia. Although effective in lowering cholesterol levels, statin use has been associated with muscle weakness, myopathies and, in rare cases, rhabdomyolysis. The mechanisms underlying toxicity have not been elucidated, however, cerivastatin, a compound removed from the market due to reports of rhabdomyolysis, has been shown to trigger Ca2+ release from isolated skeletal sarcoplasmic reticulum (SR) vesicles (Inoue et al., 2003). To investigate whether statins can directly activate RyR channels, we incorporated single sheep cardiac RyR2 or mouse skeletal RyR1 channels into planar phospholipid bilayers under voltage-clamp conditions and added simvastatin, a commonly prescribed statin, to the cytosolic channel side. In the presence of 10 μM cytosolic [Ca2+], 10 μM simvastatin (carboxylate form), significantly increased RyR1 open probability (Po) from 0.020±0.013 to 0.269±0.068 (SEM, n=13, p<0.001). This effect was fully reversible after washout of simvastatin (0.033±0.029, SEM, n=6). The ability of simvastatin to activate RyR1 was further highlighted by our experiments showing that simvastatin is more effective than caffeine at stimulating [3H]ryanodine binding to skeletal SR (n=3, p<0.01). Unexpectedly, we found that 10 µM simvastatin did not activate RyR2 but that higher concentrations were required to increase Po. Low concentrations of simvastatin (1 µM) tended to reduce Po. These results may explain why the most frequently reported adverse effects of statins are of skeletal rather than cardiac origin. Confirmation that the direct modulation of RyR1 and RyR2 observed in bilayer experiments is relevant in cells is shown in permeabilised cardiac and skeletal myocytes where simvastatin increased the frequency of Ca2+ sparks in skeletal cells but decreased frequency in cardiac cells (see poster by Lotteau et al.).Funded by the British Heart Foundation.

The EF-hand Ca2+ Binding Domain Is Not Required for Cytosolic Ca2+ Activation of the Cardiac Ryanodine Receptor

Activation of the cardiac ryanodine receptor (RyR2) by elevating cytosolic Ca2+ is a central step in the process of Ca2+-induced Ca2+ release, but the molecular basis of RyR2 activation by cytosolic Ca2+ is poorly defined. It has been proposed recently that the putative Ca2+ binding domain encompassing a pair of EF-hand motifs (EF1 and EF2) in the skeletal muscle ryanodine receptor (RyR1) functions as a Ca2+ sensor that regulates the gating of RyR1. Although the role of the EF-hand domain in RyR1 function has been studied extensively, little is known about the functional significance of the corresponding EF-hand domain in RyR2. Here we investigate the effect of mutations in the EF-hand motifs on the Ca2+ activation of RyR2. We found that mutations in the EF-hand motifs or deletion of the entire EF-hand domain did not affect the Ca2+-dependent activation of [3H]ryanodine binding or the cytosolic Ca2+ activation of RyR2. On the other hand, deletion of the EF-hand domain markedly suppressed the luminal Ca2+ activation of RyR2 and spontaneous Ca2+ release in HEK293 cells during store Ca2+ overload or store overload-induced Ca2+ release (SOICR). Furthermore, mutations in the EF2 motif, but not EF1 motif, of RyR2 raised the threshold for SOICR termination, whereas deletion of the EF-hand domain of RyR2 increased both the activation and termination thresholds for SOICR. These results indicate that, although the EF-hand domain is not required for RyR2 activation by cytosolic Ca2+, it plays an important role in luminal Ca2+ activation and SOICR.

The H29D Mutation Does Not Enhance Cytosolic Ca2+ Activation of the Cardiac Ryanodine Receptor.

The N-terminal domain of the cardiac ryanodine receptor (RyR2) harbors a large number of naturally occurring mutations that are associated with stress-induced ventricular tachyarrhythmia and sudden death. Nearly all these disease-associated N-terminal mutations are located at domain interfaces or buried within domains. Mutations at these locations would alter domain-domain interactions or the stability/folding of domains. Recently, a novel RyR2 mutation H29D associated with ventricular arrhythmia at rest was found to enhance the activation of single RyR2 channels by diastolic levels of cytosolic Ca2+. Unlike other N-terminal disease-associated mutations, the H29D mutation is located on the surface of the N-terminal domain. It is unclear how this surface-exposed H29D mutation that does not appear to interact with other parts of the RyR2 structure could alter the intrinsic properties of the channel. Here we carried out detailed functional characterization of the RyR2-H29D mutant at the molecular and cellular levels. We found that the H29D mutation has no effect on the basal level or the Ca2+ dependent activation of [3H]ryanodine binding to RyR2, the cytosolic Ca2+ activation of single RyR2 channels, or the cytosolic Ca2+- or caffeine-induced Ca2+ release in HEK293 cells. In addition, the H29D mutation does not alter the propensity for spontaneous Ca2+ release or the thresholds for Ca2+ release activation or termination. Furthermore, the H29D mutation does not have significant impact on the thermal stability of the N-terminal region (residues 1–547) of RyR2. Collectively, our data show that the H29D mutation exerts little or no effect on the function of RyR2 or on the folding stability of the N-terminal region. Thus, our results provide no evidence that the H29D mutation enhances the cytosolic Ca2+ activation of RyR2.

Distinctive malfunctions of calmodulin mutations associated with heart RyR2-mediated arrhythmic disease

Calmodulin (CaM) is a cytoplasmic calcium sensor that interacts with the cardiac ryanodine receptor (RyR2), a large Ca2+ channel complex that mediates Ca2+ efflux from the sarcoplasmic reticulum (SR) to activate cardiac muscle contraction. Direct CaM association with RyR2 is an important physiological regulator of cardiac muscle excitation–contraction coupling and defective CaM–RyR2 protein interaction has been reported in cases of heart failure. Recent genetic studies have identified CaM missense mutations in patients with a history of severe cardiac arrhythmogenic disorders that present divergent clinical features, including catecholaminergic polymorphic ventricular tachycardia (CPVT), long QT syndrome (LQTS) and idiopathic ventricular fibrillation (IVF). Herein, we describe how two CPVT- (N54I & N98S) and three LQTS-associated (D96V, D130G & F142L) CaM mutations result in alteration of their biochemical and biophysical properties. Ca2+-binding studies indicate that the CPVT-associated CaM mutations, N54I & N98S, exhibit the same or a 3-fold reduced Ca2+-binding affinity, respectively, versus wild-type CaM, whereas the LQTS-associated CaM mutants, D96V, D130G & F142L, display more profoundly reduced Ca2+-binding affinity. In contrast, all five CaM mutations confer a disparate RyR2 interaction and modulation of [3H]ryanodine binding to RyR2, regardless of CPVT or LQTS association. Our findings suggest that the clinical presentation of CPVT or LQTS associated with these five CaM mutations may involve both altered intrinsic Ca2+-binding as well as defective interaction with RyR2.

Channel Gating Dependence on Pore Lining Helix Glycine Residues in Skeletal Muscle Ryanodine Receptor

Type 1 ryanodine receptors (RyR1s) release Ca(2+) from the sarcoplasmic reticulum to initiate skeletal muscle contraction. The role of RyR1-G4934 and -G4941 in the pore-lining helix in channel gating and ion permeation was probed by replacing them with amino acid residues of increasing side chain volume. RyR1-G4934A, -G4941A, and -G4941V mutant channels exhibited a caffeine-induced Ca(2+) release response in HEK293 cells and bound the RyR-specific ligand [(3)H]ryanodine. In single channel recordings, significant differences in the number of channel events and mean open and close times were observed between WT and RyR1-G4934A and -G4941A. RyR1-G4934A had reduced K(+) conductance and ion selectivity compared with WT. Mutations further increasing the side chain volume at these positions (G4934V and G4941I) resulted in reduced caffeine-induced Ca(2+) release in HEK293 cells, low [(3)H]ryanodine binding levels, and channels that were not regulated by Ca(2+) and did not conduct Ca(2+) in single channel measurements. Computational predictions of the thermodynamic impact of mutations on protein stability indicated that although the G4934A mutation was tolerated, the G4934V mutation decreased protein stability by introducing clashes with neighboring amino acid residues. In similar fashion, the G4941A mutation did not introduce clashes, whereas the G4941I mutation resulted in intersubunit clashes among the mutated isoleucines. Co-expression of RyR1-WT with RyR1-G4934V or -G4941I partially restored the WT phenotype, which suggested lessening of amino acid clashes in heterotetrameric channel complexes. The results indicate that both glycines are important for RyR1 channel function by providing flexibility and minimizing amino acid clashes.