Calsequestrin interacts directly with the cardiac ryanodine receptor luminal domain.

Cardiac muscle contraction requires sarcoplasmic reticulum (SR) Ca2+ release mediated by the quaternary complex comprising the ryanodine receptor 2 (RyR2), calsequestrin 2 (CSQ2), junctin (encoded by ASPH) and triadin. Here, we demonstrate that a direct interaction exists between RyR2 and CSQ2. Topologically, CSQ2 binding occurs at the first luminal loop of RyR2. Co-expression of RyR2 and CSQ2 in a human cell line devoid of the other quaternary complex proteins results in altered Ca2+-release dynamics compared to cells expressing RyR2 only. These findings provide a new perspective for understanding the SR luminal Ca2+ sensor and its involvement in cardiac physiology and disease.

The mechanism of flecainide action in CPVT does not involve a direct effect on RyR2.

RATIONALE: Flecainide, a class 1c antiarrhythmic, has emerged as an effective therapy in preventing arrhythmias in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT) refractory to ß-adrenergic receptor blockade. It has been proposed that the clinical efficacy of flecainide in CPVT is because of the combined actions of direct blockade of ryanodine receptors (RyR2) and Na(+) channel inhibition. However, there is presently no direct evidence to support the notion that flecainide blocks RyR2 Ca(2+) flux in the physiologically relevant (luminal-to-cytoplasmic) direction. The mechanism of flecainide action remains controversial. OBJECTIVE: To examine, in detail, the effect of flecainide on the human RyR2 channel and to establish whether the direct blockade of physiologically relevant RyR2 ion flow by the drug contributes to its therapeutic efficacy in the clinical management of CPVT. METHODS AND RESULTS: Using single-channel analysis, we show that, even at supraphysiological concentrations, flecainide did not inhibit the physiologically relevant, luminal-to-cytosolic flux of cations through the channel. Moreover, flecainide did not alter RyR2 channel gating and had negligible effect on the mechanisms responsible for the sarcoplasmic reticulum charge-compensating counter current. Using permeabilized cardiac myocytes to eliminate any contribution of plasmalemmal Na(+) channels to the observed actions of the drug at the cellular level, flecainide did not inhibit RyR2-dependent sarcoplasmic reticulum Ca(2+) release. CONCLUSIONS: The principal action of flecainide in CPVT is not via a direct interaction with RyR2. Our data support a model of flecainide action in which Na(+)-dependent modulation of intracellular Ca(2+) handling attenuates RyR2 dysfunction in CPVT.

N-terminus oligomerization regulates the function of cardiac ryanodine receptors.

The ryanodine receptor (RyR) is an ion channel composed of four identical subunits mediating calcium efflux from the endo/sarcoplasmic reticulum of excitable and non-excitable cells. We present several lines of evidence indicating that the RyR2 N-terminus is capable of self-association. A combination of yeast two-hybrid screens, co-immunoprecipitation analysis, chemical crosslinking and gel filtration assays collectively demonstrate that a RyR2 N-terminal fragment possesses the intrinsic ability to oligomerize, enabling apparent tetramer formation. Interestingly, N-terminus tetramerization mediated by endogenous disulfide bond formation occurs in native RyR2, but notably not in RyR1. Disruption of N-terminal inter-subunit interactions within RyR2 results in dysregulation of channel activation at diastolic Ca(2+) concentrations from ryanodine binding and single channel measurements. Our findings suggest that the N-terminus interactions mediating tetramer assembly are involved in RyR channel closure, identifying a crucial role for this structural association in the dynamic regulation of intracellular Ca(2+) release.

Insights into the gating mechanism of the ryanodine-modified human cardiac Ca2+-release channel (ryanodine receptor 2).

Ryanodine receptors (RyRs) are intracellular membrane channels playing key roles in many Ca(2+) signaling pathways and, as such, are emerging novel therapeutic and insecticidal targets. RyRs are so named because they bind the plant alkaloid ryanodine with high affinity and although it is established that ryanodine produces profound changes in all aspects of function, our understanding of the mechanisms underlying altered gating is minimal. We address this issue using detailed single-channel gating analysis, mathematical modeling, and energetic evaluation of state transitions establishing that, with ryanodine bound, the RyR pore adopts an extremely stable open conformation. We demonstrate that stability of this state is influenced by interaction of divalent cations with both activating and inhibitory cytosolic sites and, in the absence of activating Ca(2+), trans-membrane voltage. Comparison of the conformational stability of ryanodine- and Imperatoxin A-modified channels identifies significant differences in the mechanisms of action of these qualitatively similar ligands.

Investigations of the contribution of a putative glycine hinge to ryanodine receptor channel gating.

Ryanodine receptor channels (RyR) are key components of striated muscle excitation-contraction coupling, and alterations in their function underlie both inherited and acquired disease. A full understanding of the disease process will require a detailed knowledge of the mechanisms and structures involved in RyR function. Unfortunately, high-resolution structural data, such as exist for K(+)-selective channels, are not available for RyR. In the absence of these data, we have used modeling to identify similarities in the structural elements of K(+) channel pore-forming regions and postulated equivalent regions of RyR. This has identified a sequence of residues in the cytosolic cavity-lining transmembrane helix of RyR (G(4864)LIIDA(4869) in RyR2) analogous to the glycine hinge motif present in many K(+) channels. Gating in these K(+) channels can be disrupted by substitution of residues for the hinge glycine. We investigated the involvement of glycine 4864 in RyR2 gating by monitoring properties of recombinant human RyR2 channels in which this glycine is replaced by residues that alter gating in K(+) channels. Our data demonstrate that introducing alanine at position 4864 produces no significant change in RyR2 function. In contrast, function is altered when glycine 4864 is replaced by either valine or proline, the former preventing channel opening and the latter modifying both ion translocation and gating. Our studies reveal novel information on the structural basis of RyR gating, identifying both similarities with, and differences from, K(+) channels. Glycine 4864 is not absolutely required for channel gating, but some flexibility at this point in the cavity-lining transmembrane helix is necessary for normal RyR function.

Disparities in the association of the ryanodine receptor and the FK506-binding proteins in mammalian heart.

The FK506-binding proteins (FKBP12 and FKBP12.6; also known as FKBP1A and FKBP1B, respectively) are accessory subunits of the ryanodine receptor (RyR) Ca(2+) release channel. Aberrant RyR2-FKBP12.6 interactions have been proposed to be the underlying cause of channel dysfunction in acquired and inherited cardiac disease. However, the stoichiometry of the RyR2 association with FKBP12 or FKBP12.6 in mammalian heart is currently unknown. Here, we describe detailed quantitative analysis of cardiac stoichiometry between RyR2 and FKBP12 or FKBP12.6 using immunoblotting and [(3)H]ryanodine-binding assays, revealing striking disparities between four mammalian species. In mouse and pig heart, RyR2 is found complexed with both FKBP12 and FKBP12.6, although the former is the most abundant isoform. In rat heart, RyR2 is predominantly associated with FKBP12.6, whereas in rabbit it is associated with FKBP12 only. Co-immunoprecipitation experiments demonstrate RyR2-specific interaction with both FKBP isoforms in native cardiac tissue. Assuming four FKBP-binding sites per RyR2 tetramer, only a small proportion of available sites are occupied by endogenous FKBP12.6. FKBP interactions with RyR2 are very strong and resistant to drug (FK506, rapamycin and cyclic ADPribose) and redox (H(2)O(2) and diamide) treatment. By contrast, the RyR1-FKBP12 association in skeletal muscle is readily disrupted under oxidative conditions. This is the first study to directly assess association of endogenous FKBP12 and FKBP12.6 with RyR2 in native cardiac tissue. Our results challenge the widespread perception that RyR2 associates exclusively with FKBP12.6 to near saturation, with important implications for the role of the FK506-binding proteins in RyR2 pathophysiology and cardiac disease.

Techniques and methodologies to study the ryanodine receptor at the molecular, subcellular and cellular level.

In excitable tissues, the ryanodine receptor Ca(2+) release channel (RyR) protein complex regulates excitation-contraction coupling, exocytosis, gene expression and apoptosis. Defects in RyR function, in genetic or acquired pathologies, lead to massive disruptions of Ca(2+) release that can be lethal. Therefore, RyR has emerged as a putative therapeutic target and an increasing number of RyR-targeting drugs are currently being tested.Nonetheless this large-size channel is still a mystery in terms of structure, which hinders full characterization of the properties of this central protein. This chapter is dedicated to the methods available to examine RyR structure and function. The aim of the article is to concentrate on contemporary methodologies rather than focusing overtly on the progress that has been achieved using these techniques. Here we review a series of reliable approaches that are routinely employed to investigate this channel. Technical limitations are discussed, and technological developments are presented. This work is not a handbook, but it can be used as a resource and a starting point for the investigation of RyR at different levels of resolution.

Changes in negative charge at the luminal mouth of the pore alter ion handling and gating in the cardiac ryanodine-receptor.

We have tested the hypothesis that a high density of negative charge at the luminal mouth of the RyR2 pore plays a pivotal role in the high cation conductance and limited selectivity observed in this channel by introducing into each monomer a double point mutation to neutralize acidic residues in this region of the mouse RyR2 channel. The resultant channel, ED4832AA, is capable of functioning as a calcium-release channel in situ. Consistent with our hypothesis, the ED4832AA mutation altered the ion handling characteristics of single RyR2 channels. The mutant channel retains the ability to discriminate between cations and anions but cation conductance is altered significantly. Unitary K+ conductance is reduced at low levels of activity but increases dramatically as activity is raised and shows little sign of saturation. ED4832AA no longer discriminates between divalent and monovalent cations. In addition, the gating characteristics of single RyR2 channels are altered markedly by residue neutralization. Open probability in the ED4832AA channel is substantially higher than that of the wild-type channel. Moreover, at holding potentials in excess of +/-50 mV several subconductance states become apparent in ED4832AA and are more prevalent at very high holding potentials. These observations are discussed within the structural framework provided by a previously developed model of the RyR2 pore. Our data indicates that neutralization of acidic residues in the luminal mouth of the pore produces wide-ranging changes in the electric field in the pore, the interaction energies of permeant ions in the pore and the stability of the selectivity filter region of the pore, which together contribute to the observed changes ion handling and gating.

Myocardial stunning is associated with impaired calcium uptake by sarcoplasmic reticulum.

Myocardial stunning (temporary post-ischaemic contractile dysfunction) may be caused by oxidative stress and/or impaired myocyte calcium homeostasis. Regional myocardial stunning was induced in open-chest pigs (segment shortening reduced to 68.3+/-4.7% of baseline) by repetitive brief circumflex coronary occlusion (I/R). Reduced glutathione was depleted in stunned myocardium (1.34+/-0.06 vs. 1.77+/-0.11 nmol/mg, p=0.02 vs. remote myocardium) indicating regional oxidant stress, but no regional differences were observed in protein-bound 3-nitrotyrosine or S-nitrosothiol content. Repetitive I/R did not affect myocardial quantities of the sarcolemmal sodium-calcium exchanger, L-type channel, SR calcium ATPase and phospholamban, or the kinetics of ligand binding to L-type channels and SR calcium release channels. However, initial rates of oxalate-supported (45)Ca uptake by SR were impaired in stunned myocardium (41.3+/-13.5 vs. 73.0+/-15.6 nmol/min/mg protein, p=0.03). The ability of SR calcium ATPase to sequester cytosolic calcium is impaired in stunned myocardium. This is a potential mechanism underlying contractile dysfunction.

Ryanodine receptor regulation by intramolecular interaction between cytoplasmic and transmembrane domains.

Ryanodine receptors (RyR) function as Ca(2+) channels that regulate Ca(2+) release from intracellular stores to control a diverse array of cellular processes. The massive cytoplasmic domain of RyR is believed to be responsible for regulating channel function. We investigated interaction between the transmembrane Ca(2+)-releasing pore and a panel of cytoplasmic domains of the human cardiac RyR in living cells. Expression of eGFP-tagged RyR constructs encoding distinct transmembrane topological models profoundly altered intracellular Ca(2+) handling and was refractory to modulation by ryanodine, FKBP12.6 and caffeine. The impact of coexpressing dsRed-tagged cytoplasmic domains of RyR2 on intracellular Ca(2+) phenotype was assessed using confocal microscopy coupled with parallel determination of in situ protein: protein interaction using fluorescence resonance energy transfer (FRET). Dynamic interactions between RyR cytoplasmic and transmembrane domains were mediated by amino acids 3722-4610 (Interacting or "I"-domain) which critically modulated intracellular Ca(2+) handling and restored RyR sensitivity to caffeine activation. These results provide compelling evidence that specific interaction between cytoplasmic and transmembrane domains is an important mechanism in the intrinsic modulation of RyR Ca(2+) release channels.

Massive Accumulation of Myofibroblasts in the Critical Isthmus Is Associated With Ventricular Tachycardia Inducibility in Post-Infarct Swine Heart.

OBJECTIVES: In this study the authors determined the extent of cellular infiltration and dispersion, and regional vascularization in electrophysiologically (EP) defined zones in post-myocardial infarction (MI) swine ventricle. BACKGROUND: The critical isthmus (CI) in post-MI re-entrant ventricular tachycardia (VT) is a target for catheter ablation. In vitro evidence suggests that myofibroblasts (MFB) within the scar border zone (BZ) may increase the susceptibility to slow conduction and VT, but whether this occurs in vivo remains unproven. METHODS: Six weeks after mid-left anterior descending coronary artery occlusion, EP catheter-based mapping was used to assess susceptibility to VT induction. EP data were correlated with detailed cellular profiling of ventricular zones using immunohistochemistry and spatial distribution analysis of cardiomyocytes, fibroblasts, MFB, and vascularization. RESULTS: In pigs with induced sustained monomorphic VT (mean cycle length: 353 ± 89 ms; n = 6) the area of scar that consisted of the BZ (i.e., between the normal and the low-voltage area identified by substrate mapping) was greater in VT-inducible hearts (iVT) than in noninducible hearts (non-VT) (p < 0.05). Scar in iVT hearts was characterized by MFB accumulation in the CI (>100 times that in normal myocardium and >5 times higher than that in the BZ in non-VT hearts) and by a 1.7-fold increase in blood vessel density within the dense scar region extending towards the CI. Sites of local abnormal ventricular activity potentials exhibited cellularity and vascularization that were intermediate to the CI in iVT and BZ in non-VT hearts. CONCLUSIONS: The authors reported the first cellular analysis of the VT CI following an EP-based zonal analysis of iVT and non-VT hearts in pigs post-MI. The data suggested that VT susceptibility was defined by a remarkable number of MFB in the VT CI, which appeared to bridge the few remaining dispersed clusters of cardiomyocytes. These findings define the cellular substrate for the proarrhythmic slow conduction pathway.

Unambiguous observation of blocked states reveals altered, blocker-induced, cardiac ryanodine receptor gating.

The flow of ions through membrane channels is precisely regulated by gates. The architecture and function of these elements have been studied extensively, shedding light on the mechanisms underlying gating. Recent investigations have focused on ion occupancy of the channel's selectivity filter and its ability to alter gating, with most studies involving prokaryotic K+ channels. Some studies used large quaternary ammonium blocker molecules to examine the effects of altered ionic flux on gating. However, the absence of blocking events that are visibly distinct from closing events in K+ channels makes unambiguous interpretation of data from single channel recordings difficult. In this study, the large K+ conductance of the RyR2 channel permits direct observation of blocking events as distinct subconductance states and for the first time demonstrates the differential effects of blocker molecules on channel gating. This experimental platform provides valuable insights into mechanisms of blocker-induced modulation of ion channel gating.

Structural insights into Ca(2+)-activated long-range allosteric channel gating of RyR1.

Ryanodine receptors (RyRs) are a class of giant ion channels with molecular mass over 2.2 mega-Daltons. These channels mediate calcium signaling in a variety of cells. Since more than 80% of the RyR protein is folded into the cytoplasmic assembly and the remaining residues form the transmembrane domain, it has been hypothesized that the activation and regulation of RyR channels occur through an as yet uncharacterized long-range allosteric mechanism. Here we report the characterization of a Ca(2+)-activated open-state RyR1 structure by cryo-electron microscopy. The structure has an overall resolution of 4.9 Å and a resolution of 4.2 Å for the core region. In comparison with the previously determined apo/closed-state structure, we observed long-range allosteric gating of the channel upon Ca(2+) activation. In-depth structural analyses elucidated a novel channel-gating mechanism and a novel ion selectivity mechanism of RyR1. Our work not only provides structural insights into the molecular mechanisms of channel gating and regulation of RyRs, but also sheds light on structural basis for channel-gating and ion selectivity mechanisms for the six-transmembrane-helix cation channel family.

Effect of flecainide derivatives on sarcoplasmic reticulum calcium release suggests a lack of direct action on the cardiac ryanodine receptor.

BACKGROUND AND PURPOSE: Flecainide is a use-dependent blocker of cardiac Na(+) channels. Mechanistic analysis of this block showed that the cationic form of flecainide enters the cytosolic vestibule of the open Na(+) channel. Flecainide is also effective in the treatment of catecholaminergic polymorphic ventricular tachycardia but, in this condition, its mechanism of action is contentious. We investigated how flecainide derivatives influence Ca(2) (+) -release from the sarcoplasmic reticulum through the ryanodine receptor channel (RyR2) and whether this correlates with their effectiveness as blockers of Na(+) and/or RyR2 channels. EXPERIMENTAL APPROACH: We compared the ability of fully charged (QX-FL) and neutral (NU-FL) derivatives of flecainide to block individual recombinant human RyR2 channels incorporated into planar phospholipid bilayers, and their effects on the properties of Ca(2) (+) sparks in intact adult rat cardiac myocytes. KEY RESULTS: Both QX-FL and NU-FL were partial blockers of the non-physiological cytosolic to luminal flux of cations through RyR2 channels but were significantly less effective than flecainide. None of the compounds influenced the physiologically relevant luminal to cytosol cation flux through RyR2 channels. Intracellular flecainide or QX-FL, but not NU-FL, reduced Ca(2) (+) spark frequency. CONCLUSIONS AND IMPLICATIONS: Given its inability to block physiologically relevant cation flux through RyR2 channels, and its lack of efficacy in blocking the cytosolic-to-luminal current, the effect of QX-FL on Ca(2) (+) sparks is likely, by analogy with flecainide, to result from Na(+) channel block. Our data reveal important differences in the interaction of flecainide with sites in the cytosolic vestibules of Na(+) and RyR2 channels.

An anionic ryanoid, 10-O-succinoylryanodol, provides insights into the mechanisms governing the interaction of ryanoids and the subsequent altered function of ryanodine-receptor channels.

We have investigated the interactions of a novel anionic ryanoid, 10-O-succinoylryanodol, with individual mammalian cardiac muscle ryanodine receptor channels under voltage clamp conditions. As is the case for all ryanoids so far examined, the interaction of 10-O-succinoylryanodol with an individual RyR channel produces profound alterations in both channel gating and rates of ion translocation. In the continued presence of the ryanoid the channel fluctuates between periods of normal and modified gating, indicating a reversible interaction of the ligand with its receptor. Unlike the majority of ryanoids, we observe a range of different fractional conductance states of RyR in the presence of 10-O-succinoylryanodol. We demonstrate that 10-O-succinoylryanodol is a very flexible molecule and propose that each fractional conductance state arises from the interaction of a different conformer of the ryanoid molecule with the RyR channel. The probability of channel modification by 10-O-succinoylryanodol is dependent on the transmembrane holding potential. Comparison of the voltage dependence of channel modification by this novel anionic ryanoid with previous data obtained with cationic and neutral ryanoids reveals that the major influence of transmembrane potential on the probability of RyR channel modification by ryanoids results from an alteration in receptor affinity. These investigations also demonstrate that the charge of the ryanoid has a major influence on the rate of association of the ligand with its receptor indicating that ionic interactions are likely to be involved in this reaction.

The cardiac ryanodine receptor (calcium release channel): emerging role in heart failure and arrhythmia pathogenesis.

The cardiac sarcoplasmic reticulum calcium release channel, commonly referred to as the ryanodine receptor, is a key component in cardiac excitation-contraction coupling, where it is responsible for the release of calcium from the sarcoplasmic reticulum. As our knowledge of the ryanodine receptor has advanced an appreciation that this key E-C coupling component may have a role in the pathogenesis of human cardiac disease has emerged. Heart failure and arrhythmia generation are both pathophysiological states that can result from deranged excitation-contraction coupling. Evidence is now emerging that hyperphosphorylation of the cardiac ryanodine receptor is an important event in chronic heart failure, contributing to impaired contraction and the generation of triggered ventricular arrhythmias. Furthermore the therapeutic benefits of beta blockers in heart failure appear to be partly explained through a reversal of this phenomenon. Two rare inherited arrhythmogenic conditions, which can cause sudden death in children, have also been shown to result from mutations in the cardiac ryanodine receptor. These conditions, catecholaminergic polymorphic ventricular tachycardia and arrhythmogenic right ventricular cardiomyopathy (subtype 2), further implicate the ryanodine receptor as a potentially arrhythmogenic substrate and suggest that this channel may offer a new therapeutic target in the treatment of both cardiac arrhythmias and heart failure.

Dantrolene rescues aberrant N-terminus intersubunit interactions in mutant pro-arrhythmic cardiac ryanodine receptors.

AIMS: The ryanodine receptor (RyR2) is an intracellular Ca(2+) release channel essential for cardiac excitation-contraction coupling. Abnormal RyR2 channel function results in the generation of arrhythmias and sudden cardiac death. The present study was undertaken to investigate the mechanistic basis of RyR2 dysfunction in inherited arrhythmogenic cardiac disease. METHODS AND RESULTS: We present several lines of complementary evidence, indicating that the arrhythmia-associated L433P mutation disrupts RyR2 N-terminus self-association. A combination of yeast two-hybrid, co-immunoprecipitation, and chemical cross-linking assays collectively demonstrate that a RyR2 N-terminal fragment carrying the L433P mutation displays substantially reduced self-interaction compared with wild type. Moreover, sucrose density gradient centrifugation reveals that the L433P mutation impairs tetramerization of the full-length channel. [(3)H]Ryanodine-binding assays demonstrate that disrupted N-terminal intersubunit interactions within RyR2(L433P) confer an altered sensitivity to Ca(2+) activation. Calcium imaging of RyR2(L433P)-expressing cells reveals substantially prolonged Ca(2+) transients and reduced Ca(2+) store content indicating defective channel closure. Importantly, dantrolene treatment reverses the L433P mutation-induced impairment and restores channel function. CONCLUSION: The N-terminus domain constitutes an important structural determinant for the functional oligomerization of RyR2. Our findings are consistent with defective N-terminus self-association as a molecular mechanism underlying RyR2 channel deregulation in inherited arrhythmogenic cardiac disease. Significantly, the therapeutic action of dantrolene may occur via the restoration of normal RyR2 N-terminal intersubunit interactions.

A Systemized Approach to Investigate Ca(2+) Synchronization in Clusters of Human Induced Pluripotent Stem-Cell Derived Cardiomyocytes.

Induced pluripotent stem cell-derived cardiomyocytes (IPS-CM) are considered by many to be the cornerstone of future approaches to repair the diseased heart. However, current methods for producing IPS-CM typically yield highly variable populations with low batch-to-batch reproducibility. The underlying reasons for this are not fully understood. Here we report on a systematized approach to investigate the effect of maturation in embryoid bodies (EB) vs. "on plate" culture on spontaneous activity and regional Ca(2+) synchronization in IPS-CM clusters. A detailed analysis of the temporal and spatial organization of Ca(2+) spikes in IPS-CM clusters revealed that the disaggregation of EBs between 0.5 and 2 weeks produced IPS-CM characterized by spontaneous beating and high levels of regional Ca(2+) synchronization. These phenomena were typically absent in IPS-CM obtained from older EBs (>2 weeks). The maintenance of all spontaneously active IPS-CM clusters under "on plate" culture conditions promoted the progressive reduction in regional Ca(2+) synchronization and the loss of spontaneous Ca(2+) spiking. Raising the extracellular [Ca(2+)] surrounding these quiescent IPS-CM clusters from ~0.4 to 1.8 mM unmasked discrete behaviors typified by either (a) long-lasting Ca(2+) elevation that returned to baseline or (b) persistent, large-amplitude Ca(2+) oscillations around an increased cytoplasmic [Ca(2+)]. The different responses of IPS-CM to elevated extracellular [Ca(2+)] could be traced back to their routes of derivation. The data point to the possibility of predictably influencing IPS-CM phenotype and response to external activation via defined interventions at early stages in their maturation.