RyR2 Activity Research Articles

Mitochondrial Abnormalities in a Mouse CPVT Model with RyR2 Loss-Of-Function Mutation

Mitochondria exchange matrix content in a unique and dynamic way by one of two mechanisms: a short-range direct interaction via closely spaced kissing junctions or long-range propagation of signal by the extrusion of extended nanotubular extensions (nanotunneling) (Huang et al 2013. Proc Natl Acad Sci U S A, 110(8): 2846-51). Both are normal, but infrequent features of cardiac myocytes. It is interesting to know whether this behavior may be affected by changes in Ca2+ homeostasis. We evaluated the cardiac ultrastructure in 7-month old heterozygous mice with a cardiac ryanodine receptor (RyR2) mutation (RyR2-A4860G) that depresses channel function and leads to Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) without hypertrophy (Zhao et al., submitted). The mutation is embryonic lethal in mice homozygous for the mutation. In ventricular myocytes of heterozygotes mice (RyR2-A4860G+/−) it has a dual effect on SR Ca2+ release: an initial reduction due to lower RyR2 activity, linked to random bursts due to SR Ca2+ overload. The most striking and only structural alteration in cardiac myocytes in the mutant left ventricle is a clear increase in the frequency of long thin nano-tunneling mitochondrial extensions. The frequency of nanotunneling was ∼3 fold higher in mutant than in WT mice. Numerous small mitochondrial profiles are more frequent in mutant mice resulting in an average surface area of 0.17± 0.15 µm2 in mutant versus 0.28 ± 0.22 µm2 in WT (p<0.00001). This decrease is mostly due to sections through the thin tunneling extensions. The increased nanotunneling frequency would indicate an enhanced rate of long range intermitochondrial signal transfer. Interestingly, no activation of nanotunneling has been reported by EM in other CPVT models and/or in other alterations affecting proteins of CRU and Ca2+ homeostasis, making this a unique mitochondrial response.

Mechanisms of SR calcium release in healthy and failing human hearts.

Normal heart contraction and rhythm relies on the proper flow of calcium ions (Ca2+) into cardiac cells and between their intracellular organelles, and any disruption can lead to arrhythmia and sudden cardiac death. Electrical excitation of the surface membrane activates voltage-dependent L-type Ca2+ channels to open and allow Ca2+ to enter the cytoplasm. The subsequent increase in cytoplasmic Ca2+ concentration activates calcium release channels (RyR2) located at specialised Ca2+ release sites in the sarcoplasmic reticulum (SR), which serves as an intracellular Ca2+ store. Animal models have provided valuable insights into how intracellular Ca2+ transport mechanisms are altered in human heart failure. The aim of this review is to examine how Ca2+ release sites are remodelled in heart failure and how this affects intracellular Ca2+ transport with an emphasis on Ca2+ release mechanisms in the SR. Current knowledge on how heart failure alters the regulation of RyR2 by Ca2+ and Mg2+ and how these mechanisms control the activity of RyR2 in the confines of the Ca2+ release sites is reviewed.

Multiple modes of ryanodine receptor 2 inhibition by flecainide.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) causes sudden cardiac death due to mutations in cardiac ryanodine receptors (RyR2), calsequestrin, or calmodulin. Flecainide, a class I antiarrhythmic drug, inhibits Na(+) and RyR2 channels and prevents CPVT. The purpose of this study is to identify inhibitory mechanisms of flecainide on RyR2. RyR2 were isolated from sheep heart, incorporated into lipid bilayers, and investigated by single-channel recording under various activating conditions, including the presence of cytoplasmic ATP (2 mM) and a range of cytoplasmic [Ca(2+)], [Mg(2+)], pH, and [caffeine]. Flecainide applied to either the cytoplasmic or luminal sides of the membrane inhibited RyR2 by two distinct modes: 1) a fast block consisting of brief substate and closed events with a mean duration of ∼1 ms, and 2) a slow block consisting of closed events with a mean duration of ∼1 second. Both inhibition modes were alleviated by increasing cytoplasmic pH from 7.4 to 9.5 but were unaffected by luminal pH. The slow block was potentiated in RyR2 channels that had relatively low open probability, whereas the fast block was unaffected by RyR2 activation. These results show that these two modes are independent mechanisms for RyR2 inhibition, both having a cytoplasmic site of action. The slow mode is a closed-channel block, whereas the fast mode blocks RyR2 in the open state. At diastolic cytoplasmic [Ca(2+)] (100 nM), flecainide possesses an additional inhibitory mechanism that reduces RyR2 burst duration. Hence, multiple modes of action underlie RyR2 inhibition by flecainide.

An explant muscle model to examine the refinement of the synaptic landscape.

Signals from nerve and muscle regulate the formation of synapses. Transgenic mouse models and muscle cell cultures have elucidated the molecular mechanisms required for aggregation and stabilization of synaptic structures. However, far less is known about the molecular pathways involved in redistribution of muscle synaptic components. Here we established a physiologically viable whole-muscle embryonic explant system, in the presence or absence of the nerve, which demonstrates the synaptic landscape is dynamic and malleable. Manipulations of factors intrinsic to the muscle or extrinsically provided by the nerve illustrate vital functions during formation, redistribution and elimination of acetylcholine receptor (AChR) clusters. In particular, RyR1 activity is an important mediator of these functions. This physiologically relevant and readily accessible explant system provides a new approach to genetically uncouple nerve-derived signals and for manipulation via signaling molecules, drugs, and electrical stimulation to examine early formation of the neuromuscular circuit.

Differential rescue of spatial memory deficits in aged rats by L-type voltage-dependent calcium channel and ryanodine receptor antagonism

Age-associated memory impairments may result as a consequence of neuroinflammatory induction of intracellular calcium (Ca+2) dysregulation. Altered L-type voltage-dependent calcium channel (L-VDCC) and ryanodine receptor (RyR) activity may underlie age-associated learning and memory impairments. Various neuroinflammatory markers are associated with increased activity of both L-VDCCs and RyRs, and increased neuroinflammation is associated with normal aging. In vitro, pharmacological blockade of L-VDCCs and RyRs has been shown to be anti-inflammatory. Here, we examined whether pharmacological blockade of L-VDCCs or RyRs with the drugs nimodipine and dantrolene, respectively, could improve spatial memory and reduce age-associated increases in microglia activation. Dantrolene and nimodipine differentially attenuated age-associated spatial memory deficits but were not anti-inflammatory in vivo. Furthermore, RyR gene expression was inversely correlated with spatial memory, highlighting the central role of Ca+2 dysregulation in age-associated memory deficits.

Long-term simulated microgravity causes cardiac RyR2 phosphorylation and arrhythmias in mice

BackgroundLong-term exposure to microgravity during space flight may lead to cardiac remodeling and rhythm disturbances. In mice, hindlimb unloading (HU) mimics the effects of microgravity and stimulates physiological adaptations, including cardiovascular deconditioning. Recent studies have demonstrated an important role played by changes in intracellular Ca handling in the pathogenesis of heart failure and arrhythmia. In this study, we tested the hypothesis that cardiac remodeling following HU in mice involves abnormal intracellular Ca regulation through the cardiac ryanodine receptor (RyR2). Methods and resultsMice were subjected to HU by tail suspension for 28 to 56days in order to induce cardiac remodeling (n=15). Control mice (n=19) were treated equally, with the exception of tail suspension. Echocardiography revealed cardiac enlargement and depressed contractility starting at 28days post-HU versus control. Moreover, mice were more susceptible to pacing-induced ventricular arrhythmias after HU. Ventricular myocytes isolated from HU mice exhibited an increased frequency of spontaneous sarcoplasmic reticulum (SR) Ca release events and enhanced SR Ca leak via RyR2. Western blotting revealed increased RyR2 phosphorylation at S2814, and increased CaMKII auto-phosphorylation at T287, suggesting that CaMKII activation of RyR2 might underlie enhanced SR Ca release in HU mice. ConclusionThese data suggest that abnormal intracellular Ca handling, likely due to increased CaMKII phosphorylation of RyR2, plays a role in cardiac remodeling following simulated microgravity in mice.

Adverse effects of doxorubicin and its metabolic product on cardiac RyR2 and SERCA2A.

The use of anthracycline chemotherapeutic drugs is restricted owing to potentially fatal cardiotoxic side effects. It has been hypothesized that anthracycline metabolites have a primary role in this cardiac dysfunction; however, information on the molecular interactions of these compounds in the heart is scarce. Here we provide novel evidence that doxorubicin and its metabolite, doxorubicinol, bind to the cardiac ryanodine receptor (RyR2) and to the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA2A) and deleteriously alter their activity. Both drugs (0.01 μM-2.5 μM) activated single RyR2 channels, and this was reversed by drug washout. Both drugs caused a secondary inhibition of RyR2 activity that was not reversed by drug washout. Preincubation with the reducing agent dithiothreitol (DTT, 1 mM) prevented drug-induced inhibition of channel activity. Doxorubicin and doxorubicinol reduced the abundance of thiol groups on RyR2, further indicating that oxidation reactions may be involved in the actions of the compounds. Ca(2+) uptake into sarcoplasmic reticulum vesicles by SERCA2A was inhibited by doxorubicinol, but not doxorubicin. Unexpectedly, in the presence of DTT, doxorubicinol enhanced the rate of Ca(2+) uptake by SERCA2A. Together the evidence provided here shows that doxorubicin and doxorubicinol interact with RyR2 and SERCA2A in similar ways, but that the metabolite acts with greater efficacy than the parent compound. Both compounds modify RyR2 and SERCA2A activity by binding to the proteins and also act via thiol oxidation to disrupt SR Ca(2+) handling. These actions would have severe consequences on cardiomyocyte function and contribute to clinical symptoms of acute anthracycline cardiotoxicity.

CaMKII regulation of cardiac ryanodine receptors and inositol triphosphate receptors.

Ryanodine receptors (RyRs) and inositol triphosphate receptors (InsP3Rs) are structurally related intracellular calcium release channels that participate in multiple primary or secondary amplified Ca2+ signals, triggering muscle contraction and oscillatory Ca2+ waves, or activating transcription factors. In the heart, RyRs play an indisputable role in the process of excitation–contraction coupling as the main pathway for Ca2+ release from sarcoplasmic reticulum (SR), and a less prominent role in the process of excitation–transcription coupling. Conversely, InsP3Rs are believed to contribute in subtle ways, only, to contraction of the heart, and in more important ways to regulation of transcription factors. Because uncontrolled activity of either RyRs or InsP3Rs may elicit life-threatening arrhythmogenic and/or remodeling Ca2+ signals, regulation of their activity is of paramount importance for normal cardiac function. Due to their structural similarity, many regulatory factors, accessory proteins, and post-translational processes are equivalent for RyRs and InsP3Rs. Here we discuss regulation of RyRs and InsP3Rs by CaMKII phosphorylation, but touch on other kinases whenever appropriate. CaMKII is emerging as a powerful modulator of RyR and InsP3R activity but interestingly, some of the complexities and controversies surrounding phosphorylation of RyRs also apply to InsP3Rs, and a clear-cut effect of CaMKII on either channel eludes investigators for now. Nevertheless, some effects of CaMKII on global cellular activity, such as SR Ca2+ leak or force-frequency potentiation, appear clear now, and this constrains the limits of the controversies and permits a more tractable approach to elucidate the effects of phosphorylation at the single channel level.

Oxidative stress and the impact of prenatal chronic hypoxia on ryanodine receptor generated calcium responses in fetal pulmonary arterial myocytes (1089.11)

Ryanodine receptors (RyRs) are sensitive to reactive oxygen species in that this stress increases leak of Ca2+ through the channels. Our recent work shows RyR‐generated Ca2+ sparks are aberrant following prenatal chronic hypoxia (CH) in pulmonary arterial myocytes of fetal sheep. Other evidence suggests that the enhanced Ca2+ leak due to oxidative stress can cause loss of Ca2+ from the sarcoplasmic reticulum (SR); our Ca2+ spark data are consistent with such a loss in SR Ca2+ and CH can cause oxidative stress. To address potential Ca2+ loss we quantified 10 mM caffeine (CAF) elicited Ca2+ release from the SR, as RyR activation provides an index of Ca2+ storage. This was performed in pulmonary arterial myocytes of intact arteries using Fluo‐4 and confocal imaging approaches of fetal sheep that lived in normoxia at low altitude (FN) or fetal sheep that lived in CH at (3,801 m) for &gt;100 days (FH). Using customized analysis software, the data show that under control conditions prenatal CH does not impact the area under the curve (AUC) due to CAF. Thus, the changes in spark activity previously observed likely arise from something other than alterations in SR Ca2+ storage. Pretreating PA with 1 mM tert‐butyl H2O2 resulted in a ~ 31% decrease in CAF‐elicited AUC in FN and a 6% increase in FH. These data suggest that sheep exposed to prenatal CH may adapt, which limits decrements in total SR Ca2+ storage in an attempt to preserve SR function.Grant Funding Source: NSF MRI 0923559, NIH HD‐069746, P01HD031226, R01HD003807, 5P20 MD‐006988, LLUSOM

Type 2 ryanodine receptors are highly sensitive to alcohol.

Exposure to ethanol levels reached in circulation during alcohol intoxication (>10mM) constricts cerebral arteries in rats and humans. Remarkably, targets and mechanisms underlying this action remain largely unidentified. Artery diameter is regulated by myocyte Ca(2+) sparks, a vasodilatory signal contributed to by type 2 ryanodine receptors (RyR2). Using laser confocal microscopy in rat cerebral arteries and bilayer electrophysiology we unveil that ethanol inhibits both Ca(2+) spark and RyR2 activity with IC50<20 mM, placing RyR2 among the ion channels that are most sensitive to ethanol. Alcohol directly targets RyR2 and its lipid microenvironment, leading to stabilization of RyR2 closed states.

Regulation of the Skeletal Muscle Ryanodine Receptor/Ca2+-release Channel RyR1 by S-Palmitoylation

The ryanodine receptor/Ca(2+)-release channels (RyRs) of skeletal and cardiac muscle are essential for Ca(2+) release from the sarcoplasmic reticulum that mediates excitation-contraction coupling. It has been shown that RyR activity is regulated by dynamic post-translational modifications of Cys residues, in particular S-nitrosylation and S-oxidation. Here we show that the predominant form of RyR in skeletal muscle, RyR1, is subject to Cys-directed modification by S-palmitoylation. S-Palmitoylation targets 18 Cys within the N-terminal, cytoplasmic region of RyR1, which are clustered in multiple functional domains including those implicated in the activity-governing protein-protein interactions of RyR1 with the L-type Ca(2+) channel CaV1.1, calmodulin, and the FK506-binding protein FKBP12, as well as in "hot spot" regions containing sites of mutations implicated in malignant hyperthermia and central core disease. Eight of these Cys have been identified previously as subject to physiological S-nitrosylation or S-oxidation. Diminishing S-palmitoylation directly suppresses RyR1 activity as well as stimulus-coupled Ca(2+) release through RyR1. These findings demonstrate functional regulation of RyR1 by a previously unreported post-translational modification and indicate the potential for extensive Cys-based signaling cross-talk. In addition, we identify the sarco/endoplasmic reticular Ca(2+)-ATPase 1A and the α1S subunit of the L-type Ca(2+) channel CaV1.1 as S-palmitoylated proteins, indicating that S-palmitoylation may regulate all principal governors of Ca(2+) flux in skeletal muscle that mediates excitation-contraction coupling.

β1a490–508, a 19-Residue Peptide from C-Terminal Tail of Cav1.1 β1a Subunit, Potentiates Voltage-Dependent Calcium Release in Adult Skeletal Muscle Fibers

The α1 and β1a subunits of the skeletal muscle calcium channel, Cav1.1, as well as the Ca2+ release channel, ryanodine receptor (RyR1), are essential for excitation-contraction coupling. RyR1 channel activity is modulated by the β1a subunit and this effect can be mimicked by a peptide (β1a490–524) corresponding to the 35-residue C-terminal tail of the β1a subunit. Protein-protein interaction assays confirmed a high-affinity interaction between the C-terminal tail of the β1a and RyR1. Based on previous results using overlapping peptides tested on isolated RyR1, we hypothesized that a 19-amino-acid residue peptide (β1a490–508) is sufficient to reproduce activating effects of β1a490–524. Here we examined the effects of β1a490–508 on Ca2+ release and Ca2+ currents in adult skeletal muscle fibers subjected to voltage-clamp and on RyR1 channel activity after incorporating sarcoplasmic reticulum vesicles into lipid bilayers. β1a490–508 (25 nM) increased the peak Ca2+ release flux by 49% in muscle fibers. Considerably fewer activating effects were observed using 6.25, 100, and 400 nM of β1a490–508 in fibers. β1a490–508 also increased RyR1 channel activity in bilayers and Cav1.1 currents in fibers. A scrambled form of β1a490–508 peptide was used as negative control and produced negligible effects on Ca2+ release flux and RyR1 activity. Our results show that the β1a490–508 peptide contains molecular components sufficient to modulate excitation-contraction coupling in adult muscle fibers.

The ryanodine receptor store-sensing gate controls Ca2+ waves and Ca2+-triggered arrhythmias

Spontaneous Ca(2+) release from intracellular stores is important for various physiological and pathological processes. In cardiac muscle cells, spontaneous store overload-induced Ca(2+) release (SOICR) can result in Ca(2+) waves, a major cause of ventricular tachyarrhythmias (VTs) and sudden death. The molecular mechanism underlying SOICR has been a mystery for decades. Here we show that a point mutation, E4872A, in the helix bundle crossing region (the proposed gate) of the cardiac ryanodine receptor (RyR2) completely abolishes luminal, but not cytosolic, Ca(2+) activation of RyR2. The introduction of metal-binding histidines at this site converts RyR2 into a luminal Ni(2+)-gated channel. Mouse hearts harboring a heterozygous RyR2 mutation at this site (E4872Q) are resistant to SOICR and are completely protected against Ca(2+)-triggered VTs. These data show that the RyR2 gate directly senses luminal (store) Ca(2+), explaining the regulation of RyR2 by luminal Ca(2+), the initiation of Ca(2+) waves and Ca(2+)-triggered arrhythmias. This newly identified store-sensing gate structure is conserved in all RyR and inositol 1,4,5-trisphosphate receptor isoforms.

PCB 136 Atropselectively Alters Morphometric and Functional Parameters of Neuronal Connectivity in Cultured Rat Hippocampal Neurons via Ryanodine Receptor-Dependent Mechanisms

We recently demonstrated that polychlorinated biphenyl (PCB) congeners with multiple ortho chlorine substitutions sensitize ryanodine receptors (RyRs), and this activity promotes Ca²⁺-dependent dendritic growth in cultured neurons. Many ortho-substituted congeners display axial chirality, and we previously reported that the chiral congener PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl) atropselectively sensitizes RyRs. Here, we test the hypothesis that PCB 136 atropisomers differentially alter dendritic growth and other parameters of neuronal connectivity influenced by RyR activity. (-)-PCB 136, which potently sensitizes RyRs, enhances dendritic growth in primary cultures of rat hippocampal neurons, whereas (+)-PCB 136, which lacks RyR activity, has no effect on dendritic growth. The dendrite-promoting activity of (-)-PCB 136 is observed at concentrations ranging from 0.1 to 100 nM and is blocked by pharmacologic RyR antagonism. Neither atropisomer alters axonal growth or cell viability. Quantification of PCB 136 atropisomers in hippocampal cultures indicates that atropselective effects on dendritic growth are not due to differential partitioning of atropisomers into cultured cells. Imaging of hippocampal neurons loaded with Ca²⁺-sensitive dye demonstrates that (-)-PCB 136 but not (+)-PCB 136 increases the frequency of spontaneous Ca²⁺ oscillations. Similarly, (-)-PCB 136 but not (+)-PCB 136 increases the activity of hippocampal neurons plated on microelectrode arrays. These data support the hypothesis that atropselective effects on RyR activity translate into atropselective effects of PCB 136 atropisomers on neuronal connectivity, and suggest that the variable atropisomeric enrichment of chiral PCBs observed in the human population may be a significant determinant of individual susceptibility for adverse neurodevelopmental outcomes following PCB exposure.

InsP3R Activation Facilitates Ca2+ Wave Propagation in Ventricular Myocytes

The contribution and significance of sarcoplasmic reticulum (SR) Ca2+ release driven by InsP3-R activation (IP3ICR) in cardiac excitation-contraction coupling (ECC) is still a matter of debate. In particular it's role in the Ca2+-induced Ca2+ release (CICR) mechanism in non-hypertrophic ventricular myocytes seems to be ambiguous. Here we characterize the contribution of IP3ICR in Ca2+ wave propagation driven by CICR in ventricular myocytes. We hypothesized a functional crosstalk and/or cooperativity between both SR-Ca2+ release channels, e.g. IP3ICR may trigger or facilitate CICR via RyR activation and/or RyR sensitization. IP3ICR was activated in a highly targeted way by UV-flash photolysis of a membrane-permeant caged InsP3 while simultaneously imaging Ca2+ with laser-scanning confocal microscopy in ventricular myocytes acutely isolated from mouse hearts. Recently, it has been shown that cellular ECC remodeling under pathophysiological stress leads to increased expression of InsP3-Rs and IP3ICR. To boost this situation to the extreme and in order to unmask a potential role of IP3ICR in ECC we used an InsP3-R overexpressing mouse model. Photolytic InsP3 release resulted in an immediate increase in Ca2+ wave propagation (from 63±7.3 μm/s in control to 79±3.7 μm/s after flash, n=10). This response was sensitive to the InsP3-R blocker Xestospongin C and was not seen in WT mice. After repetitive photolytic InsP3 release in a time window of >5 min, Ca2+ wave propagation reverted back to control. In addition, spontaneous Ca2+ wave appearance decreased or dissapeared. This suggests that Ca2+ wave propagation under conditions of high functional InsP3-R expression are accelerated by regional RyR sensitization mediated by IP3ICR. However, over long term RyR Ca2+ desensitisation and SR-Ca2+ store depletion by InsP3-Rs openings may lead to Ca2+ wave termination. Supported by SNF and Novartis Res. Foundation.

Cardiac ryanodine receptor activation by high Ca2+ store load is reversed in a reducing cytoplasmic redox environment

ABSTRACTHere, we report the impact of redox potential on isolated cardiac ryanodine receptor (RyR2) channel activity and its response to physiological changes in luminal [Ca2+]. Basal leak from the sarcoplasmic reticulum is required for normal Ca2+ handling, but excess diastolic Ca2+ leak attributed to oxidative stress is thought to lower the threshold of RyR2 for spontaneous sarcoplasmic reticulum Ca2+ release, thus inducing arrhythmia in pathological situations. Therefore, we examined the RyR2 response to luminal [Ca2+] under reducing or oxidising cytoplasmic redox conditions. Unexpectedly, as luminal [Ca2+] increased from 0.1 to 1.5 mM, RyR2 activity declined when pretreated with cytoplasmic 1 mM DTT or buffered with GSH∶GSSG to a normal reduced cytoplasmic redox potential (−220 mV). Conversely, with 20 µM cytoplasmic 4,4′-DTDP or buffering of the redox potential to an oxidising value (−180 mV), RyR2 activity increased with increasing luminal [Ca2+]. The luminal redox potential was constant at −180 mV in each case. These responses to luminal [Ca2+] were maintained with cytoplasmic 2 mM Na2ATP or 5 mM MgATP (1 mM free Mg2+). Overall, the results suggest that the redox potential in the RyR2 junctional microdomain is normally more oxidised than that of the bulk cytoplasm.

Phosphorylation of Maurocalcine Strongly Modifies its Effect on Type 1 Ryanodine Receptor

Maurocalcine (MCa) is a 33-amino acid peptide isolated from the venom of Scorpio maurus palmatus, a Tunisian scorpion. It possesses efficient cell penetration properties and has been shown to strongly modify ryanodine receptor (RyR1) channel behavior by increasing open probability and inducing a long-lasting subconductance state. The amino acid residue threonine at position 26 belongs to a putative phosphorylation site within MCa sequence. We investigated the effect of a) T26 phosphorylation (MCa T26Phospho) by protein kinase A and b) replacement of T26 by glutamic acid residue to mimic phosphorylation on the effect of MCa on RyR1 properties. Using [3H]ryanodine ([3H]Ry) binding and single channel voltage-clamp measurements, we show that both MCa T26Phospho and MCa T26E analogs almost completely lose the ability to induce RyR1 activation. Neither MCa T26Phospho nor MCa T26E (up to 2µM) increase [3H]rya binding. Small increase (∼1.65-fold activation; p<0.01) was observed with high concentrations (10µM) of either MCa T26Phospho or MCa T26E. Single channel measurements revealed that neither MCa T26E nor MCa T26Phospho (up to 2µM) were able to induce the characteristic long-lasting subconductance state of RyR1 observed in presence of wild-type MCa. MCa T26Phospho altered RyR1 channel gating in a time-dependent biphasic manner with initial activation of Po followed by almost complete inactivation. These effects of MCa T26Phospho could be reversed by 200nM wt MCa. At concentrations ≥2μM, MCa T26E enhances Po∼3 fold by shortening τc without altering τo. These results show that single phosphorylation of MCa modifies RyR1 activity.1P01 AR52354.

Modulation of DHPR Inactivation by RyR1 Activity in Mouse Skeletal Muscle Fibers

Malignant hyperthermia is a potentially fatal hypermetabolic state originating from excessive release of calcium stored in the sarcoplasmic reticulum (SR) of skeletal muscle. In most cases, MH susceptibility results from mutations in the type 1 ryanodine receptor (RyR1). In previous work (Andronache et al., PNAS 2009) we reported that heterozygous murine carriers of MH mutation Y524S (human Y522S) exhibit changes in steady state inactivation of the dihydropyridine receptor (DHPR), the sensor of the transverse tubular (TT) membrane potential. Availability curves were left shifted along the voltage axis suggesting that a feedback signal from RyR1 modulates DHPR inactivation. In the present study we investigated the hypothesis that junctional fluctuations of free Ca2+ concentration are involved in the feedback mechanism. We performed two-electrode voltage clamp experiments on enzymatically isolated toe muscle fibers of both WT and mutant mice (Y524S+/−) and measured L-type Ca2+ current and optical signals from fluorescent Ca2+ indicators. To test the hypothesis we applied conditions that would modify junctional Ca2+ levels. Millimolar concentrations of caffeine led to a left shift in the availability curve for L-type current indicating that drug-induced RyR1 hyperactivity can mimic the effect of the mutation. On the other hand, internal dialysis with an artificial solution containing 10 mM of BAPTA to effectively reduce the local Ca2+ transients near open RyR1 channels had little effect on the difference in steady state inactivation between WT and mutant fibers. We conclude that the altered inactivation depends on RyR1 hyperactivity but does not require the continuous presence of local Ca2+ fluctuations within the junctional gap separating TT and SR.

Ligand-Dependent Conformational Changes in the Cardiac Ryanodine Receptor

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).

The mechanism of hetero-synaptic interaction based on spatiotemporal intracellular calcium dynamics.

The mechanism of hetero-synaptic interaction based on spatiotemporal intracellular calcium dynamics.