RyR Isoforms Research Articles

Rapid small-scale nanobody-assisted purification of ryanodine receptors for cryo-EM

Ryanodine receptors (RyRs) are large Ca2+ release channels residing in the endoplasmic or sarcoplasmic reticulum membrane. Three isoforms of RyRs have been identified in mammals, the disfunction of which has been associated with a series of life-threatening diseases. The need for large amounts of native tissue or eukaryotic cell cultures limits advances in structural studies of RyRs. Here, we report a method that utilizes nanobodies to purify RyRs from only 5 mg of total protein. The purification process, from isolated membranes to cryo-EM grade protein, is achieved within four hours on the bench, yielding protein usable for cryo-EM analysis. This is demonstrated by solving the structures of rabbit RyR1, solubilized in detergent, reconstituted into lipid nanodiscs or liposomes, and bovine RyR2 reconstituted in nanodisc, and mouse RyR2 in detergent. The reported method facilitates structural studies of RyRs directed toward drug development and is useful in cases where the amount of starting material is limited.

Molecular Aspects Implicated in Dantrolene Selectivity with Respect to Ryanodine Receptor Isoforms

Dantrolene is an intra-cellularly acting skeletal muscle relaxant used for the treatment of the rare genetic disorder, malignant hyperthermia (MH). In most cases, MH susceptibility is caused by dysfunction of the skeletal ryanodine receptor (RyR1) harboring one of nearly 230 single-point MH mutations. The therapeutic effect of dantrolene is the result of a direct inhibitory action on the RyR1 channel, thus suppressing aberrant Ca2+ release from the sarcoplasmic reticulum. Despite the almost identical dantrolene-binding sequence exits in all three mammalian RyR isoforms, dantrolene appears to be an isoform-selective inhibitor. Whereas RyR1 and RyR3 channels are competent to bind dantrolene, the RyR2 channel, predominantly expressed in the heart, is unresponsive. However, a large body of evidence suggests that the RyR2 channel becomes sensitive to dantrolene-mediated inhibition under certain pathological conditions. Although a consistent picture of the dantrolene effect emerges from in vivo studies, in vitro results are often contradictory. Hence, our goal in this perspective is to provide the best possible clues to the molecular mechanism of dantrolene's action on RyR isoforms by identifying and discussing potential sources of conflicting results, mainly coming from cell-free experiments. Moreover, we propose that, specifically in the case of the RyR2 channel, its phosphorylation could be implicated in acquiring the channel responsiveness to dantrolene inhibition, interpreting functional findings in the structural context.

Kinetics and mapping of calcium-driven calmodulin conformations on ryanodine receptors.

Calmodulin (CaM) is an important Ca2+-sensor for Ca2+ cycling between the sarcoplasmic reticulum and cytoplasm during relaxation and contraction in cardiac and skeletal muscle. Although several high-resolution cryo-EM structures have been published recently, the physiologically relevant mechanism by which CaM modulates the SR Ca2+ release channel (ryanodine receptor, RyR) is still not completely understood. Using distances calculated from fluorescence lifetime (FLT)-detected FRET between RyR-bound FKBP to CaM, we have trilaterated the structural states of CaM and mapped Ca2+-driven shifts in the conformation of CaM bound to RyR. As the locations of both the N- and C-lobes can be determined, we found that CaM becomes more compact in contracting high-Ca2+ relative to the relaxing low-Ca2+ and that skeletal and cardiac RyR isoforms show different CaM-RyR conformations and binding. These structural observations correlate with the experimentally determined modulatory action of CaM. By integrating our FLT-FRET system in a stopped-flow experiment, we resolved CaM binding kinetics and millisecond Ca2+-driven structural transitions of CaM-bound to RyR. Distinct binding kinetics of CaM to RyR1 and RyR2 were observed, suggesting different CaM-RyR interactions. Specifically, CaM association to RyR2 is three-fold faster vs. RyR1 at 30 nM and 30 μM. Upon rapid Ca2+ transition from μM to nM, the structural transition is four-fold slower for CaM bound to RyR2 relative to RyR1. The results provide mechanistic insight into CaM's action to transduce Ca2+ signals for RyR channel function during muscle contraction, and furthermore the technology developed holds promise for determining structure-function relationships of other RyR modulators. This work was supported by NIH grants R01HL92097 (RLC, DMB) and R37AG26160 (DDT), and by American Heart Association Fellowship 16POST31010019 (RTR).

Expression of Concern: RyR1 and RyR3 isoforms provide distinct intracellular Ca2+ signals in HEK 293 cells.

There was an error published in Fig. 6 in J. Cell Sci. (2002) 115, 2497-2504 (doi:10.1242/jcs.115.12.2497).There is a duplication of a segment of signal in Fig. 6A. According to the authors, this was due to an error in the process of assembling the trace. They made every effort to find the original data, but, as the work dates back more than twenty years, they were unable to recover the original files.The authors state that this error does not significantly impact the interpretation of the article, the data reliability or the reader's understanding of the paper and that the scholarly integrity of the article remains intact. “Data reported in Fig. 6A and B aimed to illustrate variability of spontaneous Ca2+ signals in two RyR3-expressing cells; spontaneous Ca2+ release events are also reported in Fig. 5 and duplication of signal in Fig. 6Ab does not impact on the main interpretation of the article which was that HEK293 cells expressing RyR3 display spontaneous Ca2+ release events and those expressing RyR1 do not. This suggested that, at least in these cells, isoform-specific functional properties may contribute to generation of intracellular Ca2+ signals.”Without the original data, it is difficult to determine how the issues arose, so the journal is publishing this note to alert readers to our concerns, with the agreement of the authors.

Structural Insight Into Ryanodine Receptor Channelopathies.

The ryanodine receptors (RyRs) are large cation-selective ligand-gated channels that are expressed in the sarcoplasmic reticulum (SR) membrane. They mediate the controlled release of Ca2+ from SR and play an important role in many cellular processes. The mutations in RyRs are associated with several skeletal muscle and cardiac conditions, including malignant hyperthermia (MH), central core disease (CCD), catecholaminergic polymorphic ventricular tachycardia (CPVT), and arrhythmogenic right ventricular dysplasia (ARVD). Recent breakthroughs in structural biology including cryo-electron microscopy (EM) and X-ray crystallography allowed the determination of a number of near-atomic structures of RyRs, including wildtype and mutant structures as well as the structures in complex with different modulating molecules. This allows us to comprehend the physiological gating and regulatory mechanisms of RyRs and the underlying pathological mechanisms of the disease-causing mutations. In this review, based on the insights gained from the available high-resolution structures of RyRs, we address several questions: 1) what are the gating mechanisms of different RyR isoforms; 2) how RyRs are regulated by multiple channel modulators, including ions, small molecules, and regulatory proteins; 3) how do disease-causing mutations affect the structure and function of RyRs; 4) how can these structural information aid in the diagnosis of the related diseases and the development of pharmacological therapies.

Neglected wardens: T lymphocyte ryanodine receptors.

Ryanodine receptors (RyRs) are intracellular Ca2+ release channels ubiquitously expressed in various cell types. RyRs were extensively studied in striated muscle cells due to their crucial role in muscle contraction. In contrast, the role of RyRs in Ca2+ signalling and functions in non-excitable cells, such as T lymphocytes, remains poorly understood. Expression of different isoforms of RyRs was shown in primary T cells and T cell lines. In T cells, RyRs co-localize with the plasmalemmal store-operated Ca2+ channels of the Orai family and endoplasmic reticulum Ca2+ sensing Stim family proteins and are activated by store-operated Ca2+ entry and pyridine nucleotide metabolites, the intracellular second messengers generated upon stimulation of T cell receptors. Experimental data indicate that together with d-myo-inositol 1,4,5-trisphosphate receptors, RyRs regulate intercellular Ca2+ dynamics by controlling Ca2+ concentration within the lumen of the endoplasmic reticulum and, consequently, store-operated Ca2+ entry. Gain-of-function mutations, genetic deletion or pharmacological inhibition of RyRs alters T cell Ca2+ signalling and effector functions. The picture emerging from the collective data shows that RyRs are the essential regulators of T cell Ca2+ signalling and can be potentially used as molecular targets for immunomodulation or T cell-based diagnostics of the disorders associated with RyRs dysregulation.

RyR1 Mutation Associated with Malignant Hyperthermia Induces Cardiac Arrythmia via Mitochondrial Calcium Overload

Introduction The “leaky” gain-of-function mutations of the ryanodine receptor type 1 (RyR1) are linked to skeletal muscle disorders, including malignant hyperthermia (MH), which occurs in response to particular medications used during general anesthesia. In addition to a severe reaction to particular anesthetic drugs, sudden cardiac death (SCD) in MH patients has been also reported to occur in non-anesthesia conditions. However, the molecular mechanism of SCD associated with MH remains unresolved. Although the major RyR isoform expressed in the heart is RyR2, we previously reported a low level of RyR1 expression in cardiac mitochondria but not in the sarcoplasmic reticulum (SR). Hypothesis Chronic mitochondrial Ca2+ overload through the ‘leaky’ RyR1 expressed in the cardiac mitochondria induces mitochondrial injury and oxidative stress leading to cardiac arrhythmia. Methods We utilized a knock-in mouse model carrying a human RyR1 MH mutation Y522S (YS) and assessed in vivo and ex vivo cardiac function. Cardiac mitochondria and ventricular myocytes were isolated from heterozygous YS and wild type (WT) mouse hearts for in situ assays. Results Both WT and heterozygous YS hearts possessed RyR1 expression in the mitochondria but not in SR, but only the heterozygous YS hearts showed disrupted mitochondrial morphology, damaged myofibrils, and high cellular oxidative state. In addition, isolated YS mitochondria showed higher basal mitochondrial Ca2+ concentration, and depolarized mitochondrial membrane potential. However, isolated YS ventricular myocytes possessed higher levels of mitochondrial reactive oxygen species and slower cytosolic Ca2+ clearance compared to controls. However, pretreating YS mitochondria/ ventricular myocytes with a clinically approved anti-MH drug and a RyR blocker, dantrolene, prevented these changes and normalized their Ca2+ handling profiles. Although the in vivo basal cardiac function was comparable to the WT, the YS heart was insensitive to isoproterenol-induced positive inotropy and chronotropy due to a higher level of catecholamine signaling at baseline. However, ex vivo YS (but not WT) hearts frequently developed multiple ventricular extrasystoles in response to isoproterenol bolus. Optical mapping of YS hearts showed increased early after-depolarization (EAD) and premature ventricular contractions (PVC) in response to isoproterenol, which was effectively abolished by dantrolene treatment. Conclusion: Chronic mitochondrial Ca2+ overload via ‘leaky’ RyR1 disrupts cardiac mitochondrial functions and structures and increases the vulnerability to ventricular arrhythmia by catecholamine.

Neuronal junctophilins recruit specific CaV and RyR isoforms to ER-PM junctions and functionally alter CaV2.1 and CaV2.2.

Junctions between the endoplasmic reticulum and plasma membrane that are induced by the neuronal junctophilins are of demonstrated importance, but their molecular architecture is still poorly understood and challenging to address in neurons. This is due to the small size of the junctions and the multiple isoforms of candidate junctional proteins in different brain areas. Using colocalization of tagged proteins expressed in tsA201 cells, and electrophysiology, we compared the interactions of JPH3 and JPH4 with different calcium channels. We found that JPH3 and JPH4 caused junctional accumulation of all the tested high-voltage-activated CaV isoforms, but not a low-voltage-activated CaV. Also, JPH3 and JPH4 noticeably modify CaV2.1 and CaV2.2 inactivation rate. RyR3 moderately colocalized at junctions with JPH4, whereas RyR1 and RyR2 did not. By contrast, RyR1 and RyR3 strongly colocalized with JPH3, and RyR2 moderately. Likely contributing to this difference, JPH3 binds to cytoplasmic domain constructs of RyR1 and RyR3, but not of RyR2.

Ginkgolide B Maintains Calcium Homeostasis in Hypoxic Hippocampal Neurons by Inhibiting Calcium Influx and Intracellular Calcium Release

Ginkgolide B (GB), a terpene lactone and active ingredient of Ginkgo biloba, shows protective effects in neuronal cells subjected to hypoxia. We investigated whether GB might protect neurons from hypoxic injury through regulation of neuronal Ca2+ homeostasis. Primary hippocampal neurons subjected to chemical hypoxia (0.7 mM CoCl2) in vitro exhibited an increase in cytoplasmic Ca2+ (measured from the fluorescence of fluo-4), but this effect was significantly diminished by pre-treatment with 0.4 mM GB. Electrophysiological recordings from the brain slices of rats exposed to hypoxia in vivo revealed increases in spontaneous discharge frequency, action potential frequency and calcium current magnitude, and all these effects of hypoxia were suppressed by pre-treatment with 12 mg/kg GB. Western blot analysis demonstrated that hypoxia was associated with enhanced mRNA and protein expressions of Cav1.2 (a voltage-gated Ca2+ channel), STIM1 (a regulator of store-operated Ca2+ entry) and RyR2 (isoforms of Ryanodine Receptor which mediates sarcoplasmic reticulum Ca2+ release), and these actions of hypoxia were suppressed by GB. Taken together, our in vitro and in vivo data suggest that GB might protect neurons from hypoxia, in part, by regulating Ca2+ influx and intracellular Ca2+ release to maintain Ca2+ homeostasis.

Aging alters spontaneous and neurotransmitter-mediated Ca2+ signaling in smooth muscle cells of mouse mesenteric arteries.

Aging impairs MA dilation by reducing the ability of sensory nerves to counteract sympathetic vasoconstriction. This study tested whether altered SMC Ca2+ signals to sympathetic (NE) and sensory (CGRP) neurotransmitters underlie aging-related deficits in vasodilation. MAs from young and old mice were pressurized and loaded with Fluo-4 dye for confocal measurement of SMC Ca2+ sparks and waves. Endothelial denudation resolved the influence of ECs. SMCs were immunolabeled for RyR isoforms and compared with transcript levels for RyRs and CGRP receptor components. SMCs from young vs old mice exhibited more spontaneous Ca2+ spark sites with no difference in Ca2+ waves. NE reduced spark sites and increased waves for both groups; addition of CGRP restored sparks and reduced waves only for young mice. Endothelial denudation attenuated Ca2+ responses to CGRP for young but not old mice, which were already attenuated, suggesting a diminished role for ECs with aging. CGRP receptor expression was similar between ages with increased serum CGRP in old mice, where RyR1 expression was replaced by RyR3. With aging, we suggest that altered RyR expression in SMCs contributes to impaired ability of sensory neurotransmission to restore Ca2+ signaling underlying vasomotor control during sympathetic activation.

A Cryo-EM Based Study of An Equivalent N Terminal Domain Mutation in Skeletal and Cardiac Ryanodine Receptor (RyR)

Mutations in ryanodine receptors (RyRs), large intracellular Ca2+ channels on the sarcoplasmic reticulum, are linked to disorders such as malignant hyperthermia and central core disease in the case of skeletal RyR1 isoform and catecholaminergic polymorphic ventricular tachycardia (CPVT) in the case of cardiac RyR2 isoform. RyRs are complex molecular machines whose activity is under precise regulation of interdomain interactions between the various subdomains as well as external binding partners. We examined equivalent single point mutations in the N-terminal A (NTD-A) domain of skeletal and cardiac RyR isoforms using cryo-EM to study their effect on the channel conformation and shed light on the possible underlying mechanism of leakiness. We resolved the 3D structures of RyR1 R164C and RyR2 R176Q at 3.5 Å and 3.9 Å, respectively. 3D analysis suggests that mutation R164C results in a tilting of the rhomboid structure (composed of NTDs, SPRY1-3, P1, HD1 and HD2 domains) indicative of a “primed” state in the absence of any activator ligands (such as Ca2+ or caffeine) and could be a result of disruption of the NTD-A interaction with the neighboring central domain. The effects of mutation R176Q in RyR2 were milder, suggesting isoform-specific effects on channel conformation despite the mutation being a loss of positively charged residue, arginine, in both isoforms. Analysis of these and other long-range conformational changes provides insight into the leaky phenotype of RyR1 and RyR2 produced by mutations situated far away (>80 Å) from the pore. Supported by grants AHA 19POST34430178 (K.A.I) and AHA 14GRNT19660003, MDA352845, NIH R01 AR068431, and NIH U24GM116789 (M.S.).

Resolved Structural States of Calmodulin in Regulation of Skeletal Muscle Calcium Release

Calmodulin (CaM) is proposed to modulate activity of the skeletal muscle sarcoplasmic reticulum (SR) calcium release channel (ryanodine receptor, RyR1 isoform) via a mechanism dependent on the conformation of RyR1-bound CaM. However, the correlation between CaM structure and functional regulation of RyR in physiologically relevant conditions is largely unknown. Here, we have used time-resolved fluorescence resonance energy transfer (TR-FRET) to study structural changes in CaM that may play a role in the regulation of RyR1. We covalently labeled each lobe of CaM (N and C) with fluorescent probes and used intramolecular TR-FRET to assess interlobe distances when CaM is bound to RyR1 in SR membranes, purified RyR1, or a peptide corresponding to the CaM-binding domain of RyR (RyRp). TR-FRET resolved an equilibrium between two distinct structural states (conformations) of CaM, each characterized by an interlobe distance and Gaussian distribution width (disorder). In isolated CaM, at low Ca2+, the two conformations of CaM are resolved, centered at 5 nm (closed) and 7 nm (open). At high Ca2+, the equilibrium shifts to favor the open conformation. In the presence of RyRp at high Ca2+, the closed conformation shifts to a more compact conformation and is the major component. When CaM is bound to full-length RyR1, either purified or in SR membranes, strikingly different results were obtained: 1) the two conformations are resolved and more ordered, 2) the open state is the major component, and 3) Ca2+ stabilized the closed conformation by a factor of two. We conclude that the Ca2+-dependent structural distribution of CaM bound to RyR1 is distinct from that of CaM bound to RyRp. We propose that the function of RyR1 is tuned to the Ca2+-dependent structural dynamics of bound CaM.

Molecular Insights into Calcium Dependent Regulation of Ryanodine Receptor Calcium Release Channels.

Ryanodine receptor calcium release channels (RyRs) play central roles in controlling intracellular calcium concentrations in excitable and non-excitable cells. RyRs are located in the sarcoplasmic or endoplasmic reticulum, intracellular Ca2+ storage compartment, and release Ca2+ during cellular action potentials or in response to other cellular stimuli. Mammalian cells express three structurally related isoforms of RyR. RyR1 and RyR2 are the major RyR isoforms in skeletal and cardiac muscle, respectively, and RyR3 is expressed in various tissues along with the other two isoforms. A prominent feature of RyRs is that the Ca2+ release channel activities of RyRs are regulated by calcium ions; therefore, intracellular Ca2+ release controls positive- and negative-feedback phenomena through the RyRs. RyR channel activities are also regulated by Ca2+ indirectly, i.e. through Ca2+ binding proteins at both cytosolic and sarco/endoplasmic reticulum luminal sides. Here, I summarize Ca2+-dependent feedback regulation of RyRs including recent progress in the structure/function aspects.

Crystal structure of diamondback moth ryanodine receptor Repeat34 domain reveals insect-specific phosphorylation sites

BackgroundRyanodine receptor (RyR), a calcium-release channel located in the sarcoplasmic reticulum membrane of muscles, is the target of insecticides used against a wide range of agricultural pests. Mammalian RyRs have been shown to be under the regulatory control of several kinases and phosphatases, but little is known about the regulation of insect RyRs by phosphorylation.ResultsHere we present the crystal structures of wild-type and phospho-mimetic RyR Repeat34 domain containing PKA phosphorylation sites from diamondback moth (DBM), a major lepidopteran pest of cruciferous vegetables. The structure has unique features, not seen in mammalian RyRs, including an additional α-helix near the phosphorylation loop. Using tandem mass spectrometry, we identify several PKA sites clustering in the phosphorylation loop and the newly identified α-helix. Bioinformatics analysis shows that this α-helix is only present in Lepidoptera, suggesting an insect-specific regulation. Interestingly, the specific phosphorylation pattern is temperature-dependent. The thermal stability of the DBM Repeat34 domain is significantly lower than that of the analogous domain in the three mammalian RyR isoforms, indicating a more dynamic domain structure that can be partially unfolded to facilitate the temperature-dependent phosphorylation. Docking the structure into the cryo-electron microscopy model of full-length RyR reveals that the interface between the Repeat34 and neighboring HD1 domain is more conserved than that of the phosphorylation loop region that might be involved in the interaction with SPRY3 domain. We also identify an insect-specific glycerol-binding pocket that could be potentially targeted by novel insecticides to fight the current resistance crisis.ConclusionsThe crystal structures of the DBM Repeat34 domain reveals insect-specific temperature-dependent phosphorylation sites that may regulate insect ryanodine receptor function. It also reveals insect-specific structural features and a potential ligand-binding site that could be targeted in an effort to develop green pesticides with high species-specificity.

Evidence of functional ryanodine receptors in rat mesenteric collecting lymphatic vessels.

In the current study, the potential contributions of ryanodine receptors (RyRs) to intrinsic pumping and responsiveness to substance P (SP) were investigated in isolated rat mesenteric collecting lymphatic vessels. Responses to SP were characterized in lymphatic vessels in the absence or presence of pretreatment with nifedipine to block L-type Ca2+ channels, caffeine to block normal release and uptake of Ca2+ from the sarcoplasmic reticulum, ryanodine to block all RyR isoforms, or dantrolene to more selectively block RyR1 and RyR3. RyR expression and localization in lymphatics was also assessed by quantitative PCR and immunofluorescence confocal microscopy. The results show that SP normally elicits a significant increase in contraction frequency and a decrease in end-diastolic diameter. In the presence of nifedipine, phasic contractions stop, yet subsequent SP treatment still elicits a strong tonic contraction. Caffeine treatment gradually relaxes lymphatics, causing a loss of phasic contractions, and prevents subsequent SP-induced tonic contraction. Ryanodine also gradually diminishes phasic contractions but without causing vessel relaxation and significantly inhibits the SP-induced tonic contraction. Dantrolene treatment did not significantly impair lymphatic contractions nor the response to SP. The mRNA for all RyR isoforms is detectable in isolated lymphatics. RyR2 and RyR3 proteins are found predominantly in the collecting lymphatic smooth muscle layer. Collectively, the data suggest that SP-induced tonic contraction requires both extracellular Ca2+ plus Ca2+ release from internal stores and that RyRs play a role in the normal contractions and responsiveness to SP of rat mesenteric collecting lymphatics.NEW & NOTEWORTHY The mechanisms that govern contractions of lymphatic vessels remain unclear. Tonic contraction of lymphatic vessels caused by substance P was blocked by caffeine, which prevents normal uptake and release of Ca2+ from internal stores, but not nifedipine, which blocks L-type channel-mediated Ca2+ entry. Ryanodine, which also disrupts normal sarcoplasmic reticulum Ca2+ release and reuptake, significantly inhibited substance P-induced tonic contraction. Ryanodine receptors 2 and 3 were detected within the smooth muscle layer of collecting lymphatic vessels.

Role of Ryanodine Type 2 Receptors in Elementary Ca2+ Signaling in Arteries and Vascular Adaptive Responses.

BackgroundHypertension is the major risk factor for cardiovascular disease, the most common cause of death worldwide. Resistance arteries are capable of adapting their diameter independently in response to pressure and flow‐associated shear stress. Ryanodine receptors (RyRs) are major Ca2+‐release channels in the sarcoplasmic reticulum membrane of myocytes that contribute to the regulation of contractility. Vascular smooth muscle cells exhibit 3 different RyR isoforms (RyR1, RyR2, and RyR3), but the impact of individual RyR isoforms on adaptive vascular responses is largely unknown. Herein, we generated tamoxifen‐inducible smooth muscle cell–specific RyR2‐deficient mice and tested the hypothesis that vascular smooth muscle cell RyR2s play a specific role in elementary Ca2+ signaling and adaptive vascular responses to vascular pressure and/or flow.Methods and ResultsTargeted deletion of the Ryr2 gene resulted in a complete loss of sarcoplasmic reticulum–mediated Ca2+‐release events and associated Ca2+‐activated, large‐conductance K+ channel currents in peripheral arteries, leading to increased myogenic tone and systemic blood pressure. In the absence of RyR2, the pulmonary artery pressure response to sustained hypoxia was enhanced, but flow‐dependent effects, including blood flow recovery in ischemic hind limbs, were unaffected.ConclusionsOur results establish that RyR2‐mediated Ca2+‐release events in VSCMs specifically regulate myogenic tone (systemic circulation) and arterial adaptation in response to changes in pressure (hypoxic lung model), but not flow. They further suggest that vascular smooth muscle cell–expressed RyR2 deserves scrutiny as a therapeutic target for the treatment of vascular responses in hypertension and chronic vascular diseases.

Investigation of Mutant Ryanodine Receptor Channel Activity using Functional Analysis and Molecular Dynamics

Membrane depolarization is translated into intracellular Ca2+ signals, and ryanodine receptors (RyRs), located in the sarcoplasmic/endoplasmic reticulum membrane, play a key role in intracellular Ca2+ release. There are three isoforms of RyRs, of which type 1 RyR (RyR1) is dominantly expressed in the skeletal muscle. Mutations in the RyR1 gene (RyR1) cause severe muscle diseases, such as malignant hyperthermia (MH), which is a disorder of Ca2+-induced Ca2+ release via the RyR1 in skeletal muscle. So far, more than 300 mutations have been reported in RyR1 of patients with MH, and most of those mutations have been found in three “hot spot” regions of RyR1. However, due to lack of comprehensive analysis of the structure-function relationship of mutant RyR1 Ca2+ release channels, the mechanism remains largely unknown. Here, we combined functional studies and molecular dynamics (MD) simulation of RyR1 channels carrying disease-associated mutations at the N-terminal region. When expressed in HEK293 cells, those mutant RyR1 Ca2+ release channels caused abnormalities in Ca2+ homeostasis. MD simulation of the mutant RyR1 revealed that hydrogen bonds/salt bridges between subdomains strongly correlate with the channel function of RyR1. Especially, the mutations of Arg402, which plays key role in connecting three subdomains (A, B, C) of the N-terminal region, exhibit interesting result. Indeed, B and C subdomains together rotate clockwise with respect to A subdomain in the mutants. This movement may increase the open probability of the channel, and this increase may be the main cause of MH in the Arg402 mutants.

Linking the heart and the brain: Neurodevelopmental disorders in patients with catecholaminergic polymorphic ventricular tachycardia

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an uncommon inherited arrhythmia disorder characterized by adrenergically evoked ventricular arrhythmias. Mutations in the cardiac calcium release channel/ryanodine receptor gene (RYR2) are identified in the majority of patients with CPVT. RyR2 is also the major RyR isoform expressed in the brain. The purpose of this study was to estimate the prevalence of intellectual disability (ID) and other neurodevelopmental disorders (NDDs) in RYR2-associated CPVT (CPVT1) and to study the characteristics of these patients. We reviewed the medical records of all CPVT1 patients from 12 international centers and analyzed the characteristics of all CPVT1 patients with concomitant NDDs. We functionally characterized the mutations to assess their response to caffeine activation. We did not correct for potential confounders. Among 421 CPVT1 patients, we identified 34 patients with ID (8%; 95% confidence interval 6%-11%). Median age at diagnosis was 9.3 years (interquartile range 7.0-14.5). Parents for 24 of 34 patients were available for genetic testing, and 13 of 24 (54%) had a de novo mutation. Severity of ID ranged from mild to severe and was accompanied by other NDDs in 9 patients (26%). Functionally, the ID-associated mutations showed a markedly enhanced response of RyR2 to activation by caffeine. Seventeen patients (50%) also had supraventricular arrhythmias. During median follow-up of 8.4 years (interquartile range 1.8-12.4), 15 patients (45%) experienced an arrhythmic event despite adequate therapy. Our study indicates that ID is more prevalent among CPVT1 patients (8%) than in the general population (1%-3%). This subgroup of CPVT1 patients reveals a malignant cardiac phenotype with marked supraventricular and ventricular arrhythmias.

Identification of Ca2+ signaling components in neural stem/progenitor cells during differentiation into neurons and glia in intact and dissociated zebrafish neurospheres

The development of the CNS in vertebrate embryos involves the generation of different sub-types of neurons and glia in a complex but highly-ordered spatio-temporal manner. Zebrafish are commonly used for exploring the development, plasticity and regeneration of the CNS, and the recent development of reliable protocols for isolating and culturing neural stem/progenitor cells (NSCs/NPCs) from the brain of adult fish now enables the exploration of mechanisms underlying the induction/specification/differentiation of these cells. Here, we refined a protocol to generate proliferating and differentiating neurospheres from the entire brain of adult zebrafish. We demonstrated via RT-qPCR that some isoforms of ip3r, ryr and stim are upregulated/downregulated significantly in differentiating neurospheres, and via immunolabelling that 1,4,5-inositol trisphosphate receptor (IP3R) type-1 and ryanodine receptor (RyR) type-2 are differentially expressed in cells with neuron- or radial glial-like properties. Furthermore, ATP but not caffeine (IP3R and RyR agonists, respectively), induced the generation of Ca2+ transients in cells exhibiting neuron- or glial-like morphology. These results indicate the differential expression of components of the Ca2+-signaling toolkit in proliferating and differentiating cells. Thus, given the complexity of the intact vertebrate brain, neurospheres might be a useful system for exploring neurodegenerative disease diagnosis protocols and drug development using Ca2+ signaling as a read-out.

Role of Ryanodine Type 2 Receptors in Elementary Ca2+ Signaling in Arteries and Vascular Adaptive Responses

ObjectiveCardiovascular disease is the most common cause of death worldwide, and hypertension is the major risk factor. Resistance arteries can adapt their vessel diameter independently by pressure and by flow. Ryanodine receptors (RyRs) are major Ca2+ release channels in the sarcoplasmic reticulum (SR) membrane of myocytes contributing to contractility. Vascular smooth muscle cells (VSMCs) exhibit three different RyR isoforms (RyR1, 2, 3), but the impact of individual RyR isoforms on vascular adaptive responses is largely unknown.MethodsWe generated tamoxifen‐inducible smooth muscle cell (SM) specific RyR2 deficient mice and tested the hypothesis that VSMC RyR2s play a specific role in elementary Ca2+ signaling and adaptive vascular responses to vascular pressure and/or flow.ResultsTargeted deletion of the Ryr2 gene resulted in a complete loss of SR released Ca2+ sparks and the associated Ca2+ activated potassium (BKCa) channel currents, leading to increased myogenic arterial tone in peripheral arteries and systemic blood pressure. RyR2 deficiency enhanced the pulmonary artery pressure response to sustained hypoxia, but had no effect on flow‐dependent effects, including blood flow recovery in ischemic hind limbs.ConclusionOur results identify a specific contribution of RyR2 in mediating VSMC Ca2+ sparks relevant for regulating myogenic tone (systemic circulation) and arterial adaptation in response to changes in pressure (hypoxic lung model), but not flow. VSMC expressed RyR2 deserves scrutiny as a therapeutic target for the treatment of vascular responses in hypertension and chronic vascular diseases.Support or Funding InformationThis study was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) to M.G and M.W. (SFB‐TR84 C3 and C6), and the Deutsche Akademische Austauschdienst (DAAD) to M.G. and I.S, and the NIH (R37 DK053832, R01 HL121706, PO1 HL095488, R01 HL131181), Fondation Leducq, and the Totman Medical Research Trust to M.T.N., and by the DZHK (German Centre for Cardiovascular Research) and by the BMBF (German Ministry of Education and Research) to M.G. and M.K. We thank Dr. Kirill Essin for help in Ca2+ spark recordings using line scan imaging. CFG‐P is recipient of Ministerio de Educación Cultura y Deporte, Fundación San Pablo‐CEU and Banco Santander fellowships.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.