ent-Verticilide B1 Inhibits Type 2 Ryanodine Receptor Channels and is Antiarrhythmic in Casq2 -/- Mice.

Intracellular Ca2+ leak from cardiac ryanodine receptor (RyR2) is an established mechanism of sudden cardiac death (SCD), whereby dysregulated Ca2+ handling causes ventricular arrhythmias. We previously discovered the RyR2-selective inhibitor ent-(+)-verticilide (ent-1), a 24-membered cyclooligomeric depsipeptide that is the enantiomeric form of a natural product (nat-(-)-verticilide). Here, we examined its 18-membered ring-size oligomer (ent-verticilide B1; "ent-B1") in RyR2 single channel and [3H]ryanodine binding assays, and in Casq2 -/- cardiomyocytes and mice, a gene-targeted model of SCD. ent-B1 inhibited RyR2 single channels and RyR2-mediated spontaneous Ca2+ release in Casq2 -/- cardiomyocytes with sub-micromolar potency. ent-B1 was a partial RyR2 inhibitor, with maximal inhibitory efficacy of less than 50%. ent-B1 was stable in plasma, with a peak plasma concentration of 1460 ng/ml at 10 minutes and half-life of 45 minutes after intraperitoneal administration of 3 mg/kg in mice. In vivo, ent-B1 significantly reduced catecholamine-induced ventricular arrhythmias in Casq2 -/- mice in a dose-dependent manner. Hence, we have identified a novel chemical entity - ent-B1 - that preserves the mechanism of action of a hit compound and shows therapeutic efficacy. These findings strengthen RyR2 as an antiarrhythmic drug target and highlight the potential of investigating the mirror-image isomers of natural products to discover new therapeutics. SIGNIFICANCE STATEMENT: The cardiac ryanodine receptor (RyR2) is an untapped target in the stagnant field of antiarrhythmic drug development. We have confirmed RyR2 as an antiarrhythmic target in a mouse model of sudden cardiac death and shown the therapeutic efficacy of a second enantiomeric natural product.

RYR2 Channel Inhibition Is the Principal Mechanism of Flecainide Action in CPVT.

RATIONALE: The class Ic antiarrhythmic drug flecainide prevents ventricular tachyarrhythmia in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), a disease caused by hyperactive RyR2 (cardiac ryanodine receptor) mediated calcium (Ca) release. Although flecainide inhibits single RyR2 channels in vitro, reports have claimed that RyR2 inhibition by flecainide is not relevant for its mechanism of antiarrhythmic action and concluded that sodium channel block alone is responsible for flecainide's efficacy in CPVT. OBJECTIVE: To determine whether RyR2 block independently contributes to flecainide's efficacy for suppressing spontaneous sarcoplasmic reticulum Ca release and for preventing ventricular tachycardia in vivo. METHODS AND RESULTS: We synthesized N-methylated flecainide analogues (QX-flecainide and N-methyl flecainide) and showed that N-methylation reduces flecainide's inhibitory potency on RyR2 channels incorporated into artificial lipid bilayers. N-methylation did not alter flecainide's inhibitory activity on human cardiac sodium channels expressed in HEK293T cells. Antiarrhythmic efficacy was tested utilizing a Casq2 (cardiac calsequestrin) knockout (Casq2-/-) CPVT mouse model. In membrane-permeabilized Casq2-/- cardiomyocytes-lacking intact sarcolemma and devoid of sodium channel contribution-flecainide, but not its analogues, suppressed RyR2-mediated Ca release at clinically relevant concentrations. In voltage-clamped, intact Casq2-/- cardiomyocytes pretreated with tetrodotoxin to inhibit sodium channels and isolate the effect of flecainide on RyR2, flecainide significantly reduced the frequency of spontaneous sarcoplasmic reticulum Ca release, while QX-flecainide and N-methyl flecainide did not. In vivo, flecainide effectively suppressed catecholamine-induced ventricular tachyarrhythmias in Casq2-/- mice, whereas N-methyl flecainide had no significant effect on arrhythmia burden, despite comparable sodium channel block. CONCLUSIONS: Flecainide remains an effective inhibitor of RyR2-mediated arrhythmogenic Ca release even when cardiac sodium channels are blocked. In mice with CPVT, sodium channel block alone did not prevent ventricular tachycardia. Hence, RyR2 channel inhibition likely constitutes the principal mechanism of antiarrhythmic action of flecainide in CPVT.

Measures of Maximal Tactile Pressures during a Sustained Grasp Task Using a TactArray Device Have Satisfactory Reliability and Concurrent Validity in People with Stroke.

Sensor-based devices can record pressure or force over time during grasping and therefore offer a more comprehensive approach to quantifying grip strength during sustained contractions. The objectives of this study were to investigate the reliability and concurrent validity of measures of maximal tactile pressures and forces during a sustained grasp task using a TactArray device in people with stroke. Participants with stroke (n = 11) performed three trials of sustained maximal grasp over 8 s. Both hands were tested in within- and between-day sessions, with and without vision. Measures of maximal tactile pressures and forces were measured for the complete (8 s) grasp duration and plateau phase (5 s). Tactile measures are reported using the highest value among three trials, the mean of two trials, and the mean of three trials. Reliability was determined using changes in mean, coefficients of variation, and intraclass correlation coefficients (ICCs). Pearson correlation coefficients were used to evaluate concurrent validity. This study found that measures of reliability assessed by changes in means were good, coefficients of variation were good to acceptable, and ICCs were very good for maximal tactile pressures using the average pressure of the mean of three trials over 8 s in the affected hand with and without vision for within-day sessions and without vision for between-day sessions. In the less affected hand, changes in mean were very good, coefficients of variations were acceptable, and ICCs were good to very good for maximal tactile pressures using the average pressure of the mean of three trials over 8 s and 5 s, respectively, in between-day sessions with and without vision. Maximal tactile pressures had moderate correlations with grip strength. The TactArray device demonstrates satisfactory reliability and concurrent validity for measures of maximal tactile pressures in people with stroke.

Mammalian TRP ion channels are insensitive to membrane stretch.

TRP channels of the transient receptor potential ion channel superfamily are involved in a wide variety of mechanosensory processes, including touch sensation, pain, blood pressure regulation, bone loading and detection of cerebrospinal fluid flow. However, in many instances it is unclear whether TRP channels are the primary transducers of mechanical force in these processes. In this study, we tested stretch activation of eleven TRP channels from six mammalian subfamilies. We found that these TRP channels were insensitive to short membrane stretches in cellular systems. Furthermore, we purified TRPC6 and demonstrated its insensitivity to stretch in liposomes, an artificial bilayer system free from cellular components. Additionally, we demonstrated that, when expressed in C. elegans neurons, mouse TRPC6 restores the mechanoresponse of a touch insensitive mutant but requires diacylglycerol for activation. These results strongly suggest that the mammalian members of the TRP ion channel family are insensitive to tension induced by cell membrane stretching and, thus, are more likely to be activated by cytoplasmic tethers or downstream components and to act as amplifiers of cellular mechanosensory signaling cascades.

Calmodulin inhibition of human RyR2 channels requires phosphorylation of RyR2-S2808 or RyR2-S2814.

Calmodulin (CaM) is a Ca-binding protein that binds to, and can directly inhibit cardiac ryanodine receptor calcium release channels (RyR2). Animal studies have shown that RyR2 hyperphosphorylation reduces CaM binding to RyR2 in failing hearts, but data are lacking on how CaM regulates human RyR2 and how this regulation is affected by RyR2 phosphorylation. Physiological concentrations of CaM (100 nM) inhibited the diastolic activity of RyR2 isolated from failing human hearts by ~50% but had no effect on RyR2 from healthy human hearts. Using FRET between donor-FKBP12.6 and acceptor-CaM bound to RyR2, we determined that CaM binds to RyR2 from healthy human heart with a Kd = 121 ±â€¯14 nM. Ex-vivo phosphorylation/dephosphorylation experiments suggested that the divergent CaM regulation of healthy and failing human RyR2 was caused by differences in RyR2 phosphorylation by protein kinase A and Ca-CaM-dependent kinase II. Ca2+-spark measurements in murine cardiomyocytes harbouring RyR2 phosphomimetic or phosphoablated mutants at S2814 and S2808 suggest that phosphorylation of residues corresponding to either human RyR2-S2808 or S2814 is both necessary and sufficient for RyR2 regulation by CaM. Our results challenge the current concept that CaM universally functions as a canonical inhibitor of RyR2 across species. Rather, CaM's biological action on human RyR2 appears to be more nuanced, with inhibitory activity only on phosphorylated RyR2 channels, which occurs during exercise or in patients with heart failure.

Measures of maximal tactile pressures of a sustained grasp task using a TactArray device have satisfactory reliability and validity in healthy people.

Objective: To investigate the reliability and the concurrent validity of maximal tactile pressures and forces of a sustained grasp task using a TactArray device in healthy adults.Methods: Healthy participants (n = 18, mean age: 62.2 ± 9.9 years) performed three repeat trials of sustained maximal grasp over 8 seconds. Both hands were tested in within-day and between-day sessions, with vision and without vision. Measures of maximal tactile pressures and forces were measured for the complete grasp duration (8s) and for the plateau phase (5s). Measures of maximal tactile pressures and forces were reported using the highest value among three repeat trials, the mean of two repeat trials, and the mean of three repeat trials. Reliability was determined using changes in mean, coefficients of variation and intraclass correlation coefficients (ICCs). Pearson correlation coefficients were used to evaluate concurrent validity.Results: Changes in mean were very good, coefficients of variation were good to acceptable and ICCs were very good to good for maximal tactile pressures using the highest value among three repeat trials and the mean of three repeat trials for the complete grasp duration (8s) and for the plateau phase (5s) in the dominant hand with and without vision and in the non-dominant hand without vision for within-day and between-day sessions. Maximal tactile pressures had moderate to large correlations with grip strength.Conclusion: The TactArray device demonstrates satisfactory reliability for maximal tactile pressures during a sustained grasp for within-day and between-day testing sessions in both hands. Validity was satisfactory with grip strength in both hands.

Nerve-induced responses of mouse vaginal smooth muscle.

Neural and agonist-induced contractions of proximal (i.e. upper half adjacent to the cervix) and distal mouse vaginal smooth muscle strips were investigated. We hypothesised that nerve-mediated vaginal contractions arise through activity of cholinergic nerves. Nerve activation by bursts of electrical field stimulation (EFS) caused a primary transient contraction often accompanied by a secondary transient contraction, both larger in proximal than distal tissues (i.e. primary: 7-fold larger; secondary: 3-fold larger). Our hypothesis was supported as we found that cholinergic nerves mediated the primary transient contraction in both proximal and distal vaginal strips, as EFS responses were enhanced by neostigmine an anticholinesterase, massively inhibited by the competitive muscarinic receptor antagonist atropine and not affected by the non-selective α-adrenergic receptor antagonist phentolamine. Primary transient contractions were halved in amplitude by the L-type Ca2+ channel blocker nifedipine and markedly inhibited by the sarco-endoplasmic reticulum calcium ATPase (SERCA) inhibitor cyclopiazonic acid (CPA). Resultant secondary transient contractions were abolished by nifedipine. Notably, the selective α1-adrenergic receptor agonist phenylephrine caused tonic contracture in distal but not proximal strips. Low-frequency EFS often initiated recurrent transient contractions similar to those elicited by CCh. Immunohistochemical studies demonstrated innervation of the smooth muscle. Findings of enhanced proximal cholinergic nerve-induced transient contractions, evidence that maintained nerve stimulation could cause recurrent contractions and the finding of distal phenylephrine-mediated tonic contraction have implications on insemination.

Cardiac Calcium Release Channel (Ryanodine Receptor 2) Regulation by Halogenated Anesthetics.

BACKGROUND: Halogenated anesthetics activate cardiac ryanodine receptor 2-mediated sarcoplasmic reticulum Ca release, leading to sarcoplasmic reticulum Ca depletion, reduced cardiac function, and providing cell protection against ischemia-reperfusion injury. Anesthetic activation of ryanodine receptor 2 is poorly defined, leaving aspects of the protective mechanism uncertain. METHODS: Ryanodine receptor 2 from the sheep heart was incorporated into artificial lipid bilayers, and their gating properties were measured in response to five halogenated anesthetics. RESULTS: Each anesthetic rapidly and reversibly activated ryanodine receptor 2, but only from the cytoplasmic side. Relative activation levels were as follows: halothane (approximately 4-fold; n = 8), desflurane and enflurane (approximately 3-fold,n = 9), and isoflurane and sevoflurane (approximately 1.5-fold, n = 7, 10). Half-activating concentrations (Ka) were in the range 1.3 to 2.1 mM (1.4 to 2.6 minimum alveolar concentration [MAC]) with the exception of isoflurane (5.3 mM, 6.6 minimum alveolar concentration). Dantrolene (10 µM with 100 nM calmodulin) inhibited ryanodine receptor 2 by 40% but did not alter the Ka for halothane activation. Halothane potentiated luminal and cytoplasmic Ca activation of ryanodine receptor 2 but had no effect on Mg inhibition. Halothane activated ryanodine receptor 2 in the absence and presence (2 mM) of adenosine triphosphate (ATP). Adenosine, a competitive antagonist to ATP activation of ryanodine receptor 2, did not antagonize halothane activation in the absence of ATP. CONCLUSIONS: At clinical concentrations (1 MAC), halothane desflurane and enflurane activated ryanodine receptor 2, whereas isoflurane and sevoflurane were ineffective. Dantrolene inhibition of ryanodine receptor 2 substantially negated the activating effects of anesthetics. Halothane acted independently of the adenine nucleotide-binding site on ryanodine receptor 2. The previously observed adenosine antagonism of halothane activation of sarcoplasmic reticulum Ca release was due to competition between adenosine and ATP, rather than between halothane and ATP.

The emerging role of calmodulin regulation of RyR2 in controlling heart rhythm, the progression of heart failure and the antiarrhythmic action of dantrolene.

Cardiac output and rhythm depend on the release and the take-up of calcium from the sarcoplasmic reticulum (SR). Excessive diastolic calcium leak from the SR due to dysfunctional calcium release channels (RyR2) contributes to the formation of delayed after-depolarizations, which underlie the fatal arrhythmias that occur in heart failure and inherited syndromes. Calmodulin (CaM) is a calcium-binding protein that regulates target proteins and acts as a calcium sensor. CaM is comprised of two calcium-binding EF-hand domains and a flexible linker. CaM is an accessory protein that partially inhibits RyR2 channel activity. CaM is critical for normal cardiac function, and altered CaM binding and efficacy may contribute to defects in SR calcium release. The present paper reviews CaM binding to RyR2 and how it regulates RyR2 channel activity. It then goes on to review how mutations in the CaM amino acid sequence give rise to inherited syndromes such as Catecholaminergic Polymorphic Ventricular Tachychardia (CPVT) and long QT syndrome (LQTS). In addition, the role of reduced CaM binding to RyR2 that results from RyR2 phosphorylation or from oxidation of either RyR2 or CaM contributes to the progression of heart failure is reviewed. Finally, this manuscript reviews recent evidence that CaM binding to RyR2 is required for the inhibitory action of a pharmaceutical agent (dantrolene) on RyR2. Dantrolene is a clinically used muscle relaxant that has recently been found to exert antiarrhythmic effects against SR Ca2+ overload arrhythmias.

Divergent regulation of ryanodine receptor 2 calcium release channels by arrhythmogenic human calmodulin missense mutants.

RATIONALE: Calmodulin (CaM) mutations are associated with an autosomal dominant syndrome of ventricular arrhythmia and sudden death that can present with divergent clinical features of catecholaminergic polymorphic ventricular tachycardia (CPVT) or long QT syndrome (LQTS). CaM binds to and inhibits ryanodine receptor (RyR2) Ca release channels in the heart, but whether arrhythmogenic CaM mutants alter RyR2 function is not known. OBJECTIVE: To gain mechanistic insight into how human CaM mutations affect RyR2 Ca channels. METHODS AND RESULTS: We studied recombinant CaM mutants associated with CPVT (N54I and N98S) or LQTS (D96V, D130G, and F142L). As a group, all LQTS-associated CaM mutants (LQTS-CaMs) exhibited reduced Ca affinity, whereas CPVT-associated CaM mutants (CPVT-CaMs) had either normal or modestly lower Ca affinity. In permeabilized ventricular myocytes, CPVT-CaMs at a physiological intracellular concentration (100 nmol/L) promoted significantly higher spontaneous Ca wave and spark activity, a typical cellular phenotype of CPVT. Compared with wild-type CaM, CPVT-CaMs caused greater RyR2 single-channel open probability and showed enhanced binding affinity to RyR2. Even a 1:8 mixture of CPVT-CaM:wild-type-CaM activated Ca waves, demonstrating functional dominance. In contrast, LQTS-CaMs did not promote Ca waves and exhibited either normal regulation of RyR2 single channels (D96V) or lower RyR2-binding affinity (D130G and F142L). None of the CaM mutants altered Ca/CaM binding to CaM-kinase II. CONCLUSIONS: A small proportion of CPVT-CaM is sufficient to evoke arrhythmogenic Ca disturbances, whereas LQTS-CaMs do not. Our findings explain the clinical presentation and autosomal dominant inheritance of CPVT-CaM mutations and suggest that RyR2 interactions are unlikely to explain arrhythmogenicity of LQTS-CaM mutations.

Essential Role of Calmodulin in RyR Inhibition by Dantrolene.

Dantrolene is the first line therapy of malignant hyperthermia. Animal studies suggest that dantrolene also protects against heart failure and arrhythmias caused by spontaneous Ca(2+) release. Although dantrolene inhibits Ca(2+) release from the sarcoplasmic reticulum of skeletal and cardiac muscle preparations, its mechanism of action has remained controversial, because dantrolene does not inhibit single ryanodine receptor (RyR) Ca(2+) release channels in lipid bilayers. Here we test the hypothesis that calmodulin (CaM), a physiologic RyR binding partner that is lost during incorporation into lipid bilayers, is required for dantrolene inhibition of RyR channels. In single channel recordings (100 nM cytoplasmic [Ca(2+)] + 2 mM ATP), dantrolene caused inhibition of RyR1 (rabbit skeletal muscle) and RyR2 (sheep) with a maximal inhibition of Po (Emax) to 52 ± 4% of control only after adding physiologic [CaM] = 100 nM. Dantrolene inhibited RyR2 with an IC50 of 0.16 ± 0.03 µM. Mutant N98S-CaM facilitated dantrolene inhibition with an IC50 = 5.9 ± 0.3 nM. In mouse cardiomyocytes, dantrolene had no effect on cardiac Ca(2+) release in the absence of CaM, but reduced Ca(2+) wave frequency (IC50 = 0.42 ± 0.18 µM, Emax = 47 ± 4%) and amplitude (IC50 = 0.19 ± 0.04 µM, Emax = 66 ± 4%) in the presence of 100 nM CaM. We conclude that CaM is essential for dantrolene inhibition of RyR1 and RyR2. Its absence explains why dantrolene inhibition of single RyR channels has not been previously observed.

Polarized and persistent Ca²âº plumes define loci for formation of wall ingrowth papillae in transfer cells.

Transfer cell morphology is characterized by a polarized ingrowth wall comprising a uniform wall upon which wall ingrowth papillae develop at right angles into the cytoplasm. The hypothesis that positional information directing construction of wall ingrowth papillae is mediated by Ca(2+) signals generated by spatiotemporal alterations in cytosolic Ca(2+) ([Ca(2+)]cyt) of cells trans-differentiating to a transfer cell morphology was tested. This hypothesis was examined using Vicia faba cotyledons. On transferring cotyledons to culture, their adaxial epidermal cells synchronously trans-differentiate to epidermal transfer cells. A polarized and persistent Ca(2+) signal, generated during epidermal cell trans-differentiation, was found to co-localize with the site of ingrowth wall formation. Dampening Ca(2+) signal intensity, by withdrawing extracellular Ca(2+) or blocking Ca(2+) channel activity, inhibited formation of wall ingrowth papillae. Maintenance of Ca(2+) signal polarity and persistence depended upon a rapid turnover (minutes) of cytosolic Ca(2+) by co-operative functioning of plasma membrane Ca(2+)-permeable channels and Ca(2+)-ATPases. Viewed paradermally, and proximal to the cytosol-plasma membrane interface, the Ca(2+) signal was organized into discrete patches that aligned spatially with clusters of Ca(2+)-permeable channels. Mathematical modelling demonstrated that these patches of cytosolic Ca(2+) were consistent with inward-directed plumes of elevated [Ca(2+)]cyt. Plume formation depended upon an alternating distribution of Ca(2+)-permeable channels and Ca(2+)-ATPase clusters. On further inward diffusion, the Ca(2+) plumes coalesced into a uniform Ca(2+) signal. Blocking or dispersing the Ca(2+) plumes inhibited deposition of wall ingrowth papillae, while uniform wall formation remained unaltered. A working model envisages that cytosolic Ca(2+) plumes define the loci at which wall ingrowth papillae are deposited.

Extraction of sub-microscopic Ca fluxes from blurred and noisy fluorescent indicator images with a detailed model fitting approach.

The release of Ca from intracellular stores is key to cardiac muscle function; however, the molecular control of intracellular Ca release remains unclear. Depletion of the intracellular Ca store (sarcoplasmic reticulum, SR) may play an important role, but the ability to measure local SR Ca with fluorescent Ca indicators is limited by the microscope optical resolution and properties of the indicator. This leads to an uncertain degree of spatio-temporal blurring, which is not easily corrected (by deconvolution methods) due to the low signal-to-noise ratio of the recorded signals. In this study, a 3D computer model was constructed to calculate local Ca fluxes and consequent dye signals, which were then blurred by a measured microscope point spread function. Parameter fitting was employed to adjust a release basis function until the model output fitted recorded (2D) Ca spark data. This 'forward method' allowed us to obtain estimates of the time-course of Ca release flux and depletion within the sub-microscopic local SR associated with a number of Ca sparks. While variability in focal position relative to Ca spark sites causes more out-of-focus events to have smaller calculated fluxes (and less SR depletion), the average SR depletion was to 20±10% (s.d.) of the resting level. This focus problem implies that the actual SR depletion is likely to be larger and the five largest depletions analyzed were to 8±6% of the resting level. This profound depletion limits SR release flux during a Ca spark, which peaked at 8±3 pA and declined with a half time of 7±2 ms. By comparison, RyR open probability declined more slowly, suggesting release termination is dominated by neither SR Ca depletion nor intrinsic RyR gating, but results from an interaction of these processes.

Computationally Efficient Simulation of Calcium Signaling in Cardiomyocytes.

The objective of this paper is to develop a computationally efficient simulation model of Calcium signalling in cardiomyocytes. The model considered here consists of more than two million stiff, nonlinear, and stochastic systems, each of which is composed of 62 state equations. The size of the model, combined with the broad numerical scale, non-continuous stochastic state-transitions, and underlying physiological constraints, presents a significant implementation challenge. The method involves development of specialised algorithms for parallelisation, which include fully-implicit Runge-Kutta integration with both L-stability and step-size control, Newton's root finding method with exception handling, and Conjugate Residual Squared for solving linear systems not of full-rank within available computational precision. Parallelisation of the problem across the systems is employed to allow for practical scaling with computing resources. The results produce sparks and waves akin to those observed in actual laboratory experiments within an acceptable timeframe. Performance measures of the simulation model with respect to accuracy and computation time are also given. The conclusion is that the methodologies utilised in this work are can simulate cardiomyocyte's calcium signalling in a computationally efficient manner with the results produced replicating those in the laboratory. The significance of this paper is that computational models such as the one developed here provide a way to simulate and understand the complex biological interactions operating in organisms. Accurate simulations are extremely computationally intensive and this pursuit is considered as the grand challenge for computational science into the 21st century.

Dantrolene inhibition of ryanodine channels (RyR2) in artificial lipid bilayers depends on FKBP12.6.

Dantrolene is a neutral hydantoin that is clinically used as a skeletal muscle relaxant to prevent overactivation of the skeletal muscle calcium release channel (RyR1) in response to volatile anesthetics. Dantrolene has aroused considerable recent interest as a lead compound for stabilizing calcium release due to overactive cardiac calcium release channels (RyR2) in heart failure. Previously, we found that dantrolene produces up to a 45% inhibition RyR2 with an IC50 of 160 nM, and that this inhibition requires the physiological association between RyR2 and CaM. In this study, we tested the hypothesis that dantrolene inhibition of RyR2 in the presence of CaM is modulated by RyR2 phosphorylation at S2808 and S2814. Phosphorylation was altered by incubations with either exogenous phosphatase (PP1) or kinases; PKA to phosphorylate S2808 or endogenous CaMKII to phosphorylate S2814. We found that PKA caused selective dissociation of FKBP12.6 from the RyR2 complex and a loss of dantrolene inhibition. Rapamycin-induced FKBP12.6 dissociation from RyR2 also resulted in the loss of dantrolene inhibition. Subsequent incubations of RyR2 with exogenous FKBP12.6 reinstated dantrolene inhibition. These findings indicate that the inhibitory action of dantrolene on RyR2 depends on RyR2 association with FKBP12.6 in addition to CaM as previously found.

ent -Verticilide B1 inhibits type 2 ryanodine receptor channels and is antiarrhythmic in Casq2-/- mice.

Ca 2+ leak from cardiac ryanodine receptor (RyR2) is an established mechanism of sudden cardiac death (SCD), whereby dysregulated Ca 2+ handling causes ventricular arrhythmias. We previously discovered the RyR2-selective inhibitor ent- (+)-verticilide ( ent -1), a 24-membered cyclooligomeric depsipeptide that is the enantiomeric form of a natural product ( nat -(-)-verticilide). Here, we examined its 18-membered ring-size oligomer ( ent -verticilide B1; " ent -B1") in single RyR2 channel assays, [ 3 H]ryanodine binding assays, and in Casq2 -/- cardiomyocytes and mice, a gene-targeted model of SCD. ent -B1 inhibited RyR2 single-channels and [ 3 H]ryanodine binding with low micromolar potency, and RyR2-mediated spontaneous Ca 2+ release in Casq2-/- cardiomyocytes with sub-micromolar potency. ent -B1 was a partial RyR2 inhibitor, with maximal inhibitory efficacy of less than 50%. ent -B1 was stable in plasma, with a peak plasma concentration of 1460 ng/ml at 10 min and half-life of 45 min after intraperitoneal administration of 3 mg/kg in mice. Both 3 mg/kg and 30 mg/kg ent -B1 significantly reduced catecholamine-induced ventricular arrhythmia in Casq2-/- mice. Hence, we have identified a novel chemical entity - ent -B1 - that preserves the mechanism of action of a hit compound and shows therapeutic efficacy. These findings strengthen RyR2 as an antiarrhythmic drug target and highlight the potential of investigating the mirror-image isomers of natural products to discover new therapeutics. Significance statement: The cardiac ryanodine receptor (RyR2) is an untapped target in the stagnant field of antiarrhythmic drug development. We have confirmed RyR2 as an antiarrhythmic target in a mouse model of sudden cardiac death and shown the therapeutic efficacy of a second enantiomeric natural product.

A bivalent remipede toxin promotes calcium release via ryanodine receptor activation.

Multivalent ligands of ion channels have proven to be both very rare and highly valuable in yielding unique insights into channel structure and pharmacology. Here, we describe a bivalent peptide from the venom of Xibalbanus tulumensis, a troglobitic arthropod from the enigmatic class Remipedia, that causes persistent calcium release by activation of ion channels involved in muscle contraction. The high-resolution solution structure of φ-Xibalbin3-Xt3a reveals a tandem repeat arrangement of inhibitor-cysteine knot (ICK) domains previously only found in spider venoms. The individual repeats of Xt3a share sequence similarity with a family of scorpion toxins that target ryanodine receptors (RyR). Single-channel electrophysiology and quantification of released Ca2+ stores within skinned muscle fibers confirm Xt3a as a bivalent RyR modulator. Our results reveal convergent evolution of RyR targeting toxins in remipede and scorpion venoms, while the tandem-ICK repeat architecture is an evolutionary innovation that is convergent with toxins from spider venoms.

Ion currents through Kir potassium channels are gated by anionic lipids.

Ion currents through potassium channels are gated. Constriction of the ion conduction pathway at the inner helix bundle, the textbook gate of Kir potassium channels, has been shown to be an ineffective permeation control, creating a rift in our understanding of how these channels are gated. Here we present evidence that anionic lipids act as interactive response elements sufficient to gate potassium conduction. We demonstrate the limiting barrier to K+ permeation lies within the ion conduction pathway and show that this gate is operated by the fatty acyl tails of lipids that infiltrate the conduction pathway via fenestrations in the walls of the pore. Acyl tails occupying a surface groove extending from the cytosolic interface to the conduction pathway provide a potential means of relaying cellular signals, mediated by anionic lipid head groups bound at the canonical lipid binding site, to the internal gate.

Three independent mechanisms contribute to tetracaine inhibition of cardiac calcium release channels.

Tetracaine is a tertiary amine local anaesthetic which inhibits ryanodine receptors (RyRs), the calcium release channels of the sarcoplasmic reticulum (SR). Tetracaine has been extensively used to study the role of the SR Ca(2+) fluxes in muscle cells, yet a detailed understanding of tetracaine action on RyR channels is lacking. Here we investigate tetracaine effects in single channel recording of sheep cardiac RyRs in lipid bilayers. Tetracaine decreased channel conductance (block) and open probability (inhibition). The IC(50) for inhibition had complex dependencies on membrane voltage and cytoplasmic [ATP], [Ca(2+)] and pH. We identify three mechanisms underlying these actions. First, a voltage-dependent, slow inhibition in which luminal and cytoplasmic tetracaine compete for a common neutral/cation binding site within the trans-membrane RyR domain to induce long closed events (~100 ms). The apparent binding rate is proportional to the RyR closed probability, indicating that it only operates on closed channels. Second, a voltage-independent, pH sensitive fast inhibition in which cytoplasmic and luminal tetracaine compete for a site located on the cytoplasmic domain of the RyR to induce fast closed events (~2 ms). Its IC(50) is not dependent on the open/closed conformation of RyR. Finally, a voltage-dependent block of the channel by cytoplasmic tetracaine reduced channel conductance. We develop a model for tetracaine inhibition which predicts that under diastolic conditions, i.e. when RyRs are mainly closed, the slow mechanism has the highest potency (IC(50)~200 µM) of the three mechanisms and is therefore the dominant form of inhibition. However, during periods of Ca(2+) release, i.e. when RyRs are open, the slow mechanism becomes ineffective, leaving the fast inhibition (IC(50)~2 mM) as the dominant effect. Because of this closed state inhibition property, tetracaine loses its efficacy when RyRs open. This has the effect of increasing the feedback on SR Ca(2+) release generated by cytoplasmic and luminal Ca(2+).

SR Ca2+ store refill--a key factor in cardiac pacemaking.

This study presents a theoretical analysis of the role of store Ca(2+) uptake on sinoatrial node (SAN) cell pacemaking. Two mechanisms have been shown to be involved in SAN pacemaking, these being: 1) the membrane oscillator model where rhythm generation is based on the interaction of voltage-dependent membrane ion channels and, 2) the store oscillator model where cyclical release of Ca(2+) from intracellular Ca(2+) stores depolarizes the membrane through activation of the sodium-calcium exchanger (NCX). The relative roles of these oscillators in generation and modulation of pacemaker rate have been vigorously debated and have many consequences. The main new outcomes of our study are: 1) uptake of Ca(2+) by intracellular Ca(2+) stores increases the maximum diastolic potential (MDP) by reducing the cytosolic Ca(2+) concentration [Ca(2+)](c) and hence decreasing the NCX current; 2) this hyperpolarization enhances recruitment of key pacemaker currents (e.g. the hyperpolarization-activated HCN current (I(f)) and T-type Ca(2+) current (I(T-Ca))); 3) the resultant enhanced Ca(2+) entry during the pacemaker depolarization increases [Ca(2+)](c) causing advancement of the store Ca(2+) release cycle and increased NCX current. In overview, the novel feature of our study is an investigation of the role of store Ca(2+) uptake on SAN pacemaking. This occurs during the early diastolic period and causes enhanced I(f), I(T-Ca) and store release (and hence I(NCX)) during the later diastolic period. There is thus a symbiotic interaction between the two pacemaker "clocks" over the entire diastolic period, this providing robust and highly malleable SAN pacemaking. Accounting for store Ca(2+) uptake also provides insight into hitherto unexplained SAN behaviour, as we exemplify for the sinus bradycardia exhibited in catecholaminergic polymorphic ventricular tachycardia (CPVT).