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The canonical mechanistic model explaining potassium channel gating is of a conformational change that alternately dilates and constricts a collar-like intracellular entrance to the pore. It is based on the premise that K+ ions maintain a complete hydration shell while passing between the transmembrane cavity and cytosol, which must be accommodated. To put the canonical model to the test, we locked the conformation of a Kir K+ channel to prevent widening of the narrow collar. Unexpectedly, conduction was unimpaired in the locked channels. In parallel, we employed all-atom molecular dynamics to simulate K+ ions moving along the conduction pathway between the lower cavity and cytosol. During simulations, the constriction did not significantly widen. Instead, transient loss of some water molecules facilitated K+ permeation through the collar. The low free energy barrier to partial dehydration in the absence of conformational change indicates Kir channels are not gated by the canonical mechanism., The transition between conducting and non-conducting states of K+ channels has been explained by conformational changes at the intracellular entrance to the conduction pathway. Here authors demonstrate that control over K+ currents in Kir channels is not explained by the canonical pore-gating model, as conduction is not impaired by a constricted inner helix bundle.

Phenytoin is a hydantoin derivative that is used clinically for the treatment of epilepsy and has been reported to have antiarrhythmic actions on the heart. In a failing heart, the elevated diastolic Ca2+ leak from the sarcoplasmic reticulum can be normalized by the cardiac ryanodine receptor 2 (RyR2) inhibitor, dantrolene, without inhibiting Ca2+ release during systole or affecting Ca2+ release in normal healthy hearts. Unfortunately, dantrolene is hepatotoxic and unsuitable for chronic long-term administration. Because phenytoin and dantrolene belong to the hydantoin class of compounds, we test the hypothesis that dantrolene and phenytoin have similar inhibitory effects on RyR2 using a single-channel recording of RyR2 activity in artificial lipid bilayers. Phenytoin produced a reversible inhibition of RyR2 channels from sheep and human failing hearts. It followed a hyperbolic dose response with maximal inhibition of ∼50%, Hill coefficient ∼1, and IC50 ranging from 10 to 20 µM. It caused inhibition at diastolic cytoplasmic [Ca2+] but not at Ca2+ levels in the dyadic cleft during systole. Notably, phenytoin inhibits RyR2 from failing human heart but not from healthy heart, indicating that phenytoin may selectively target defective RyR2 channels in humans. We conclude that phenytoin could effectively inhibit RyR2-mediated release of Ca2+ in a manner paralleling that of dantrolene. Moreover, the IC50 of phenytoin in RyR2 is at least threefold lower than for other ion channels and clinically used serum levels, pointing to phenytoin as a more human-safe alternative to dantrolene for therapies against heart failure and cardiac arrythmias. SIGNIFICANCE STATEMENT: We show that phenytoin, a Na channel blocker used clinically for treatment of epilepsy, is a diastolic inhibitor of cardiac calcium release channels [cardiac ryanodine receptor 2 (RyR2)] at doses threefold lower than its current therapeutic levels. Phenytoin inhibits RyR2 from failing human heart and not from healthy heart, indicating that phenytoin may selectively target defective RyR2 channels in humans and pointing to phenytoin as a more human-safe alternative to dantrolene for therapies against heart failure and cardiac arrhythmias.

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 K(d) = 121 ± 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. Ca(2+)-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.

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 the first 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.

BACKGROUND: Phenytoin has long been used to treat epilepsy and for some time as an antiarrhythmic drug (AAD). It is known that the diastolic calcium leakage through dysfunctional cardiac ryanodine receptors (RyR2) is a mechanism for arrhythmias in heart failure. Recent evidence suggests that phenytoin inhibits dysfunctional RyR2, reduces the calcium leak during diastole in heart failure, and may improve cardiac systolic function. This indicates the potential for repurposing phenytoin as an AAD in patients with heart failure.METHODS: A systematic search of MEDLINE, Embase, and the Cochrane Library databases was performed in March 2019. The search was limited to the studies published in the English language from 1946 to 2019. Studies on the antiarrhythmic effects of phenytoin in adults compared to no treatment or other AADs were included. Studies were excluded if there was insufficient clinical data regarding antiarrhythmic effects, dosing and administration of phenytoin and other ADDs. Conference abstracts, editorials, case studies and review articles were also excluded. RESULTS: A total of 157 non-duplicate titles were screened, and 25 articles underwent full-text review. 13 studies met the inclusion criteria, representing a total of 985 patients. Phenytoin was found to be effective in treating arrhythmias associated with digitalis toxicity, and in suppressing premature ventricular contractions (PVCs). In a recent animal study, phenytoin inhibited diastolic calcium leak through dysfunctional RyR2 in failing sheep hearts and improved cardiac systolic function without affecting normal functional RyR2.CONCLUSION: Phenytoin has an acceptable safety profile when used as an AAD. It has some utility in treating digitalis-induced arrhythmias and suppressing PVCs, however, further study is needed to determine its efficacy as an antiarrhythmic in heart failure patients given new evidence of its RyR2 stabilising properties.TRIAL REGISTRATION NUMBER: PROSPERO database (CRD42019129125).

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.

Cyclic calcium release and uptake in heart cells has an important role in heart rhythm and contraction, and it is known that the malregulation of calcium release is a predictor for cardiac arrhythmia. A model of this calcium release process was proposed, which consists of a large number of discrete calcium release sites, each involving stiff, stochastic, and nonlinear systems. In this paper we have developed a simulation of this calcium release model, which is parallel across the problem. The simulation is developed in a CUDA framework to be solved using GPUs. Computational efficiency is enhanced by using a DIIRK solver and taking advantage of the sparsity of the Jacobian. The output is shown to display behaviour similar to empirical observations, in particular displaying behaviour known as calcium waves.

Ryanodine receptors (RyRs) are the Ca2+ release channels in the sarcoplasmic reticulum in striated muscle which play an important role in excitation-contraction coupling and cardiac pacemaking. Single channel recordings have revealed a wealth of information about ligand regulation of RyRs from mammalian skeletal and cardiac muscle (RyR1 and RyR2, respectively). RyR subunit has a Ca2+ activation site located in the luminal and cytoplasmic domains of the RyR. These sites synergistically feed into a common gating mechanism for channel activation by luminal and cytoplasmic Ca2+. RyRs also possess two inhibitory sites in their cytoplasmic domains with Ca2+ affinities of the order of 1 μM and 1 mM. Magnesium competes with Ca2+ at these sites to inhibit RyRs and this plays an important role in modulating their Ca2+-dependent activity in muscle. This review focuses on how these sites lead to RyR modulation by Ca2+ and Mg2+ and how these mechanisms control Ca2+ release in excitation-contraction coupling and cardiac pacemaking.

Circulating SN (secretoneurin) concentrations are increased in patients with myocardial dysfunction and predict poor outcome. Because SN inhibits CaMKIIδ (CaCirculating levels of SN and other biomarkers were assessed in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT; n=8) and in resuscitated patients after ventricular arrhythmia-induced cardiac arrest (n=155). In vivo effects of SN were investigated in CPVT mice (RyR2 [ryanodine receptor 2]-R2474S) using adeno-associated virus-9-induced overexpression. Interactions between SN and CaMKIIδ were mapped using pull-down experiments, mutagenesis, ELISA, and structural homology modeling. Ex vivo actions were tested in Langendorff hearts and effects on CaSN levels were elevated in patients with CPVT and following ventricular arrhythmia-induced cardiac arrest. In contrast to NT-proBNP (N-terminal pro-B-type natriuretic peptide) and hs-TnT (high-sensitivity troponin T), circulating SN levels declined after resuscitation, as the risk of a new arrhythmia waned. Myocardial pro-SN expression was also increased in CPVT mice, and further adeno-associated virus-9-induced overexpression of SN attenuated arrhythmic induction during stress testing with isoproterenol. Mechanistic studies mapped SN binding to the substrate binding site in the catalytic region of CaMKIIδ. Accordingly, SN attenuated isoproterenol induced autophosphorylation of Thr287-CaMKIIδ in Langendorff hearts and inhibited CaMKIIδ-dependent RyR phosphorylation. In line with CaMKIIδ and RyR inhibition, SN treatment decreased CaSN production is upregulated in conditions with cardiomyocyte Ca

Highlights • Human failing heart ryanodine receptor 2 (RyR2) Ca2+ release channels display an altered regulation to cytoplasmic Ca2+. • Alterations in failing heart RyR2 function are correlated with higher RyR2 phosphorylation and thiol modification, and lower FKBP association. • Observed changes in RyR2 function and protein modifications would contribute to the diastolic leak phenotype in heat failure. Abstract Rationale Heart failure is a multimodal disorder, of which disrupted Ca2+ homeostasis is a hallmark. Central to Ca2+ homeostasis is the major cardiac Ca2+ release channel – the ryanodine receptor (RyR2) – whose activity is influenced by associated proteins, covalent modification and by Ca2+ and Mg2 +. That RyR2 is remodelled and its function disturbed in heart failure is well recognized, but poorly understood. Objective To assess Ca2+ and Mg2 + regulation of RyR2 from left ventricles of healthy, cystic fibrosis and failing hearts, and to correlate these functional changes with RyR2 modifications and remodelling. Methods and results The function of RyR2 from left ventricular samples was assessed using lipid bilayer single-channel measurements, whilst RyR2 modification and protein:protein interactions were determined using Western Blots and co-immunoprecipitation. In all failing hearts there was an increase in RyR2 activity at end-diastolic cytoplasmic Ca2+ (100 nM), a decreased cytoplasmic [Ca2+] required for half maximal activation (Ka) and a decrease in inhibition by cytoplasmic Mg2 +. This was accompanied by significant hyperphosphorylation of RyR2 S2808 and S2814, reduced free thiol content and a reduced interaction with FKBP12.0 and FKBP12.6. Either dephosphorylation of RyR2 using PP1 or thiol reduction using DTT eliminated any significant difference in the activity of RyR2 from healthy and failing hearts. We also report a subgroup of RyR2 in failing hearts that were not responsive to regulation by intracellular Ca2+ or Mg2 +. Conclusion Despite different aetiologies, disrupted RyR2 Ca2+ sensitivity and biochemical modification of the channel are common constituents of failing heart RyR2 and may underlie the pathological disturbances in intracellular Ca2+ signalling.

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.

Calmodulin (CaM) is a calcium-binding protein that can directly inhibit cardiac ryanodine receptor calcium release channels (ryanodine receptor 2 [RyR2]) [(1)][1]. CaM mutations can cause an autosomal-dominant form of catecholaminergic polymorphic ventricular tachycardia (CPVT), a syndrome

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., Summary: Mammalian TRP ion channels are insensitive to membrane stretch, suggesting that they function as secondary – rather than primary – mechanotransducers in cellular mechanotransduction processes.

Using a spatially discretised model structure to represent the behaviour of calcium release sites in a cell, this paper presents a parallel solution algorithm which treats each release site as an independent sub-system, and manages inter-site data communication on a global timestep. When compared to the equivalent single-thread solution algorithm, the parallel method features a negligible reduction in accuracy, and improves computation time scaling from a quadratic, O( n 2 ) to a linear, O ( n ), with respect to the number of release sites, n , in the model.

The mammalian brain is a mechanosensitive organ that responds to different mechanical forces ranging from intrinsic forces implicated in brain morphogenesis to extrinsic forces that can cause concussion and traumatic brain injury. However, little is known of the mechanosensors that transduce these forces. In this study we use cell-attached patch recording to measure single mechanically-gated (MG) channel currents and their affects on spike activity in identified neurons in neonatal mouse brain slices. We demonstrate that both neocortical and hippocampal pyramidal neurons express stretch-activated MG cation channels that are activated by suctions of ~25mm Hg, have a single channel conductance for inward current of 50-70pS and show weak selectivity for alkali metal cations (i.e., Na(+)

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, it is unclear in many instances 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 subfamilies. We found that these TRP channels were insensitive to short membrane stretch 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 they are more likely activated by cytoplasmic tethers or downstream components and act as amplifiers of cellular mechanosensory signaling cascades.

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 Ca2+ signals generated by spatiotemporal alterations in cytosolic Ca2+ ([Ca2+]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 Ca2+ signal, generated during epidermal cell trans-differentiation, was found to co-localize with the site of ingrowth wall formation. Dampening Ca2+ signal intensity, by withdrawing extracellular Ca2+ or blocking Ca2+ channel activity, inhibited formation of wall ingrowth papillae. Maintenance of Ca2+ signal polarity and persistence depended upon a rapid turnover (minutes) of cytosolic Ca2+ by co-operative functioning of plasma membrane Ca2+-permeable channels and Ca2+-ATPases. Viewed paradermally, and proximal to the cytosol–plasma membrane interface, the Ca2+ signal was organized into discrete patches that aligned spatially with clusters of Ca2+-permeable channels. Mathematical modelling demonstrated that these patches of cytosolic Ca2+ were consistent with inward-directed plumes of elevated [Ca2+]cyt. Plume formation depended upon an alternating distribution of Ca2+-permeable channels and Ca2+-ATPase clusters. On further inward diffusion, the Ca2+ plumes coalesced into a uniform Ca2+ signal. Blocking or dispersing the Ca2+ plumes inhibited deposition of wall ingrowth papillae, while uniform wall formation remained unaltered. A working model envisages that cytosolic Ca2+ plumes define the loci at which wall ingrowth papillae are deposited.

Regulation of the cardiac ryanodine receptor (RyR2) by intracellular Ca(2+) and Mg(2+) plays a key role in determining cardiac contraction and rhythmicity, but their role in regulating the human RyR2 remains poorly defined. The Ca(2+)- and Mg(2+)-dependent regulation of human RyR2 was recorded in artificial lipid bilayers in the presence of 2 mM ATP and compared with that in two commonly used animal models for RyR2 function (rat and sheep). Human RyR2 displayed cytoplasmic Ca(2+) activation (K(a) = 4 µM) and inhibition by cytoplasmic Mg(2+) (K(i) = 10 µM at 100 nM Ca(2+)) that was similar to RyR2 from rat and sheep obtained under the same experimental conditions. However, in the presence of 0.1 mM Ca(2+), RyR2s from human were 3.5-fold less sensitive to cytoplasmic Mg(2+) inhibition than those from sheep and rat. The K(a) values for luminal Ca(2+) activation were similar in the three species (35 µM for human, 12 µM for sheep, and 10 µM for rat). From the relationship between open probability and luminal [Ca(2+)], the peak open probability for the human RyR2 was approximately the same as that for sheep, and both were ~10-fold greater than that for rat RyR2. Human RyR2 also showed the same sensitivity to luminal Mg(2+) as that from sheep, whereas rat RyR2 was 10-fold more sensitive. In all species, modulation of RyR2 gating by luminal Ca(2+) and Mg(2+) only occurred when cytoplasmic [Ca(2+)] was

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.

The factors responsible for the regulation of regenerative calcium-induced calcium release (CICR) during Ca2+ spark evolution remain unclear. Cardiac ryanodine receptor (RyR) gating in rats and sheep was recorded at physiological Ca2+, Mg2+, and ATP levels and incorporated into a 3D model of the cardiac dyad, which reproduced the time course of Ca2+ sparks, Ca2+ blinks, and Ca2+ spark restitution. The termination of CICR by induction decay in the model principally arose from the steep Ca2+ dependence of RyR closed time, with the measured sarcoplasmic reticulum (SR) lumen Ca2+ dependence of RyR gating making almost no contribution. The start of CICR termination was strongly dependent on the extent of local depletion of junctional SR Ca2+, as well as the time course of local Ca2+ gradients within the junctional space. Reducing the dimensions of the dyad junction reduced Ca2+ spark amplitude by reducing the strength of regenerative feedback within CICR. A refractory period for Ca2+ spark initiation and subsequent Ca2+ spark amplitude restitution arose from 1), the extent to which the regenerative phase of CICR can be supported by the partially depleted junctional SR, and 2), the availability of releasable Ca2+ in the junctional SR. The physical organization of RyRs within the junctional space had minimal effects on Ca2+ spark amplitude when more than nine RyRs were present. Spark amplitude had a nonlinear dependence on RyR single-channel Ca2+ flux, and was approximately halved by reducing the flux from 0.6 to 0.2 pA. Although rat and sheep RyRs had quite different Ca2+ sensitivities, Ca2+ spark amplitude was hardly affected. This suggests that moderate changes in RyR gating by second-messenger systems will principally alter the spatiotemporal properties of SR release, with smaller effects on the amount released.

Background Halogenated anesthetics activate cardiac ryanodine receptor 2–mediated sarcoplasmic reticulum Ca2+ release, leading to sarcoplasmic reticulum Ca2+ 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 Ca2+ activation of ryanodine receptor 2 but had no effect on Mg2+ 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 Ca2+ release was due to competition between adenosine and ATP, rather than between halothane and ATP.

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.

1. Mechanisms underlying the generation and propagation of gastrointestinal slow wave depolarizations have long been controversial. The present review aims to collate present knowledge on this subject with specific reference to slow waves in gastric smooth muscle. 2. At present, there is strong agreement that interstitial cells of Cajal (ICC) are the pacemaker cells that generate slow waves. What has been less clear is the relative role of primary types of ICC, including the network in the myenteric plexus (ICC-MY) and the intramuscular network (ICC-IM). It is concluded that both ICC-MY and ICC-IM are likely to serve a major role in slow wave generation and propagation. 3. There has been long-standing controversy as to how slow waves 'propagate' circumferentially and down the gastrointestinal tract. Two mechanisms have been proposed, one being action potential (AP)-like conduction and the other phase wave-based 'propagation' resulting from an interaction of coupled oscillators. Studies made on single bundle gastric strips indicate that both mechanisms apply with relative dominance depending on conditions; the phase wave mechanism is dominant under circumstances of rhythmically generating slow waves and the AP-like propagation is dominant when the system is perturbed. 4. The phase wave mechanism (termed Ca(2+) phase wave) uses cyclical Ca(2+) release as the oscillator, with coupling between oscillators mediated by several factors, including: (i) store-induced depolarization; (ii) resultant electrical current flow/depolarization through the pacemaker cell network; and (iii) depolarization-induced increase in excitability of downstream Ca(2+) stores. An analogy is provided by pendulums in an array coupled together by a network of springs. These, when randomly activated, entrain to swing at the same frequency but with a relative delay along the row giving the impression of a propagating wave. 5. The AP-like mechanism (termed voltage-accelerated Ca(2+) wave) propagates sequentially like a conducting AP. However, it is different in that it depends on regenerative store Ca(2+) release and resultant depolarization rather than regenerative activation of voltage-dependent channels in the cell membrane. 6. The applicability of these mechanisms to describing propagation in large intact gastrointestinal tissues, where voltage-dependent Ca(2+) entry is also likely to be functional, is discussed.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a potentially lethal inherited arrhythmia syndrome in which drug therapy is often ineffective. We discovered that flecainide prevents arrhythmias in a mouse model of CPVT by inhibiting cardiac ryanodine receptor-mediated Ca(2+) release and thereby directly targeting the underlying molecular defect. Flecainide completely prevented CPVT in two human subjects who had remained highly symptomatic on conventional drug therapy, indicating that this currently available drug is a promising mechanism-based therapy for CPVT.

The ryanodine receptors form the calcium release channel in the membrane of the sarcoplasmic reticulum (SR, the main intracellular Ca(2+) store). The importance of ryanodine receptors (RyRs) to cardiac pacemaking and rhythmicity is highlighted by more than 69 mutations, RyR mutations, which underlie arrhythmias and sudden cardiac death. Although most of these mutations lie in cytoplasmic domains, they all cause increased RyR activation by Ca(2+) in the SR lumen. Presented here is a review of the mechanisms by which cytoplasmic domains of the RyR can determine luminal activation.

In cardiac muscle, intracellular Ca(2+) and Mg(2+) are potent regulators of calcium release from the sarcoplasmic reticulum (SR). It is well known that the free [Ca(2+)] in the SR ([Ca(2+)](L)) stimulates the Ca(2+) release channels (ryanodine receptor [RYR]2). However, little is known about the action of luminal Mg(2+), which has not been regarded as an important regulator of Ca(2+) release. The effects of luminal Ca(2+) and Mg(2+) on sheep RYR2 were measured in lipid bilayers. Cytoplasmic and luminal Ca(2+) produced a synergistic increase in the opening rate of RYRs. A novel, high affinity inhibition of RYR2 by luminal Mg(2+) was observed, pointing to an important physiological role for luminal Mg(2+) in cardiac muscle. At diastolic [Ca(2+)](C), luminal Mg(2+) inhibition was voltage independent, with K(i) = 45 microM at luminal [Ca(2+)] ([Ca(2+)](L)) = 100 microM. Luminal and cytoplasmic Mg(2+) inhibition was alleviated by increasing [Ca(2+)](L) or [Ca(2+)](C). Ca(2+) and Mg(2+) on opposite sides of the bilayer exhibited competitive effects on RYRs, indicating that they can compete via the pore for common sites. The data were accurately fitted by a model based on a tetrameric RYR structure with four Ca(2+)-sensing mechanisms on each subunit: activating luminal L-site (40-microM affinity for Mg(2+) and Ca(2+)), cytoplasmic A-site (1.2 microM for Ca(2+) and 60 microM for Mg(2+)), inactivating cytoplasmic I(1)-site (approximately 10 mM for Ca(2+) and Mg(2+)), and I(2)-site (1.2 microM for Ca(2+)). Activation of three or more subunits will cause channel opening. Mg(2+) inhibition occurs primarily by Mg(2+) displacing Ca(2+) from the L- and A-sites, and Mg(2+) fails to open the channel. The model predicts that under physiological conditions, SR load-dependent Ca(2+) release (1) is mainly determined by Ca(2+) displacement of Mg(2+) from the L-site as SR loading increases, and (2) depends on the properties of both luminal and cytoplasmic activation mechanisms.

The clustering of cardiac RyR mutations, linked to sudden cardiac death (SCD), into several regions in the amino acid sequence underlies the hypothesis that these mutations interfere with stabilising interactions between different domains of the RyR2. SCD mutations cause increased channel sensitivity to cytoplasmic and luminal Ca(2+). A synthetic peptide corresponding to part of the central domain (DPc10:(2460)G-P(2495)) was designed to destabilise the interaction of the N-terminal and central domains of wild-type RyR2 and mimic the effects of SCD mutations. With Ca(2+) as the sole regulating ion, DPc10 caused increased channel activity which could be reversed by removal of the peptide whereas in the presence of ATP DPc10 caused no activation. In support of the domain destablising hypothesis, the corresponding peptide (DPc10-mut) containing the CPVT mutation R2474S did not affect channel activity under any circumstances. DPc10-induced activation was due to a small increase in RyR2 sensitivity to cytoplasmic Ca(2+) and a large increase in the magnitude of luminal Ca(2+) activation. The increase in the luminal Ca(2+) response appeared reliant on the luminal-to-cytoplasmic Ca(2+) flux in the channel, indicating that luminal Ca(2+) was activating the RyR2 via its cytoplasmic Ca(2+) sites. DPc10 had no significant effect on the RyR2 gating associated with luminal Ca(2+) sensing sites. The results were fitted by the luminal-triggered Ca(2+) feed-through model and the effects of DPc10 were explained entirely by perturbations in cytoplasmic Ca(2+)-activation mechanism.

The free [Ca2+] in endoplasmic/sarcoplasmic reticulum Ca2+ stores regulates excitability of Ca2+ release by stimulating the Ca2+ release channels. Just how the stored Ca2+ regulates activation of these channels is still disputed. One proposal attributes luminal Ca2+-activation to luminal facing regulatory sites, whereas another envisages Ca2+ permeation to cytoplasmic sites. This study develops a unified model for luminal Ca2+ activation for single cardiac ryanodine receptors (RyR2) and RyRs in coupled clusters in artificial lipid bilayers. It is shown that luminal regulation of RyR2 involves three modes of action associated with Ca2+ sensors in different parts of the molecule; a luminal activation site (L-site, 60 microM affinity), a cytoplasmic activation site (A-site, 0.9 microM affinity), and a novel cytoplasmic inactivation site (I2-site, 1.2 microM affinity). RyR activation by luminal Ca2+ is demonstrated to occur by a multistep process dubbed luminal-triggered Ca2+ feedthrough. Ca2+ binding to the L-site initiates brief openings (1 ms duration at 1-10 s(-1)) allowing luminal Ca2+ to access the A-site, producing up to 30-fold prolongation of openings. The model explains a broad data set, reconciles previous conflicting observations and provides a foundation for understanding the action of pharmacological agents, RyR-associated proteins, and RyR2 mutations on a range of Ca2+-mediated physiological and pathological processes.

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.

We examined the interaction of GABA and the competitive inhibitor SR95531 at human alpha1beta1gamma2S and alpha1beta1 GABA(A) receptors expressed in Sf9 cells. The efficacy and potency of inhibition depended on the relative timing of the GABA and SR95531 applications. In saturating (10 mM) GABA, the half-inhibitory concentrations of SR95531 (IC50) when coapplied with GABA to alpha1beta1gamma2S or alpha1beta1 receptors were 49 and 210 microM for the peak and 18 and 130 microM for the plateau current, respectively. Our data are explained by an inhibition mechanism in which SR95531 and GABA bind to two sites on the receptor where the binding of GABA allows channel opening but SR95531 does not. The SR95531 affinity for both receptor types was approximately 200 nM and the binding rate was found to be 10-fold faster than that for GABA. The dual binding-site model gives insights into the differential effects of GABA and SR95531 on the peak and plateau currents. The model predicts the effect of SR95531 on GABA currents in the synapse (GABA concentration approximately mM) and at extrasynaptic (GABA concentration < or = microM) sites. The IC50 (50-100 nM) for the synaptic response to SR95531 was insensitive to the GABA affinity of the receptors whereas the IC50 (50-800 nM) for extrasynaptic inhibition correlated with the GABA affinity.

Contraction in skeletal and cardiac muscle occurs when Ca(2+) is released from the sarcoplasmic reticulum (SR) through ryanodine receptor (RyR) Ca(2+) release channels. Several isoforms of the RyR exist throughout the animal kingdom, which are modulated by ATP, Ca(2+) and Mg(2+) in the cytoplasm and by Ca(2+) in the lumen of the SR. This review brings to light recent findings on their mechanisms of action in the mammalian isoforms RyR-1 and RyR-2 with an emphasis on RyR-1 from skeletal muscle. Cytoplasmic Mg(2+) is a potent RyR antagonist that binds to two classes of cytoplasmic site, identified as low-affinity, non-specific inhibition sites and high-affinity Ca(2+) activation sites (A-sites). Mg(2+) inhibition at the A-sites is very sensitive to the cytoplasmic and luminal milieu. Cytoplasmic Ca(2+), Mg(2+) and monovalent cations compete for the A-sites. In isolated RyRs, luminal Ca(2+) alters the Mg(2+) affinity of the A-site by an allosteric mechanism mediated by luminal sites. However, in close-packed RyR arrays luminal Ca(2+) can also compete with cytoplasmic ions for the A-site. Activation of RyRs by luminal Ca(2+) has been attributed to either Ca(2+) feedthrough to A-sites or to Ca(2+) regulatory sites on the luminal side of the RyR. As yet there is no consensus on just how luminal Ca(2+) alters RyR activation. Recent evidence indicates that both mechanisms operate and are likely to be important. Allosteric regulation of A-site Mg(2+) affinity could trigger Ca(2+) release, which is reinforced by Ca(2+) feedthrough.

In resting muscle, cytoplasmic Mg(2+) is a potent inhibitor of Ca(2+) release from the sarcoplasmic reticulum (SR). It is thought to inhibit calcium release channels (RyRs) by binding both to low affinity, low specificity sites (I-sites) and to high affinity Ca(2+) sites (A-sites) thus preventing Ca(2+) activation. We investigate the effects of luminal and cytoplasmic Ca(2+) on Mg(2+) inhibition at the A-sites of skeletal RyRs (RyR1) in lipid bilayers, in the presence of ATP or modified by ryanodine or DIDS. Mg(2+) inhibits RyRs at the A-site in the absence of Ca(2+), indicating that Mg(2+) is an antagonist and does not simply prevent Ca(2+) activation. Cytoplasmic Ca(2+) and Cs(+) decreased Mg(2+) affinity by a competitive mechanism. We describe a novel mechanism for luminal Ca(2+) regulation of Ca(2+) release whereby increasing luminal [Ca(2+)] decreases the A-site affinity for cytoplasmic Mg(2+) by a noncompetitive, allosteric mechanism that is independent of Ca(2+) flow. Ryanodine increases the Ca(2+) sensitivity of the A-sites by 10-fold, which is insufficient to explain the level of activation seen in ryanodine-modified RyRs at nM Ca(2+), indicating that ryanodine activates independently of Ca(2+). We describe a model for ion binding at the A-sites that predicts that modulation of Mg(2+) inhibition by luminal Ca(2+) is a significant regulator of Ca(2+) release from the SR. We detected coupled gating of RyRs due to luminal Ca(2+) permeating one channel and activating neighboring channels. This indicated that the RyRs existed in stable close-packed rafts within the bilayer. We found that luminal Ca(2+) and cytoplasmic Mg(2+) did not compete at the A-sites of single open RyRs but did compete during multiple channel openings in rafts. Also, luminal Ca(2+) was a stronger activator of multiple openings than single openings. Thus it appears that RyRs are effectively "immune" to Ca(2+) emanating from their own pore but sensitive to Ca(2+) from neighboring channels.

Calsequestrin is by far the most abundant Ca(2+)-binding protein in the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle. It allows the Ca2+ required for contraction to be stored at total concentrations of up to 20mM, while the free Ca2+ concentration remains at approximately 1mM. This storage capacity confers upon muscle the ability to contract frequently with minimal run-down in tension. Calsequestrin is highly acidic, containing up to 50 Ca(2+)-binding sites, which are formed simply by clustering of two or more acidic residues. The Kd for Ca2+ binding is between 1 and 100 microM, depending on the isoform, species and the presence of other cations. Calsequestrin monomers have a molecular mass of approximately 40 kDa and contain approximately 400 residues. The monomer contains three domains each with a compact alpha-helical/beta-sheet thioredoxin fold which is stable in the presence of Ca2+. The protein polymerises when Ca2+ concentrations approach 1mM. The polymer is anchored at one end to ryanodine receptor (RyR) Ca2+ release channels either via the intrinsic membrane proteins triadin and junctin or by binding directly to the RyR. It is becoming clear that calsequestrin has several functions in the lumen of the SR in addition to its well-recognised role as a Ca2+ buffer. Firstly, it is a luminal regulator of RyR activity. When triadin and junctin are present, calsequestrin maximally inhibits the Ca2+ release channel when the free Ca2+ concentration in the SR lumen is 1mM. The inhibition is relieved when the Ca2+ concentration alters, either because of small changes in the conformation of calsequestrin or its dissociation from the junctional face membrane. These changes in calsequestrin's association with the RyR amplify the direct effects of luminal Ca2+ concentration on RyR activity. In addition, calsequestrin activates purified RyRs lacking triadin and junctin. Further roles for calsequestrin are indicated by the kinase activity of the protein, its thioredoxin-like structure and its influence over store operated Ca2+ entry. Clearly, calsequestrin plays a major role in calcium homeostasis that extends well beyond its ability to buffer Ca2+ ions.

Calcium-induced calcium release (CICR) is an inherently regenerative process due to the Ca(2+)-dependent gating of ryanodine receptors (RyRs) in the sarco/endoplasmic reticulum (SR) and is critical for cardiac excitation-contraction coupling. This process is seen as Ca(2+) sparks, which reflect the concerted gating of groups of RyRs in the dyad, a specialised junctional signalling domain between the SR and surface membrane. However, the mechanism(s) responsible for the termination of regenerative CICR during the evolution of Ca(2+) sparks remain uncertain. Rat cardiac RyR gating was recorded at physiological Ca(2+), Mg(2+) and ATP levels and incorporated into a 3D model of the cardiac dyad which reproduced the time-course of Ca(2+) sparks, Ca(2+) blinks and Ca(2+) spark restitution. Model CICR termination was robust, relatively insensitive to the number of dyadic RyRs and automatic. This emergent behaviour arose from the rapid development and dissolution of nanoscopic Ca(2+) gradients within the dyad. These simulations show that CICR does not require intrinsic inactivation or SR calcium sensing mechanisms for stability and cessation of regeneration that arises from local control at the molecular scale via a process we call 'induction decay'.

The actions of peptide C, corresponding to (724)Glu-Pro(760) of the II-III loop of the skeletal dihydropyridine receptor, on ryanodine receptor (RyR) channels incorporated into lipid bilayers with the native sarcoplasmic reticulum membrane show that the peptide is a high-affinity activator of native skeletal RyRs at cytoplasmic concentrations of 100 nM-10 microM. In addition, we found that peptide C inhibits RyRs in a voltage-independent manner when added for longer times or at higher concentrations (up to 150 microM). Peptide C had a random-coil structure indicating that it briefly assumes a variety of structures, some of which might activate and others which might inhibit RyRs. The results suggest that RyR activation and inhibition by peptide C arise from independent stochastic processes. A rate constant of 7.5 x 10(5) s(-1).M(-1) was obtained for activation and a lower estimate for the rate constant for inhibition of 5.9 x 10(3) s(-1).M(-1). The combined actions of peptide C and peptide A (II-III loop sequence (671)Thr-Leu(690)) showed that peptide C prevented activation but not blockage of RyRs by peptide A. We suggest that the effects of peptide C indicate functional interactions between a part of the dihydropyridine receptor and the RyR. These interactions could reflect either dynamic changes that occur during excitation-contraction coupling or interactions between the proteins at rest.

Activation of skeletal muscle ryanodine receptors (RyRs) by suramin and disulfonic stilbene derivatives (Diisothiocyanostilbene-2',2'-disulfonic acid (DIDS), 4,4'-dibenzamidostilbene-2,2'-disulfonic acid (DBDS),and 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS)) was investigated using planar bilayers. One reversible and two nonreversible mechanisms were identified. K(a) for reversible activation (approximately 100 micro M) depended on cytoplasmic [Ca(2+)] and the bilayer composition. Replacement of neutral lipids by negative phosphatidylserine increased K(a) fourfold, suggesting that reversible binding sites are near the bilayer surface. Suramin and the stilbene derivatives adsorbed to neutral bilayers with maximal mole fractions between 1-8% and with affinities approximately 100 micro M but did not adsorb to negative lipids. DIDS activated RyRs by two nonreversible mechanisms, distinguishable by their disparate DIDS binding rates (10(5) and 60 M(-1) s(-1)) and actions. Both mechanisms activated RyRs via several jumps in open probability, indicating several DIDS binding events. The fast and slow mechanisms are independent of each other, the reversible mechanism and ATP binding. The fast mechanism confers DIDS sensitivity approximately 1000-fold greater than previously reported, increases Ca(2+) activation and increases K(i) for Ca(2+)/Mg(2+) inhibition 10-fold. The slow mechanism activates RyRs in the absence of Ca(2+) and ATP, increases ATP activation without altering K(a), and slightly increases activity at pH < 6.5. These findings explain how different types of DIDS activation are observed under different conditions.

Dantrolene is a muscle relaxant that has been used clinically as the treatment for malignant hyperthermia. Dantrolene acts on skeletal and cardiac muscle by inhibiting Ca2+ release from the SR. However, single channel studies fail to see any effect of dantrolene on RyRs. Calmodulin (CaM) regulates RyR2 directly by binding to RyR2 and dantrolene influences CaM binding to RyR2. CaM can dissociate from RyR2 within ∼1 min so when RyR2 are isolated form cells and examined in artificial lipid bilayers, CaM is not usually present. Thus, we hypothesized that CaM is the missing protein required for dantrolene inhibition of RyR2.Single channel recordings were obtained from RyR2 isolated from sheep heart and incorporated into artificial lipid bilayers. Control RyR2 activity was measured for periods of 1 min, and then during 1 min exposure to dantrolene and then again after washout. This was repeated in the absence and presence of exogenous 100 nM CaM. In diastolic cytoplasmic [Ca2+] (100 nM), dantrolene (50 µM) reduced the mean open probability of RyR2 to 45 ± 6 % in the presence of CaM (n = 7, p < 0.05, student paired t-test) but had no significant effect when CaM was absent (95 ± 9 %, n=20, p = 0.24). Dantrolene exhibited a hyperbolic dose-response in the presence of CaM with an IC50 of 0.16 ± 0.03 µM and with a saturating relative Po (Emax) of 52 ± 4 %. Emax increased to one as cytoplasmic Ca2+ was increased to levels above 1 µM.In saponin permeablized mouse cardiomyocytes supplemented with 100 nM CaM, dantrolene reduced the frequency and amplitude of Ca2+ waves with an IC50 of 0.3 µM. However, when cells were depleted of CaM, dantrolene had no effect. Thus CaM is essential for inhibitory action of dantrolene.

We provide novel evidence that the sarcoplasmic reticulum calcium binding protein, calsequestrin, inhibits native ryanodine receptor calcium release channel activity. Calsequestrin dissociation from junctional face membrane was achieved by increasing luminal (trans) ionic strength from 250 to 500 mM with CsCl or by exposing the luminal side of ryanodine receptors to high [Ca(2+)] (13 mM) and dissociation was confirmed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Calsequestrin dissociation caused a 10-fold increase in the duration of ryanodine receptor channel opening in lipid bilayers. Adding calsequestrin back to the luminal side of the channel after dissociation reversed this increased activity. In addition, an anticalsequestrin antibody added to the luminal solution reduced ryanodine receptor activity before, but not after, calsequestrin dissociation. A population of ryanodine receptors (approximately 35%) may have initially lacked calsequestrin, because their activity was high and was unaffected by increasing ionic strength or by anticalsequestrin antibody: their activity fell when purified calsequestrin was added and they then responded to antibody. In contrast to native ryanodine receptors, purified channels, depleted of triadin and calsequestrin, were not inhibited by calsequestrin. We suggest that calsequestrin reduces ryanodine receptor activity by binding to a coprotein, possibly to the luminal domain of triadin.

Highlight A persistent and polarized cytosolic Ca2+ signal, formed into plumes by co-operative activities of plasma membrane Ca2+ channels and Ca2+-ATPase clusters, directs papillate wall ingrowth deposition in trans-differentiating 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 Ca2+ signals generated by spatiotemporal alterations in cytosolic Ca2+ ([Ca2+]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 Ca2+ signal, generated during epidermal cell trans-differentiation, was found to co-localize with the site of ingrowth wall formation. Dampening Ca2+ signal intensity, by withdrawing extracellular Ca2+ or blocking Ca2+ channel activity, inhibited formation of wall ingrowth papillae. Maintenance of Ca2+ signal polarity and persistence depended upon a rapid turnover (minutes) of cytosolic Ca2+ by co-operative functioning of plasma membrane Ca2+-permeable channels and Ca2+-ATPases. Viewed paradermally, and proximal to the cytosol–plasma membrane interface, the Ca2+ signal was organized into discrete patches that aligned spatially with clusters of Ca2+-permeable channels. Mathematical modelling demonstrated that these patches of cytosolic Ca2+ were consistent with inward-directed plumes of elevated [Ca2+]cyt. Plume formation depended upon an alternating distribution of Ca2+-permeable channels and Ca2+-ATPase clusters. On further inward diffusion, the Ca2+ plumes coalesced into a uniform Ca2+ signal. Blocking or dispersing the Ca2+ plumes inhibited deposition of wall ingrowth papillae, while uniform wall formation remained unaltered. A working model envisages that cytosolic Ca2+ plumes define the loci at which wall ingrowth papillae are deposited.

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.