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Recent data on structure of dyads in cardiac myocytes indicate variable clustering of RyR calcium release channels. The question arises as to how geometric factors of RyR arrangement translate to their role in formation of calcium release events (CRE). Since this question is not experimentally testable in situ, we performed in silico experiments on a large set of calcium release site (CRS) models. The models covered the range of RyR spatial distributions observed in dyads, and included gating of RyRs with open probability dependent on Ca2+ and Mg2+ concentration. The RyR single-channel calcium current, varied in the range of previously reported values, was set constant in the course of CRE simulations. Other known features of dyads were omitted in the model formulation for clarity. CRE simulations initiated by a single random opening of one of the RyRs in a CRS produced spark-like responses with characteristics that varied with RyR vicinity, a newly defined parameter quantifying spatial distribution of RyRs in the CRSs, and with the RyR single-channel calcium current. The CRE characteristics followed the law of mass action with respect to a CRS state variable, defined as a weighed product of RyR vicinity and RyR single-channel calcium current. The results explained the structure-function relations among determinants of cardiac dyads on synergy principles and thus allowed to evolve the concept of CRS as a dynamic unit of cardiac dyad.

Iaparov et al. use in silico modeling to test the effect of changes in the geometric arrangement of RYR channels on calcium release in cardiac myocytes. Their simulations predict a coupling between RYR distribution at the calcium release site and dyad function., The dyads of cardiac myocytes contain ryanodine receptors (RYRs) that generate calcium sparks upon activation. To test how geometric factors of RYR distribution contribute to the formation of calcium sparks, which cannot be addressed experimentally, we performed in silico simulations on a large set of models of calcium release sites (CRSs). Our models covered the observed range of RYR number, density, and spatial arrangement. The calcium release function of CRSs was modeled by RYR openings, with an open probability dependent on concentrations of free Ca2+ and Mg2+ ions, in a rapidly buffered system, with a constant open RYR calcium current. We found that simulations of spontaneous sparks by repeatedly opening one of the RYRs in a CRS produced three different types of calcium release events (CREs) in any of the models. Transformation of simulated CREs into fluorescence signals yielded calcium sparks with characteristics close to the observed ones. CRE occurrence varied broadly with the spatial distribution of RYRs in the CRS but did not consistently correlate with RYR number, surface density, or calcium current. However, it correlated with RYR coupling strength, defined as the weighted product of RYR vicinity and calcium current, so that CRE characteristics of all models followed the same state-response function. This finding revealed the synergy between structure and function of CRSs in shaping dyad function. Lastly, rearrangements of RYRs simulating hypothetical experiments on splitting and compaction of a dyad revealed an increased propensity to generate spontaneous sparks and an overall increase in calcium release in smaller and more compact dyads, thus underlying the importance and physiological role of RYR arrangement in cardiac myocytes.

Cardiac excitation-contraction coupling relies on dyads, the intracellular calcium synapses of cardiac myocytes, where the plasma membrane contacts sarcoplasmic reticulum and where electrical excitation triggers calcium release. The morphology of dyads and dynamics of local calcium release vary substantially. To better understand the correspondence between the structure and the functionality of dyads, we estimated incidences of structurally different dyads and of kinetically different calcium release sites and tested their responsiveness to experimental myocardial injury in left ventricular myocytes of rats. According to the structure of dyads estimated in random electron microscopic images of myocardial tissue, the dyads were sorted into ‘compact’ or ‘loose’ types. The calcium release fluxes, triggered at local calcium release sites in patch-clamped ventricular myocytes and recorded by laser scanning confocal fluorescence microscopy, were decomposed into ‘early’ and ‘late’ components. ANOVA tests revealed very high correlation between the relative amplitudes of early and late calcium release flux components and the relative occurrences of compact and loose dyads in the control and in the injured myocardium. This finding ascertained the relationship between the structure of dyads and the functionality of calcium release sites and the responsiveness of calcium release sites to physical load in cardiac myocytes.

We established that Endosidin2 (ES2) affected the trafficking routes of both newly synthesized and endocytic pools of PIN-FORMED2 (PIN2) in Arabidopsis root epidermal cells. PIN2 populations accumulated in separated patches, which gradually merged into large and compact ES2 aggregates (ES2As). FM4-64 endocytic tracer labeled ES2As as well. Both PIN2 pools also appeared in vacuoles. Accelerated endocytosis of PIN2, its aggregation in the cytoplasm, and redirection of PIN2 flows to vacuoles led to a substantial reduction of the abundance of this protein in the plasma membrane. Whereas PIN-FORMED3 and PIN-FORMED4 also aggregated in the cytoplasm, SYT1 was not sensitive to ES2 treatment and did not appear either in the cytoplasmic aggregates or vacuoles. Ultrastructural analysis revealed that ES2 affects the Golgi apparatus so that stacks acquired cup-shape and even circular shape surrounded by several vesicles. Abnormally shaped Golgi stacks, stack remnants, multi-lamellar structures, separated Golgi cisterna rings, tubular structures, and vesicles formed discrete clusters.

Developing cardiac myocytes undergo substantial structural and functional changes transforming the mechanism of excitation-contraction coupling from the embryonic form, based on calcium influx through sarcolemmal DHPR calcium channels, to the adult form, relying on local calcium release through RYR calcium channels of sarcoplasmic reticulum stimulated by calcium influx. We characterized day-by-day the postnatal development of the structure of sarcolemma, using techniques of confocal fluorescence microscopy, and the development of the calcium current, measured by the whole-cell patch-clamp in isolated rat ventricular myocytes. We characterized the appearance and expansion of the t-tubule system and compared it with the appearance and progress of the calcium current inactivation induced by the release of calcium ions from sarcoplasmic reticulum as structural and functional measures of direct DHPR-RYR interaction. The release-dependent inactivation of calcium current preceded the development of the t-tubular system by several days, indicating formation of the first DHPR-RYR couplons at the surface sarcolemma and their later spreading close to contractile myofibrils with the growing t-tubules. Large variability of both of the measured parameters among individual myocytes indicates uneven maturation of myocytes within the growing myocardium.

The function of ion channels to mediate the flux of ions through membranes of living cells depends on their number, conductance, and open probability. The open probability, PO, characterizes gating of channels that is sensitive to experimental conditions and that can be determined in single-channel experiments. Individual experimental records and even whole series of single-channel activity measurements represent random samples of the stochastic gating continuous in time. The aim of this study was to understand the relationship between the accuracy (trueness and precision) of PO determination and the method of single-channel activity data collection. We used simulated single-channel experiments with variable settings of data collection for a range of open probability values. We found that at low PO, the trueness of PO determination depends on the average number of channel openings per record, while the precision of PO determination depends on the total number of channel openings in the whole dataset and on the distribution of open and closed times. We derived relationships that allow planning of single-channel experiments for the required accuracy of PO determination over a large span of open probabilities. (C) 2020 Elsevier Ltd. All rights reserved.

Calcium release sites (CRSs) play a key role in excitation–contraction coupling of cardiac myocytes. Recent studies based on electron tomography and super-resolution imaging revealed that CRSs are not completely filled with ryanodine receptors (RyRs) and that the spatial arrangement of RyRs is neither uniform nor static. In this work, we studied the effect of spatial arrangement of RyRs on RyR activation using simulations based on Monte Carlo (MC) and mean-field (MF) approaches. Both approaches showed that activation of RyRs is sensitive to the arrangement of RyRs in the CRS. However, the MF simulations did not reproduce results of MC simulations for non-compact CRSs, suggesting that the approximations used in the MF approach are not suitable for simulation studies of RyRs arrangements observed experimentally. MC simulations revealed the importance of realistic spatial arrangement of RyRs for adequate modelling of calcium release in cardiac myocytes.

Calcium waves can form and propagate at low frequencies of spontaneous calcium sparks if the calcium dependence of spark frequency is sufficiently steep, or the number of open RyRs is sufficiently large., The role of cardiac ryanodine receptor (RyR) gating in the initiation and propagation of calcium waves was investigated using a mathematical model comprising a stochastic description of RyR gating and a deterministic description of calcium diffusion and sequestration. We used a one-dimensional array of equidistantly spaced RyR clusters, representing the confocal scanning line, to simulate the formation of calcium sparks. Our model provided an excellent description of the calcium dependence of the frequency of diastolic calcium sparks and of the increased tendency for the production of calcium waves after a decrease in cytosolic calcium buffering. We developed a hypothesis relating changes in the propensity to form calcium waves to changes of RyR gating and tested it by simulation. With a realistic RyR gating model, increased ability of RyR to be activated by Ca2+ strongly increased the propensity for generation of calcium waves at low (0.05–0.1-µM) calcium concentrations but only slightly at high (0.2–0.4-µM) calcium concentrations. Changes in RyR gating altered calcium wave formation by changing the calcium sensitivity of spontaneous calcium spark activation and/or the average number of open RyRs in spontaneous calcium sparks. Gating changes that did not affect RyR activation by Ca2+ had only a weak effect on the propensity to form calcium waves, even if they strongly increased calcium spark frequency. Calcium waves induced by modulating the properties of the RyR activation site could be suppressed by inhibiting the spontaneous opening of the RyR. These data can explain the increased tendency for production of calcium waves under conditions when RyR gating is altered in cardiac diseases.

In mammalian cardiac myocytes, the elementary calcium releases triggered by step voltage stimuli manifest either as solitary or as twin spikes that vary widely in kinetics and amplitude for unknown reasons. Here we examined the variability of calcium spikes measured using line-scanning confocal microscopy in patch-clamped rat ventricular myocytes. Amplitude distributions of the single and of the first of twin spikes were broader than those of the second spikes. All could be best approximated by a sum of a few elementary Gaussian probability distribution functions. The latency distributions of the single and the first spikes were identical, much shorter and less variable than those of the second spikes. The multimodal distribution of spike amplitudes and the probability of occurrence of twin spikes were stochastically congruent with activation of only a few of the many RyR2 channels present in the release site cluster. The occurrence of twin release events was rare due to refractoriness of release, induced with a probability proportional to the number of RyR2s activated in the primary release event. We conclude that the variability of the elementary calcium release events supports a calcium signalling mechanism that arises from stochastics of RyR2 gating and from inactivation of local origin.

X-ray and solution structures of the human RyR2 N-terminal region were obtained under near-physiological conditions. The structure exhibits a unique network of interactions between its three domains, revealing an important stabilizing role of the central helix., Human ryanodine receptor 2 (hRyR2) mediates calcium release from the sarcoplasmic reticulum, enabling cardio­myocyte contraction. The N-terminal region of hRyR2 (amino acids 1–606) is the target of >30 arrhythmogenic mutations and contains a binding site for phosphoprotein phosphatase 1. Here, the solution and crystal structures determined under near-physiological conditions, as well as a homology model of the hRyR2 N-terminal region, are presented. The N-terminus is held together by a unique network of interactions among its three domains, A, B and C, in which the central helix (amino acids 410–437) plays a prominent stabilizing role. Importantly, the anion-binding site reported for the mouse RyR2 N-terminal region is notably absent from the human RyR2. The structure concurs with the differential stability of arrhythmogenic mutations in the central helix (R420W, I419F and I419F/R420W) which are owing to disparities in the propensity of mutated residues to form energetically favourable or unfavourable contacts. In solution, the N-terminus adopts a globular shape with a prominent tail that is likely to involve residues 545–606, which are unresolved in the crystal structure. Docking the N-terminal domains into cryo-electron microscopy maps of the closed and open RyR1 conformations reveals Cα atom movements of up to 8 Å upon channel gating, and predicts the location of the leucine–isoleucine zipper segment and the interaction site for spinophilin and phosphoprotein phosphatase 1 on the RyR surface.

The principal role of calcium current in the triggering of calcium release in cardiac myocytes is well recognized. The mechanism of how calcium current (ICa) controls the intensity of calcium release is not clear because of the stochastic nature of voltage-dependent gating of calcium channels (DHPRs) and of calcium-dependent gating of ryanodine receptors (RyRs). To disclose the relation between DHPR openings and the probability of calcium release, local calcium release activation by ICa was investigated in rat ventricular myocytes using patch-clamp and confocal microscopy. Calcium spikes were activated by temporally synchronized DHPR calcium current triggers, generated by instantaneous ‘tail’ICa and modulated by prepulse duration, by tail potential, and by the DHPR agonist BayK 8644. The DHPR–RyR coupling fidelity was determined from the temporal distribution of calcium spike latencies using a model based on exponentially distributed DHPR open times. The analysis provided a DHPR mean open time of ∼0.5 ms, RyR activation time constant of ∼0.6 ms, and RyR activation kinetics of the 4th order. The coupling fidelity was low due to the inherent prevalence of very short DHPR openings but was increased when DHPR openings were prolonged by BayK 8644. The probability of calcium release activation was high, despite low coupling fidelity, due to the activation of many DHPRs at individual release sites. We conclude that the control of calcium release intensity by physiological stimuli can be achieved by modulating the number and duration of DHPR openings at low coupling fidelity, thus avoiding the danger of inadvertently triggering calcium release events.

The effect of a domain peptide DPCPVTc from the central region of the RYR2 on ryanodine receptors from rat heart has been examined in planar lipid bilayers. At a zero holding potential and at 8 mmol L−1 luminal Ca2+ concentration, DPCPVTc induced concentrationdependent activation of the ryanodine receptor that led up to 20-fold increase of PO at saturating DPCPVTc concentrations. DPCPVTc prolonged RyR2 openings and increased RyR2 opening frequency. At all peptide concentrations the channels displayed large variability in open probability, open time and frequency of openings. With increasing peptide concentration, the fraction of high open probability records increased together with their open time. The closed times of neither low- nor high-open probability records depended on peptide concentration. The concentration dependence of all gating parameters had EC50 of 20 μmol L−1 and a Hill slope of 2. Comparison of the effects of DPCPVTc with the effects of ATP and cytosolic Ca2+ suggests that activation does not involve luminal feed-through and is not caused by modulation of the cytosolic activation A-site. The data suggest that although “domain unzipping” by DPCPVTc occurs in both modes of RyR activity, it affects RyR gating only when the channel resides in the H-mode of activity.

Proper function of cardiac myocytes requires a reliable mechanism for regulation of calcium influx during the contraction-relaxation cycle. The most effective is negative feedback based on the calcium-dependent inactivation of calcium current. Calcium release activated by L-type Ca-current causes rapid and extensive inactivation of the Ca-current. The aim of this study was to characterize the effect of local Ca-release on the extent of calcium release-dependent inactivation (RDI) using characteristics of calcium spikes.Calcium release was induced in isolated rat ventricular myocytes in the whole-cell patch-clamp configuration, using a two pulse protocol [1]. A short pre-pulse of varying length or voltage was followed by a standard test-pulse (0 mV, 80 ms). The extent of RDI was assessed as the fraction of peak Ca-current remaining in the test-pulse relative to peak current in the absence of the pre-pulse. Local calcium release fluxes (Ca-spikes) were measured by laser scanning fluorescence confocal microscopy using 0.1 mM fluo-3 and 1 mM EGTA in the patch-pipette solution. The latency and the amplitude were determined for individual Ca-spikes. Probability of Ca-release activation (PA) was calculated as the fraction of activated calcium release sites. Synchrony of Ca-release was calculated as the inverse of standard deviation of the latency.The extent of RDI showed linear correlation with PA (R = 0.95) and amplitude (R = 0.89), while latency and synhrony did not affect the extent of RDI. The extent of RDI showed sigmoidal dependence on the product of amplitude and PA that reflects the amount of released calcium. We conclude that RDI of L-type calcium currents results from the local character of calcium release and local calcium-dependent inactivation of L-type calcium channels.Support: VEGA 2/0095/15 and VEGA 2/0147/14[1] Zahradnikova et al., 2004. Am J Physiol Cell Physiol (286): 330-341

The synergic effect of luminal Ca2+, cytosolic Ca2+, and cytosolic adenosine triphosphate (ATP) on activation of cardiac ryanodine receptor (RYR2) channels was examined in planar lipid bilayers. The dose–response of RYR2 gating activity to ATP was characterized at a diastolic cytosolic Ca2+ concentration of 100 nM over a range of luminal Ca2+ concentrations and, vice versa, at a diastolic luminal Ca2+ concentration of 1 mM over a range of cytosolic Ca2+ concentrations. Low level of luminal Ca2+ (1 mM) significantly increased the affinity of the RYR2 channel for ATP but without substantial activation of the channel. Higher levels of luminal Ca2+ (8–53 mM) markedly amplified the effects of ATP on the RYR2 activity by selectively increasing the maximal RYR2 activation by ATP, without affecting the affinity of the channel to ATP. Near-diastolic cytosolic Ca2+ levels (

Cytosolic calcium concentration in resting cardiac myocytes locally fluctuates as a result of spontaneous microscopic Ca(2+) releases or abruptly rises as a result of an external trigger. These processes, observed as calcium sparks, are fundamental for proper function of cardiac muscle. In this study, we analyze how the characteristics of spontaneous and triggered calcium sparks are related to cardiac ryanodine receptor (RYR) gating. We show that the frequency of spontaneous sparks and the probability distribution of calcium release flux quanta of triggered sparks correspond quantitatively to predictions of an allosteric homotetrameric model of RYR gating. This model includes competitive binding of Ca(2+) and Mg(2+) ions to the RYR activation sites and allosteric interaction between divalent ion binding and channel opening. It turns out that at rest, RYRs are almost fully occupied by Mg(2+). Therefore, spontaneous sparks are most frequently evoked by random openings of the highly populated but rarely opening Mg(4)RYR and CaMg(3)RYR forms, whereas triggered sparks are most frequently evoked by random openings of the less populated but much more readily opening Ca(2)Mg(2)RYR and Ca(3)MgRYR forms. In both the spontaneous and the triggered sparks, only a small fraction of RYRs in the calcium release unit manages to open during the spark because of the limited rate of Mg(2+) unbinding. This mechanism clarifies the unexpectedly low calcium release flux during elementary release events and unifies the theory of calcium signaling in resting and contracting cardiac myocytes.

The local calcium release flux signals (calcium spikes) evoked by membrane depolarization were recorded at high temporal resolution (2000 lines s(-1)) in isolated ventricular myocytes of male rats, using combination of scanning confocal microscopy and the patch-clamp technique. The kinetic properties of calcium spikes were investigated. The time course of calcium spike activation could be described reliably by a model with higher-order (n = 3) kinetics, but not by a first-order exponential process. A model of calcium spike with calcium release termination coupled to its activation was preferential to a model with the release termination independent of its activation. Three fluorescent calcium dyes (OG-5N, fluo-3, and fluo-4) were compared for calcium spike measurements. Experimental measurements as well as simulations showed that the occurrence and latency of calcium spikes could be measured faithfully with all indicators, while the kinetics of calcium spikes was reliably traced only with OG-5N. Calcium spikes evoked by a step depolarization from -50 to 0 mV commenced with a mean latency of 4.1 +/- 0.2 ms and peaked 6.7 +/- 0.2 ms later. Their full amplitudes were normally distributed. The activation time constant of calcium spikes was 3.1 +/- 0.1 ms, and the time constant of termination was 5.5 +/- 0.2 ms. A negative correlation was observed between the observed amplitude of calcium spikes and their time constant of activation, but there was no correlation between their observed amplitude and time constant of termination, in agreement with the concept of steep calcium-dependent activation and fateful inactivation of calcium release flux.

Despite its importance and abundance of experimental data, the molecular mechanism of RyR2 activation by calcium is poorly understood. Recent experimental studies involving coexpression of wild-type (WT) RyR2 together with a RyR2 mutant deficient in calcium-dependent activation (Li, P., and S.R. Chen. 2001. J. Gen. Physiol. 118:33–44) revealed large variations of calcium sensitivity of the RyR tetramers with their monomer composition. Together with previous results on kinetics of Ca activation (Zahradníková, A., I. Zahradník, I. Györke, and S. Györke. 1999. J. Gen. Physiol. 114:787–798), these data represent benchmarks for construction and testing of RyR models that would reproduce RyR behavior and be structurally realistic as well. Here we present a theoretical study of the effects of RyR monomer substitution by a calcium-insensitive mutant on the calcium dependence of RyR activation. Three published models of tetrameric RyR channels were used either directly or after adaptation to provide allosteric regulation. Additionally, two alternative RyR models with Ca binding sites created jointly by the monomers were developed. The models were modified for description of channels composed of WT and mutant monomers. The parameters of the models were optimized to provide the best approximation of published experimental data. For reproducing the observed calcium dependence of RyR tetramers containing mutant monomers (a) single, independent Ca binding sites on each monomer were preferable to shared binding sites; (b) allosteric models were preferable to linear models; (c) in the WT channel, probability of opening to states containing a Ca2+-free monomer had to be extremely low; and (d) models with fully Ca-bound closed states, additional to those of an Monod-Wyman-Changeaux model, were preferable to models without such states. These results provide support for the concept that RyR activation is possible (albeit vanishingly small in WT channels) in the absence of Ca2+ binding. They also suggest further avenues toward understanding RyR gating.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a genetic disease characterized by stress-induced syncope and/or sudden death in young individuals with structurally normal heart. More than 150 mutations located in the cardiac Ca2+ release channel (type-2 ryanodine receptor, RyR2) gene are related to CPVT. Besides ventricular tachycardia (VT) under stress, sinoatrial node (SAN) dysfunction is frequently observed in CPVT patients. However, the cellular mechanisms remain underexplored. We created a KI mice model bearing a mutation in the N-terminal portion of the RyR2 found in a CPVT family, RyR2(R420Q). ECGs were recorded in KI and WT littermates in resting condition and after epinephrine/caffeine (2/120 mg/kg) challenge (i.p. injection). All KI mice (n=7) showed sustained bidirectional VT, validating the model. Moreover, resting heart rhythm was slower in KI mice during rest period (525.3±9.0 bpm n=4 and 498.5±4.9 bpm n=3, P

Intracellular calcium signals are studied by laser-scanning confocal fluorescence microscopy. The required spatio-temporal resolution makes description of calcium signals difficult because of the low signal-to-noise ratio. We designed a new procedure of calcium spike analysis based on their fitting with a model. The accuracy and precision of calcium spike description were tested on synthetic datasets generated either with randomly varied spike parameters and Gaussian noise of constant amplitude, or with constant spike parameters and Gaussian noise of various amplitudes. Statistical analysis was used to evaluate the performance of spike fitting algorithms. The procedure was optimized for reliable estimation of calcium spike parameters and for dismissal of false events. A new algorithm was introduced that corrects the acquisition time of pixels in line-scan images that is in error due to sequential acquisition of individual pixels along the space coordinate. New software was developed in Matlab and provided for general use. It allows interactive dissection of temporal profiles of calcium spikes from x-t images, their fitting with predefined function(s) and acceptance of results on statistical grounds, thus allowing efficient analysis and reliable description of calcium signaling in cardiac myocytes down to the in situ function of ryanodine receptors.

Local calcium release via RYRs, observed in the form of calcium sparks ([Cheng et al., 1993][1]), is generally accepted to be a principal mediator of calcium homeostasis and excitation–contraction coupling in cardiac myocytes. However, investigation of local calcium release signals is extremely

In the heart, depolarization during the action potential activates voltage-dependent Ca2+ channels that mediate a small, localized Ca2+ influx (ICa). This small Ca2+ signal activates specialized Ca2+ release channels, the ryanodine receptors (RyRs), in the sarcoplasmic reticulum (SR). This process

The local control concept of excitation–contraction coupling in the heart postulates that the activity of the sarcoplasmic reticulum ryanodine receptor channels (RyR) is controlled by Ca2+entry through adjoining sarcolemmal single dihydropyridine receptor channels (DHPRs). One unverified premise of this hypothesis is that the RyR must be fast enough to track the brief (2+elevations accompanying single DHPR channel openings. To define the kinetic limits of effective trigger Ca2+signals, we recorded activity of single cardiac RyRs in lipid bilayers during rapid and transient increases in Ca2+generated by flash photolysis of DM-nitrophen. Application of such Ca2+spikes (amplitude ∼10–30 μM, duration ∼0.1–0.4 ms) resulted in activation of the RyRs with a probability that increased steeply (apparent Hill slope ∼2.5) with spike amplitude. The time constants of RyR activation were 0.07–0.27 ms, decreasing with spike amplitude. To fit the rising portion of the open probability, a single exponential function had to be raised to a powern∼ 3. We show that these data could be adequately described with a gating scheme incorporating four sequential Ca2+-sensitive closed states between the resting and the first open states. These results provide evidence that brief Ca2+triggers are adequate to activate the RyR, and support the possibility that RyR channels are governed by single DHPR openings. They also provide evidence for the assumption that RyR activation requires binding of multiple Ca2+ions in accordance with the tetrameric organization of the channel protein.

NO donors were found to reduce the rate of Ca2+ release from isolated skeletal muscle sarcoplasmic reticulum (SR) and the open probability of single ryanodine receptor Ca2+ release channels (RyRCs) in planar lipid bilayers, and these effects were prevented by the NO quencher hemoglobin and reversed by 2-mercaptoethanol. Ca2+ release assessed in skeletal muscle homogenates was also reduced by NO that was generated in situ from l-arginine by endogenous, nitro-l-arginine methylester-sensitive NO-synthase. The effect of NO on the RyRC might explain NO-induced depression of contractile force in striated muscles and, since both RyRC isoforms and NOS isoenzymes are ubiquitous, may represent a wide-spread feedback mechanism in Ca2+ signaling; i.e. Ca-dependent activation of NO production and NO-evoked reduction of Ca2+ release from intracellular Ca2+ stores.

We have constructed a three-dimensional reaction-diffusion model of the mammalian cardiac calcium release unit. We analyzed effects of diffusion coefficients, single channel current amplitude, density of RyR channels, and reaction kinetics of ATP(2-) with Ca(2+) and Mg(2+) ions on spatiotemporal concentration profiles of Ca(2+), Mg(2+), and ATP(2-) in the dyadic cleft during Ca(2+) release. The model revealed that Ca(2+) concentration gradients persist near RyRs in the steady state. Even with low number of open RyRs, peak [Ca(2+)] in the dyadic space reached values similar to estimates of luminal [Ca(2+)] in approximately 1 ms, suggesting that during calcium release the Ca(2+) gradient moves from the cisternal membrane towards the boundary of the dyadic space with the cytosol. The released Ca(2+) bound to ATP(2-), and thus substantially decreased ATP(2-) concentration in the dyadic space. The released Ca(2+) could also replace Mg(2+) in its complex with ATP(2-) during first milliseconds of release if dissociation of MgATP was fast. The results suggest that concentration changes of Ca(2+), Mg(2+), and ATP(2-) might be large and fast enough to reduce dyadic RyR activity. Thus, under physiological conditions, termination of calcium release may be facilitated by the synergic effect of the construction and chemistry of mammalian cardiac dyads.

In mammalian cardiac myocytes, calcium released into the dyadic space rapidly inactivates calcium current ( ICa). We used this Ca2+ release-dependent inactivation (RDI) of ICa as a local probe of sarcoplasmic reticulum Ca2+ release activation. In whole cell patch-clamped rat ventricular myocytes, Ca2+ entry induced by short prepulses from —50 mV to positive voltages caused suppression of peak ICa during a test pulse. The negative correlation between peak ICa suppression and ICa inactivation during the test pulse indicated that RDI evoked by the prepulse affected only calcium channels in those dyads in which calcium release was activated. Ca2+ ions injected during the prepulse and during the subsequent tail current suppressed peak ICa in the test pulse to a different extent. Quantitative analysis indicated that equal Ca2+ charge was 3.5 times less effective in inducing release when entering during the prepulse than when entering during the tail. Tail Ca2+ charge injected by the first voltage-dependent calcium channel (DHPR) openings was three times less effective than that injected by DHPR reopenings. These findings suggest that calcium release activation can be profoundly influenced by the recent history of L-type Ca2+ channel activity due to potentiation of ryanodine receptors (RyRs) by previous calcium influx. This conclusion was confirmed at the level of single RyRs in planar lipid bilayers: using flash photolysis of the calcium cage NP-EGTA to generate two sequential calcium stimuli, we showed that RyR activation in response to the second stimulus was four times higher than that in response to the first stimulus.

Human cardiac ryanodine receptor (hRyR2) is a channel mediating Ca2+ release from the sarcoplasmic reticulum during excitation-contraction coupling. The N-terminal (1-655) and central (2100-2500) regions of hRyR2 are thought to be involved in regulating channel gating. Mutations linked to several heart diseases are clustered within these two, as well as in the channel pore-containing C-terminal regions. High resolution structures of key regions involved in the regulation of RyR2 activity could further the understanding of the gating mechanism of hRyR2 and of its malfunction in disease.

The effect of a domain peptide DPCPVTc from the central region of the RYR2 on ryanodine receptors isolated from rat heart was examined in planar lipid bilayers. At a zero holding potential and at 100 nM cytosolic and 8 mM luminal Ca2+ concentration, DPCPVTc induced concentration-dependent activation of the ryanodine receptor that led up to 20-fold increase of open probability at saturating DPCPVTc concentrations. The effect of the peptide appeared within 30 s after addition to the experimental chamber. At all DPCPVTc concentrations RyR2 channels displayed large variability in open probability, open time and opening frequency. DPCPVTc prolonged RyR2 openings up to 8× and increased RyR2 opening frequency by up to 100%. With increasing DPCPVTc concentration, the fraction of high open probability records increased up to 5×, and their open time increased up to 4×. The closed times did not depend on DPCPVTc concentration either in low- or high-open probability records. The DPCPVTc concentration dependence of all gating parameters had EC50 of 20 µM and a Hill slope of 2. Comparison of the effects of DPCPVTc with those of ATP [1] and cytosolic Ca2+ [2] suggests that activation does not involve luminal feed-through and is not caused by modulation of the cytosolic activation A-site. The data suggest that although “domain unzipping” by DPCPVTc occurs in both modes of RyR activity, it affects RyR gating only when the channel resides in the H-mode of activity.Supported in part by the European Union Contract No. LSHM-CT-2005-018802/CONTICA and by grants APVV-LPP-0441-09, APVV-0628-10 and VEGA 2/0197/11.[1] Tencerova B, Zahradnikova A, Gaburjakova J, Gaburjakova M. J Gen Physiol 140: 93, 2012.[2] Gaburjakova J, Gaburjakova M. J Membr Biol 212: 17, 2006.

Channel adaptation is a basic property of the sarcoplasmic reticulum Ca2+-release channels/ryanodine receptors (RyRs). It allows channel activity to decay during sustained increases in the concentration of activating Ca2+. Despite the potential physiological importance of this self-confining process, its molecular mechanism is not well understood. To define the mechanism of adaptation we studied the dynamics of cardiac Ca2+-release channel (RyR) gating using the planar lipid bilayer technique in combination with photolysis of caged Ca2+ (DM-nitrophen). Channels activated by rapid and sustained increases in Ca2+ concentration (from 0.1 to 0.5 micromol/l) displayed three distinct gating modes, manifested as current records with frequent and long openings (H-mode), with rare and short openings (L-mode), and with no openings (I-mode). H-mode channel activity occurred primarily at early times while L- and I-modes predominated at late times after the rapid Ca2+ concentration increase. The decrease in probability of H-mode, mirrored by an increase in the probability of the I-mode, proceeded with a time constant similar to that observed for spontaneous decay in channel activity (i.e., adaptation) in ensemble average records. These results indicate that RyR adaptation transpires by a shift of channel gating from a high open probability mode to low open probability and inactivated modes of the channel.

1. We used the planar lipid bilayer method to study single ryanodine receptor Ca2+ release channels (RyRCs) from fast skeletal muscle of the rabbit. We found that changes in membrane voltage directly induced gating transitions of the RyRC: (i) in the steady state, even at activating Ca2+ concentrations (20 microM), at a constant membrane potential the channels resided in a low open probability (Po) state (inactivated-, I-mode), and (ii) upon abrupt changes of voltage, the apparent inactivation of the RyRCs was relieved, resulting in a rapid and transient increase in Po. 2. The magnitude of the Po increase was a function of both the duration and the amplitude of the applied prepulse, but was independent of the channel activity during the prepulse. 3. The voltage-induced Po increase probably involved major conformational changes of the channel, as it resulted in substantial alterations in the gating pattern of the channels: the voltage change-induced increase in Po was accompanied by the rapid appearance of two types of channel activity (high (H) and low (L) open probability modes). 4. The response of the RyRC to voltage changes raises the interesting possibility that the activation of RyRC in situ might involve electrical events, i.e. a possible dipole-dipole coupling between the release channel and the voltage sensor.

The kinetics of Ca2+ release from canine cerebellum and rabbit skeletal muscle microsomes, mediated by the inositol 1,4,5-trisphosphate (IP3) receptor (IRC) and the ryanodine receptor (RyRC), respectively, were analyzed by a model, which considers that Ca2+ release channels undergo spontaneous inactivation. We found that: (i) both the initial rate of release (vo) and the rate of inactivation (v1) were saturable functions of the activating ligand concentration (CL); and (ii) the ratio of vi/vo, termed the relative tendency for inactivation, decreased with increasing CL. Equilibrium [3H]-IP3 binding studies, on the other hand, revealed the presence of one single class of non-co-operative IP3 sites in cerebellum membranes (Kdeq = 47 nM and Hill coefficient = 1.1). Based on the above vi−vo relationship and the IP3-binding data, we propose that quantal Ca2+ release through IRCs might be a result of spontaneous channel inactivation, whose rate is controlled by the ratio of IP3-occupied/free monomers in the tetrameric release channel units. Furthermore, because of the kinetic similarities between the IRC- and RyRC-mediated Ca2+ release processes, as well as between quantal Ca2+ release and channel adaptation, the same mechanism is also proposed to apply to the RyRC-mediated Ca2+ release as well as to constitute the basis of release channel adaptation.

We have recently reported [Meszaros L.G., Minarovic I., Zahradnikova A. Inhibition of the skeletal muscle ryanodine receptor calcium release channel by nitric oxide. FEBS Lett 1996; 380: 49-52] that nitric oxide (NO) reduces the activity of the skeletal muscle ryanodine receptor Ca2+ release channel (RyRC), a principal component of the excitation-contraction coupling machinery in striated muscles. Since (i) as shown here, we have obtained evidence which indicates that the NO synthase (eNOS) of cardiac muscle origin co-purified with RyRC-containing sarcoplasmic reticulum (SR) fractions; and (ii) the effects of NO donors on the release channel, as well as on cardiac function, appear somewhat contradictory, we have made an attempt to investigate the response of the cardiac RyRC to NO that is generated in situ from L-arginine in the NOS reaction. We found that L-arginine-derived NO inactivates Ca2+ release from cardiac SR and reduces the steady-state activity (i.e. open probability) of single RyRCs fused into a planar lipid bilayer. This reduction was prevented by NOS inhibitors and the NO quencher hemoglobin and was reversed by 2-mercaptoethanol. We thus conclude that: (i) in isolated SR preparations, it is possible to assess the effects of NO that is generated from L-arginine in the NOS reaction; and (ii) cardiac RyRc responds to NO in a manner which is identical to that we have previously found with the skeletal channel. These findings suggest that the direct modulation of the RyRC by NO is a signaling mechanism which likely participates in earlier demonstrated NO-induced myocardial contractility changes.

ADP-ribose (ADPR) was found to decrease the rate of Ca 2+ release from isolated cardiac sarcoplasmic reticulum (SR) vesicles, which was limited to a maximum of 46 ± 8% inhibition and was in accordance with our results obtained with single cardiac ryanodine receptor Ca 2+ release channels (RyRC) incorporated into planar lipid bilayers: Out of 23 separate single channels, 9 responded to ADPR by a complete closure, while 14 channels showed no response at all, resulting in a reduction in overall open probability in the presence of ADPR (relative to control channels) by 39.7%. Although the ADPR-responsive and unresponsive single channels showed no differences in their respective open times, current amplitudes or relative occurrences of dwell levels, the bare existence of two types of response to ADPR together with the 50%-limited inhibition of cardiac SR Ca 2+ release by ADPR indicates a heterogeneity of RyRCs in cardiac SR, which is likely due to protein(s) that interact(s) with the channel and are present in substoichiometric mole ratios.

The mechanism of activation of the cardiac ryanodine receptor (RyR2) by luminal Ca2+ in the presence of ATP was examined in planar lipid bilayers. The dose response of the RyR2 channel to ATP was characterized at a range of cytosolic (100-400 nM) and luminal (0.01 - 53 mM) Ca2+ concentrations. Luminal Ca2+ markedly increased the maximal open probability in the presence of ATP (POmax) and markedly decreased EC50 for ATP (EC50ATP). Cytosolic Ca2+, without substantially activating the RyR2 channel in the absence of ATP, greatly amplified the effects of 1 mM [Ca2+]L on POmax and EC50ATP. An allosteric model of RyR2 interaction with ATP showed that the increase in RyR2 open probability by luminal Ca2+ in the presence of ATP is induced by a decrease of the ATP dissociation constant (KATP) at low [Ca2+]L, by an increase in the positive allosteric effect of ATP on channel opening (decrease of fATP) at intermediate [Ca2+]L, and by an increase in the stability of the ATP-free RyR2 open state (decrease of KO0) at the highest [Ca2+]L. Increasing [Ca2+]C did not affect KATP but led to a parallel decrease of KO0 and fATP. These results suggest that the allosteric effect of ATP might be mediated by the energy of the ATP-free open state, i.e., indirectly by the effect of the occupancy of the channel by Ca2+ at the cytosolic and luminal sites on the stability of the open state. The increase of RyR2 open probability at diastolic levels of cytosolic Ca2+ by elevated luminal Ca2+ may play a role in the calcium overload induced Ca2+ release.Supported by APVV-0721-10, APVV-0628-10, VEGA2/0118/09 and VEGA 2/0190/10

The effects of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) on individual cardiac sarcoplasmic reticulum Ca2+ release channels have been examined in planar lipid bilayers. A sudden step-like increase in open probability (Po) induced by DIDS was observed. At zero holding potential it proceeded with an apparent time constant of 84 s and Po was increased 15 times in the steady state. Independent of membrane voltage, DIDS induced a long-lived open state with tau o = 15 ms at 0 mV and tau o = 5 ms at -50 mV (cis (cytoplasmic) side negative). While at 0 mV the modified channel spent most of the time in this long-lived state, at -50 mV the occupancy of the long-lived state was very low and most openings occurred to the short and medium open state, leading to deactivation of the channel to only 2-3 times of control. Voltage-induced changes in the activity of modified channels were reversible and proceeded with time constants of 14 s for deactivation and 12 s for activation, that is, faster than onset of activation by DIDS. As no changes in single channel conductance of the channel were observed in the presence of DIDS and its abrupt activating action could not be removed, a covalent modification of an amino group residing in the gating structure of the channel by DIDS can be considered.

The effects of the Ca(2+)-induced Ca2+ release blocker procaine on individual sarcoplasmic reticulum Ca2+ release channels have been examined in planar lipid bilayers. Procaine did not reduce the single channel conductance nor appreciably shorten the mean open times of the channel; rather, it increased the longest closed time. These results indicated that procaine interacted selectively with a closed state of the channel rather than with an open state. Gating of the sarcoplasmic reticulum Ca2+ release channel was described by a modified scheme of Ashley and Williams (1990. J. Gen. Physiol. 95:981–1005), including an additional long-lived closed state. Computer simulations determined that procaine was more likely to interact with this long-lived Ca(2+)-bound closed state of the channel rather than with other states of the channel. Simulations with the same model were also able to reproduce a prominent Ca(2+)-sensitive transition between "random" and "bursting" forms of gating of the channel, variations of which may account for "gearshift" behavior reported in studies with this and other single channels.

The planar lipid bilayer and vesicle release experiments are two alternative approaches used to study the function of the ryanodine receptor (RyR) channel at the subcellular level. In this work, we combine models of gating (Zahradnikova and Zahradnik, Biophys. J. 71 (1996) 2996–3012) and permeation (Tinker et al., J. Gen. Physiol. 100 (1992) 495–517) of the cardiac RyR channel to simulate calcium release experiments on sarcoplasmic reticulum vesicles. The resulting model and real experimental data agreed well within the experimental scatter, confirming indistinguishable properties of the RyRC in the vesicle preparation and in the planar lipid bilayer. The previously observed differences in calcium dependencies of the release and the gating processes can be explained by binding of calcium within the RyRC conducting pore. A novel method of analysis of calcium dependence of calcium release was developed and tested. Three gating models of the RyRC, showing, respectively, an increase, no change, and a decrease in calcium sensitivity over time, were compared. The described method of analysis enabled determination of temporal changes in calcium sensitivity, giving potential for detection of the adaptation/inactivation phenomena of the RyRC in both vesicle and in situ release experiments.

Integral calcium release fluxes in mammalian cardiac myocytes increase in amplitude not only due to the increased recruitment of individual release sites but also due to their increased synchronization (1). Here we characterize the voltage dependence of synchronization of individual release sites at high temporal resolution. Calcium spikes and calcium currents elicited in isolated rat ventricular myocytes (2) by 80 ms voltage pulses from −50 to −40 - +50 mV were recorded using Leica TCS AOBS confocal microscope (2 kHz line scan mode) and Axopatch 200B patch-clamp amplifier, respectively. Calcium spikes were identified and analyzed by a new Matlab-based software that highly increased the productivity and reliability of approximation of calcium spikes by the kinetic model of local calcium release (2). Calcium spikes were described by their amplitude, onset latency, time-to-peak, FDHM, time constants of activation and termination, and fractional probability of activation. The synchrony of calcium spikes increased with depolarization and could be fully explained by the voltage dependence of their onset latency that reached a lower limit of about 4 ms at depolarizations between 0 and 40 mV. The rate of calcium spike activation after its onset was independent of the applied voltage or of calcium current amplitude, while the probability of calcium spike activation was related to the voltage dependence of the integral of calcium current. We conclude that both the synchrony of local calcium release activation and recruitment of individual release sites are fully controlled by the activation probability of DHPR calcium channels and by the DHPR-RYR coupling fidelity.Supported by APVV-0721-10, VEGA 2/0203/11, VEGA 2/0917/11.1. Song LS, Sham JS, Stern MD, Lakatta EG, Cheng H. J Physiol. 512:677-91, 1998.2. Zahradnikova A Jr, Polakova E, Zahradnik I, Zahradnikova A. J Physiol. 578:677-91, 2007.

Calcium release units (CRUs) in cardiac myocytes contain a large number of ryanodine receptors (RyRs), but it seems that only a small fraction of RyRs is being activated during a single calcium release event. Repetitive activity of CRUs is not well understood from the point of refractoriness. We used direct measurement of Ca-spikes activated by calcium current (ICa) to unveil the refractoriness of CRU firing from the properties and occurrence of calcium spikes under control conditions and in the presence of the DHPR activator FPL.ICa was activated by voltage pulses from 50 to 0 mV under whole-cell patch-clamp. Ca-spikes were measured by confocal microscopy using Ca2+ indicators (Fluo-3 and Oregon Green BAPTA-5N) in the presence of EGTA. A large majority of CRUs responded to stimulation by a single Ca-spike, while 13.4 % produced two subsequent (twin) Ca-spikes. Early Ca-spikes (single spikes and the first of twin Ca-spikes) had similar latencies, while the second Ca-spikes were significantly delayed. Amplitude distribution of the early Ca-spikes consisted of four quantal levels with equal amplitudes and binomially distributed frequency of occurrence, supporting the hypothesis that different Ca-spike amplitudes are due to a different number of independent quantal levels, likely individual RYR openings. The probability of occurrence of second Ca-spikes was inversely proportional to the quantal size of the early Ca-spike. In the presence of FPL, the probability of occurrence of twin spikes was decreased relative to control, despite persistent activation of DHPR receptors.We conclude that refractoriness of local calcium release does not result from Ca-dependent DHPR inactivation, but rather it is caused by inactivation of the release unit due to local depletion of the SR.Supported by APVV-0721-10, APVV-0441-09, VEGA 2/0203/11, VEGA 2/0917/11, and VEGA 2/0190/10.