Multiscale modeling of calcium cycling in cardiac ventricular myocyte: macroscopic consequences of microscopic dyadic function

In cardiac ventricular myocytes, calcium (Ca) release occurs at distinct structures (dyads) along t-tubules, where L-type Ca channels (LCCs) appose sarcoplasmic reticulum (SR) Ca release channels (RyR2s). We developed a model of the cardiac ventricular myocyte that simulates local stochastic Ca release processes. At the local Ca release level, the model reproduces Ca spark properties. At the whole-cell level, the model reproduces the action potential, Ca currents, and Ca transients. Changes in microscopic dyadic properties (e.g., during detubulation in heart failure) affect whole-cell behavior in complex ways, which we investigated by simulating changes in the dyadic volume and number of LCCs/RyR2s in the dyad, and effects of calsequestrin (CSQN) as a Ca buffer (CSQN buffer) or a luminal Ca sensor (CSQN regulator). We obtained the following results: 1), Increased dyadic volume and reduced LCCs/RyR2s decrease excitation-contraction coupling gain and cause asynchrony of SR Ca release, and interdyad coupling partially compensates for the reduced synchrony. 2), Impaired CSQN buffer depresses Ca transients without affecting the synchrony of SR Ca release. 3), When CSQN regulator function is impaired, interdyad coupling augments diastolic Ca release activity to form Ca waves and long-lasting Ca release events.     

Dependency of Calcium Alternans on Ryanodine Receptor Refractoriness

Background

Rapid pacing rates induce alternations in the cytosolic calcium concentration caused by fluctuations in calcium released from the sarcoplasmic reticulum (SR). However, the relationship between calcium alternans and refractoriness of the SR calcium release channel (RyR2) remains elusive.

Methodology/Principal Findings

To investigate how ryanodine receptor (RyR2) refractoriness modulates calcium handling on a beat-to-beat basis using a numerical rabbit cardiomyocyte model. We used a mathematical rabbit cardiomyocyte model to study the beat-to-beat calcium response as a function of RyR2 activation and inactivation. Bi-dimensional maps were constructed depicting the beat-to-beat response. When alternans was observed, a novel numerical clamping protocol was used to determine whether alternans was caused by oscillations in SR calcium loading or by RyR2 refractoriness. Using this protocol, we identified regions of RyR2 gating parameters where SR calcium loading or RyR2 refractoriness underlie the induction of calcium alternans, and we found that at the onset of alternans both mechanisms contribute. At low inactivation rates of the RyR2, calcium alternans was caused by alternation in SR calcium loading, while at low activation rates it was caused by alternation in the level of available RyR2s.

Conclusions/Significance

We have mapped cardiomyocyte beat-to-beat responses as a function of RyR2 activation and inactivation, identifying domains where SR calcium load or RyR2 refractoriness underlie the induction of calcium alternans. A corollary of this work is that RyR2 refractoriness due to slow recovery from inactivation can be the cause of calcium alternans even when alternation in SR calcium load is present.     

Calsequestrin mediates changes in spontaneous calcium release profiles

Calsequestrin (CSQ) is the primary calcium buffer within the sarcoplasmic reticulum (SR) of cardiac cells. It has also been identified as a regulator of Ryanodine receptor (RyR) calcium release channels by serving as a SR luminal sensor. When calsequestrin is free and unbound to calcium, it can bind to RyR and desensitize the channel from cytoplasmic calcium activation. In this paper, we study the role of CSQ as a buffer and RyR luminal sensor using a mechanistic model of RyR-CSQ interaction. By using various asymptotic approximations and mean first exit time calculation, we derive a minimal model of a calcium release unit which includes CSQ dependence. Using this model, we then analyze the effect of changing CSQ expression on the calcium release profile and the rate of spontaneous calcium release. We show that because of its buffering capability, increasing CSQ increases the spark duration and size. However, because of luminal sensing effects, increasing CSQ depresses the basal spark rate and increases the critical SR level for calcium release termination. Finally, we show that with increased bulk cytoplasmic calcium concentration, the CRU model exhibits deterministic oscillations.     

Alternans of cardiac calcium cycling in a cluster of ryanodine receptors: a simulation study

Mechanical alternans in cardiac muscle is associated with intracellular Ca(2+) alternans. Mechanisms underlying intracellular Ca(2+) alternans are unclear. In previous experimental studies, we produced alternans of systolic Ca(2+) under voltage clamp, either by partially inhibiting the Ca(2+) release mechanism, or by applying small depolarizing pulses. In each case, alternans relied on propagating waves of Ca(2+) release. The aim of this study is to investigate by computer modeling how alternans of systolic Ca(2+) is produced. A mathematical model of a cardiac cell with 75 coupled elements is developed, with each element contains L-type Ca(2+) current, a subspace into which Ca release takes place, a cytoplasmic space, sarcoplasmic reticulum (SR) release channels [ryanodine receptor (RyR)], and uptake sites (SERCA). Interelement coupling is via Ca(2+) diffusion between neighboring subspaces via cytoplasmic spaces and network SR spaces. Small depolarizing pulses were simulated by step changes of cell membrane potential (20 mV) with random block of L-type channels. Partial inhibition of the release mechanism is mimicked by applying a reduction of RyR open probability in response to full stimulation by L-type channels. In both cases, systolic alternans follow, consistent with our experimental observations, being generated by propagating waves of Ca(2+) release and sustained through alternation of SR Ca(2+) content. This study provides novel and fundamental insights to understand mechanisms that may underlie intracellular Ca(2+) alternans without the need for refractoriness of L-type Ca or RyR channels under rapid pacing.     

Mice null for calsequestrin 1 exhibit deficits in functional performance and sarcoplasmic reticulum calcium handling

In skeletal muscle, the release of calcium (Ca(2+)) by ryanodine sensitive sarcoplasmic reticulum (SR) Ca(2+) release channels (i.e., ryanodine receptors; RyR1s) is the primary determinant of contractile filament activation. Much attention has been focused on calsequestrin (CASQ1) and its role in SR Ca(2+) buffering as well as its potential for modulating RyR1, the L-type Ca(2+) channel (dihydropyridine receptor, DHPR) and other sarcolemmal channels through sensing luminal [Ca(2+)]. The genetic ablation of CASQ1 expression results in significant alterations in SR Ca(2+) content and SR Ca(2+) release especially during prolonged activation. While these findings predict a significant loss-of-function phenotype in vivo, little information on functional status of CASQ1 null mice is available. We examined fast muscle in vivo and in vitro and identified significant deficits in functional performance that indicate an inability to sustain contractile activation. In single CASQ1 null skeletal myofibers we demonstrate a decrease in voltage dependent RyR Ca(2+) release with single action potentials and a collapse of the Ca(2+) release with repetitive trains. Under voltage clamp, SR Ca(2+) release flux and total SR Ca(2+) release are significantly reduced in CASQ1 null myofibers. The decrease in peak Ca(2+) release flux appears to be solely due to elimination of the slowly decaying component of SR Ca(2+) release, whereas the rapidly decaying component of SR Ca(2+) release is not altered in either amplitude or time course in CASQ1 null fibers. Finally, intra-SR [Ca(2+)] during ligand and voltage activation of RyR1 revealed a significant decrease in the SR[Ca(2+)](free) in intact CASQ1 null fibers and a increase in the release and uptake kinetics consistent with a depletion of intra-SR Ca(2+) buffering capacity. Taken together we have revealed that the genetic ablation of CASQ1 expression results in significant functional deficits consistent with a decrease in the slowly decaying component of SR Ca(2+) release.     

Superresolution Modeling of Calcium Release in the Heart

Stable calcium-induced calcium release (CICR) is critical for maintaining normal cellular contraction during cardiac excitation-contraction coupling. The fundamental element of CICR in the heart is the calcium (Ca²⁺) spark, which arises from a cluster of ryanodine receptors (RyR). Opening of these RyR clusters is triggered to produce a local, regenerative release of Ca²⁺ from the sarcoplasmic reticulum (SR). The Ca²⁺ leak out of the SR is an important process for cellular Ca²⁺ management, and it is critically influenced by spark fidelity, i.e., the probability that a spontaneous RyR opening triggers a Ca²⁺ spark. Here, we present a detailed, three-dimensional model of a cardiac Ca²⁺ release unit that incorporates diffusion, intracellular buffering systems, and stochastically gated ion channels. The model exhibits realistic Ca²⁺ sparks and robust Ca²⁺ spark termination across a wide range of geometries and conditions. Furthermore, the model captures the details of Ca²⁺ spark and nonspark-based SR Ca²⁺ leak, and it produces normal excitation-contraction coupling gain. We show that SR luminal Ca²⁺-dependent regulation of the RyR is not critical for spark termination, but it can explain the exponential rise in the SR Ca²⁺ leak-load relationship demonstrated in previous experimental work. Perturbations to subspace dimensions, which have been observed in experimental models of disease, strongly alter Ca²⁺ spark dynamics. In addition, we find that the structure of RyR clusters also influences Ca²⁺ release properties due to variations in inter-RyR coupling via local subspace Ca²⁺ concentration ([Ca²⁺]_ss). These results are illustrated for RyR clusters based on super-resolution stimulated emission depletion microscopy. Finally, we present a believed-novel approach by which the spark fidelity of a RyR cluster can be predicted from structural information of the cluster using the maximum eigenvalue of its adjacency matrix. These results provide critical insights into CICR dynamics in heart, under normal and pathological conditions.     

Altered function and regulation of cardiac ryanodine receptors in cardiac disease

In cardiac muscle, the ryanodine receptor (RyR2) on the sarcoplasmic reticulum (SR) releases the calcium required for muscle contraction. The magnitude of Ca²⁺ release by RyR2, which is subject to regulation by several physiological mediators, determines cardiac contractility. In heart failure, chronic stimulation of the β-adrenergic signaling pathway leads to hyperphosphorylation of RyR2 by protein kinase A, which dissociates calstabin2 (FKBP12.6) from the receptor. Calstabin2-depleted channels display altered channel gating and can cause diastolic Ca²⁺ release from the SR. This release depletes the SR Ca²⁺ stores, leading to reduced myocardial contractility. Mutant RyR2, found in patients with catecholaminergic polymorphic ventricular tachycardia, has decreased calstabin2 binding affinity, which can trigger ventricular arrhythmias and sudden cardiac death after stress and exercise. Thus, defects in RyR2 have been linked to heart failure and exercise-induced sudden cardiac death and might provide novel therapeutic targets for the treatment of these common diseases of the heart.     

The role of calsequestrin, triadin, and junctin in conferring cardiac ryanodine receptor responsiveness to luminal calcium

The level of Ca inside the sarcoplasmic reticulum (SR) is an important determinant of functional activity of the Ca release channel/ryanodine receptor (RyR) in cardiac muscle. However, the molecular basis of RyR regulation by luminal Ca remains largely unknown. In the present study, we investigated the potential role of the cardiac SR luminal auxiliary proteins calsequestrin (CSQ), triadin 1, and junctin in forming the luminal calcium sensor for the cardiac RyR. Recordings of single RyR channels incorporated into lipid bilayers, from either SR vesicle or purified RyR preparations, were performed in the presence of MgATP using Cs+ as the charge carrier. Raising luminal [Ca] from 20 microM to 5 mM increased the open channel probability (Po) of native RyRs in SR vesicles, but not of purified RyRs. Adding CSQ to the luminal side of the purified channels produced no significant changes in Po, nor did it restore the ability of RyRs to respond to luminal Ca. When triadin 1 and junctin were added to the luminal side of purified channels, RyR Po increased significantly; however, the channels still remained unresponsive to changes in luminal [Ca]. In RyRs reassociated with triadin 1 and junctin, adding luminal CSQ produced a significant decrease in activity. After reassociation with all three proteins, RyRs responded to rises of luminal [Ca] by increasing their Po. These results suggest that a complex of CSQ, triadin 1, and junctin confer RyR luminal Ca sensitivity. CSQ apparently serves as a luminal Ca sensor that inhibits the channel at low luminal [Ca], whereas triadin 1 and/or junctin may be required to mediate interactions of CSQ with RyR.     

Sarcoplasmic Reticulum Structure and Functional Properties that Promote Long-Lasting Calcium Sparks

Calcium (Ca) sparks are the fundamental sarcoplasmic reticulum (SR) Ca release events in cardiac myocytes, and they have a typical duration of 20–40 ms. However, when a fraction of ryanodine receptors (RyRs) are blocked by tetracaine or ruthenium red, Ca sparks lasting hundreds of milliseconds have been observed experimentally. The fundamental mechanism underlying these extremely prolonged Ca sparks is not understood. In this study, we use a physiologically detailed mathematical model of subcellular Ca cycling to examine how Ca spark duration is influenced by the number of functional RyRs in a junctional cluster (which is reduced by tetracaine or ruthenium red) and other SR Ca handling properties. One RyR cluster contains a few to several hundred RyRs, and we use a four-state Markov RyR gating model. Each RyR opens stochastically and is regulated by cytosolic and luminal Ca. We varied the number of functional RyRs in the single cluster, diffusion within the SR network, diffusion between network and junctional SR, cytosolic Ca diffusion, SERCA uptake activity, and RyR open probability. For long-lasting Ca release events, opening events within the cluster must occur continuously because the typical open time of the RyR is only a few milliseconds. We found the following: 1) if the number of RyRs is too small, it is difficult to maintain consecutive openings and stochastic attrition terminates the release; 2) if the number of RyRs is too large, the depletion of Ca from the junctional SR terminates the release; and 3) very long release events require relatively small-sized RyR clusters (reducing flux as seen experimentally with tetracaine) and sufficiently rapid intra-SR Ca diffusion, such that local junctional intra-SR [Ca] can be maintained by intra-SR diffusion and overall SR Ca reuptake.     

Ryanodine Receptor Luminal Ca Regulation: Swapping Calsequestrin and Channel Isoforms

Sarcoplasmic reticulum (SR) Ca²⁺ release in striated muscle is mediated by a multiprotein complex that includes the ryanodine receptor (RyR) Ca²⁺ channel and the intra-SR Ca²⁺ buffering protein calsequestrin (CSQ). Besides its buffering role, CSQ is thought to regulate RyR channel function. Here, CSQ-dependent luminal Ca²⁺ regulation of skeletal (RyR1) and cardiac (RyR2) channels is explored. Skeletal (CSQ1) or cardiac (CSQ2) calsequestrin were systematically added to the luminal side of single RyR1 or RyR2 channels. The luminal Ca²⁺ dependence of open probability (Po) over the physiologically relevant range (0.05-1 mM Ca²⁺) was defined for each of the four RyR/CSQ isoform pairings. We found that the luminal Ca²⁺ sensitivity of single RyR2 channels was substantial when either CSQ isoform was present. In contrast, no significant luminal Ca²⁺ sensitivity of single RyR1 channels was detected in the presence of either CSQ isoform. We conclude that CSQ-dependent luminal Ca²⁺ regulation of single RyR2 channels lacks CSQ isoform specificity, and that CSQ-dependent luminal Ca²⁺ regulation in skeletal muscle likely plays a relatively minor (if any) role in regulating the RyR1 channel activity, indicating that the chief role of CSQ1 in this tissue is as an intra-SR Ca²⁺ buffer.     

Recruiting RyRs to Open in a Ca2+ Release Unit: Single-RyR Gating Properties Make RyR Group Dynamics

In cardiac myocytes, clusters of type-2 ryanodine receptors (RyR2s) release Ca²⁺ from the sarcoplasmic reticulum (SR) via a positive feedback mechanism in which fluxed Ca²⁺ activates nearby RyRs. Although the general principles of this are understood, less is known about how single-RyR gating properties define the RyR group dynamics in an array of many channels. Here, we examine this using simulations with three models of RyR gating that have identical open probabilities: the commonly used two-state Markov gating model, one that utilizes multiple exponentials to fit single-channel open time (OT) and closed time (CT) distributions, and an extension of this multiexponential model that also includes experimentally measured correlations between single-channel OTs and CTs. The simulations of RyR clusters that utilize the multiexponential gating model produce infrequent Ca²⁺ release events with relatively few open RyRs. Ca²⁺ release events become even smaller when OT/CT correlations are included. This occurs because the correlations produce a small but consistent bias against recruiting more RyRs to open during the middle of a Ca²⁺ release event, between the initiation and termination phases (which are unaltered compared to the uncorrelated simulations). In comparison, the two-state model produces frequent, large, and long Ca²⁺ release events because it had a recruitment bias in favor of opening more RyRs. This difference stems from the two-state model’s single-RyR OT and CT distributions being qualitatively different from the experimental ones. Thus, the details of single-RyR gating can profoundly affect SR Ca²⁺ release even if open probability and mean OTs and CTs are identical. We also show that Ca²⁺ release events can terminate spontaneously without any reduction in SR [Ca²⁺], luminal regulation, Ca²⁺-dependent inactivation, or physical coupling between RyRs when Ca²⁺ flux is below a threshold value. This supports and extends the pernicious attrition/induction decay hypothesis that SR Ca²⁺ release events terminate below a threshold Ca²⁺ flux.     

Life and death of a cardiac calcium spark

Calcium sparks in cardiac myocytes are brief, localized calcium releases from the sarcoplasmic reticulum (SR) believed to be caused by locally regenerative calcium-induced calcium release (CICR) via couplons, clusters of ryanodine receptors (RyRs). How such regeneration is terminated is uncertain. We performed numerical simulations of an idealized stochastic model of spark production, assuming a RyR gating scheme with only two states (open and closed). Local depletion of calcium in the SR was inevitable during a spark, and this could terminate sparks by interrupting CICR, with or without assumed modulation of RyR gating by SR lumenal calcium. Spark termination by local SR depletion was not robust: under some conditions, sparks could be greatly and variably prolonged, terminating by stochastic attrition–a phenomenon we dub “spark metastability.” Spark fluorescence rise time was not a good surrogate for the duration of calcium release. Using a highly simplified, deterministic model of the dynamics of a couplon, we show that spark metastability depends on the kinetic relationship of RyR gating and junctional SR refilling rates. The conditions for spark metastability resemble those produced by known mutations of RyR2 and CASQ2 that cause life-threatening triggered arrhythmias, and spark metastability may be mitigated by altering the kinetics of the RyR in a manner similar to the effects of drugs known to prevent those arrhythmias. The model was unable to explain the distributions of spark amplitudes and rise times seen in chemically skinned cat atrial myocytes, suggesting that such sparks may be more complex events involving heterogeneity of couplons or local propagation among sub-clusters of RyRs.     

Activation of cardiac ryanodine receptors by cardiac glycosides

This study investigated the effects of cardiac glycosides on single-channel activity of the cardiac sarcoplasmic reticulum (SR) Ca2+ release channels or ryanodine receptor (RyR2) channels and how this action might contribute to their inotropic and/or toxic actions. Heavy SR vesicles isolated from canine left ventricle were fused with artificial planar lipid bilayers to measure single RyR2 channel activity. Digoxin and actodigin increased single-channel activity at low concentrations normally associated with therapeutic plasma levels, yielding a 50% of maximal effect of approximately 0.2 nM for each agent. Channel activation by glycosides did not require MgATP and occurred only when digoxin was applied to the cytoplasmic side of the channel. Similar results were obtained in human RyR2 channels; however, neither the crude skeletal nor the purified cardiac channel was activated by glycosides. Channel activation was dependent on [Ca2+] on the luminal side of the bilayer with maximal stimulation occurring between 0.3 and 10 mM. Rat RyR2 channels were activated by digoxin only at 1 microM, consistent with the lower sensitivity to glycosides in rat heart. These results suggest a model in which RyR2 channel activation by digoxin occurs only when luminal [Ca2+] was increased above 300 microM (in the physiological range). Consequently, increasing SR load (by Na+ pump inhibition) serves to amplify SR release by promoting direct RyR2 channel activation via a luminal Ca2+-sensitive mechanism. This high-affinity effect of glycosides could contribute to increased SR Ca2+ release and might play a role in the inotropic and/or toxic actions of glycosides in vivo.     

Modulation of SR Ca release by luminal Ca and calsequestrin in cardiac myocytes: effects of CASQ2 mutations linked to sudden cardiac death

Cardiac calsequestrin (CASQ2) is an intrasarcoplasmic reticulum (SR) low-affinity Ca-binding protein, with mutations that are associated with catecholamine-induced polymorphic ventricular tachycardia (CPVT). To better understand how CASQ2 mutants cause CPVT, we expressed two CPVT-linked CASQ2 mutants, a truncated protein (at G112+5X, CASQ2^DEL) or CASQ2 containing a point mutation (CASQ2^R33Q), in canine ventricular myocytes and assessed their effects on Ca handling. We also measured CASQ2-CASQ2 variant interactions using fluorescence resonance transfer in a heterologous expression system, and evaluated CASQ2 interaction with triadin. We found that expression of CASQ2^DEL or CASQ2^R33Q altered myocyte Ca signaling through two different mechanisms. Overexpressing CASQ2^DEL disrupted the CASQ2 polymerization required for high capacity Ca binding, whereas CASQ2^R33Q compromised the ability of CASQ2 to control ryanodine receptor (RyR2) channel activity. Despite profound differences in SR Ca buffering strengths, local Ca release terminated at the same free luminal [Ca] in control cells, cells overexpressing wild-type CASQ2 and CASQ2^DEL-expressing myocytes, suggesting that a decline in [Ca]_SR is a signal for RyR2 closure. Importantly, disrupting interactions between the RyR2 channel and CASQ2 by expressing CASQ2^R33Q markedly lowered the [Ca]_SR threshold for Ca release termination. We conclude that CASQ2 in the SR determines the magnitude and duration of Ca release from each SR terminal by providing both a local source of releasable Ca and by effects on luminal Ca-dependent RyR2 gating. Furthermore, two CPVT-inducing CASQ2 mutations, which cause mechanistically different defects in CASQ2 and RyR2 function, lead to increased diastolic SR Ca release events and exhibit a similar CPVT disease phenotype.     

Model of intracellular calcium cycling in ventricular myocytes

We present a mathematical model of calcium cycling that takes into account the spatially localized nature of release events that correspond to experimentally observed calcium sparks. This model naturally incorporates graded release by making the rate at which calcium sparks are recruited proportional to the whole cell L-type calcium current, with the total release of calcium from the sarcoplasmic reticulum (SR) being just the sum of local releases. The dynamics of calcium cycling is studied by pacing the model with a clamped action potential waveform. Experimentally observed calcium alternans are obtained at high pacing rates. The results show that the underlying mechanism for this phenomenon is a steep nonlinear dependence of the calcium released from the SR on the diastolic SR calcium concentration (SR load) and/or the diastolic calcium level in the cytosol, where the dependence on diastolic calcium is due to calcium-induced inactivation of the L-type calcium current. In addition, the results reveal that the calcium dynamics can become chaotic even though the voltage pacing is periodic. We reduce the equations of the model to a two-dimensional discrete map that relates the SR and cytosolic concentrations at one beat and the previous beat. From this map, we obtain a condition for the onset of calcium alternans in terms of the slopes of the release-versus-SR load and release-versus-diastolic-calcium curves. From an analysis of this map, we also obtain an understanding of the origin of chaotic dynamics.     

CaMKIIδC slows [Ca]i decline in cardiac myocytes by promoting Ca sparks

Acute activation of calcium/calmodulin-dependent protein kinase (CaMKII) in permeabilized phospholamban knockout (PLN-KO) mouse myocytes phosphorylates ryanodine receptors (RyRs) and activates spontaneous local sarcoplasmic reticulum (SR) Ca release events (Ca sparks) even at constant SR Ca load. To assess how CaMKII regulates SR Ca release in intact myocytes (independent of SR Ca content changes or PLN effects), we compared Ca sparks in PLN-KO versus mice, which also have transgenic cardiac overexpression of CaMKIIδC in the PLN-KO background (KO/TG). Compared with PLN-KO mice, these KO/TG cardiomyocytes exhibited 1), increased twitch Ca transient and fractional release (both by ～35%), but unaltered SR Ca load; 2), increased resting Ca spark frequency (300%) despite a lower diastolic [Ca]i, which also slowed twitch [Ca]i decline (suggesting CaMKII-dependent RyR Ca sensitization); 3), elevated Ca spark amplitude and rate of Ca release (which might indicate that more RyR channels participate in a single spark); 4), prolonged Ca spark rise time (which implies that CaMKII either delays RyR closure or prolongs the time when openings can occur); 5), more frequent repetitive sparks at single release sites. Analysis of repetitive sparks from individual Ca release sites indicates that CaMKII enhanced RyR Ca sensitivity, but did not change the time course of SR Ca refilling. These results demonstrate that there are dramatic CaMKII-mediated effects on RyR Ca release that occur via regulation of both RyR activation and termination processes.     

调控剂的电子传递性质对骨骼肌型钙释放通道的调控效应研究

肌质网(sarcoplasmic reticulum,SR)中的钙释放通道利阿诺定受体(ryanodine receptor,RyR)是调控胞浆钙离子浓度的重要蛋白,其活性受多种调控剂影响．调控剂的不同电子传递性质可能作用于RyR的功能性巯基,进而影响其门控状态．了解具有不同电子传递性质的调控剂影响钙通道的作用机制具有重要意义．本研究采用光子相关光谱法(PCS)、CPM(7-二乙 基-3-(4′-马来酰亚胺苯基)4-甲基香豆素)荧光标记法及[³H]-ryanodine结合等实验,分别检测多种调控剂对RyR1的蛋白及复合体粒度分布、自由巯基量及对通道状态的影响,利用光漂白法检测各调控剂的电子传递性质．结果显示,激活剂和巯基氧化剂具有类似电子受体的性质并产生相似作用,即自由蛋白粒度增加,自由巯基量减少,具有激活通道作用;抑制剂和巯基还原剂则具有类似电子供体的性质,作用效果相反．     

Luminal Ca2+-regulated Mg2+ inhibition of skeletal RyRs reconstituted as isolated channels or coupled clusters

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.     

Electron-conformational model of ryanodine receptor lattice dynamics

We propose a simple, physically reasonable electron-conformational model for the ryanodine receptor (RyR) and, on that basis, present a theory to describe RyR lattice responses to L-type channel triggering as an induced non-equilibrium phase transition. Each RyR is modelled with a single open and a single closed (electronic) state only, described utilizing a s=12 pseudospin approach. In addition to the fast electronic degree of freedom, the RyR channel is characterized by a slow classical conformational coordinate, Q, which specifies the RyR channel calcium conductance and provides a multimodal continuum of possible RyR states. The cooperativity in the RyR lattice is assumed to be determined by inter-channel conformational coupling. Given a threshold sarcoplasmic reticulum (SR) calcium load, the RyR lattice fires due to a nucleation process with a step-by-step domino-like opening of a fraction of lattice channels, providing for a sufficient release to generate calcium sparks. The optimal mode of RyR lattice functioning during calcium-induced calcium release implies a fractional release with a robust termination due to a decrease in SR calcium load, accompanied by a respective change in effective conformational strain of the lattice. SR calcium overload is shown to result in excitation of RyR lattice auto-oscillations with spontaneous RyR channel opening and closure.     

Coupled gating between individual cardiac ryanodine calcium release channels

In order to study interactions between ryanodine receptor calcium release (RyR2) channels during excitation-contraction coupling in cardiac muscle, we used bilayer lipid membrane (BLM) and improved the method of cardiac sarcoplasmic vesicle fusion into BLM. We increased fusion gradient for the vesicles, used chloride ions for fusion up to concentration of 1.2 mol/l and fused the vesicles by adding them directly to the forming BLM. Under these conditions, increased probability of fusion of vesicles containing 2-7 ryanodine channels into BLM was observed. Interestingly about 10% of the channels did not gate into BLM independently, but their gating was coupled. At 53 mmol/l calcium solution, two coupled gating channels had double conductance (191 +/- 15 pS) in comparison with the noncoupled channels (93 +/- 10 pS). Activities of the coupled channels were decreased by 5 micromol/l ryanodine and inhibited by 10 micromol/l ruthenium red similarly as single RyR2 channels. We suppose that cardiac sarcoplasmic vesicles contain single as well as coupled RyR2 channels.