Invited review: significance of spatial and temporal heterogeneity of calcium transients in smooth muscle.

The multiplicity of mechanisms involved in regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) in smooth muscle results in both intra- and intercellular heterogeneities in [Ca(2+)](i). Heterogeneity in [Ca(2+)](i) regulation is reflected by the presence of spontaneous, localized [Ca(2+)](i) transients (Ca(2+) sparks) representing Ca(2+) release through ryanodine receptor (RyR) channels. Ca(2+) sparks display variable spatial Ca(2+) distributions with every occurrence within and across cellular regions. Individual sparks are often grouped, and fusion of sparks produces large local elevations in [Ca(2+)](i) that occasionally trigger propagating [Ca(2+)](i) waves. Ca(2+) sparks may modulate membrane potential and thus smooth muscle contractility. Sparks may also be the target of other regulatory factors in smooth muscle. Agonists induce propagating [Ca(2+)](i) oscillations that originate from foci with high spark incidence and also represent Ca(2+) release through RyR channels. With increasing agonist concentration, the peak of regional [Ca(2+)](i) oscillations remains relatively constant, whereas both frequency and propagation velocity increase. In contrast, the global cellular response appears as a concentration-dependent increase in peak as well as mean cellular [Ca(2+)](i), representing a spatial and temporal integration of the oscillations. The significance of agonist-induced [Ca(2+)](i) oscillations lies in the establishment of a global [Ca(2+)](i) level for slower Ca(2+)-dependent physiological processes.     

Caffeine-induced Ca(2+) sparks in mouse ventricular myocytes

Ca(2+) sparks are spatially localized intracellular Ca(2+) release events that were first described in 1993. Sparks have been ascribed to sarcoplasmic reticulum Ca(2+) release channel (ryanodine receptor, RyR) opening induced by Ca(2+) influx via L-type Ca(2+) channels or by spontaneous RyR openings and have been thought to reflect Ca(2+) release from a cluster of RyR. Here we describe a pharmacological approach to study sparks by exposing ventricular myocytes to caffeine with a rapid solution-switcher device. Sparks under these conditions have properties similar to naturally occurring sparks in terms of size and intracellular Ca(2+) concentration ([Ca(2+)](i)) amplitude. However, after the diffusion of caffeine, sparks first appear close to the cell surface membrane before coalescing to produce a whole cell transient. Our results support the idea that a whole cell [Ca(2+)](i) transient consists of the summation of sparks and that Ca(2+) sparks consist of the opening of a cluster of RyR and confirm that characteristics of the cluster rather than the L-type Ca(2+) channel-RyR relation determine spark properties.     

Calcium-induced calcium release in smooth muscle: loose coupling between the action potential and calcium release

Calcium-induced calcium release (CICR) has been observed in cardiac myocytes as elementary calcium release events (calcium sparks) associated with the opening of L-type Ca(2+) channels. In heart cells, a tight coupling between the gating of single L-type Ca(2+) channels and ryanodine receptors (RYRs) underlies calcium release. Here we demonstrate that L-type Ca(2+) channels activate RYRs to produce CICR in smooth muscle cells in the form of Ca(2+) sparks and propagated Ca(2+) waves. However, unlike CICR in cardiac muscle, RYR channel opening is not tightly linked to the gating of L-type Ca(2+) channels. L-type Ca(2+) channels can open without triggering Ca(2+) sparks and triggered Ca(2+) sparks are often observed after channel closure. CICR is a function of the net flux of Ca(2+) ions into the cytosol, rather than the single channel amplitude of L-type Ca(2+) channels. Moreover, unlike CICR in striated muscle, calcium release is completely eliminated by cytosolic calcium buffering. Thus, L-type Ca(2+) channels are loosely coupled to RYR through an increase in global [Ca(2+)] due to an increase in the effective distance between L-type Ca(2+) channels and RYR, resulting in an uncoupling of the obligate relationship that exists in striated muscle between the action potential and calcium release.     

Altered Ca2+ sparks and gating properties of ryanodine receptors in aging cardiomyocytes

To investigate the cellular mechanisms for altered cardiac function in senescence, we measured Ca(2+) transients and Ca(2+) sparks in ventricular cardiomyocytes from 6- to 24-month-old Fisher 344 (F344) rat hearts. The single channel properties of ryanodine receptors from adult and senescent hearts were also studied. In senescent myocytes, we observed a decreased peak [Ca(2+)](i) amplitude and an increased time constant for decay (tau), both of which correlated with a reduced Ca(2+) content of the sarcoplasmic reticulum (SR). Our studies also revealed that senescent cardiomyocytes had an increased frequency of Ca(2+) sparks and a slight but statistically significant decrease in average amplitude, full-width-at-half-maximum (FWHM) and full-duration-at-half-maximum (FDHM). Single channel recordings of ryanodine receptors (RyR2) demonstrated that in aging hearts, the open probability (P(o)) of RyR2 was increased but the mean open time was shorter, providing a molecular correlate for the increased frequency of Ca(2+) sparks and decreased size of sparks, respectively. Thus, modifications of normal RyR2 gating properties may play a role in the altered Ca(2+) homeostasis observed in senescent myocytes.     

beta(1)-Subunit of BK channels regulates arterial wall[Ca(2+)] and diameter in mouse cerebral arteries.

Mice with a disrupted beta(1) (BK beta(1))-subunit of the large-conductance Ca(2+)-activated K(+) (BK) channel gene develop systemic hypertension and cardiac hypertrophy, which is likely caused by uncoupling of Ca(2+) sparks to BK channels in arterial smooth muscle cells. However, little is known about the physiological levels of global intracellular Ca(2+) concentration ([Ca(2+)](i)) and its regulation by Ca(2+) sparks and BK channel subunits. We utilized a BK beta(1) knockout C57BL/6 mouse model and studied the effects of inhibitors of ryanodine receptor and BK channels on the global [Ca(2+)](i) and diameter of small cerebral arteries pressurized to 60 mmHg. Ryanodine (10 microM) or iberiotoxin (100 nM) increased [Ca(2+)](i) by approximately 75 nM and constricted +/+ BK beta(1) wild-type arteries (pressurized to 60 mmHg) with myogenic tone by approximately 10 microm. In contrast, ryanodine (10 microM) or iberiotoxin (100 nM) had no significant effect on [Ca(2+)](i) and diameter of -/- BK beta(1)-pressurized (60 mmHg) arteries. These results are consistent with the idea that Ca(2+) sparks in arterial smooth muscle cells limit myogenic tone through activation of BK channels. The activation of BK channels by Ca(2+) sparks reduces the voltage-dependent Ca(2+) influx and [Ca(2+)](i) through tonic hyperpolarization. Deletion of BK beta(1) disrupts this negative feedback mechanism, leading to increased arterial tone through an increase in global [Ca(2+)](i).     

Ablation of skeletal muscle triadin impairs FKBP12/RyR1 channel interactions essential for maintaining resting cytoplasmic Ca2+

Previously, we have shown that lack of expression of triadins in skeletal muscle cells results in significant increase of myoplasmic resting free Ca(2+) ([Ca(2+)](rest)), suggesting a role for triadins in modulating global intracellular Ca(2+) homeostasis. To understand this mechanism, we study here how triadin alters [Ca(2+)](rest), Ca(2+) release, and Ca(2+) entry pathways using a combination of Ca(2+) microelectrodes, channels reconstituted in bilayer lipid membranes (BLM), Ca(2+), and Mn(2+) imaging analyses of myotubes and RyR1 channels obtained from triadin-null mice. Unlike WT cells, triadin-null myotubes had chronically elevated [Ca(2+)](rest) that was sensitive to inhibition with ryanodine, suggesting that triadin-null cells have increased basal RyR1 activity. Consistently, BLM studies indicate that, unlike WT-RyR1, triadin-null channels more frequently display atypical gating behavior with multiple and stable subconductance states. Accordingly, pulldown analysis and fluorescent FKBP12 binding studies in triadin-null muscles revealed a significant impairment of the FKBP12/RyR1 interaction. Mn(2+) quench rates under resting conditions indicate that triadin-null cells also have higher Ca(2+) entry rates and lower sarcoplasmic reticulum Ca(2+) load than WT cells. Overexpression of FKBP12.6 reverted the null phenotype, reducing resting Ca(2+) entry, recovering sarcoplasmic reticulum Ca(2+) content levels, and restoring near normal [Ca(2+)](rest). Exogenous FKBP12.6 also reduced the RyR1 channel P(o) but did not rescue subconductance behavior. In contrast, FKBP12 neither reduced P(o) nor recovered multiple subconductance gating. These data suggest that elevated [Ca(2+)](rest) in triadin-null myotubes is primarily driven by dysregulated RyR1 channel activity that results in part from impaired FKBP12/RyR1 functional interactions and a secondary increased Ca(2+) entry at rest.     

Caffeine-induced Release of Intracellular Ca2+ from Chinese Hamster Ovary Cells Expressing Skeletal Muscle Ryanodine Receptor : Effects on Full-Length and Carboxyl-Terminal Portion of Ca2+Release Channels

The ryanodine receptor (RyR)/Ca²⁺ release channel is an essential component of excitation–contraction coupling in striated muscle cells. To study the function and regulation of the Ca²⁺ release channel, we tested the effect of caffeine on the full-length and carboxyl-terminal portion of skeletal muscle RyR expressed in a Chinese hamster ovary (CHO) cell line. Caffeine induced openings of the full length RyR channels in a concentration-dependent manner, but it had no effect on the carboxyl-terminal RyR channels. CHO cells expressing the carboxyl-terminal RyR proteins displayed spontaneous changes of intracellular [Ca²⁺]. Unlike the native RyR channels in muscle cells, which display localized Ca²⁺ release events (i.e., “Ca²⁺ sparks” in cardiac muscle and “local release events” in skeletal muscle), CHO cells expressing the full length RyR proteins did not exhibit detectable spontaneous or caffeine-induced local Ca²⁺ release events. Our data suggest that the binding site for caffeine is likely to reside within the amino-terminal portion of RyR, and the localized Ca²⁺ release events observed in muscle cells may involve gating of a group of Ca²⁺ release channels and/or interaction of RyR with muscle-specific proteins.     

Evidence for a role of C-terminus in Ca(2+) inactivation of skeletal muscle Ca(2+) release channel (ryanodine receptor).

Six chimeras of the skeletal muscle (RyR1) and cardiac muscle (RyR2) Ca(2+) release channels (ryanodine receptors) previously used to identify RyR1 dihydropyridine receptor interactions [Nakai et al. (1998) J. Biol. Chem. 273, 13403] were expressed in HEK293 cells to assess their Ca(2+) dependence in [(3)H]ryanodine binding and single channel measurements. The results indicate that the C-terminal one-fourth has a major role in Ca(2+) activation and inactivation of RyR1. Further, our results show that replacement of RyR1 regions with corresponding RyR2 regions can result in loss and/or reduction of [(3)H]ryanodine binding affinity while maintaining channel activity.     

Phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) potentiates cardiac contractility via activation of the ryanodine receptor

Phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) is the most recently identified phosphoinositide, and its functions have yet to be fully elucidated. Recently, members of our muscle group have shown that PI(3,5)P2 plays an important role in skeletal muscle function by altering Ca(2+) homeostasis. Therefore, we hypothesized that PI(3,5)P2 may also modulate cardiac muscle contractility by altering intracellular Ca(2+) ([Ca(2+)](i)) in cardiac myocytes. We first confirmed that PI(3,5)P2 was present and increased by insulin treatment of cardiomyocytes via immunohistochemistry. To examine the acute effects of PI(3,5)P2 treatment, electrically paced left ventricular muscle strips were incubated with PI(3,5)P2. Treatment with PI(3,5)P2 increased the magnitude of isometric force, the rate of force development, and the area associated with the contractile waveforms. These enhanced contractile responses were also observed in MIP/Mtmr14(-/-) mouse hearts, which we found to have elevated levels of PI(3,5)P2. In cardiac myocytes loaded with fura-2, PI(3,5)P2 produced a robust elevation in [Ca(2+)](i). The PI(3,5)P2-induced elevation of [Ca(2+)](i) was not present in conditions free of extracellular Ca(2+) and was completely blocked by ryanodine. We investigated whether the phosphoinositide acted directly with the Ca(2+) release channels of the sarcoplasmic reticulum (ryanodine receptors; RyR2). PI(3,5)P2 increased [(3)H]ryanodine binding and increased the open probability (P(o)) of single RyR2 channels reconstituted in lipid bilayers. This strongly suggests that the phosphoinositide binds directly to the RyR2 channel. Thus, we provide inaugural evidence that PI(3,5)P2 is a powerful activator of sarcoplasmic reticulum Ca(2+) release and thereby modulates cardiac contractility.     

The predicted TM10 transmembrane sequence of the cardiac Ca2+ release channel (ryanodine receptor) is crucial for channel activation and gating

The predicted TM10 transmembrane sequence, (4844)IIFDITFFFFVIVILLAIIQGLII(4867), has been proposed to be the pore inner helix of the ryanodine receptor (RyR) and to play a crucial role in channel activation and gating, as with the inner helix of bacterial potassium channels. However, experimental evidence for the involvement of the TM10 sequence in RyR channel activation and gating is lacking. In the present study, we have systematically investigated the effects of mutations of each residue within the 24-amino acid TM10 sequence of the mouse cardiac ryanodine receptor (RyR2) on channel activation by caffeine and Ca(2+). Intracellular Ca(2+) release measurements in human embryonic kidney 293 cells expressing the RyR2 wild type and TM10 mutants revealed that several mutations in the TM10 sequence either abolished caffeine response or markedly reduced the sensitivity of the RyR2 channel to activation by caffeine. By assessing the Ca(2+) dependence of [(3)H]ryanodine binding to RyR2 wild type and TM10 mutants we also found that mutations in the TM10 sequence altered the sensitivity of the channel to activation by Ca(2+) and enhanced the basal activity of [(3)H]ryanodine binding. Furthermore, single I4862A mutant channels exhibited considerable channel openings and altered gating at very low concentrations of Ca(2+). Our data indicate that the TM10 sequence constitutes an essential determinant for channel activation and gating, in keeping with the proposed role of TM10 as an inner helix of RyR. Our results also shed insight into the orientation of the TM10 helix within the RyR channel pore.     

Role of amino-terminal half of the S4-S5 linker in type 1 ryanodine receptor (RyR1) channel gating

The type 1 ryanodine receptor (RyR1) is a Ca(2+) release channel found in the sarcoplasmic reticulum of skeletal muscle and plays a pivotal role in excitation-contraction coupling. The RyR1 channel is activated by a conformational change of the dihydropyridine receptor upon depolarization of the transverse tubule, or by Ca(2+) itself, i.e. Ca(2+)-induced Ca(2+) release (CICR). The molecular events transmitting such signals to the ion gate of the channel are unknown. The S4-S5 linker, a cytosolic loop connecting the S4 and S5 transmembrane segments in six-transmembrane type channels, forms an α-helical structure and mediates signal transmission in a wide variety of channels. To address the role of the S4-S5 linker in RyR1 channel gating, we performed alanine substitution scan of N-terminal half of the putative S4-S5 linker (Thr(4825)-Ser(4829)) that exhibits high helix probability. The mutant RyR1 was expressed in HEK cells, and CICR activity was investigated by caffeine-induced Ca(2+) release, single-channel current recordings, and [(3)H]ryanodine binding. Four mutants (T4825A, I4826A, S4828A, and S4829A) had reduced CICR activity without changing Ca(2+) sensitivity, whereas the L4827A mutant formed a constitutive active channel. T4825I, a disease-associated mutation for malignant hyperthermia, exhibited enhanced CICR activity. An α-helical wheel representation of the N-terminal S4-S5 linker provides a rational explanation to the observed activities of the mutants. These results suggest that N-terminal half of the S4-S5 linker may form an α-helical structure and play an important role in RyR1 channel gating.     

RyRCa2+ leak limits cardiac Ca2+ window current overcoming the tonic effect of calmodulinin mice

Ca(2+) mediates the functional coupling between L-type Ca(2+) channel (LTCC) and sarcoplasmic reticulum (SR) Ca(2+) release channel (ryanodine receptor, RyR), participating in key pathophysiological processes. This crosstalk manifests as the orthograde Ca(2+)-induced Ca(2+)-release (CICR) mechanism triggered by Ca(2+) influx, but also as the retrograde Ca(2+)-dependent inactivation (CDI) of LTCC, which depends on both Ca(2+) permeating through the LTCC itself and on SR Ca(2+) release through the RyR. This latter effect has been suggested to rely on local rather than global Ca(2+) signaling, which might parallel the nanodomain control of CDI carried out through calmodulin (CaM). Analyzing the CICR in catecholaminergic polymorphic ventricular tachycardia (CPVT) mice as a model of RyR-generated Ca(2+) leak, we evidence here that increased occurrence of the discrete local SR Ca(2+) releases through the RyRs (Ca(2+) sparks) cause a depolarizing shift in activation and a hyperpolarizing shift in isochronic inactivation of cardiac LTCC current resulting in the reduction of window current. Both increasing fast [Ca(2+)](i) buffer capacity or depleting SR Ca(2+) store blunted these changes, which could be reproduced in WT cells by RyRCa(2+) leak induced with Ryanodol and CaM inhibition.Our results unveiled a new paradigm for CaM-dependent effect on LTCC gating and further the nanodomain Ca(2+) control of LTCC, emphasizing the importance of spatio-temporal relationships between Ca(2+) signals and CaM function.     

Single ryanodine receptor channel basis of caffeine's action on Ca2+ sparks

Caffeine (1, 3, 7-trimethylxanthine) is a widely used pharmacological agonist of the cardiac ryanodine receptor (RyR2) Ca(2+) release channel. It is also a well-known stimulant that can produce adverse side effects, including arrhythmias. Here, the action of caffeine on single RyR2 channels in bilayers and Ca(2+) sparks in permeabilized ventricular cardiomyocytes is defined. Single RyR2 caffeine activation depended on the free Ca(2+) level on both sides of the channel. Cytosolic Ca(2+) enhanced RyR2 caffeine affinity, whereas luminal Ca(2+) essentially scaled maximal caffeine activation. Caffeine activated single RyR2 channels in diastolic quasi-cell-like solutions (cytosolic MgATP, pCa 7) with an EC(50) of 9.0 ± 0.4 mM. Low-dose caffeine (0.15 mM) increased Ca(2+) spark frequency ～75% and single RyR2 opening frequency ～150%. This implies that not all spontaneous RyR2 openings during diastole are associated with Ca(2+) sparks. Assuming that only the longest openings evoke sparks, our data suggest that a spark may result only when a spontaneous single RyR2 opening lasts >6 ms.     

Ca2+ responses of pulmonary arterial myocytes to acute hypoxia require release from ryanodine and inositol trisphosphate receptors in sarcoplasmic reticulum

In pulmonary arterial smooth muscle cells (PASMC), acute hypoxia increases intracellular Ca(2+) concentration ([Ca(2+)](i)) by inducing Ca(2+) release from the sarcoplasmic reticulum (SR) and Ca(2+) influx through store- and voltage-operated Ca(2+) channels in sarcolemma. To evaluate the mechanisms of hypoxic Ca(2+) release, we measured [Ca(2+)](i) with fluorescent microscopy in primary cultures of rat distal PASMC. In cells perfused with Ca(2+)-free Krebs Ringer bicarbonate solution (KRBS), brief exposures to caffeine (30 mM) and norepinephrine (300 μM), which activate SR ryanodine and inositol trisphosphate receptors (RyR, IP(3)R), respectively, or 4% O(2) caused rapid transient increases in [Ca(2+)](i), indicating intracellular Ca(2+) release. Preexposure of these cells to caffeine, norepinephrine, or the SR Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA; 10 μM) blocked subsequent Ca(2+) release to caffeine, norepinephrine, and hypoxia. The RyR antagonist ryanodine (10 μM) blocked Ca(2+) release to caffeine and hypoxia but not norepinephrine. The IP(3)R antagonist xestospongin C (XeC, 0.1 μM) blocked Ca(2+) release to norepinephrine and hypoxia but not caffeine. In PASMC perfused with normal KRBS, acute hypoxia caused a sustained increase in [Ca(2+)](i) that was abolished by ryanodine or XeC. These results suggest that in rat distal PASMC 1) the initial increase in [Ca(2+)](i) induced by hypoxia, as well as the subsequent Ca(2+) influx that sustained this increase, required release of Ca(2+) from both RyR and IP(3)R, and 2) the SR Ca(2+) stores accessed by RyR, IP(3)R, and hypoxia functioned as a common store, which was replenished by a CPA-inhibitable Ca(2+)-ATPase.     

Role of FKBP12.6 in hypoxia- and norepinephrine-induced Ca2+ release and contraction in pulmonary artery myocytes

The cellular and molecular processes underlying the regulation of ryanodine receptor (RyR) Ca(2+) release in smooth muscle cells (SMCs) are incompletely understood. Here we show that FKBP12.6 proteins are expressed in pulmonary artery (PA) smooth muscle and associated with type-2 RyRs (RyR2), but not RyR1, RyR3, or IP(3) receptors (IP(3)Rs) in PA sarcoplasmic reticulum. Application of FK506, which binds to FKBPs and dissociates these proteins from RyRs, induced an increase in [Ca(2+)](i) and Ca(2+)-activated Cl(-) and K(+) currents in freshly isolated PASMCs, whereas cyclosporin, an agent known to inhibit calcineurin but not to interact with FKBPs, failed to induce an increase in [Ca(2+)](i). FK506-induced [Ca(2+)](i) increase was completely blocked by the RyR antagonist ruthenium red and ryanodine, but not the IP(3)R antagonist heparin. Hypoxic Ca(2+) response and hypoxic vasoconstriction were significantly enhanced in FKBP12.6 knockout mouse PASMCs. FK506 or rapamycin pretreatment also enhanced hypoxic increase [Ca(2+)](i), but did not alter caffeine-induced Ca(2+) release (SR Ca(2+) content) in PASMCs. Norepinephrine-induced Ca(2+) release and force generation were also markedly enhanced in PASMCs from FKBP12.6 null mice. These findings suggest that FKBP12.6 plays an important role in hypoxia- and neurotransmitter-induced Ca(2+) and contractile responses by regulating the activity of RyRs in PASMCs.     

Contribution of ryanodine receptor type 3 to Ca(2+) sparks in embryonic mouse skeletal muscle.

The kinetic behavior of Ca(2+) sparks in knockout mice lacking a specific ryanodine receptor (RyR) isoform should provide molecular information on function and assembly of clusters of RyRs. We examined resting Ca(2+) sparks in RyR type 3-null intercostal myotubes from embryonic day 18 (E18) mice and compared them to Ca(2+) sparks in wild-type (wt) mice of the same age and to Ca(2+) sparks in fast-twitch muscle cells from the foot of wt adult mice. Sparks from RyR type 3-null embryonic cells (368 events) were significantly smaller, briefer, and had a faster time to peak than sparks from wt cells (280 events) of the same age. Sparks in adult cells (220 events) were infrequent, yet they were highly reproducible with population means smaller than those in embryonic RyR type 3-null cells but similar to those reported in adult amphibian skeletal muscle fibers. Three-dimensional representations of the spark peak intensity (DeltaF/Fo) vs. full width at half-maximal intensity (FWHM) vs. full duration at half-maximal intensity (FTHM) showed that wt embryonic sparks were considerably more variable in size and kinetics than sparks in adult muscle. In all cases, tetracaine (0.2 mM) abolished Ca(2+) spark activity, whereas caffeine (0.1 mM) lengthened the spark duration in wt embryonic and adult cells but not in RyR type 3-null cells. These results confirmed that sparks arose from RyRs. The low caffeine sensitivity of RyR type 3-null cells is entirely consistent with observations by other investigators. There are three conclusions from this study: i) RyR type-1 engages in Ca(2+) spark activity in the absence of other RyR isoforms in RyR type 3-null myotubes; ii) Ca(2+) sparks with parameters similar to those reported in adult amphibian skeletal muscle can be detected, albeit at a low frequency, in adult mammalian skeletal muscle cells; and iii) a major contributor to the unusually large Ca(2+) sparks observed in normal (wt) embryonic muscle is RyR type 3. To explain the reduction in the size of sparks in adult compared to embryonic skeletal muscle, we suggest that in embryonic muscle, RyR type 1 and RyR type 3 channels co-contribute to Ca(2+) release during the same spark and that Ca(2+) sparks undergo a maturation process which involves a decrease in RyR type 3.     

Ryanodine sensitizes the Ca(2+) release channel (ryanodine receptor) to Ca(2+) activation

Ryanodine, a plant alkaloid, is one of the most widely used pharmacological probes for intracellular Ca(2+) signaling in a variety of muscle and non-muscle cells. Upon binding to the Ca(2+) release channel (ryanodine receptor), ryanodine causes two major changes in the channel: a reduction in single-channel conductance and a marked increase in open probability. The molecular mechanisms underlying these alterations are not well understood. In the present study, we investigated the gating behavior and Ca(2+) dependence of the wild type (wt) and a mutant cardiac ryanodine receptor (RyR2) after being modified by ryanodine. Single-channel studies revealed that the ryanodine-modified wt RyR2 channel was sensitive to inhibition by Mg(2+) and to activation by caffeine and ATP. In the presence of Mg(2+), the ryanodine-modified single wt RyR2 channel displayed a sigmoidal Ca(2+) dependence with an EC(50) value of 110 nm, whereas the ryanodine-unmodified single wt channel exhibited an EC(50) of 120 microm for Ca(2+) activation, indicating that ryanodine is able to increase the sensitivity of the wt RyR2 channel to Ca(2+) activation by approximately 1,000-fold. Furthermore, ryanodine is able to restore Ca(2+) activation and ligand response of the E3987A mutant RyR2 channel that has been shown to exhibit approximately 1,000-fold reduction in Ca(2+) sensitivity to activation. The E3987A mutation, however, affects neither [(3)H]ryanodine binding to, nor the stimulatory and inhibitory effects of ryanodine on, the RyR2 channel. These results demonstrate that ryanodine does not "lock" the RyR channel into an open state as generally believed; rather, it sensitizes dramatically the channel to activation by Ca(2+).     

Thermodynamically irreversible gating of ryanodine receptors in situ revealed by stereotyped duration of release in Ca(2+) sparks

For a single or a group of Markov channels gating reversibly, distributions of open and closed times should be the sum of positively weighted decaying exponentials. Violation of this microscopic reversibility has been demonstrated previously on a number of occasions at the single channel level, and has been attributed to possible channel coupling to external sources of free energy. Here we show that distribution of durations of Ca(2+) release underlying Ca(2+) sparks in intact cardiac myocytes exhibits a prominent mode at approximately 8 ms. Analysis of the cycle time for repetitive sparks at hyperactive sites revealed no intervals briefer than approximately 35 ms and a mode at approximately 90 ms. These results indicate that, regardless of whether Ca(2+) sparks are single-channel or multi-channel in origin, they are generated by thermodynamically irreversible stochastic processes. In contrast, data from planar lipid bilayer experiments were consistent with reversible gating of RyR under asymmetric cis (4 microM) and trans Ca(2+) (10 mM), suggesting that the irreversibility for Ca(2+) spark genesis may reside at a supramolecular level. Modeling suggests that Ca(2+)-induced Ca(2+) release among adjacent RyRs may couple the external energy derived from Ca(2+) gradients across the SR to RyR gating in situ, and drive the irreversible generation of Ca(2+) sparks.     

Store-operated Ca2+ influx causes Ca2+ release from the intracellular Ca2+ channels that is required for T cell activation

The precise control of many T cell functions relies on cytosolic Ca(2+) dynamics that is shaped by the Ca(2+) release from the intracellular store and extracellular Ca(2+) influx. The Ca(2+) influx activated following T cell receptor (TCR)-mediated store depletion is considered to be a major mechanism for sustained elevation in cytosolic Ca(2+) concentration ([Ca(2+)](i)) necessary for T cell activation, whereas the role of intracellular Ca(2+) release channels is believed to be minor. We found, however, that in Jurkat T cells [Ca(2+)](i) elevation observed upon activation of the store-operated Ca(2+) entry (SOCE) by passive store depletion with cyclopiazonic acid, a reversible blocker of sarco-endoplasmic reticulum Ca(2+)-ATPase, inversely correlated with store refilling. This indicated that intracellular Ca(2+) release channels were activated in parallel with SOCE and contributed to global [Ca(2+)](i) elevation. Pretreating cells with (-)-xestospongin C (10 microM) or ryanodine (400 microM), the antagonists of inositol 1,4,5-trisphosphate receptor (IP3R) or ryanodine receptor (RyR), respectively, facilitated store refilling and significantly reduced [Ca(2+)](i) elevation evoked by the passive store depletion or TCR ligation. Although the Ca(2+) release from the IP3R can be activated by TCR stimulation, the Ca(2+) release from the RyR was not inducible via TCR engagement and was exclusively activated by the SOCE. We also established that inhibition of IP3R or RyR down-regulated T cell proliferation and T-cell growth factor interleukin 2 production. These studies revealed a new aspect of [Ca(2+)](i) signaling in T cells, that is SOCE-dependent Ca(2+) release via IP3R and/or RyR, and identified the IP3R and RyR as potential targets for manipulation of Ca(2+)-dependent functions of T lymphocytes.