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.     

Halothane modulation of skeletal muscle ryanodine receptors: dependence on Ca2+, Mg2+, and ATP

Malignant hyperthermia (MH) susceptibility is a genetic disorder of skeletal muscle associated with mutations in the ryanodine receptor isoform 1 (RyR1) of sarcoplasmic reticulum (SR). In MH-susceptible skeletal fibers, RyR1-mediated Ca(2+) release is highly sensitive to activation by the volatile anesthetic halothane. Indeed, studies with isolated RyR1 channels (using simple Cs(+) solutions) found that halothane selectively affects mutated but not wild-type RyR1 function. However, studies in skeletal fibers indicate that halothane can also activate wild-type RyR1-mediated Ca(2+) release. We hypothesized that endogenous RyR1 agonists (ATP, lumenal Ca(2+)) may increase RyR1 sensitivity to halothane. Consequently, we studied how these agonists affect halothane action on rabbit skeletal RyR1 reconstituted into planar lipid bilayers. We found that cytosolic ATP is required for halothane-induced activation of the skeletal RyR1. Unlike RyR1, cardiac RyR2 (much less sensitive to ATP) responded to halothane even in the absence of this agonist. ATP-dependent halothane activation of RyR1 was enhanced by cytosolic Ca(2+) (channel agonist) and counteracted by Mg(2+) (channel inhibitor). Dantrolene, a muscle relaxant used to treat MH episodes, did not affect RyR1 or RyR2 basal activity and did not interfere with halothane-induced activation. Studies with skeletal SR microsomes confirmed that halothane-induced RyR1-mediated SR Ca(2+) release is enhanced by high ATP-low Mg(2+) in the cytosol and by increased SR Ca(2+) load. Thus, physiological or pathological processes that induce changes in cellular levels of these modulators could affect RyR1 sensitivity to halothane in skeletal fibers, including the outcome of halothane-induced contracture tests used to diagnose MH susceptibility.     

Activation of cardiac ryanodine receptors by the calcium channel agonist FPL-64176

We investigated the possibility that the Ca(2+) channel agonist FPL-64176 (FPL) might also activate the cardiac sarcoplasmic reticulum (SR) Ca(2+) release channel ryanodine receptor (RyR). The effects of FPL were tested on single channel activity of purified and crude vesicular RyR (RyR2) isolated from human and dog hearts using the planar lipid bilayer technique. FPL (100-200 microM) increased single channel open probability (P(o)) when added to the cytoplasmic side of the channel (P(o) = 0.070 +/- 0.021 in control RyR2; 0.378 +/- 0.086 in 150 microM FPL, n = 9, P < 0.01) by prolonging open times and decreasing closed times without changing current magnitude. FPL had no effect on P(o) when added to the trans (luminal) side of the bilayer (P(o) = 0.079 +/- 0.036 in control and 0.103 +/- 0.066 in FPL, n = 4, no significant difference). The bell-shaped [Ca(2+)] dependence of [(3)H]ryanodine binding and of P(o) was altered by FPL, suggesting that the mechanism by which FPL increases channel activity is by an increase in Ca(2+)-induced activation at low [Ca(2+)] (without a change in threshold) and suppression of Ca(2+)-induced inactivation at high [Ca(2+)]. However, the fact that inactivation was restored at elevated [Ca(2+)] suggests a competitive interaction between Ca(2+) and FPL on inactivation. FPL had no effect on RyR skeletal channels (RyR1), where P(o) was 0.039 +/- 0.005 in control versus 0.030 +/- 0.006 in 150 microM FPL (no significant difference). These results suggest that, in addition to its ability to activate the L-type Ca(2+) channels, FPL activates cardiac RyR2 primarily by reducing the Ca(2+) sensitivity of inactivation.     

The 30 S lobster skeletal muscle Ca2+ release channel (ryanodine receptor) has functional properties distinct from the mammalian channel proteins.

The 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps)-solubilized ryanodine receptor (RyR) of lobster skeletal muscle has been isolated by rate density centrifugation as a 30 S protein complex. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the purified 30 S receptor revealed a single high molecular weight protein band with a mobility intermediate between those of the mammalian skeletal and cardiac M(r) 565,000 RyR polypeptides. Immunoblot analysis showed no or only minimal cross-reactivity with the rabbit skeletal and canine cardiac RyR polypeptides. By immunofluorescence the lobster RyR was localized to the junctions of the A-I bands. Following planar lipid bilayer reconstitution of the purified 30 S lobster RyR, single channel K+ and Ca2+ currents were observed which were modified by ryanodine and optimally activated by millimolar concentrations of cis (cytoplasmic) Ca2+. Vesicle-45Ca2+ flux measurements also indicated an optimal activation of the lobster Ca2+ channel by millimolar Ca2+, whereas 45Ca2+ efflux from mammalian skeletal and cardiac muscle sarcoplasmic reticulum (SR) vesicles is optimally activated by micromolar Ca2+. Further, mammalian muscle SR Ca2+ release activity is modulated by Mg2+ and ATP, whereas neither ligand appreciably affected 45Ca2+ efflux from lobster SR vesicles. These results suggested that lobster and mammalian muscle express immunologically and functionally distinct SR Ca2+ release channel protein complexes.     

A postulated role of the near amino-terminal domain of the ryanodine receptor in the regulation of the sarcoplasmic reticulum Ca(2+) channel

To test the hypothesis that interactions among several putative domains of the ryanodine receptor (RyR) are involved in the regulation of its Ca(2+) release channel, we synthesized several peptides corresponding to selected NH(2)-terminal regions of the RyR. We then examined their effects on ryanodine binding and Ca(2+) release activities of the sarcoplasmic reticulum isolated from skeletal and cardiac muscle. Peptides 1-2s, 1-2c, and 1 enhanced ryanodine binding to cardiac RyR and induced a rapid Ca(2+) release from cardiac SR in a dose-dependent manner. The order of the potency for the activation of the Ca(2+) release channel was 1-2c > 1 > 1-2s. Interestingly, these peptides produced significant activation of the cardiac RyR at near zero or subactivating [Ca(2+)], indicating that the peptides enhanced the Ca(2+) sensitivity of the channel. Peptides 1-2c, 1-2s, and 1 had virtually no effect on skeletal RyR, although occasional and variable extents of activation were observed in ryanodine binding assays performed at 36 degrees C. Peptide 3 affected neither cardiac nor skeletal RyR. We propose that domains 1 and 1-2 of the RyR, to which these activating peptides correspond, would interact with one or more other domains within the RyR (including presumably the Ca(2+)-binding domain) to regulate the Ca(2+) channel.     

FKBP12 activates the cardiac ryanodine receptor Ca2+-release channel and is antagonised by FKBP12.6

Changes in FKBP12.6 binding to cardiac ryanodine receptors (RyR2) are implicated in mediating disturbances in Ca(2+)-homeostasis in heart failure but there is controversy over the functional effects of FKBP12.6 on RyR2 channel gating. We have therefore investigated the effects of FKBP12.6 and another structurally similar molecule, FKBP12, which is far more abundant in heart, on the gating of single sheep RyR2 channels incorporated into planar phospholipid bilayers and on spontaneous waves of Ca(2+)-induced Ca(2+)-release in rat isolated permeabilised cardiac cells. We demonstrate that FKBP12 is a high affinity activator of RyR2, sensitising the channel to cytosolic Ca(2+), whereas FKBP12.6 has very low efficacy, but can antagonise the effects of FKBP12. Mathematical modelling of the data shows the importance of the relative concentrations of FKBP12 and FKBP12.6 in determining RyR2 activity. Consistent with the single-channel results, physiological concentrations of FKBP12 (3 μM) increased Ca(2+)-wave frequency and decreased the SR Ca(2+)-content in cardiac cells. FKBP12.6, itself, had no effect on wave frequency but antagonised the effects of FKBP12.We provide a biophysical analysis of the mechanisms by which FK-binding proteins can regulate RyR2 single-channel gating. Our data indicate that FKBP12, in addition to FKBP12.6, may be important in regulating RyR2 function in the heart. In heart failure, it is possible that an alteration in the dual regulation of RyR2 by FKBP12 and FKBP12.6 may occur. This could contribute towards a higher RyR2 open probability, 'leaky' RyR2 channels and Ca(2+)-dependent arrhythmias.     

Ryanodine receptor regulation by intramolecular interaction between cytoplasmic and transmembrane domains

Ryanodine receptors (RyR) function as Ca(2+) channels that regulate Ca(2+) release from intracellular stores to control a diverse array of cellular processes. The massive cytoplasmic domain of RyR is believed to be responsible for regulating channel function. We investigated interaction between the transmembrane Ca(2+)-releasing pore and a panel of cytoplasmic domains of the human cardiac RyR in living cells. Expression of eGFP-tagged RyR constructs encoding distinct transmembrane topological models profoundly altered intracellular Ca(2+) handling and was refractory to modulation by ryanodine, FKBP12.6 and caffeine. The impact of coexpressing dsRed-tagged cytoplasmic domains of RyR2 on intracellular Ca(2+) phenotype was assessed using confocal microscopy coupled with parallel determination of in situ protein: protein interaction using fluorescence resonance energy transfer (FRET). Dynamic interactions between RyR cytoplasmic and transmembrane domains were mediated by amino acids 3722-4610 (Interacting or "I"-domain) which critically modulated intracellular Ca(2+) handling and restored RyR sensitivity to caffeine activation. These results provide compelling evidence that specific interaction between cytoplasmic and transmembrane domains is an important mechanism in the intrinsic modulation of RyR Ca(2+) release channels.     

Calcium uptake studies of 1,4-dihydropyridine agonists into rabbit aortic smooth muscle cells in culture

The effects of the three dihydropyridine calcium channel agonists (+/-)BAY K 8644, (+)202-791 and (+/-)CGP 28392 on 45Ca++ uptake were studied in cultures of rabbit aortic smooth muscle cells. At 10(-7) M each agonist enhanced 45Ca++ uptake in 15-50 mM K+ but had no effect on the basal 45Ca++ uptake at 5 mM K+. At the uptake threshold of 15 mM K+ each agonist potentiated 45Ca++ uptake in a dose-dependent manner with half maximal effects at 2.4 nM for (+/-)BAY K 8644, 22 nM for (+)202-791 and 18 nM for (+/-)CGP 28392. The agonists showed no significant antagonistic activity. Responses were antagonized competitively by nifedipine and non-competitively by (+/-)D-600. The 45Ca++ uptake dose-response curves and the half maximal effects of the three agonists were over the same range of concentrations as their inhibition of [3H]nitrendipine binding to rat ventricular receptor membrane preparations. The data suggest that these cells mimic the calcium uptake by the intact aorta better than commercial vascular smooth muscle lines or cardiac cells.     

Functional calcium release channel formed by the carboxyl-terminal portion of ryanodine receptor.

The ryanodine receptor (RyR) is one of the key proteins involved in excitation-contraction (E-C) coupling in skeletal muscle, where it functions as a Ca2+ release channel in the sarcoplasmic reticulum (SR) membrane. RyR consists of a single polypeptide of approximately 560 kDa normally arranged in a homotetrameric structure, which contains a carboxyl (C)-terminal transmembrane domain and a large amino (N)-terminal cytoplasmic domain. To test whether the carboxyl-terminal portion of RyR is sufficient to form a Ca2+ release channel, we expressed the full-length (RyR-wt) and C-terminal (RyR-C, approximately 130 kDa) RyR proteins in a Chinese hamster ovary (CHO) cell line, and measured their Ca2+ release channel functions in planar lipid bilayer membranes. The single-channel properties of RyR-wt were found to be similar to those of RyR from skeletal muscle SR. The RyR-C protein forms a cation-selective channel that shares some of the channel properties with RyR-wt, including activation by cytoplasmic Ca2+ and regulation by ryanodine. Unlike RyR-wt, which exhibits a linear current-voltage relationship and inactivates at millimolar Ca2+, the channels formed by RyR-C display significant inward rectification and fail to close at high cytoplasmic Ca2+. Our results show that the C-terminal portion of RyR contains structures sufficient to form a functional Ca2+ release channel, but the N-terminal portion of RyR also affects the ion-conduction and calcium-dependent regulation of the Ca2+ release channel.     

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+).     

FKBP12.6 deficiency and defective calcium release channel (ryanodine receptor) function linked to exercise-induced sudden cardiac death

Arrhythmias, a common cause of sudden cardiac death, can occur in structurally normal hearts, although the mechanism is not known. In cardiac muscle, the ryanodine receptor (RyR2) on the sarcoplasmic reticulum releases the calcium required for muscle contraction. The FK506 binding protein (FKBP12.6) stabilizes RyR2, preventing aberrant activation of the channel during the resting phase of the cardiac cycle. We show that during exercise, RyR2 phosphorylation by cAMP-dependent protein kinase A (PKA) partially dissociates FKBP12.6 from the channel, increasing intracellular Ca(2+) release and cardiac contractility. FKBP12.6(-/-) mice consistently exhibited exercise-induced cardiac ventricular arrhythmias that cause sudden cardiac death. Mutations in RyR2 linked to exercise-induced arrhythmias (in patients with catecholaminergic polymorphic ventricular tachycardia [CPVT]) reduced the affinity of FKBP12.6 for RyR2 and increased single-channel activity under conditions that simulate exercise. These data suggest that "leaky" RyR2 channels can trigger fatal cardiac arrhythmias, providing a possible explanation for CPVT.     

Transmembrane redox sensor of ryanodine receptor complex

Inositol 1,4,5-trisphosphate receptors (IP(3)R) and ryanodine receptors (RyR) mediate the release of endoplasmic and sarcoplasmic reticulum (ER/SR) Ca(2+) stores and regulate Ca(2+) entry through voltage-dependent or ligand-gated channels of the plasma membrane. A prominent property of ER/SR Ca(2+) channels is exquisite sensitivity to sulfhydryl-modifying reagents. A plausible role for sulfhydryl chemistry in physiologic regulation of Ca(2+) release channels and the fidelity of Ca(2+) release from ER/SR is lacking. This study reveals the existence of a transmembrane redox sensor within the RyR1 channel complex that confers tight regulation of channel activity in response to changes in transmembrane redox potential produced by cytoplasmic and luminal glutathione. A transporter selective for glutathione is co-localized with RyR1 within the SR membrane to maintain local redox potential gradients consistent with redox regulation of ER/SR Ca(2+) release. Hyperreactive sulfhydryls previously shown to reside within the RyR1 complex (Liu, G., and Pessah, I. N. (1994) J. Biol. Chem. 269, 33028-33034) are an essential biochemical component of a transmembrane redox sensor. Transmembrane redox sensing may represent a fundamental mechanism by which ER/SR Ca(2+) channels respond to localized changes in transmembrane glutathione redox potential produced by physiologic and pathophysiologic modulators of Ca(2+) release from stores.     

Ryanodine receptor function in newborn rat heart

The role of ryanodine receptor (RyR) in cardiac excitation-contraction (E-C) coupling in newborns (NB) is not completely understood. To determine whether RyR functional properties change during development, we evaluated cellular distribution and functionality of sarcoplasmic reticulum (SR) in NB rats. Sarcomeric arrangement of immunostained SR Ca(2+)-ATPase (SERCA2a) and the presence of sizeable caffeine-induced Ca2+ transients demonstrated that functional SR exists in NB. E-C coupling properties were then defined in NB and compared with those in adult rats (AD). Ca2+ transients in NB reflected predominantly sarcolemmal Ca2+ entry, whereas the RyR-mediated component was approximately 13%. Finally, the RyR density and functional properties at the single-channel level in NB were compared with those in AD. Ligand binding assays revealed that in NB, RyR density can be up to 36% of that found in AD, suggesting that some RyRs do not contribute to the Ca2+ transient. To test the hypothesis that RyR functional properties change during development, we incorporated single RyRs into lipid bilayers. Our results show that permeation and gating kinetics of NB RyRs are identical to those of AD. Also, endogenous ligands had similar effects on NB and AD RyRs: sigmoidal Ca2+ dependence, stronger Mg(2+)-induced inhibition at low cytoplasmic Ca2+ concentrations, comparable ATP-activating potency, and caffeine sensitivity. These observations indicate that NB rat heart contains fully functional RyRs and that the smaller contribution of RyR-mediated Ca2+ release to the intracellular Ca2+ transient in NB is not due to different single RyR channel properties or to the absence of functional intracellular Ca2+ stores.     

Is ryanodine receptor a calcium or magnesium channel? Roles of K+ and Mg2+ during Ca2+ release

The ryanodine receptor (RyR) is a poorly selective channel that mediates Ca(2+) release from intracellular Ca(2+) stores. How RyR's selectivity between the physiological cations K(+), Mg(2+), and Ca(2+) affects single-channel Ca(2+) current amplitude is examined using a recent model of RyR permeation. It is found that K(+) provides the vast majority of the countercurrent (through RyR itself) that is needed to prevent the sarcoplasmic reticulum (SR) membrane potential from changing and stopping Ca(2+) release. Moreover, intra-pore competition between Ca(2+) and Mg(2+) defines single RyR Ca(2+) current amplitude. Since both [Mg(2+)] and [Ca(2+)](SR) can change during pathophysiological conditions, the RyR unitary Ca(2+) current amplitude during Ca(2+) release may change significantly due to this Ca(2+)/Mg(2+) competition. Compared to the classic action of Mg(2+) on RyR open probability, these Ca(2+) current amplitude changes have as large or larger effects on overall RyR Ca(2+) mobilization. A new aspect of RyR divalent versus monovalent selectivity is also identified where this kind of selectivity decreases as divalent concentration increases.     

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.     

The Ryanodine Receptor Pore Blocker Neomycin also Inhibits Channel Activity via a Previously Undescribed High-Affinity Ca2+ Binding Site

In this study, we present evidence for the mechanism of neomycin inhibition of skeletal ryanodine receptors (RyRs). In single-channel recordings, neomycin produced monophasic inhibition of RyR open probability and biphasic inhibition of [(3)H]ryanodine binding. The half-maximal inhibitory concentration (IC(50)) for channel blockade by neomycin was dependent on membrane potential and cytoplasmic [Ca(2+)], suggesting that neomycin acts both as a pore plug and as a competitive antagonist at a cytoplasmic Ca(2+) binding site that causes allosteric inhibition. This novel Ca(2+)/neomycin binding site had a neomycin affinity of 100 nM: and a Ca(2+) affinity of 35 nM,: which is 30-fold higher than that of the well-described cytoplasmic Ca(2+) activation site. Therefore, a new high-affinity class of Ca(2+) binding site(s) on the RyR exists that mediates neomycin inhibition. Neomycin plugging of the channel pore induced brief (1-2 ms) conductance substates at 30% of the fully open conductance, whereas allosteric inhibition caused complete channel closure with durations that depended on the neomycin concentration. We quantitatively account for these results using a dual inhibition model for neomycin that incorporates voltage-dependent pore plugging and Ca(2+)-dependent allosteric inhibition.     

Defective Ca(2+) handling proteins regulation during heart failure

Abnormal release of Ca(2+) from sarcoplasmic reticulum (SR) via the cardiac ryanodine receptor (RyR2) may contribute to contractile dysfunction in heart failure (HF). We previously demonstrated that RyR2 macromolecular complexes from HF rat were significantly more depleted of FK506 binding protein (FKBP12.6). Here we assessed expression of key Ca(2+) handling proteins and measured SR Ca(2+) content in control and HF rat myocytes. Direct measurements of SR Ca(2+) content in permeabilized cardiac myocytes demonstrated that SR luminal [Ca(2+)] is markedly lowered in HF (HF: DeltaF/F(0) = 26.4+/-1.8, n=12; control: DeltaF/F(0) = 49.2+/-2.9, n=10; P<0.01). Furthermore, we demonstrated that the expression of RyR2 associated proteins (including calmodulin, sorcin, calsequestrin, protein phosphatase 1, protein phosphatase 2A), Ca(2+) ATPase (SERCA2a), PLB phosphorylation at Ser16 (PLB-S16), PLB phosphorylation at Thr17 (PLB-T17), L-type Ca(2+) channel (Cav1.2) and Na(+)- Ca(2+) exchanger (NCX) were significantly reduced in rat HF. Our results suggest that systolic SR reduced Ca(2+) release and diastolic SR Ca(2+) leak (due to defective protein-protein interaction between RyR2 and its associated proteins) along with reduced SR Ca(2+) uptake (due to down-regulation of SERCA2a, PLB-S16 and PLB-T17), abnormal Ca(2+) extrusion (due to down-regulation of NCX) and defective Ca(2+) -induced Ca(2+) release (due to down-regulation of Cav1.2) could contribute to HF.     

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.     

Markovian models of low and high activity levels of cardiac ryanodine receptors

The modal gating behavior of single sheep cardiac sarcoplasmic reticulum (SR) Ca2+-release/ryanodine receptor (RyR) channels was assessed. We find that the gating of RyR channels spontaneously shifts between high (H) and low (L) levels of activity and inactive periods where no channel openings are detected (I). Moreover, we find that there is evidence for multiple gating modes within H activity, which we term H1 and H2 mode. Our results demonstrate that the underlying mechanisms regulating gating are similar in native and purified channels. Dwell-time distributions of L activity were best fitted by three open and five closed significant exponential components whereas dwell-time distributions of H1 activity were best fitted by two to three open and four closed significant exponential components. Increases in cytosolic [Ca2+] cause an increase in open probability (Po) within L activity and an increase in the probability of occurrence of H activity. Open lifetime distributions within L activity were Ca2+ independent whereas open lifetime distributions within H activity were Ca2+ dependent. This study is the first attempt to estimate RyR single-channel kinetic parameters from sequences of idealized dwell-times and to develop kinetic models of RyR gating using the criterion of maximum likelihood. We propose distinct kinetic schemes for L, H1, and H2 activity that describe the major features of sheep cardiac RyR channel gating at these levels of activity.     

Expression and functional characterization of the cardiac muscle ryanodine receptor Ca(2+) release channel in Chinese hamster ovary cells.

To study the function and regulation of the cardiac ryanodine receptor (RyR2) Ca(2+) release channel, we expressed the RyR2 proteins in a Chinese hamster ovary (CHO) cell line, and assayed its function by single channel current recording and confocal imaging of intracellular Ca(2+) ([Ca(2+)](i)). The 16-kb cDNA encoding the full-length RyR2 was introduced into CHO cells using lipofectAmine and electroporation methods. Incorporation of microsomal membrane vesicles isolated from these transfected cells into lipid bilayer membrane resulted in single Ca(2+) release channel activities similar to those of the native Ca(2+) release channels from rabbit cardiac muscle SR membranes, both in terms of gating kinetics, conductance, and ryanodine modification. The expressed RyR2 channels were found to exhibit more frequent transitions to subconductance states than the native RyR2 channels and RyR1 expressed in CHO cells. Caffeine, an exogenous activator of RyR, induced release of [Ca(2+)](i) from these cells. Confocal imaging of cells expressing RyR2 did not detect spontaneous or caffeine-induced local Ca(2+) release events (i.e., "Ca(2+) sparks") typically seen in cardiac muscle. Our data show that the RyR2 expressed in CHO cells forms functional Ca(2+) release channels. Furthermore, the lack of localized Ca(2+) release events in these cells suggests that Ca(2+) sparks observed in cardiac muscle may involve cooperative gating of a group of Ca(2+) release channels and/or their interaction with muscle-specific proteins.