Imperatoxin a enhances Ca(2+) release in developing skeletal muscle containing ryanodine receptor type 3

Most adult mammalian skeletal muscles contain only one isoform of ryanodine receptor (RyR1), whereas neonatal muscles contain two isoforms (RyR1 and RyR3). Membrane depolarization fails to evoke calcium release in muscle cells lacking RyR1, demonstrating an essential role for this isoform in excitation-contraction coupling. In contrast, the role of RyR3 is unknown. We studied the participation of RyR3 in calcium release in wild type (containing both RyR1 and RyR3 isoforms) and RyR3-/- (containing only RyR1) myotubes in the presence or absence of imperatoxin A (IpTxa), a high-affinity agonist of ryanodine receptors. IpTxa significantly increased the amplitude and the rate of release only in wild-type myotubes. Calcium currents, recorded simultaneously with the transients, were not altered with IpTxa treatment. [(3)H]ryanodine binding to RyR1 or RyR3 was significantly increased in the presence of IpTxa. Additionally, IpTxa modified the gating and conductance level of single RyR1 or RyR3 channels when studied in lipid bilayers. Our data show that IpTxa can interact with both RyRs and that RyR3 is functional in myotubes and it can amplify the calcium release signal initiated by RyR1, perhaps through a calcium-induced mechanism. In addition, our data indicate that when RyR3-/- myotubes are voltage-clamped, the effect of IpTxa is not detected because RyR1s are under the control of the dihydropyridine receptor.     

Slow calcium signals after tetanic electrical stimulation in skeletal myotubes

The fluorescent calcium signal from rat myotubes in culture was monitored after field-stimulation with tetanic protocols. After the calcium signal sensitive to ryanodine and associated to the excitation-contraction coupling, a second long-lasting calcium signal refractory to ryanodine was consistently found. The onset kinetics of this slow signal were slightly modified in nominally calcium-free medium, as were both the frequency and number of pulses during tetanus. No signal was detected in the presence of tetrodotoxin. The participation of the dihydropyridine receptor (DHPR) as the voltage sensor for this signal was assessed by treatment with agonist and antagonist dihydropyridines (Bay K 8644 and nifedipine), showing an enhanced and inhibitory response, respectively. In the dysgenic GLT cell line, which lacks the alpha1(S) subunit of the DHPR, the signal was absent. Transfection of these cells with the alpha1(S) subunit restored the slow signal. In myotubes, the inositol 1,4,5-trisphosphate (IP(3)) mass increase induced by a tetanus protocol preceded in time the slow calcium signal. Both an IP(3) receptor blocker and a phospholipase C inhibitor (xestospongin C and U73122, respectively) dramatically inhibit this signal. Long-lasting, IP(3)-generated slow calcium signals appear to be a physiological response to activity-related fluctuations in membrane potential sensed by the DHPR.     

Focal sarcoplasmic reticulum calcium stores and diffuse inositol 1,4,5-trisphosphate and ryanodine receptors in human myometrium

Intracellular calcium stores of human uterine myocytes in primary and second passage cell culture were visualized using the low-affinity calcium-sensitive fluorescent dye, fluo-3FF. The calcium stores appeared as numerous small (0.2-0.5 microm diameter) focal fluorescences. The stores were not depleted by exposing the cells to oxytocin or ryanodine under standard conditions. The stores were rapidly depleted by oxytocin or ryanodine exposure when sarcoplasmic reticulum (SR) calcium re-uptake was inhibited by pretreatment with thapsigargin. Immunofluorescence experiments indicated that both ryanodine and inositol 1,4,5-trisphosphate (IP(3)) receptors were smoothly distributed throughout the SR, and neither receptor co-localized with the calcium stores. Since IP(3) and ryanodine calcium channels are tightly associated with their receptor, these results suggest that SR calcium release occurs via second messenger channels that are remote from the SR calcium stores. These observations are consistent only with a mechanism for release of calcium stores where the SR serves three functions: (1) as site of calcium storage, (2) as the structure that contains the IP(3)- and ryanodine receptors and their associated release channels, and (3) as a conduit between the calcium stores and the release channels.     

Homer modulates NFAT-dependent signaling during muscle differentiation

While changes in intracellular calcium are well known to influence muscle contraction through excitation contraction coupling, little is understood of the calcium signaling events regulating gene expression through the calcineurin/NFAT pathway in muscle. Here, we demonstrate that Ca(+2) released via the inositol trisphosphate receptor (IP3R) increases nuclear entry of NFAT in undifferentiated skeletal myoblasts, but the IP3R Ca(+2) pool in differentiated myotubes promotes nuclear exit of NFAT despite a comparable quantitative change in [Ca(+2)]i. In contrast, Ca(+2) released via ryanodine receptors (RYR) increases NFAT nuclear entry in myotubes. The scaffolding protein Homer, known to interact with both IP3R and RYR, is expressed as part of the myogenic differentiation program and enhances NFAT-dependent signaling by increasing RYR Ca(+2) release. These results demonstrate that differentiated skeletal myotubes employ discrete pools of intracellular calcium to restrain (IP3R pool) or activate (RYR pool) NFAT-dependent signaling, in a manner distinct from undifferentiated myoblasts. The selective expression of Homer proteins contributes to these differentiation-dependent features of calcium signaling.     

Role of sigma-1 receptor C-terminal segment in inositol 1,4,5-trisphosphate receptor activation: constitutive enhancement of calcium signaling in MCF-7 tumor cells

Sigma-1 receptor (sigma-1R) agonists enhance inositol 1,4,5-trisphosphate (IP3)-dependent calcium release from endoplasmic reticulum by inducing dissociation of ankyrin B 220 (ANK 220) from the IP3 receptor (IP3R-3), releasing it from inhibition. MCF-7 breast tumor cells express little or no sigma-1R and were used here to investigate the effect of receptor overexpression and the role of its N- and C-terminal segments in function. We stably expressed intact sigma-1R (amino acids (aa) 1-223; lines 11 and 41), N-fragment (aa 1-100; line K3), or C-fragment (aa 102-223; line sg101). C-fragment expressed as a peripheral membrane-bound protein that was removable from the endoplasmic reticulum membrane by chaotropic salt wash, consistent with lack of a putative transmembrane domain. The expressed sigma-1R, N-fragment, and C-fragment exhibited normal, low affinity, and no [3H](+)-pentazocine binding activity, respectively. All transfected lines showed constitutive enhancement of bradykinin (BDK)-induced calcium release, because of a decrease in BDK ED50 values. Interestingly, sigma-1R and C-fragment had high activities, whereas the N-fragment was much less active. The antagonist BD1063 behaved as an inverse agonist in sigma-1R cells, whereas C-fragment was insensitive to ligand regulation. Like BDK, vasopressin- and ATP-induced calcium release was enhanced with the same pattern in cell lines. Anti-IP3R-3 immunoprecipitates from cells expressing sigma-1R or C-fragment contained significantly less ANK 220 compared with untransfected or N-fragment cells, indicating a higher amount of ankyrin-free IP3R-3. Anti-ankyrin B immunoprecipitates contained sigma-1R or C-fragment, with markedly lower levels of N-fragment present. These results suggest that sigma-1R overexpression drives sigma agonist-independent dissociation of ANK 220 from IP3R-3, resulting in activation. The C-terminal segment plays a key role in the interaction.     

Functional expression of the calcium release channel from skeletal muscle ryanodine receptor cDNA

Combined patch-clamp and fura-2 measurements were performed to study the calcium release properties of Chinese hamster ovary (CHO) cells transfected with the rabbit skeletal muscle ryanodine receptor cDNA carried by an expression vector. Both caffeine (1-50 mM) and ryanodine (100 microM) induced release of calcium from intracellular stores of transformed CHO cells but not from control (non-transfected) CHO cells. The calcium responses to caffeine and ryanodine closely resembled those commonly observed in skeletal muscle. Repetitive applications of caffeine produced characteristic all-or-none rises in intracellular calcium. Inositol 1,4,5-trisphosphate (IP3) neither activated the ryanodine receptor channel nor interfered with the caffeine-elicited calcium release. These results indicate that functional calcium release channels are formed by expression of the ryanodine receptor cDNA.     

Calcium transients in 1B5 myotubes lacking ryanodine receptors are related to inositol trisphosphate receptors

Potassium depolarization of skeletal myotubes evokes slow calcium waves that are unrelated to contraction and involve the cell nucleus (Jaimovich, E., Reyes, R., Liberona, J. L., and Powell, J. A. (2000) Am. J. Physiol. 278, C998-C1010). Studies were done in both the 1B5 (Ry53-/-) murine "dyspedic" myoblast cell line, which does not express any ryanodine receptor isoforms (Moore, R. A., Nguyen, H., Galceran, J., Pessah, I. N., and Allen, P. D. (1998) J. Cell Biol. 140, 843-851), and C(2)C(12) cells, a myoblast cell line that expresses all three isoforms. Although 1B5 cells lack ryanodine binding, they bind tritiated inositol (1,4,5)-trisphosphate. Both type 1 and type 3 inositol trisphosphate receptors were immuno-located in the nuclei of both cell types and were visualized by Western blot analysis. After stimulation with 47 mm K(+), inositol trisphosphate mass raised transiently in both cell types. Both fast calcium increase and slow propagated calcium signals were seen in C(2)C(12) myotubes. However, 1B5 myotubes (as well as ryanodine-treated C(2)C(12) myotubes) displayed only a long-lasting, non-propagating calcium increase, particularly evident in the nuclei. Calcium signals in 1B5 myotubes were almost completely blocked by inhibitors of the inositol trisphosphate pathway: U73122, 2-aminoethoxydiphenyl borate, or xestospongin C. Results support the hypothesis that inositol trisphosphate mediates slow calcium signals in muscle cell ryanodine receptors, having a role in their time course and propagation.     

Properties of a new calcium-permeable single channel from tracheal microsomes

After the incorporation of the tracheal microsomal membrane into bilayer lipid membrane (BLM), a new single channel permeable for calcium was observed. Using the BLM conditions, 53 mM Ca2+ in trans solution versus 200 nM Ca2+ in cis solution, the single calcium channel current at 0 mV was 1.4-2.1 pA and conductance was 62-75 pS. The channel Ca2+/K+ permeability ratio was 4.8. The open probability (P-open) was in the range of 0.7-0.97. The P-open, measured at -10 mV to +30 mV (trans-cis), was not voltage dependent. The channel was neither inhibited by 10-20 microM ruthenium red, a specific blocker of ryanodine calcium release channel, nor by 10-50 microM heparin, a specific blocker of IP3 receptor calcium release channel, and its activity was not influenced by addition of 0.1 mM MgATP. We suggest that the observed new channel is permeable for calcium, and it is neither identical with the known type 1 or 2 ryanodine calcium release channel, nor type 1 or 2 IP3 receptor calcium release channel.     

Regulation of TRPC1 and TRPC4 Cation Channels Requires an ��1-Syntrophin-dependent Complex in Skeletal Mouse Myotubes

The dystrophin-associated protein complex (DAPC) is essential for skeletal muscle, and the lack of dystrophin in Duchenne muscular dystrophy results in a reduction of DAPC components such as syntrophins and in fiber necrosis. By anchoring various molecules, the syntrophins may confer a role in cell signaling to the DAPC. Calcium disorders and abnormally elevated cation influx in dystrophic muscle cells have suggested that the DAPC regulates some sarcolemmal cationic channels. We demonstrated previously that mini-dystrophin and α1-syntrophin restore normal cation entry in dystrophin-deficient myotubes and that sarcolemmal TRPC1 channels associate with dystrophin and the bound PDZ domain of α1-syntrophin. This study shows that small interfering RNA (siRNA) silencing of α1-syntrophin dysregulated cation influx in myotubes. Moreover, deletion of the PDZ-containing domain prevented restoration of normal cation entry by α1-syntrophin transfection in dystrophin-deficient myotubes. TRPC1 and TRPC4 channels are expressed at the sarcolemma of muscle cells; forced expression or siRNA silencing showed that cation influx regulated by α1-syntrophin is supported by TRPC1 and TRPC4. A molecular association was found between TRPC1 and TRPC4 channels and the α1-syntrophin-dystrophin complex. TRPC1 and TRPC4 channels may form sarcolemmal channels anchored to the DAPC, and α1-syntrophin is necessary to maintain the normal regulation of TRPC-supported cation entry in skeletal muscle. Cation channels with DAPC form a signaling complex that modulates cation entry and may be crucial for normal calcium homeostasis in skeletal muscles.     

Knocking down type 2 but not type 1 calsequestrin reduces calcium sequestration and release in C2C12 skeletal muscle myotubes

We examined the roles of type 1 and type 2 calsequestrins (CSQ1 and CSQ2) in stored Ca2+ release of C2C12 skeletal muscle myotubes. Transduction of C2C12 myoblasts with CSQ1 or CSQ2 small interfering RNAs effectively reduced the expression of targeted CSQ protein to near undetectable levels. As compared with control infected or CSQ1 knockdown myotubes, CSQ2 and CSQ1/CSQ2 knockdown myotubes had significantly reduced stored Ca2+ release evoked by activators of intracellular Ca2+ release channel/ryanodine receptor (10 mM caffeine, 200 microM 4-chloro-m-cresol, or 10 mM KCl). Thus, CSQ1 is not essential for effective stored Ca2+ release in C2C12 myotubes despite our in vitro studies suggesting that CSQ1 may enhance ryanodine receptor channel activity. To determine the basis of the reduced stored Ca2+ release in CSQ2 knockdown myotubes, we performed immunoblot analyses and found a significant reduction in both sarco/endoplasmic reticulum Ca2+-ATPase and skeletal muscle ryanodine receptor proteins in CSQ2 and CSQ1/CSQ2 knockdown myotubes. Moreover, these knockdown myotubes exhibited reduced Ca2+ uptake and reduced stored Ca2+ release by UTP (400 microM) that activates a different family of intracellular Ca2+ release channels (inositol 1,4,5-trisphosphate receptors). Taken together, our data suggest that knocking down CSQ2, but not CSQ1, leads to reduced Ca2+ storage and release in C2C12 myotubes.     

Type 1 and type 3 ryanodine receptors generate different Ca(2+) release event activity in both intact and permeabilized myotubes.

In this investigation we use a "dyspedic" myogenic cell line, which does not express any ryanodine receptor (RyR) isoform, to examine the local Ca(2+) release behavior of RyR3 and RyR1 in a homologous cellular system. Expression of RyR3 restored caffeine-sensitive, global Ca(2+) release and causes the appearance of relatively frequent, spontaneous, spatially localized elevations of [Ca(2+)], as well as occasional spontaneous, propagating Ca(2+) release, in both intact and saponin-permeabilized myotubes. Intact myotubes expressing RyR3 did not, however, respond to K(+) depolarization. Expression of RyR1 restored depolarization-induced global Ca(2+) release in intact myotubes and caffeine-induced global release in both intact and permeabilized myotubes. Both intact and permeabilized RyR1-expressing myotubes exhibited relatively infrequent spontaneous Ca(2+) release events. In intact myotubes, the frequency of occurrence and properties of these RyR1-induced events were not altered by partial K(+) depolarization or by application of nifedipine, suggesting that these RyR1 events are independent of the voltage sensor. The events seen in RyR1-expressing myotubes were spatially more extensive than those seen in RyR3-expressing myotubes; however, when analysis was limited to spatially restricted "Ca(2+) spark"-like events, events in RyR3-expressing myotubes were larger in amplitude and duration compared with those in RyR1. Thus, in this skeletal muscle context, differences exist in the spatiotemporal properties and frequency of occurrence of spontaneous release events generated by RyR1 and RyR3. These differences underscore functional differences between the Ca(2+) release behavior of RyR1 and RyR3 in this homologous expression system.     

Sodium-dependent action potentials induced by brevetoxin-3 trigger both IP3 increase and intracellular Ca2+ release in rat skeletal myotubes

Brevetoxin-3 (PbTx-3), described to increase the open probability of voltage-dependent sodium channels, caused trains of action potentials and fast oscillatory changes in fluorescence intensity of fluo-3-loaded rat skeletal muscle cells in primary culture, indicating that the toxin increased intracellular Ca(2+) levels. PbTx-3 did not elicit calcium transients in dysgenic myotubes (GLT cell line), lacking the alpha1 subunit of the dihydropyridine receptor (DHPR), but after transfection of the alpha1DHPR cDNA to GLT cells, PbTx-3 induced slow calcium transients that were similar to those of normal cells. Ca(2+) signals evoked by PbTx-3 were inhibited by blocking either IP(3) receptors, with 2-aminoethoxydiphenyl borate, or phospholipase C with U73122. PbTx-3 caused a tetrodotoxin-sensitive increase in intracellular IP(3) mass levels, dependent on extra-cellular Na(+). A similar increase in IP(3) mass was induced by high K(+) depolarization but no action potential trains (nor calcium signals) were elicited by prolonged depolarization under current clamp conditions. The increase in IP(3) mass induced by either PbTx-3 or K(+) was also detected in Ca(2+)-free medium. These results establish that the effect of the toxin on both intracellular Ca(2+) and IP(3) levels occurs via a membrane potential sensor instead of directly by Na(+) flux and supports the notion of a train of action potentials being more efficient as a stimulus than sustained depolarization, suggesting that tetanus is the physiological stimulus for the IP(3)-dependent calcium signal involved in regulation of gene expression.     

Altered stored calcium release in skeletal myotubes deficient of triadin and junctin

Triadin and junctin are integral sarcoplasmic reticulum membrane proteins that form a macromolecular complex with the skeletal muscle ryanodine receptor (RyR1) but their roles in skeletal muscle calcium homeostasis remain incompletely understood. Here we report that delivery of siRNAs specific for triadin or junctin into C2C12 skeletal myoblasts reduced the expression of triadin and junctin in 8-day-old myotubes by 80 and 100%, respectively. Knocking down either triadin or junctin in these cells reduced Ca2+ release induced by depolarization (10mM KCl) by 20-25%. Unlike triadin knockdown myotubes, junctin knockdown and junctin/triadin double knockdown myotubes also had reduced Ca2+ release induced by 400 microM 4-chloro-m-cresol, 10mM caffeine, 400 microM UTP, or 1 microM thapsigargin. Thus, knocking down junctin compromised the Ca2+ stores in the sarcoplasmic reticulum of these cells. Our subsequent studies showed that in junctin knockdown myotubes at least two sarcoplasmic reticulum proteins (RyR1 and skeletal muscle calsequestrin) were down-regulated while these proteins' mRNA expression was not affected. The results suggest that triadin has a role in facilitating KCl depolarization-induced Ca2+ release in contrast to junctin which has a role in maintaining sarcoplasmic reticulum Ca2+ store size in C2C12 myotubes.     

The role of calmodulin for inositol 1,4,5-trisphosphate receptor function

Intracellular calcium release is a fundamental signaling mechanism in all eukaryotic cells. The ryanodine receptor (RyR) and inositol 1,4,5-trisphosphate receptor (IP(3)R) are intracellular calcium release channels. Both channels can be regulated by calcium and calmodulin (CaM). In this review we will first discuss the role of calcium as an activator and inactivator of the IP(3)R, concluding that calcium is the most important regulator of the IP(3)R. In the second part we will further focus on the role of CaM as modulator of the IP(3)R, using results of the voltage-dependent Ca(2+) channels and the RyR as reference material. Here we conclude that despite the fact that different CaM-binding sites have been characterized, their function for the IP(3)R remains elusive. In the third part we will discuss the possible functional role of CaM in IP(3)-induced Ca(2+) release (IICR) by direct and indirect mechanisms. Special attention will be given to the Ca(2+)-binding proteins (CaBPs) that were shown to activate the IP(3)R in the absence of IP(3).     

Calcium mobilization is required for peroxynitrite-mediated enhancement of spontaneous transient outward currents in arteriolar smooth muscle cells

Transiently local release of Ca(2+) from the sarcoplasmic reticulum (SR) activates nearby Ca(2+)-activated K(+) channels to produce spontaneous transient outward currents (STOCs) in smooth muscle cells. The purpose of the present study was to investigate the possible effect of peroxynitrite (ONOO(-)) on STOCs in mesenteric arteriolar smooth muscle cells (ASMCs) and decide whether Ca(2+) mobilization was involved in STOCs alteration by ONOO(-). STOCs were recorded and characterized using the perforated whole-cell patch-clamp configuration. The results demonstrated that STOCs activity was greatly suppressed by removal of extracellular Ca(2+); by addition of nifedipine, a specific inhibitor of L-type voltage-gated Ca(2+) channels (VGCCs); or by addition of ryanodine, a SR ryanodine receptors (RyRs) blocker. In contrast, both caffeine, a RyR activator, and 2-aminoethoxydiphenylborate (2-APB), a membrane-permeable inositol 1,4,5-trisphosphate receptors, (IP3R) antagonist, increased STOCs activity. 3-morpholinosydnonimine (SIN-1), an ONOO(-) donor, at concentrations of 20-200 microM, induced a dose-dependent enhancement of STOCs in ASMCs and led to conspicuous increases in STOCs frequency and amplitude, which were prevented by prior exposure to low external Ca(2+) (200 nM), ryanodine (10 microM), or nifedipine (10 microM). In contrast, caffeine (0.5 mM) did not further stimulate STOCs in ASMCs preincubated with SIN-1, and pretreatment with 2-APB (50 microM) had little effect on ONOO(-) -induced STOCs activation. These findings suggest that complex Ca(2+)-mobilizing pathways, including external Ca2+ influx through VGCCs activation and subsequent internal Ca(2+) release through RyRs but not IP3Rs, are involved in ONOO(-)mediated STOCs enhancement in ASMCs.