Role of malignant hyperthermia domain in the regulation of Ca2+ release channel (ryanodine receptor) of skeletal muscle sarcoplasmic reticulum

A fusion protein encompassing Gly341 of the skeletal muscle ryanodine receptor was used to raise monoclonal antibodies; epitope mapping demonstrates that monoclonal antibody 419 (mAb419) reacts with a sequence a few residues upstream from Gly341. The mAb419 was then used to probe ryanodine receptor (RYR) functions. Our results show that upon incubation of triads vesicles with mAb419 the Ca2+-induced Ca2+ release rate at pCa 8 was increased. Equilibrium evaluation of [3H]ryanodine binding at different [Ca2+] indicates that mAb419 shifted the half-maximal [Ca2+] for stimulation of ryanodine binding to lower value (0.1 versus 1.2 microM). Such functional effects may be due to a direct action of the Ab on the Ca2+ binding domain of the RYR or to the perturbation by the Ab of the intramolecular interaction between the immunopositive region and regulatory domain of the RYR. The latter hypothesis was tested directly using the optical biosensor BIAcore (Pharmacia Biotech Inc.): we show that the immunopositive RYR polypeptide is able to interact with the native RYR complex. Ligand overlays with immunopositive digoxigenin-RYR fusion protein indicate that such an interaction might occur with a calmodulin binding domain (defined by residues 3010-3225) and with a polypeptide defined by residues 799-1172. In conclusion our results suggest that the stimulation by the mAb419 of the RYR channel activity is due to the perturbation of an intramolecular interaction between the immunopositive polypeptide and a Ca2+ regulatory site probably corresponding to a calmodulin binding domain.     

Functional properties of the ryanodine receptor type 3 (RyR3) Ca2+ release channel

Single-channel analysis of sarcoplasmic reticulum vesicles prepared from diaphragm muscle, which contains both RyR1 and RyR3 isoforms, revealed the presence of two functionally distinct ryanodine receptor calcium release channels. In addition to channels with properties typical of RyR1 channels, a second population of ryanodine-sensitive channels with properties distinct from those of RyR1 channels was observed. The novel channels displayed close-to-zero open-probability at nanomolar Ca2+ concentrations in the presence of 1 mM ATP, but were shifted to the open conformation by increasing Ca2+ to micromolar levels and were not inhibited at higher Ca2+ concentrations. These novel channels were sensitive to the stimulatory effects of cyclic adenosine 5'-diphosphoribose (cADPR). Detection of this second population of RyR channels in lipid bilayers was always associated with the presence of the RyR3 isoform in muscle preparations used for single-channel measurements and was abrogated by the knockout of the RyR3 gene in mice. Based on the above, we associated the novel population of channels with the RyR3 isoform of Ca2+ release channels. The functional properties of the RyR3 channels are in agreement with a potential qualitative contribution of this channel to Ca2+ release in skeletal muscle and in other tissues.     

Glutathione modulates ryanodine receptor from skeletal muscle sarcoplasmic reticulum. Evidence for redox regulation of the Ca2+ release mechanism

In this report, we demonstrate the ability of the cellular thiol glutathione to modulate the ryanodine receptor from skeletal muscle sarcoplasmic reticulum. Reduced glutathione (GSH) inhibited Ca2+-stimulated [3H]ryanodine binding to the sarcoplasmic reticulum and inhibited the single-channel gating activity of the reconstituted Ca2+ release channel. The effects of GSH on both the [3H]ryanodine binding and single-channel measurements were dose-dependent, exhibiting an IC50 of approximately 2.4 mM in binding experiments. Scatchard analysis demonstrated that GSH decreased the binding affinity of ryanodine for its receptor (increased Kd) and lowered the maximal binding occupancy (Bmax). In addition, GSH did not modify the Ca2+ dependence of [3H]ryanodine binding. In single-channel experiments, GSH (5-10 mM), added to the cis side of the bilayer lipid membrane, lowered the open probability (Po) of a Ca2+ (50 microM)-stimulated Ca2+ channel without modifying the single-channel conductance. Subsequent perfusion of the cis chamber with an identical buffer, containing 50 microM Ca2+ without GSH, re-established Ca2+-stimulated channel gating. GSH did not inhibit channel activity when added to the trans side of the bilayer lipid membrane. Similar to GSH, the thiol-reducing agents dithiothreitol and beta-mercaptoethanol also inhibited high affinity [3H]ryanodine binding to sarcoplasmic reticulum membranes. In contrast to GSH, glutathione disulfide (GSSG) was a potent stimulator of high affinity [3H]ryanodine binding and it also stimulated the activity of the reconstituted single Ca2+ release channel. These results provide direct evidence that glutathione interacts with reactive thiols associated with the Ca2+ release channel/ryanodine receptor complex, which are located on the cytoplasmic face of the SR, and support previous observations (Liu, G, Abramson, J. J., Zable, A. C., and Pessah, I. N. (1994) Mol. Pharmacol. 45, 189-200) that reactive thiols may be involved in the gating of the Ca2+ release channel.     

Modification of sulfhydryls of the skeletal muscle calcium release channel by organic mercurial compounds alters Ca2+ affinity of regulatory Ca2+ sites in single channel recordings and [3H]ryanodine binding

Two purified proteinaceous fungal elicitors, parasiticein (an alpha elicitin) and cryptogein (a beta elicitin), as well as a fungal cell wall-derived carbohydrate elicitor all rapidly activated a 48-kD kinase in tobacco suspension cells. The maximum activation of this kinase paralleled or preceded medium alkalization and activation of the defense gene phenylalanine ammonia-lyase (PAL). In addition, the two elicitins, which also induced hypersensitive cell death, activated a 44- and a 40-kD kinase with delayed kinetics. By contrast, the cell wall-derived elicitor only weakly activated the 44-kD kinase and failed to activate the 40-kD kinase. The size and substrate preference of the 48-kD kinase are reminiscent of the recently purified and cloned salicylic acid-induced protein (SIP) kinase, which is a member of the mitogen-activated protein kinase family. Antibodies raised against a peptide corresponding to the unique N terminus of SIP kinase immunoreacted with the 48-kD kinase activated by all three elicitors from Phytophthora spp. In addition, the cell wall elicitor and the salicylic acid-activated 48-kD kinase copurified through several chromatography steps and comigrated on two-dimensional gels. Based on these results, all three fungal elicitors appear to activate the SIP kinase. In addition, inhibition of SIP kinase activation by kinase inhibitors correlated with the suppression of cell wall elicitor-induced medium alkalization and PAL gene activation, suggesting a regulatory function for the SIP kinase in these defense responses.     

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.     

Relationship of low affinity [3]ryanodine binding sites to high affinity sites on the skeletal muscle Ca2+ release channel

Both high and low affinity binding sites for [3H]ryanodine exist in sarcoplasmic reticulum membranes derived from rabbit skeletal muscle. Negatively cooperative binding of [3H]ryanodine at one of four initially identical sites cannot account for some of the kinetic features of the binding to high and low affinity sites. The presence of excess unlabeled ryanodine greatly slows the rate at which [3H]ryanodine bound at the high affinity site dissociates. An examination of the rate of dissociation of [3H]ryanodine bound at increasing [3H]ryanodine concentrations reveals the existence of a second site, occupied only at high ligand concentrations. The occupation of this site correlates well with the conversion of the high affinity site from a site with a dissociation rate constant of approximately 0.0025 min-1 to one with a dissociation rate constant of less than 0.00025 min-1. The low affinity site itself has a dissociation rate constant of 0.013 min-1 and dissociation from this site is unaffected by the presence of 100 microM unlabeled ryanodine. These data suggest that the two binding sites are different but are either allosterically or sterically coupled. Association experiments support this interpretation. Low affinity binding sites for [3H]ryanodine exist in transverse tubule (t-tubule) as well as sarcoplasmic reticulum membranes. High concentrations of both ryanodine and ruthenium red inhibit the binding of [3H]PN200-110 to the dihydropyridine-binding protein in t-tubule membranes. Whether the low affinity site in t-tubule membranes is related to that found in sarcoplasmic reticulum membranes is not yet known.     

Influence of adrenergic agonists on the release of amino acids from rat skeletal muscle studied by microdialysis

Epidermal growth factor (EGF) has been considered to be a candidate for neurotrophic factors on the basis of the results of several in vitro studies. However, the in vivo effect of EGF on ischemic neurons as well as its mechanism of action have not been fully understood. In the present in vivo study using a gerbil ischemia-model, we examined the effects of EGF on ischemia-induced learning disability and hippocampal CA1 neuron damage. Cerebroventricular infusion of EGF (24 or 120 ng/d) for 7 days to gerbils starting 2 hours before or immediately after transient forebrain ischemia caused a significant prolongation of response latency time in a passive avoidance task in comparison with the response latency of vehicle-treated ischemic animals. Subsequent histologic examinations showed that EGF effectively prevented delayed neuronal death of CA1 neurons in the stratum pyramidale and preserved synapses intact within the strata moleculare, radiatum, and oriens of the hippocampal CA1 region. In situ detection of DNA fragmentation (TUNEL staining) revealed that ischemic animals infused with EGF contained fewer TUNEL-positive neurons in the hippocampal CA1 field than those infused with vehicle alone at the seventh day after ischemia. In primary hippocampal cultures, EGF (0.048 to 6.0 ng/mL) extended the survival of cultured neurons, facilitated neurite outgrowth, and prevented neuronal damage caused by the hydroxyl radical-producing agent FeSO4 and by the peroxynitrite-producing agent 3-morpholinosydnonimine in a dose-dependent manner. Moreover, EGF significantly attenuated FeSO4-induced lipid peroxidation of cultured neurons. These findings suggest that EGF has a neuroprotective effect on ischemic hippocampal neurons in vivo possibly through inhibition of free radical neurotoxicity and lipid peroxidation.     

Dual mechanisms of Mg2+ block of ryanodine receptor Ca2+ release channel from cardiac sarcoplasmic reticulum

We investigated the effects of cytosolic Mg2+ on ryanodine receptor Ca2+ release channel (RyR) of bovine cardiac sarcoplasmic reticulum incorporated into planar lipid bilayers recording single channel activities. Channels were activated by > or = 0.1 microM Ca2+ in the cis solution. At constant Ca2+, application of Mg2+ (0.1-1 mM) to cis side decreased channel activity in a concentration-dependent manner. A half maximal blocking concentration (Kd) was 35 microM and a complete block was obtained at 1 mM. In the presence of 1 mM free Mg2+ in cis solution, the relation between the channel open probability (Po) and concentration of free Ca2+ in cis solution ([Ca2+]cis) shifted to the right, indicating the competition of Mg2+ and Ca2+. Blocking effects of Mg2+ on RyR were antagonized by increasing [Ca2+]cis > or = 0.1 mM. In the presence of 1 m Mg2+ and 1 mM Ca2+ in cis solution, the channel conductance was markedly depressed to approximately 400 pS (n = 7) from 603 +/- 40 pS (mean +/- S.D., n = 22) in the absence of Mg2+, indicating the flickering block. These results show that Mg2+ causes a direct inhibition of RyR in cardiac SR and this inhibition may be mediated through two different mechanisms. A competition of Mg2+ and Ca2+ at a Ca2+ sensitive site on the RyR and a flickery block of the open channel by Mg2+.     

Dual regulation of the skeletal muscle ryanodine receptor by triadin and calsequestrin

Triadin, a calsequestrin-anchoring transmembrane protein of the sarcoplasmic reticulum (SR), was successfully purified from the heavy fraction of SR (HSR) of rabbit skeletal muscle with an anti-triadin immunoaffinity column. Since depletion of triadin from solubilized HSR with the column increased the [3H]ryanodine binding activity, we tested a possibility of triadin for a negative regulator of the ryanodine receptor/Ca2+ release channel (RyR). Purified triadin not only inhibited [3H]ryanodine binding to the solubilized HSR but also reduced openings of purified RyR incorporated into the planar lipid bilayers. On the other hand, calsequestrin, an endogenous activator of RyR [Kawasaki and Kasai (1994) Biochem. Biophys. Res. Commun. 199, 1120-1127; Ohkura et al. (1995) Can. J. Physiol. Pharmacol. 73, 1181-1185] potentiated [3H]ryanodine binding to the solubilized HSR. Ca2+ dependency of [3H]ryanodine binding to the solubilized HSR was reduced by triadin, whereas that was enhanced by calsequestrin. Interestingly, [3H]ryanodine binding to the solubilized HSR potentiated by calsequestrin was reduced by triadin. Immunostaining with anti-triadin antibody proved that calsequestrin inhibited the formation of oligomeric structure of triadin. These results suggest that triadin inhibits the RyR activity and that RyR is regulated by both triadin and calsequestrin, probably through an interaction between them. In this paper, triadin has been first demonstrated to have an inhibitory role in the regulatory mechanism of the RyR.     

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.     

Dantrolene inhibition of sarcoplasmic reticulum Ca2+ release by direct and specific action at skeletal muscle ryanodine receptors

The skeletal muscle relaxant dantrolene inhibits the release of Ca2+ from the sarcoplasmic reticulum during excitation-contraction coupling and suppresses the uncontrolled Ca2+ release that underlies the skeletal muscle pharmacogenetic disorder malignant hyperthermia; however, the molecular mechanism by which dantrolene selectively affects skeletal muscle Ca2+ regulation remains to be defined. Here we provide evidence of a high-affinity, monophasic inhibition by dantrolene of ryanodine receptor Ca2+ channel function in isolated sarcoplasmic reticulum vesicles prepared from malignant hyperthermia-susceptible and normal pig skeletal muscle. In media simulating resting myoplasm, dantrolene increased the half-time for 45Ca2+ release from both malignant hyperthermia and normal vesicles approximately 3.5-fold and inhibited sarcoplasmic reticulum vesicle [3H]ryanodine binding (Ki approximately 150 nM for both malignant hyperthermia and normal). Inhibition of vesicle [3H]ryanodine binding by dantrolene was associated with a decrease in the extent of activation by both calmodulin and Ca2+. Dantrolene also inhibited [3H]ryanodine binding to purified skeletal muscle ryanodine receptor protein reconstituted into liposomes. In contrast, cardiac sarcoplasmic reticulum vesicle 45Ca2+ release and [3H]ryanodine binding were unaffected by dantrolene. Together, these results demonstrate selective effects of dantrolene on skeletal muscle ryanodine receptors that are consistent with the actions of dantrolene in vivo and suggest a mechanism of action in which dantrolene may act directly at the skeletal muscle ryanodine receptor complex to limit its activation by calmodulin and Ca2+. The potential implications of these results for understanding how dantrolene and malignant hyperthermia mutations may affect the voltage-dependent activation of Ca2+ release in intact skeletal muscle are discussed.     

Role of ryanodine receptors in the assembly of calcium release units in skeletal muscle

In muscle cells, excitation-contraction (e-c) coupling is mediated by "calcium release units," junctions between the sarcoplasmic reticulum (SR) and exterior membranes. Two proteins, which face each other, are known to functionally interact in those structures: the ryanodine receptors (RyRs), or SR calcium release channels, and the dihydropyridine receptors (DHPRs), or L-type calcium channels of exterior membranes. In skeletal muscle, DHPRs form tetrads, groups of four receptors, and tetrads are organized in arrays that face arrays of feet (or RyRs). Triadin is a protein of the SR located at the SR-exterior membrane junctions, whose role is not known. We have structurally characterized calcium release units in a skeletal muscle cell line (1B5) lacking Ry1R. Using immunohistochemistry and freeze-fracture electron microscopy, we find that DHPR and triadin are clustered in foci in differentiating 1B5 cells. Thin section electron microscopy reveals numerous SR-exterior membrane junctions lacking foot structures (dyspedic). These results suggest that components other than Ry1Rs are responsible for targeting DHPRs and triadin to junctional regions. However, DHPRs in 1B5 cells are not grouped into tetrads as in normal skeletal muscle cells suggesting that anchoring to Ry1Rs is necessary for positioning DHPRs into ordered arrays of tetrads. This hypothesis is confirmed by finding a "restoration of tetrads" in junctional domains of surface membranes after transfection of 1B5 cells with cDNA encoding for Ry1R.     

FK-binding protein is associated with the ryanodine receptor of skeletal muscle in vertebrate animals

The ryanodine receptor/calcium release channel (RyR1) of sarcoplasmic reticulum from rabbit skeletal muscle terminal cisternae (TC) contains four tightly associated FK506-binding proteins (FKBP12). Dissociation and reconstitution studies have shown that RyR1 can be modulated by FKBP12, which helps to maintain the channel in the quiescent state. In this study, we found that the association of FKBP with RyR1 of skeletal muscle is common to each of the five classes of vertebrates. TC from skeletal muscle representing animals from different vertebrates, i.e. mammals (rabbit), birds (chicken), reptiles (turtle), fish (salmon and rainbow trout), and amphibians (frog), were isolated. For each, we find the following: 1) FKBP12 is localized to the TC (there are four FKBP binding sites/ryanodine receptor); 2) soluble FKBP exchanges with the bound form on RyR1 of TC; 3) release of FKBP from terminal cisternae by drug (FK590) treatment leads to a significant reduction in the net calcium loading rate, consistent with channel activation (the calcium loading rate is restored to the control value by reconstitution with FKBP12); and 4) RyR1 of skeletal muscle TC can bind to and exchange with either FKBP12 or FKBP12.6 (FKBP12.6 is the novel FKBP isoform found selectively associated with RyR2 of dog cardiac sarcoplasmic reticulum). We conclude that FKBP is an integral part of the RyR1 of skeletal muscle in each of the classes of vertebrate animals. The studies are consistent with a role for FKBP in skeletal muscle excitation-contraction coupling.     

Ca2+ release from the sarcoplasmic reticulum compared in amphibian and mammalian skeletal muscle

This paper describes and evaluates a method for quantifying the amounts of specific plasma proteins adsorbed to biomaterial surfaces. In particular, it demonstrates that macroscopic images ('stains'), that assess the spatial distribution of albumin, IgG, fibrinogen, and HMK (high molecular weight kininogen), can be obtained over areas of at least 12 cm2 using immunospecific adhesion of dyed polystyrene beads. Stain intensities, measured with a scanner and an image analysis system, were found to quantify the amount of specific protein in the solution used to coat the surfaces. Results obtained with the proposed method produced single protein isotherms for albumin, immunoglobulin G (IgG) and fibrinogen that followed Langmuir-like adsorption behavior and were similar to previously published isotherms. The HMK isotherm also exhibited Langmuir-like adsorption behavior. The proposed method also detected the presence of an expected maximum in the adsorption of fibrinogen onto glass as a function of plasma dilution. Adsorption of fibrinogen out of 6.4% plasma onto glass from a separated flow produced results indicating the quantity as well as the location of fibrinogen at the boundary of the separated region. This result confirmed the utility of the proposed method for detecting spatial distributions of specific proteins adsorbed from plasma in practical devices.     

Regulation of the cardiac ryanodine receptor channel by luminal Ca2+ involves luminal Ca2+ sensing sites

Flare and hyperalgesia after intradermal capsaicin injection in human skin. J. Neurophysiol. 80: 2801-2810, 1998. We investigated the neurovascular mechanisms that determine the flare response to intradermal capsaicin injection in humans and delineated the associated areas of mechanical and heat hyperalgesia. The flare response was monitored both visually and with infrared telethermography. The areas of mechanical and heat hyperalgesia were determined psychophysically. Thermography detected very large areas of flare. As an early event underlying the flare and before onset of the area of rubor of the skin, thermography detected the appearance of multifocal spots of increased temperature caused by dilatation of cutaneous arterioles. Repetition of capsaicin injection days apart into the same forearm induced multifocal spots of temperature elevation identical to the ones obtained in the first session, indicating dilatation of the same arterioles. Reactive hyperemia also consisted in the appearance of multifocal spots of increased temperature, which were identical to the ones reacting during the flare response, suggesting participation of the same arterioles in both events. Strips of local anesthetic placed to block cutaneous nerves prevented the spread of both the thermographic flare and associated hyperalgesia. It is inferred that the cutaneous nerve fibers responsible for the thermographic flare branch, or have coupled axons, over a long distance. The large area of flare coincided with the area of mechanical and heat hyperalgesia. Equivalence of the areas of flare and mechanical and heat hyperalgesia induced by intradermal capsaicin injection suggests that all three phenomena are the consequence of neural factors that operate peripherally.     

Regulation of mouse skeletal muscle L-type Ca2+ channel by activation of the insulin-like growth factor-1 receptor

We investigated the modulation of the skeletal muscle L-type Ca2+ channel/dihydropyridine receptor in response to insulin-like growth factor-1 receptor (IGF-1R) activation in single extensor digitorum longus muscle fibers from adult C57BL/6 mice. The L-type Ca2+ channel activity in its dual role as a voltage sensor and a selective Ca2+-conducting pore was recorded in voltage-clamp conditions. Peak Ca2+ current amplitude consistently increased after exposure to 20 ng/ml IGF-1 (EC50 = 5.6 +/- 1.8 nM). Peak IGF-1 effect on current amplitude at -20 mV was 210 +/- 18% of the control. Ca2+ current potentiation resulted from a shift in 13 mV of the Ca2+ current-voltage relationship toward more negative potentials. The IGF-1-induced facilitation of the Ca2+ current was not associated with an effect on charge movement amplitude and/or voltage distribution. These phenomena suggest that the L-type Ca2+ channel structures involved in voltage sensing are not involved in the response to the growth factor. The modulatory effect of IGF-1 on L-type Ca2+ channel was blocked by tyrosine kinase and PKC inhibitors, but not by a cAMP-dependent protein kinase inhibitor. IGF-1-dependent phosphorylation of the L-type Ca2+ channel alpha1 subunit was demonstrated by incorporation of [gamma-32P]ATP to monolayers of adult fast-twitch skeletal muscles. IGF-1 induced phosphorylation of a protein at the 165 kDa band, corresponding to the L-type Ca2+ channel alpha1 subunit. These results show that the activation of the IGF-1R facilitates skeletal muscle L-type Ca2+ channel activity via a PKC-dependent phosphorylation mechanism.     

Cyclic ADP-ribose as an endogenous regulator of the non-skeletal type ryanodine receptor Ca2+ channel

The skeletal and cardiac isoforms of the ryanodine receptor Ca2+ channel (RyRC) constitute the Ca2+ release pathway in sarcoplasmic reticulum of skeletal and cardiac muscles, respectively. A direct mechanical and a Ca(2+)-triggered mechanism (Ca(2+)-induced Ca2+ release) have been respectively proposed to explain the in situ activation of Ca2+ release in skeletal and cardiac muscle. In non-muscle cells, however, where the RyRC also participates in Ca2+ signalling, the mechanism of RyRC activation is unknown. Cyclic adenosine 5'-diphosphoribose (cADPR), which is present in many mammalian tissues, has been reported to induce Ca2+ release from ryanodine-sensitive intracellular Ca2+ stores in sea urchin eggs. Here we provide evidence that cADPR directly activates the cardiac but not the skeletal isoform of the RyRC. This, together with results on sea urchin eggs, suggests that cADPR is an endogenous activator of the non-skeletal type of RyRC and may thus have a role similar to inositol 1,4,5-trisphosphate in Ca2+ signalling.     

Regulation of glutamate dehydrogenase by branched-chain amino acids in skeletal muscle from rats and chicks

Neurotrophin-4 (NT-4) is a member of a family of neurotrophic factors, the neurotrophins, that control survival and differentiation of vertebrate neurons (2-4). Besides being the most recently discovered neurotrophin in mammals, and the least well understood, several aspects distinguish NT-4 from other members of the neurotrophin family. It is the most divergent member and, in contrast to the other neurotrophins, its expression is ubiquitous and appears to be less influenced by environmental signals. NT-4 seems to have the unique requirement of binding to the low-affinity neurotrophin receptor (p75LNGFR) for efficient signalling and retrograde transport in neurons. Moreover, while all other neurotrophin knock-outs have proven lethal during early postnatal development, mice deficient in NT-4 have so far only shown minor cellular deficits and develop normally to adulthood. Is NT-4 a recent addition to the neurotrophic factor repertoire in search of a crucial function, or is it an evolutionary relic, a kind of wisdom tooth of the neurotrophin family?     

Uridine triphosphate-sensitive pathway of Ca2+ release from the sarcoplasmic reticulum of rat skeletal muscle fibers

The pyrimidine nucleotide, uridine triphosphate (UTP), was tested with skinned skeletal muscle fibers in order to investigate the UTP-sensitive pathway of Ca2+ release from the sarcoplasmic reticulum. The presence of ryanodine (200 microM), ruthenium red (10 microM) or heparin (2.5 mg/ml) did not affect the tension elicited in the presence of UTP, demonstrating that the UTP-induced Ca2+ release involved neither ryanodine nor inositol triphosphate-sensitive channels. Drugs such as compound 48/80 or cyclopiazonic acid used to inhibit Ca2+-ATPase in its reverse function appeared to be, respectively, non-specific or without any inhibitory effect on the tension induced by UTP. Finally, the UTP-induced tension as well as the trifluoperazine-induced tension were abolished in the presence of spermidine (50 mM), supporting the hypothesis that the UTP-sensitive pathway of the SR Ca2+ release might occur through the uncoupled calcium ATPase.     

Locations of calmodulin and FK506-binding protein on the three-dimensional architecture of the skeletal muscle ryanodine receptor

Isolated skeletal muscle ryanodine receptors (RyRs) complexed with the modulatory ligands, calmodulin (CaM) or 12-kDa FK506-binding protein (FKBP12), have been characterized by electron cryomicroscopy and three-dimensional reconstruction. RyRs are composed of 4 large subunits (molecular mass 565 kDa) that assemble to form a 4-fold symmetric complex that, architecturally, comprises two major substructures, a large ( approximately 80% of the total mass) cytoplasmic assembly and a smaller transmembrane assembly. Both CaM and FKBP12 bind to the cytoplasmic assembly at sites that are 10 and 12 nm, respectively, from the putative entrance to the transmembrane ion channel. FKBP12 binds along the edge of the square-shaped cytoplasmic assembly near the face that interacts in vivo with the sarcolemma/transverse tubule membrane system, whereas CaM binds within a cleft that faces the junctional face of the sarcoplasmic reticulum membrane at the triad junction. Both ligands interact with a domain that connects directly to a cytoplasmic extension of the transmembrane assembly of the receptor, and thus might cause structural changes in the domain which in turn modulate channel gating.