Effect of temperature on [3H]ryanodine binding to sarcoplasmic reticulum from bullfrog skeletal muscle

It has been clarified that ryanodine binds to Ca2(+)-induced Ca release channels in the open state in sarcoplasmic reticulum. While the pharmacological action of ryanodine is known to be retarded at a low temperature, the Ca-releasing action of caffeine is potentiated at a low temperature. In order to obtain deeper insight into the molecular mechanism underlying Ca-release, the effect of temperature on ryanodine binding to the heavy fraction of sarcoplasmic reticulum (HFSR) from bullfrog skeletal muscle was examined. Although Ca2+ is indispensable for ryanodine binding, Ca2+ alone cannot cause ryanodine binding in a reaction medium of a salt concentration similar to that of the sarcoplasm. In addition to Ca2+, caffeine and/or beta,gamma-methylene adenosine triphosphate (AMPOPCP) are necessary. [3H]Ryanodine binding at 25 degrees C closely paralleled the Ca release activity in respect of the Ca2(+)-dependence in the presence of caffeine and/or AMPOPCP, and the effects of inhibitors. A Scatchard plot for ryanodine binding gave a straight linear line, indicating a single class of homogeneous binding sites. At 0 degrees C, the rate of ryanodine binding decreased. Q10 being about 3 on average. The affinity for ryanodine was reduced to about half that at 25 degrees C, with no change in the maximum number of binding sites. The temperature-dependent change in apparent affinity for Ca2+ on ryanodine binding is not always consistent with that in the case of Ca-release activity. The bound ryanodine may be in an occluded state because it did not dissociate for up to 90 h at 0 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of properties of Ca2+ release channels between rabbit and frog skeletal muscles

Biochemical investigation of Ca2+ release channel proteins has been carried out mainly with rabbit skeletal muscles, while frog skeletal muscles have been preferentially used for physiological investigation of Ca2+ release. In this review, we compared the properties of ryanodine receptors (RyR), Ca2+ release channel protein, in skeletal muscles between rabbit and frog. While the Ryr1 isoform is the main RyR of rabbit skeletal muscles, two isoforms, - and -RyR which are homologous to Ryr1 and Ryr3 isoforms in mammals, respectively, coexist as a homotetramer in a similar amount in frog skeletal muscles. The two isoforms in an isotonic medium show very similar property in [3H]ryanodine binding activity which is parallel to Ca2+-induced Ca2+ release (CICR) activity, and make independent contributions to the activities of the sarcoplasmic reticulum. CICR and [3H]ryanodine binding activities of rabbit and frog are qualitatively similar in stimulation by Ca2+, adenine nucleotide and caffeine, however, they showed the following quantitative differences. First, rabbit RyR showed higher Ca2+ affinity than the frog. Second, rabbit RyR showed higher activity in the presence of Ca2+ alone with less stimulation by adenine nucleotide than the frog. Third, rabbit RyR displayed less enhancement of [3H]ryanodine binding by caffeine in spite of having a similar magnitude of Ca2+ sensitization than the frog, which may explain the occasional difficulty by researchers to demonstrate caffeine contracture with mammalian skeletal muscles. Finally, but not least, rabbit RyR still showed marked inhibition of [3H]ryanodine binding in the presence of high Ca2+ concentrations in the 1 M NaCl medium, while frog RyR showed disinhibition. Other matters relevant to Ca2+ release were also discussed.     

Ryanodine Inhibits Caffeine-Evoked Ca2+ Mobilization and Catecholamine Secretion from Cultured Bovine Adrenal Chromaffin Cells

The effects of ryanodine, a selective inhibitor of the Ca(2+)-induced Ca2+ release mechanism, on caffeine-evoked changes in cytosolic Ca2+ concentration ([Ca2+]i) and catecholamine secretion were investigated using cultured bovine adrenal chromaffin cells. Caffeine (5-40 mM) caused a concentration-dependent transient rise in [Ca2+]i and catecholamine secretion in Ca2+/Mg(2+)-free medium containing 0.2 mM EGTA. Ryanodine (5 x 10(-5) M) alone had no effect on either [Ca2+]i or catecholamine secretion. Although the application of ryanodine plus caffeine caused the same increase in both [Ca2+]i and catecholamine secretion as those induced by caffeine alone, ryanodine (4 x 10(-7) - 5 x 10(-5) M) irreversibly prevented the increase in both [Ca2+]i and catecholamine secretion resulting from subsequent caffeine application over a range of concentrations. The secretory response to caffeine was markedly enhanced by replacement of Na+ with sucrose in Ca2+/Mg(2+)-free medium, and this enhanced response was also blocked by ryanodine. Caffeine was found to decrease the susceptibility of the secretory apparatus to Ca2+ in digitonin-permeabilized cells. These results indicate that caffeine mobilizes Ca2+ from intracellular stores, the function of which is irreversibly blocked by ryanodine, resulting in the increase in catecholamine secretion in the bovine adrenal chromaffin cell.     

Purification and characterization of two ryanodine-binding protein isoforms from sarcoplasmic reticulum of bullfrog skeletal muscle.

The two ryanodine-binding proteins (RyBPs) have been purified from sarcoplasmic reticulum of bullfrog skeletal muscle by Mono Q column chromatography following solubilization of SR by CHAPS and sucrose density gradient centrifugation. We conclude that the two RyBPs (alpha- and beta-RyBP) are isoforms on the basis (i) that each RyBP is distinguished by a specific polyclonal antibody and (ii) that distinct polypeptides are generated by limited tryptic digestion of the two RyBPs. Monomeric molecular weights for alpha- and beta-RyBP are estimated to be (690 +/- 10) and (570 +/- 10) kDa, respectively, as determined from mobilities on disc SDS-PAGE using the Weber-Osborn buffer system without 6 M urea, which gives an estimate of (590 +/- 10) kDa for RyBP of rabbit skeletal muscle. Similar determination in the presence of 6 M urea gave 630 kDa for alpha-RyBP and unchanged estimates for the other RyBPs. Both RyBPs show [3H]ryanodine-binding activities which are activated by Ca2+, AMPOPCP, and caffeine, and inhibited by ruthenium red, MgCl2, and procaine. beta-RyBP, however, has higher affinity for Ca2+. In the presence of Ca2+ and AMPOPCP, both RyBPs show single homogeneous binding sites for [3H]ryanodine with Kd = 2-5 nM. The values of Bmax for alpha- and beta-RyBP were 320-340 and 320-375 pmol/mg protein, respectively. These results are consistent with the conclusion that a homo-tetramer of each RyBP binds one ryanodine molecule, taking account of the estimated molecular weight.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of Ca(2+)-induced Ca2+ release channel activity from cardiac ryanodine receptor cDNA in Chinese hamster ovary cells.

We constructed an expression plasmid (pMAMCRR51) that carried the entire protein-coding sequence of the rabbit cardiac ryanodine receptor cDNA, linked to the dexamethasone-inducible mouse mammary tumor virus promoter and Escherichia coli xanthine-guanine phosphoribosyltransferase (gpt). Chinese hamster ovary (CHO) cells were transfected with pMAMCRR51 and mycophenolic acid-resistant cells showing caffeine-induced intracellular Ca2+ transients were selected. Immunoprecipitation with a monoclonal antibody against the canine cardiac ryanodine receptor revealed that the cell clones thus selected exhibited Ca(2+)-dependent [3H]ryanodine binding activity, which was stimulated by 5 mM ATP or 1 M KCl. The apparent dissociation constant (Kd) for [3H]ryanodine was 6.6 nM in 1 M KCl, which was similar to the Kd obtained with cardiac microsomes. Immunoprecipitation also demonstrated that these cell clones expressed a protein indistinguishable in M(r) from the ryanodine receptor in canine cardiac microsomes. The ryanodine binding activity expressed in CHO cells increased significantly after dexamethasone induction. In saponin-skinned CHO cells transfected with pMAMCRR51, micromolar Ca2+ or millimolar caffeine evoked rapid Ca2+ release from the intracellular Ca2+ stores. In skinned control CHO cells, we did not observe such Ca2+ release activity. These results clearly demonstrate that the cardiac ryanodine receptor is stably expressed in internal membranes of CHO cells and functions as Ca(2+)-induced Ca2+ release channels.     

Cooperative interaction between Ca2+ and beta,gamma-methylene adenosine triphosphate in their binding to fragmented sarcoplasmic reticulum from bullfrog skeletal muscle

In order to obtain a better understanding of the mechanism of the function of fragmented sarcoplasmic reticulum (FSR), we examined the binding of beta,gamma-methylene [3H]adenosine triphosphate (AMPOPCP), an unhydrolyzable ATP analogue, and 45Ca to FSR from bullfrog skeletal muscle. In medium containing 100 mM KCl and 20 mM Tris-maleate (pH 6.80) on ice, FSR has a single class of [3H]AMPOPCP-binding sites which amount to 4.4-8.6 nmol/mg protein (usually about 7 nmol/mg protein). The affinity was in the range of 6.2-12.3 X 10(3) M-1 in the absence of Ca2+. Ca2+ increased the affinity for AMPOPCP without changing the total number of binding sites, whereas Mg2+ decreased it. The change of the affinity is due to the direct effect of Ca2+ and Mg2+ on FSR. The possibility that Mg-AMPOPCP, Ca-AMPOPCP, and free AMPOPCP might have different affinities to FSR is excluded. The extent of Ca2+-induced enhancement in AMPOPCP binding is dependent not only on Ca2+ concentration but also on the concentration of AMPOPCP. The binding sites for AMPOPCP are likely to be the ATP-binding sites on Ca2+-ATPase protein on the basis of several lines of evidence, including competition between ATP, ADP, or AMP. FSR also binds 7-13 nmol Ca/mg protein (usually about 8 nmol/mg protein) with the affinity of 4-14 X 10(4) M-1 in the absence of the nucleotide in a similar medium containing 4 mM MgCl2. The ratio of Ca-binding sites to AMPOPCP-binding sites is mostly 1, but occasionally 2, corresponding to the ratio of Ca accumulated to ATP hydrolyzed by frog FSR. In the presence of a sufficient amount of the nucleotide, the affinity for Ca2+ was also increased. These findings are well explained by the random sequence binding model of Ca2+ and AMPOPCP, which bind to FSR with positive cooperative interaction between them. However, high concentrations of the nucleotide result in a negative cooperative interaction in the nucleotide binding in the presence of Ca2+, whereas no cooperativity is observed in the absence of Ca2+. Stimulation of Ca binding by AMPOPCP is also correspondingly affected. Comparative studies show that rabbit skeletal muscle FSR, in contrast to the frog one, shows negative cooperativity in its interactions with Ca2+ and AMPOPCP under some conditions and that the ratio of Ca-binding sites to AMPOPCP-binding sites is 2, corresponding to the well-known stoichiometry with ATP.     

Changes in ryanodine receptor-mediated calcium release during skeletal muscle differentiation.

We have observed a disparity between the actions of caffeine and ryanodine, two agents known to affect the same site of intracellular calcium (Ca2+) release in muscle. The site of intracellular Ca2+ release, the ryanodine receptor (RyR), is established as the route of Ca2+ movement from the sarcoplasmic reticulum (SR) to the cytosol during excitation-contraction coupling. We measured Ca2+ release fluorimetrically in both saponin-permeabilized and intact L6 cells, in response to known modulators (i.e., caffeine and ryanodine), during differentiation in vitro. The undifferentiated L6 cells showed little response to caffeine. However, a substantial caffeine-induced calcium release (caffCR) was evident by Day 3 of differentiation, and was nearly maximal by Day 7 of differentiation. By contrast, ryanodine failed to stimulate Ca2+ release until Day 4, lagging behind the caffeine response. Ryanodine-stimulated Ca2+ release was also maximal by Day 7. Higher concentrations of ryanodine, known to inhibit Ca2+ release, only began to affect caffCR at Day 4, indicating that cells were insensitive to both ryanodine stimulation and ryanodine inhibition prior to this time. Most of the results could be obtained both in permeabilized and intact cells. Using intact cells, we measured the time course of K+ -dependent (i.e., depolarization-induced) Ca2+ release. This time course matched caffeine and not ryanodine-induced Ca2+ release suggesting the action of caffeine was not due to Ca2+ release unrelated to excitation-contraction coupling. These findings suggest that ryanodine binding sites on the RyR may not be functional at early stages of muscle development, that ryanodine sensitivity is a poor indicator of Ca2+ flux through the RyR, or that other proteins are involved in Ca2+ release under certain circumstances.     

Trypsin destruction of the high affinity ryanodine binding sites of the junctional sarcoplasmic reticulum

Tryptic digestion of the junctional sarcoplasmic reticulum membranes in sucrose but not NaCl buffer leads to complete loss of ryanodine binding capacity. The presence of MgCl2 in the sucrose buffer prevents the loss of ryanodine binding by the trypsin treatment. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the treated membranes reveal that the 400-kDa protein band disappeared under all the different digestion conditions. However, the presence of 135-kDa tryptic fragment is observed only when ryanodine binding is retained. Quantitative analysis of the gels shows that the loss of ryanodine binding is well correlated with the cleavage of the 135-kDa tryptic fragment. This correlation is obtained when the cleavage was controlled either by the digestion time or by NaCl or MgCl2 concentrations. The same concentrations of MgCl2 and NaCl affect the ryanodine binding activity, the cleavage of the 135-kDa tryptic fragment, and the solubility and stability of the [3H]ryanodine-receptor complex in a detergent-containing medium. Tryptic digestion of the ryanodine receptor/junctional Ca2+ release channel, which leads to complete loss of ryanodine binding capacity, has no effect or slightly stimulates the Ca2+ accumulation activity of these membranes.     

Mechanism of anthraquinone-induced calcium release from skeletal muscle sarcoplasmic reticulum

The anthraquinones, doxorubicin, mitoxantrone, daunorubicin and rubidazone are shown to be potent stimulators of Ca2+ release from skeletal muscle sarcoplasmic reticulum (SR) vesicles and to trigger transient contractions in chemically skinned psoas muscle fibers. These effects of anthraquinones are the direct consequence of their specific interaction with the [3H] ryanodine receptor complex, which constitutes the Ca2+ release channel from the triadic junction. In the presence of adenine nucleotides and physiological Mg2+ concentrations (approximately 1.0 mM), channel activation by doxorubicin and daunorubicin exhibits a sharp dependence on submicromolar Ca2+ which is accompanied by a selective, dose-dependent increase in the apparent affinity of the ryanodine binding sites for Ca2+, in a manner similar to that previously reported with caffeine. Unlike caffeine, however, anthraquinones increase [3H]ryanodine receptor occupancy to the level observed in the presence of adenine nucleotides. A strong interaction between the anthraquinone and the caffeine binding sites on the Ca2+ release channel is also observed when monitoring Ca2+ fluxes across the SR. Millimolar caffeine both inhibits anthraquinone-stimulated Ca2+ release, and reduces anthraquinone-stimulated [3H]ryanodine receptor occupancy, without changing the effective binding constant of the anthraquinone for its binding site. The degree of cooperativity for daunorubicin activation of Ca2+ release from SR also increases in the presence of caffeine. These results demonstrate that the mechanism by which anthraquinones stimulate Ca2+ release is caused by a direct interaction with the [3H]ryanodine receptor complex, and by sensitization of the Ca2+ activator site for Ca2+.     

Synergistic release of calcium in sea urchin eggs by caffeine and ryanodine.

A transient rise in intracellular Ca2+ during fertilization is necessary for activation of the quiescent sea urchin egg. Several mechanisms contribute to the rise in Ca2+ including influx across the egg plasma membrane and release from intracellular stores. The egg contains both IP3-sensitive and -insensitive Ca2+ release mechanisms and in this study we have used single-cell spectrofluorimetry to examine the effects of caffeine and ryanodine on Ca2+ release in eggs preloaded with fura 2. Caffeine induced a small Ca2+ release that was insensitive to heparin or ruthenium red. Ca2+ liberation by caffeine could be augmented by prior treatment with thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ ATPase. Variable Ca2+ releases were observed in response to microinjection of ryanodine. The action of ryanodine appeared to be enhanced by prior injection of heparin and partially inhibited by ruthenium red. The release of Ca2+ by caffeine or ryanodine was generally insufficient to trigger cortical granule exocytosis, thus these eggs could be fertilized and a second Ca2+ release during fertilization was measured. Unlike the caffeine- and ryanodine-sensitive Ca(2+)-induced Ca2+ release mechanism in somatic cells, the graded responses in eggs suggested this caffeine- and ryanodine-sensitive release mechanism is not sensitive to sudden changes in Ca2+. Thus we could examine the combined actions of caffeine and ryanodine on Ca2+ release, which were synergistic. Caffeine treatment of ryanodine-injected eggs or ryanodine injection of caffeine-treated eggs stimulated a Ca2+ release significantly larger than the release by either drug independently. The experiments presented here suggest that sea urchin eggs liberate Ca2+ in response to caffeine and ryanodine; however, the regulation of this release differs from that described for caffeine- and ryanodine-sensitive Ca(2+)-induced Ca2+ release of somatic cells.     

Novel ryanodine-binding properties in mammalian retina

The ryanodine receptor (RyR)/Ca2+ release channel mobilizes Ca2+ from internal calcium stores to support a variety of neuronal functions. To investigate the presence of such a protein in mammalian retina, we applied ryanodine binding, PCR and antibodies against known RyRs. Surprisingly, ryanodine-binding properties of retinal endoplasmic reticulum-enriched membrane fraction were vastly different from those of skeletal and cardiac muscles ryanodine-binding proteins. In common with the skeletal and cardiac muscle, ryanodine bound with high-affinity to two or more types of binding site (Kd1 = 20.6 and Kd2 = 114 nM); binding was strongly stimulated by high concentrations of NaCl; it was inhibited by tetracaine and the protein appeared to possess an ATP-binding site. Unlike cardiac and skeletal muscle, RyRs in retina binding was Ca2+-independent; inhibited by caffeine and dantrolene; less sensitive to ruthenium red; and unaffected by La3+. Also, in retina, ryanodine rapidly associated to and dissociated from its binding sites. Furthermore, although the protein bound the ATP analog BzATP, retinal ryanodine binding was not stimulated by nucleotides. Immunostaining of bovine retinal sections with anti-RyR2 showed a strong staining of amacrine, horizontal and ganglion cells. Finally, using RT-PCR, the three known RyR isoforms were identified in retina. However, consistent with the novel binding properties, the peptide maps yielded by trypsin treatment and Western blotting demonstrate different patterns. Together, the results suggest that retina expresses a novel ryanodine-binding protein, likely to be a ryanodine receptor. Its presence in retina suggests that this protein might play a role in controlling intracellular Ca2+ concentration.     

Stimulation and inhibition of [3H]ryanodine binding to sarcoplasmic reticulum from malignant hyperthermia susceptible pigs

When compared to normal pig sarcoplasmic reticulum (SR), SR from malignant hyperthermia susceptible (MHS) porcine skeletal muscle has been shown to exhibit an increased rate of calcium release, as well as alterations in [3H]ryanodine-binding activity in the presence of microM Ca2+ (Mickelson et al., 1988, J. Biol. Chem. 263, 9310). In the present study, various stimulators (adenine nucleotides and caffeine) and inhibitors (ruthenium red and Mg2+) of the SR calcium release channel were examined for effects on MHS and normal SR [3H]ryanodine binding. The apparent affinity of the MHS SR receptor for ryanodine in the presence of 10 mM ATP (Kd = 6.0 nM) or 10 mM caffeine (Kd = 28 nM) was significantly greater than that of the normal SR (Kd = 8.5 and 65 nM in 10 mM ATP or caffeine, respectively), the Bmax (12-16 pmol/mg) was similar in all cases. The Ca2+(0.5) for inhibition of [3H]ryanodine binding in the presence of 5 mM AMPPNP (238 vs 74 microM for MHS and normal SR, respectively) and the Ca2+(0.5) for stimulation of [3H]ryanodine binding in the presence of 5 mM caffeine (0.049 vs 0.070 microM for MHS and normal SR, respectively) were also significantly different. Furthermore, in the presence of optimal Ca2+, MHS SR [3H]ryanodine binding was more sensitive to caffeine stimulation (C0.5 of 1.7 vs 3.4 mM) and was less sensitive to ruthenium red (C0.5 of 1.9 vs 1.2 microM) or Mg2+ inhibition (C0.5 of 0.34 vs 0.21 mM) than was normal SR. These results further support the hypothesis that differences in the ryanodine/receptor calcium release channel regulatory properties are responsible for the abnormal calcium releasing activity of MHS SR.     

Time- and use-dependent inhibition by ryanodine of caffeine-induced contraction of guinea-pig aortic smooth muscle

We examined how ryanodine interfered with Ca2+-releasing action of caffeine in guinea-pig aorta. Ryanodine (10 microM) decreased the caffeine-induced contraction depending on the time of pretreatment with the agent. The development of ryanodine-effect with time was neither due to a slow access to its binding site nor due to the depletion of stored Ca2+. Ryanodine more potently inhibited the second or the subsequent contraction due to caffeine than the first one even after the agent was removed from the bath after first caffeine. The enhancement of ryanodine-effect depended on the history of Ca2+ release but not on an increase in cytoplasmic Ca2+. The data suggest that an opening of Ca2+ release channel enhances the interaction of ryanodine with the channel.     

A two-gate model for the ryanodine receptor with allosteric modulation by caffeine and quercetin

We have developed a model of the tetrameric ryanodine receptor--the calcium channel of the sarcoplasmic reticulum. The model accurately describes published experimental data on channel activity at various concentrations of Ca2+, caffeine and quercetin. The proposed mechanisms involve allosteric regulation of Ca2+ affinity by both caffeine and quercetin, and the existence of two independent, A- and I-gates controlled by Ca2+ binding to an activating and an inhibitory module of the receptor. There are four different configurations of the receptor that affect ligand binding to the activation module, but not to the inhibition module. Consequently, there are four kinetic modes for the A-gate and one mode for the I-gate. At a certain moment, the receptor can be in any of the four possible conformations with equal probability. By fitting the data we are able to derive ligand affinities and Hill coefficients, to describe the observation that quercetin is an activating agent stronger than caffeine, and that caffeine and quercetin activate the channel at very low Ca2+ concentration (approximately 10(-11) M). We predict that the activation regime at saturating caffeine or quercetin should present four distinct regions at increasing Ca2+, corresponding to the four different gating modes. Another interesting prediction is the enlargement of the activity domain toward higher Ca2+ concentrations in the presence of caffeine or quercetin.     

Ionic dependence of the extracellular ATP-induced permeabilization of transformed mouse fibroblasts: role of plasma membrane activities that regulate cell volume

Extracellular ATP rendered the plasma membrane of transformed mouse fibroblasts permeable to normally impermeant molecules. This permeability change was prevented by increasing the ionic strength of the isotonic medium with NaCl. Conversely, the cells exhibited increased sensitivity to ATP when the NaCl concentration was decreased below isotonicity, when the KCl concentration was increased above 5 mM while maintaining isotonicity, and when the pH of the medium was raised above 7.0. These conditions as well as the addition of ATP itself caused cell swelling. However, the effect of ATP was independent of cell volume and dependent upon the ionic strength and not the osmolarity of the medium since 1) addition of sucrose to isotonic medium did not prevent permeabilization although media made hypertonic with either sucrose or NaCl caused a decrease in cell volume; and 2) addition of sucrose or NaCl to hypotonic media caused a decrease in cell volume, but only NaCl addition decreased the response to ATP. Conditions that have been shown to inhibit plasma membrane proteins that play a reciprocal role in cell volume regulation had reciprocal effects on the permeabilization process, even though the effect of ATP was independent of cell volume. For example, inhibition of the Na+,K+-ATPase by ouabain increased sensitivity of cells to ATP while conditions which inhibit Na+,K+,Cl- -cotransporter activity, such as treatment of the cells with the diuretics furosemide or bumetanide or replacement of sodium chloride in the medium with sodium nitrate or thiocyanate, inhibited permeabilization. The furosemide concentration that inhibited permeabilization was greater than the concentration that inhibited Na+,K+,Cl- -cotransporter-mediated 86Rb+ (K+) uptake, suggesting that the effect of furosemide on the permeabilization process may not be specific for the Na+,K+,Cl- -cotransporter.     

Interdependence of ryanodine binding, oligomeric receptor interactions, and Ca2+ release regulation in junctional sarcoplasmic reticulum

We have examined ryanodine binding to its receptor (RR) and compared its effect on Ca2+ release to the Ca2+ release triggered by Ca2+ plus ATP, using vesicular fragments of junctional terminal cisternae (JTC) obtained from skeletal muscle. Ryanodine binding is slow (taking hours or days to complete) and is highly temperature (Q10 = 4) and Ca2+ dependent. At equilibrium, the extent of binding increases as the concentration of ryanodine is raised above 10(-9) M, exhibiting negative cooperativity and reaching the stoichiometry of the 560,000-Da RR chains near 10(-5) M ryanodine. The specificity of the high affinity binding is demonstrated by competitive binding of ryanodine analogs. Kinetic studies using rapid filtration show that, in the absence of ryanodine, rapid (k = 15 s-1) release of Ca2+ follows a triggering exposure of loaded JTC vesicles to perfusion media containing Ca2+ plus ATP. Induction of this release has no lag period and displays minimal temperature dependence. In contrast, prolonged exposure of JTC vesicles to low (10(-7) M) ryanodine concentrations changes the JTC to a state permitting slow (k = 1 s-1) release of Ca2+ even in the absence of the Ca2+ plus ATP trigger. Higher (greater than microM) concentrations of ryanodine do not allow any Ca2+ release and prevent even the release normally triggered by Ca2+ plus ATP. Our data suggest that ryanodine binds to the open state of the tetrameric RR, inducing protein conformational changes and altered oligomeric interactions. Binding of the first molecule of ryanodine to one of the four binding sites on the receptor produces a partially closed and low conductance state of the Ca2+ release channel and reduces the ryanodine binding affinity of the remaining sites. Ryanodine occupancy of all four binding sites on the receptor completes closure of the Ca2+ channel and blocks the triggering action of Ca2+ plus ATP. The tetrameric association of the RR chains is demonstrated by crosslinking with bifunctional reagents, generating crosslinked tetramers that retain ryanodine binding and Ca2+ release functions.     

Inhibition of glutamate-induced delayed calcium deregulation by 2-APB and La3+ in cultured cortical neurones

Exposure of neurones in culture to excitotoxic levels of glutamate results in an initial transient spike in [Ca2+]i followed by a delayed, irreversible [Ca2+]i rise governed by rapid kinetics, with Ca2+ originating from the extracellular medium. The molecular mechanism responsible for the secondary Ca2+ rise is unknown. Here, we report that the delayed Ca2+ entry in cortical neurones is diminished by 2-aminoethoxydiphenyl borate (2-APB: IC50 = 62 +/- 9 microm) and La3+ (IC50 = 7.2 +/- 3 microm), both known to inhibit transient receptor potential (TRP) and store-operated Ca2+ (SOC) channels. Application of thapsigargin, however, failed to exacerbate the delayed Ca2+ deregulation, arguing against a store depletion event as the stimulus for induction of the secondary [Ca2+]i rise. In addition, these neurones did not exhibit SOC entry. Unexpectedly, application of ryanodine or caffeine significantly inhibited glutamate-induced delayed Ca2+ deregulation. In basal Ca2+ entry experiments, La3+ and 2-APB modulated the rapid rise in [Ca2+]i caused by exposure of neurones to Ca2+ after pre-incubating in a calcium-free medium. This basal Ca2+ influx was mitigated by extracellular Mg2+ but not aggravated by thapsigargin, ryanodine or caffeine. These results indicate that 2-APB and La3+ influence non-store-operated Ca2+ influx in cortical neurones and that this route of Ca2+ entry is involved in glutamate-induced delayed Ca2+ deregulation.     

Ryanodine receptors in liver

The ryanodine receptor has been mainly regarded as the Ca2+ release channel from sarcoplasmic reticulum controlling skeletal and cardiac muscle contraction. However, many studies have shown that it is widely expressed, with functions not restricted to muscular contraction. This study examined whether ryanodine receptor plays a role in calcium signaling in the liver. RT-PCR analysis of isolated hepatocytes showed expression of a truncated type 1 ryanodine receptor, but no type 2 or type 3 message was detected. We also detected binding sites for [3H]ryanodine in the microsomal cellular fraction and in permeabilized hepatocytes. This binding was displaced by caffeine and dantrolene, but not by ruthenium red, heparin or cyclic ADP-Ribose. Ryanodine, by itself, did not trigger Ca2+ oscillations in either primary cultured hepatocytes or hepatocytes within the intact perfused rat liver. In both preparations, however, ryanodine significantly increased the frequency of the cytosolic free [Ca2+] oscillations evoked by an alpha1 adrenergic receptor agonist. Experiments in permeabilized hepatocytes showed that both ryanodine and cyclic ADP-ribose evoked a slow Ca2+ leak from intracellular stores and were able to increase the Ca2+-released response to a subthreshold dose of inositol 1,4,5-trisphosphate. Our findings suggest the presence of a novel truncated form of the type 1 ryanodine receptor in rat hepatocytes. Ryanodine modulates the pattern of cytosolic free [Ca2+] oscillations by increasing oscillation frequency. We propose that the Ca2+ released from ryanodine receptors on the endoplasmic reticulum provides an increased pool of Ca2+ for positive feedback on inositol 1,4,5-trisphosphate receptors.     

Doxorubicin directly binds to the cardiac-type ryanodine receptor

The clinical use of doxorubicin, an antineoplasmic agent, is limited by its extensive cardiotoxicity which is mediated by the mobilization of intracellular Ca2+ from SR. In order to elucidate the mechanism of Ca2+ release, we analyzed the binding sites of doxorubicin on rabbit cardiac SR (sarcoplasmic reticulum). One of the binding sites was identified as cardiac-type ryanodine receptor (RyR2) which was purified by immunoprecipitation from solubilized cardiac SR in the presence of DTT. Ligand blot analysis revealed the direct binding of doxorubicin to RyR2. The binding of doxorubicin to RyR2 was specific and displaced by caffeine. Both doxorubicin and caffeine enhanced [3H]-ryanodine binding to RyR2 in a Ca2+ dependent manner. These results suggest that there is a doxorubicin binding site on RyR2.     

Ryanodine binding to sarcoplasmic reticulum membrane; comparison between cardiac and skeletal muscle

[3H]Ryanodine binding to skeletal muscle and cardiac sarcoplasmic reticulum (SR) vesicles was compared under experimental conditions known to inhibit or stimulate Ca2+ release. In the skeletal muscle SR, ryanodine binds to a single class of high-affinity sites (Kd of 11.3 nM). In cardiac SR vesicles, more than one class of binding sites is observed (Kd values of 3.6 and 28.1 nM). Ryanodine binding to skeletal muscle SR vesicles requires high concentrations of NaCl, whereas binding of the drug to cardiac SR is only slightly influenced by ionic strength. In the presence of 5'-adenylyl imidodiphosphate (p[NH]ppA), increased pH, and micromolar concentration of Ca2+ (which all induce Ca2+ release from SR) binding of ryanodine to SR is significantly increased in skeletal muscle, while being unchanged in cardiac muscle. Ryanodine binding to skeletal but not to cardiac muscle SR is inhibited in the presence of high Ca2+ or Mg2+ concentrations (all known to inhibit Ca2+ release from skeletal muscle SR). Ruthenium red or dicyclohexylcarbodiimide modification of cardiac and skeletal muscle SR inhibit Ca2+ release and ryanodine binding in both skeletal and cardiac membranes. These results indicate that significant differences exist in the properties of ryanodine binding to skeletal or cardiac muscle SR. Our data suggest that ryanodine binds preferably to site(s) which are accessible only when the Ca2+ release channel is in the open state.