Unique phosphorylation site on the cardiac ryanodine receptor regulates calcium channel activity.

Ryanodine receptors have recently been shown to be the Ca2+ release channels of sarcoplasmic reticulum in both cardiac muscle and skeletal muscle. Several regulatory sites are postulated to exist on these receptors, but to date, none have been definitively identified. In the work described here, we localize one of these sites by showing that the cardiac isoform of the ryanodine receptor is a preferred substrate for multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase). Phosphorylation by CaM kinase occurs at a single site encompassing serine 2809. Antibodies generated to this site react only with the cardiac isoform of the ryanodine receptor, and immunoprecipitate only cardiac [3H]ryanodine-binding sites. When cardiac junctional sarcoplasmic reticulum vesicles or partially purified ryanodine receptors are fused with planar bilayers, phosphorylation at this site activates the Ca2+ channel. In tissues expressing the cardiac isoform of the ryanodine receptor, such as heart and brain, phosphorylation of the Ca2+ release channel by CaM kinase may provide a unique mechanism for regulating intracellular Ca2+ release.     

A transverse tubule NADPH oxidase activity stimulates calcium release from isolated triads via ryanodine receptor type 1 S -glutathionylation

We report here the presence of an NADPH oxidase (NOX) activity both in intact and in isolated transverse tubules and in triads isolated from mammalian skeletal muscle, as established by immunochemical, enzymatic, and pharmacological criteria. Immunohistochemical determinations with NOX antibodies showed that the gp91(phox) membrane subunit and the cytoplasmic regulatory p47(phox) subunit co-localized in transverse tubules of adult mice fibers with the alpha1s subunit of dihydropyridine receptors. Western blot analysis revealed that isolated triads contained the integral membrane subunits gp91(phox) and p22(phox), which were markedly enriched in isolated transverse tubules but absent from junctional sarcoplasmic reticulum vesicles. Isolated triads and transverse tubules, but not junctional sarcoplasmic reticulum, also contained varying amounts of the cytoplasmic NOX regulatory subunits p47(phox) and p67(phox). NADPH or NADH elicited superoxide anion and hydrogen peroxide generation by isolated triads; both activities were inhibited by NOX inhibitors but not by rotenone. NADH diminished the total thiol content of triads by one-third; catalase or apocynin, a NOX inhibitor, prevented this effect. NADPH enhanced the activity of ryanodine receptor type 1 (RyR1) in triads, measured through [3H]ryanodine binding and calcium release kinetics, and increased significantly RyR1 S-glutathionylation over basal levels. Preincubation with reducing agents or NOX inhibitors abolished the enhancement of RyR1 activity produced by NADPH and prevented NADPH-induced RyR1 S-glutathionylation. We propose that reactive oxygen species generated by the transverse tubule NOX activate via redox modification the neighboring RyR1 Ca2+ release channels. Possible implications of this putative mechanism for skeletal muscle function are discussed.     

Direct binding of verapamil to the ryanodine receptor channel of sarcoplasmic reticulum.

Radioligand binding experiments and single channel recordings demonstrate that verapamil interacts with the ryanodine receptor Ca2+ release channel of the sarcoplasmic reticulum of rabbit skeletal muscle. In isolated triads, verapamil decreased binding of [3H]Ryanodine with an IC50 of approximately 8 microM at an optimal pH 8.5 and pCa 4.3. Nitrendipine and d-cis-diltiazem did not interfere with binding of [3H]Ryanodine to triads, suggesting that the action of verapamil does not involve the dihydropyridine receptor. Single channel recordings showed that verapamil blocked Ca2+ release channels by decreasing open probability, duration of open events, and number of events per unit time. A direct interaction of verapamil with the ryanodine receptor peptide was demonstrated after purification of the approximately 400 kDa receptor protein from Chaps-solubilized triads. The purified receptor displayed high affinity for [3H]Ryanodine with a Kd of approximately 5 nM and a Bmax of approximately 400 pmol/mg. Verapamil and D600 decreased [3H]Ryanodine binding noncompetitively by reducing the Bmax. Thus the presence of binding sites for phenylalkylamines in the Ca2+ release channel was confirmed. Verapamil blockade of Ca2+ release channels may explain some of the paralyzing effects of phenylalkylamines observed during excitation-contraction coupling of skeletal muscle.     

Functional interaction of the cytoplasmic domain of triadin with the skeletal ryanodine receptor

Triadin has been shown to co-localize with the ryanodine receptor in the sarcoplasmic reticulum membrane. We show that immunoprecipitation of solubilized sarcoplasmic reticulum membrane with antibodies directed against triadin or ryanodine receptor, leads to the co-immunoprecipitation of ryanodine receptor and triadin. We then investigated the functional importance of the cytoplasmic domain of triadin (residues 1-47) in the control of Ca2+ release from sarcoplasmic reticulum. We show that antibodies directed against a synthetic peptide encompassing residues 2-17, induce a decrease in the rate of Ca2+ release from sarcoplasmic reticulum vesicles as well as a decrease in the open probability of the ryanodine receptor Ca2+ channel incorporated in lipid bilayers. Using surface plasmon resonance spectroscopy, we defined a discrete domain (residues 18-46) of the cytoplasmic part of triadin interacting with the purified ryanodine receptor. This interaction is optimal at low Ca2+ concentration (up to pCa 5) and inhibited by increasing calcium concentration (IC50 of 300 microM). The direct molecular interaction of this triadin domain with the ryanodine receptor was confirmed by overlay assay and shown to induce the inhibition of the Ca2+ channel activity of purified RyR in bilayer. We propose that this interaction plays a critical role in the control, by triadin, of the Ca2+ channel behavior of the ryanodine receptor and therefore may represent an important step in the regulation process of excitation-contraction coupling in skeletal muscle.     

Purified ryanodine receptor from skeletal muscle sarcoplasmic reticulum is the Ca2+-permeable pore of the calcium release channel

The ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum was purified by immunoaffinity chromatography as a single approximately 450,000-Da polypeptide and it was shown to mediate single channel activity identical to that of the ryanodine-treated Ca2+ release channel of the sarcoplasmic reticulum. The purified receptor had a [3H]ryanodine binding capacity (Bmax) of 280 pmol/mg and a binding affinity (Kd) of 9.0 nM. [3H]Ryanodine binding to the purified receptor was stimulated by ATP and Ca2+ with a half-maximal stimulation at 1 mM and 8-9 microM, respectively. [3H]Ryanodine binding to the purified receptor was inhibited by ruthenium red and high concentrations of Ca2+ with an IC50 of 2.5 microM and greater than 1 mM, respectively. Reconstitution of the purified receptor in planar lipid bilayers revealed the Ca2+ channel activity of the purified receptor. Like the native sarcoplasmic reticulum Ca2+ channels treated with ryanodine, the purified receptor channels were characterized by (i) the predominance of long open states insensitive to Mg2+ and ruthenium red, (ii) a main slope conductance of approximately 35 pS and a less frequent 22 pS substate in 54 mM trans-Ca2+ or Ba2+, and (iii) a permeability ratio PBa or PCa/PTris = 8.7. The approximately 450,000-Da ryanodine receptor channel thus represents the long-term open "ryanodine-altered" state of the Ca2+ release channel from sarcoplasmic reticulum. We propose that the ryanodine receptor constitutes the physical pore that mediates Ca2+ release from the sarcoplasmic reticulum of skeletal muscle.     

Phosphorylation of the cardiac ryanodine receptor by cAMP-dependent protein kinase

The phosphorylation of canine cardiac and skeletal muscle ryanodine receptors by the catalytic subunit of cAMP-dependent protein kinase has been studied. A high-molecular-weight protein (Mr 400,000) in cardiac microsomes was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. A monoclonal antibody against the cardiac ryanodine receptor immunoprecipitated this phosphoprotein. In contrast, high-molecular-weight proteins (Mr 400,000-450,000) in canine skeletal microsomes isolated from extensor carpi radialis (fast) or superficial digitalis flexor (slow) muscle fibers were not significantly phosphorylated. In agreement with these findings, the ryanodine receptor purified from cardiac microsomes was also phosphorylated by cAMP-dependent protein kinase. Phosphorylation of the cardiac ryanodine receptor in microsomal and purified preparations occurred at the ratio of about one mol per mol of ryanodine-binding site. Upon phosphorylation of the cardiac ryanodine receptor, the levels of [3H]ryanodine binding at saturating concentrations of this ligand increased by up to 30% in the presence of Ca2+ concentrations above 1 microM in both cardiac microsomes and the purified cardiac ryanodine receptor preparation. In contrast, the Ca2+ concentration dependence of [3H]ryanodine binding did not change significantly. These results suggest that phosphorylation of the ryanodine receptor by cAMP-dependent protein kinase may be an important regulatory mechanism for the calcium release channel function in the cardiac sarcoplasmic reticulum.     

The effects of ryanodine on passive calcium fluxes across sarcoplasmic reticulum membranes

Ryanodine at concentrations of 0.01-10 microM increased, while greater concentrations of 10-300 microM decreased the calcium permeability of both rabbit fast twitch skeletal muscle junctional and canine cardiac sarcoplasmic reticulum membranes. Ryanodine did not alter calcium binding by either sarcoplasmic reticulum membranes or the calcium binding protein, calsequestrin. Therefore, the effects by this agent appear to involve only changes in membrane permeability, and the characteristics of the calcium permeability pathway affected by ryanodine were those of the calcium release channel. Consistent with this, the actions by ryanodine were localized to junctional sarcoplasmic reticulum membranes and were not observed with either longitudinal sarcoplasmic reticulum or transverse tubular membranes. In addition, passage of the junctional sarcoplasmic reticulum membranes through a French press did not diminish the effects of ryanodine indicating that intact triads were not required. Under the conditions used for the permeability studies, the binding of [3H]ryanodine to skeletal junctional sarcoplasmic reticulum membranes was specific and saturable, and Scatchard analyses indicated the presence of a single binding site with a Kd of 150-200 nM and a maximum capacity of 10.1-18.9 pmol/mg protein. [3H]ryanodine binding to this site and the increase in membrane calcium permeability caused by low concentrations of ryanodine had similar characteristics suggesting that actions at this site produce this effect. Depending on the assay conditions used, ryanodine (100-300 microM) could either increase or decrease ATP-dependent calcium accumulation by skeletal muscle junctional sarcoplasmic reticulum membranes indicating that the alterations of sarcoplasmic reticulum membrane calcium permeability caused by this agent can be determined in part by the experimental environment.     

Antibodies to junctional sarcoplasmic reticulum proteins: probes for the Ca2+-release channel.

The junctional face membrane plays a key role in excitation-contraction coupling in skeletal muscle. A protein of 350 kDa, tentatively identified as a component of the junctional feet, connects transverse tubules to terminal cisternae of sarcoplasmic reticulum [Kawamoto, Brunschwig, Kim & Caswell (1986) J. Cell Biol. 103, 1405-1414]. The membrane topology and protein composition of sarcoplasmic reticulum Ca2+-release channels of rabbit skeletal muscle were investigated using an immunological approach, with anti-(junctional face membrane) and anti-(350 kDa protein) polyclonal antibodies. Upon preincubation of the terminal cisternae with anti-(junctional face membrane) antibodies, Ca2+-ATPase and Ca2+-loading activities were not affected, whereas anti-(350 kDa protein) antibodies stimulated Ca2+-ATPase activity by 25% and inhibited Ca2+-loading activity by 50% (at an antibody/terminal cisternae protein ratio of 1:1). Specific photolabelling of terminal cisternae proteins with [14C]doxorubicin was prevented by both anti-(junctional face membrane) and anti-(350 kDa protein) antibodies. Stimulation of Ca2+ release by doxorubicin was prevented by both anti-(junctional face membrane) and anti-(350 kDa protein) antibodies. Half-maximal inhibition was obtained at an antibody/terminal cisternae protein ratio of 1:1. Kinetic measurements of Ca2+ release indicated that anti-(350 kDa protein) antibodies prevented Ca2+-induced Ca2+ release, whereas the ATP-stimulation and the inhibition by Mg2+ were not affected. These results suggest that: (i) Ca2+- and doxorubicin-induced Ca2+ release is mediated by Ca2+ channels which are selectively localized in the junctional face membrane; (ii) the 350 kDa protein is a component of the Ca2+-release channel in native terminal cisternae vesicles; and (iii) the Ca2+-activating site of the channel is separate from other allosteric sites.     

Rose bengal activates the Ca2+ release channel from skeletal muscle sarcoplasmic reticulum.

The photooxidizing xanthene dye rose bengal (10 nM to 1 microM) stimulates rapid Ca2+ release from skeletal muscle sarcoplasmic reticulum vesicles. Following fusion of sarcoplasmic reticulum (SR) vesicles to an artificial bilayer, reconstituted Ca2+ channel activity is stimulated by nanomolar concentrations of rose bengal in the presence of a broad-spectrum light source. Rose bengal does not appear to affect K+ channels present in the SR. Following reconstitution of the sulfhydryl-activated 106-kDa Ca2+ channel protein into a bilayer, rose bengal activates the isolated protein in a light-dependent manner. Ryanodine at a concentration of 10 nM is shown to lock the 106-kDa channel protein in a subconductance state which can be reversed by subsequent addition of 500 nM rose bengal. This apparent displacement of bound ryanodine by nanomolar concentrations of rose bengal is also directly observed upon measurement of [3H]ryanodine binding to JSR vesicles. These observations indicate that photooxidation of rose bengal causes a stimulation of the Ca2+ release protein from skeletal muscle sarcoplasmic reticulum by interacting with the ryanodine binding site. Furthermore, similar effects of rose bengal on isolated SR vesicles, on single channel measurements following fusion of SR vesicles, and following incorporation of the isolated 106-kDa protein strongly implicates the 106-kDa sulfhydryl-activated Ca2+ channel protein in the Ca2+ release process.     

Ryanodine-affinity chromatography purifies 106 kD Ca release channels from skeletal and cardiac sarcoplasmic reticulum

A 106 kD protein was isolated from skeletal sarcoplasmic reticulum (SR) vesicles and shown to have the properties of SR Ca2+ release channels, including blockade by 5 nM ryanodine. In view of extensive reports that the ryanodine-receptor complex consists of four 565 kD junctional feet proteins (JFPs) and is the 'physiological' Ca2+ release channel, we prepared ryanodine-affinity columns to isolate its receptor site(s). Conditions known to maximize the association and dissociation of ryanodine to SR proteins were respectively used to link, then elute, the receptor(s) from ryanodine-affinity columns. The method purified a protein at about 100 kD from both rabbit skeletal and canine cardiac SR vesicles. The skeletal and cardiac proteins isolated by ryanodine-affinity chromatography were identified as the low molecular weight Ca2+ release channel through their antigenic reaction with an anti-106 kD monoclonal antibody. Upon reconstitution in planar bilayers, both skeletal and cardiac proteins revealed the presence of functional SR Ca2+ release channels. Surprisingly, ryanodine-affinity columns did not retain JFPs but purified 106 kD Ca2+ release channels which are a minor component (0.1-0.3%) of SR proteins.     

Purification of the ryanodine receptor and identity with feet structures of junctional terminal cisternae of sarcoplasmic reticulum from fast skeletal muscle

The ryanodine receptor has been purified from junctional terminal cisternae of fast skeletal muscle sarcoplasmic reticulum (SR). The ryanodine receptor was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and stabilized by addition of phospholipids. The solubilized receptor showed the same [3H]ryanodine binding properties as the original SR vesicles in terms of affinity, Ca2+ dependence, and salt dependence. Purification of the ryanodine receptor was performed by sequential column chromatography on heparin-agarose and hydroxylapatite in the presence of CHAPS. The purified receptor bound 393 +/- 65 pmol of ryanodine/mg of protein (mean +/- S.E., n = 5). The purified receptor showed three bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Mr of 360,000, 330,000, and 175,000. Densitometry indicates that these are present in the ratio of 2/1/1, suggesting a monomer Mr of 1.225 X 10(6) and supported by gel exclusion chromatography in CHAPS. Electron microscopy of the purified preparation showed the square shape of 210 A characteristic of and comparable in size and shape to the feet structures of junctional terminal cisternae of SR, indicating that ryanodine binds directly to the feet structures. From the ryanodine binding data, the stoichiometry between ryanodine binding sites to the number of feet structures is estimated to be about 2. Since the ryanodine receptor is coupled to Ca2+ gating, the present finding suggests that the ryanodine receptor and Ca2+ release channel represent a functional unit, the structural unit being the foot structure which, in situ, is junctionally associated with the transverse tubules. It is across this triad junction that the signal for Ca2+ release is expressed. Thus, the foot structure appears to directly respond to the signal from transverse tubules, causing the release of Ca2+ from the junctional face membrane of the terminal cisternae of SR.     

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+.     

Activation of the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum by palmitoyl carnitine.

Studies of [3H]ryanodine binding, 45Ca2+ efflux, and single channel recordings in planar bilayers indicated that the fatty acid metabolite palmitoyl carnitine produced a direct stimulation of the Ca2+ release channel (ryanodine receptor) of rabbit and pig skeletal muscle junctional sarcoplasmic reticulum. At a concentration of 50 microM, palmitoyl carnitine (a) stimulated [3H]ryanodine binding 1.6-fold in a competitive manner at all pCa in the range 6 to 3; (b) released approximately 65% (30 nmol) of passively loaded 45Ca2+/mg protein; and (c) increased 7-fold the open probability of Ca2+ release channels incorporated into planar bilayers. Neither carnitine nor palmitic acid could reproduce the effect of palmitoyl carnitine on [3H]ryanodine binding, 45Ca2+ release, or channel open probability. 45Ca2+ release was induced by several long-chain acyl carnitines (C14, C16, C18) and acyl coenzyme A derivatives (C12, C14, C16), but not by the short-chain derivative C8 or by free saturated fatty acids of chain length C8 to C18, at room temperature or 36 degrees C. This newly identified interaction of esterified fatty acids and ryanodine receptors may represent a pathway by which metabolism of skeletal muscle could influence intracellular Ca2+ and may be responsible for the pathophysiology of disorders of beta-oxidation such as carnitine palmitoyl transferase II deficiency.     

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.     

Interactions between dihydropyridine receptors and ryanodine receptors in striated muscle

Excitation-contraction coupling in both skeletal and cardiac muscle depends on structural and functional interactions between the voltage-sensing dihydropyridine receptor L-type Ca²⁺ channels in the surface/transverse tubular membrane and ryanodine receptor Ca²⁺ release channels in the sarcoplasmic reticulum membrane. The channels are targeted to either side of a narrow junctional gap that separates the external and internal membrane systems and are arranged so that bi-directional structural and functional coupling can occur between the proteins. There is strong evidence for a physical interaction between the two types of channel protein in skeletal muscle. This evidence is derived from studies of excitation–contraction coupling in intact myocytes and from experiments in isolated systems where fragments of the dihydropyridine receptor can bind to the ryanodine receptors in sarcoplasmic reticulum vesicles or in lipid bilayers and alter channel activity. Although micro-regions that participate in the functional interactions have been identified in each protein, the role of these regions and the molecular nature of the protein–protein interaction remain unknown. The trigger for Ca²⁺ release through ryanodine receptors in cardiac muscle is a Ca²⁺ influx through the L-type Ca²⁺ channel. The Ca²⁺ entering through the surface membrane Ca²⁺ channels flows directly onto underlying ryanodine receptors and activates the channels. This was thought to be a relatively simple system compared with that in skeletal muscle. However, complexities are emerging and evidence has now been obtained for a bi-directional physical coupling between the proteins in cardiac as well as skeletal muscle. The molecular nature of this coupling remains to be elucidated.     

The calcium-ryanodine receptor complex of skeletal and cardiac muscle

[3H]Ryanodine binds with high affinity to saturable and Ca2+-dependent sites in heavy sarcoplasmic reticulum (SR) preparations from rabbit skeletal and cardiac muscle. Ruthenium red, known to interfere with Ca2+-induced Ca2+ release from SR vesicles, inhibits [3H]ryanodine specific binding in both skeletal and cardiac preparations whereas Mg2+, Ba2+, Cd2+ and La3+ selectively inhibit the skeletal preparation. The toxicological relevance of the [3H]ryanodine binding site is established by the correlation of binding inhibition with toxicity for seven ryanoids including two botanical insecticides. These findings provide direct evidence for Ca2+-ryanodine receptor complexes that may play a role in excitation-contraction coupling.     

Ryanodine activation and inhibition of the Ca2+ release channel of sarcoplasmic reticulum

The effect of the plant alkaloid ryanodine on the skeletal muscle sarcoplasmic reticulum Ca2+ release channel was studied by determining the Ca2+ permeability of "heavy" vesicles passively loaded with 45Ca2+ in the presence or absence of ryanodine. Depending on the experimental conditions, ryanodine either stimulated or inhibited Ca2+ efflux. Vesicles were rendered permeable to 45Ca2+ at a ryanodine concentration of 0.01 microM when diluted into a medium containing the two Ca2+ release channel inhibitors Mg2+ and ruthenium red. At ryanodine concentrations greater than 10 microM, 45Ca2+ efflux was inhibited in channel-activating (5 microM Ca2+) or -inhibiting (10 mM Mg2+ plus 10 microM ruthenium red) media. An optimal stimulatory effect was observed when vesicles were incubated with ryanodine at 37 degrees C and in media that caused partial opening of the channel. Similar results to those described above were obtained using cardiac sarcoplasmic reticulum vesicles that were capable of rapid 45Ca2+ efflux. Use of the slowly permeating molecule L-[3H]glucose allowed measurement of channel-mediated efflux rates from vesicles in the presence and absence of ryanodine. At low activating concentrations, ryanodine did not appreciably change the regulation of L-glucose efflux rates by external Ca2+, Mg2+, and adenine nucleotide. These results suggested two possible modes of action of ryanodine: 1) a change in the gating mechanism of the channel which is not readily detected using the slowly permeating molecule L-glucose or 2) a change in channel structure which prevents its complete closing.     

Calmodulin activation and inhibition of skeletal muscle Ca2+ release channel (ryanodine receptor).

The calmodulin-binding properties of the rabbit skeletal muscle Ca2+ release channel (ryanodine receptor) and the channel's regulation by calmodulin were determined at < or = 0.1 microM and micromolar to millimolar Ca2+ concentrations. [125I]Calmodulin and [3H]ryanodine binding to sarcoplasmic reticulum (SR) vesicles and purified Ca2+ release channel preparations indicated that the large (2200 kDa) Ca2+ release channel complex binds with high affinity (KD = 5-25 nM) 16 calmodulins at < or = 0.1 microM Ca2+ and 4 calmodulins at 100 microM Ca2+. Calmodulin-binding affinity to the channel showed a broad maximum at pH 6.8 and was highest at 0.15 M KCl at both < or = 0.1 MicroM and 100 microM Ca2+. Under condition closely related to those during muscle contraction and relaxation, the half-times of calmodulin dissociation and binding were 50 +/- 20 s and 30 +/- 10 min, respectively. SR vesicle-45Ca2+ flux, single-channel, and [3H]ryanodine bind measurements showed that, at < or = 0.2 microM Ca2+, calmodulin activated the Ca2+ release channel severalfold. Ar micromolar to millimolar Ca2+ concentrations, calmodulin inhibited the Ca(2+)-activated channel severalfold. Hill coefficients of approximately 1.3 suggested no or only weak cooperative activation and inhibition of Ca2+ release channel activity by calmodulin. These results suggest a role for calmodulin in modulating SR Ca2+ release in skeletal muscle at both resting and elevated Ca2+ concentrations.     

Inhibition of Ca2+ release channel (ryanodine receptor) activity by sphingolipid bases: mechanism of action

Sphingosine inhibits the activity of the skeletal muscle Ca2+ release channel (ryanodine receptor) and is a noncompetitive inhibitor of [3H]ryanodine binding (Needleman et al., Am. J. Physiol. 272, C1465-1474, 1997). To determine the contribution of other sphingolipids to the regulation of ryanodine receptor activity, several sphingolipid bases were assessed for their ability to alter [3H]ryanodine binding to sarcoplasmic reticulum (SR) membranes and to modulate the activity of the Ca2+ release channel. Three lipids, N,N-dimethylsphingosine, dihydrosphingosine, and phytosphingosine, inhibited [3H]ryanodine binding to both skeletal and cardiac SR membranes. However, the potency of these three lipids and sphingosine was lower in rabbit cardiac membranes when compared to rabbit skeletal muscle membranes and when compared to sphingosine. Like sphingosine, the lipids inhibited [3H]ryanodine binding by greatly increasing the rate of dissociation of bound [3H]ryanodine from SR membranes, indicating that these three sphingolipid bases were noncompetitive inhibitors of [3H]ryanodine binding. These bases also decreased the activity of the Ca2+ release channel incorporated into planar lipid bilayers by stabilizing a long closed state. Sphingosine-1-PO4 and C6 to C18 ceramides of sphingosine had no significant effect on [3H]ryanodine binding to cardiac or skeletal muscle SR membranes. Saturation of the double bond at positions 4-5 decreased the ability of the sphingolipid bases to inhibit [3H]ryanodine binding 2-3 fold compared to sphingosine. In summary, our data indicate that other endogenous sphingolipid bases are capable of modulating the activity of the Ca2+ release channel and as a class possess a common mechanism of inhibition.     

The 90-kDa junctional sarcoplasmic reticulum protein forms an integral part of a supramolecular triad complex in skeletal muscle.

Although it is well established that voltage-sensing of the alpha(1)-dihydropyridine receptor triggers Ca(2+)-release via the ryanodine receptor during excitation-contraction coupling in skeletal muscle fibers, it remains to be determined which junctional components are responsible for the assembly, maintenance, and stabilization of triads. Here, we analyzed the expression pattern and neighborhood relationship of a novel 90-kDa sarcoplasmic reticulum protein. This protein is highly enriched in the triad fraction and is predominantly expressed in fast-twitching muscle fibers. Chronic low-frequency electro-stimulation induced a drastic decrease in the relative abundance of this protein. Chemical crosslinking showed a potential overlap between the 90-kDa junctional face membrane protein and the ryanodine receptor Ca(2+)-release channel, suggesting tight protein-protein interactions between these two triad components. Hence, Ca(2+)-regulatory muscle proteins have a strong tendency to oligomerize and the triad region of skeletal muscle fibers forms supramolecular membrane complexes involved in the regulation of Ca(2+)-homeostasis and signal transduction.