Fatty acids modulate calcium-induced calcium release from skeletal muscle heavy sarcoplasmic reticulum fractions: implications for malignant hyperthermia

Based on studies in swine, the malignant hyperthermia syndrome has been postulated to result from an enhanced sensitivity (low threshold) of the Ca2(+)-induced Ca2(+)-release process. However, fatty acid production is elevated in homogenates of skeletal muscle from pigs and humans susceptible to malignant hyperthermia. In the present study, we demonstrate that the threshold of Ca2(+)-induced Ca2+ release is normal in susceptible humans and in susceptible swine depleted of triglycerides. Exogenously added unsaturated fatty acids decreased the threshold of Ca2(+)-induced Ca2+ release to a much greater extent in porcine and equine muscle than in human muscle. When triglyceride and free fatty acid values were reduced to about 40 and 60%, respectively, of control values, malignant hyperthermia-susceptible swine did not exhibit muscle rigidity when challenged in vivo with halothane and succinylcholine and the threshold of the Ca2(+)-induced Ca2(+)-release process in heavy sarcoplasmic reticulum fractions was normal. Despite the reduced triglyceride and fatty acid levels, these swine had a positive in vitro contracture test for malignant hyperthermia. A low Ca2(+)-induced Ca2(+)-release threshold is not essential for malignant hyperthermia susceptibility, but appears to be the result of excessive free fatty acids produced during organelle isolation.     

Increased sensitivity of the ryanodine receptor to halothane-induced oligomerization in malignant hyperthermia-susceptible human skeletal muscle.

Mutations in the skeletal muscle RyR1 isoform of the ryanodine receptor (RyR) Ca2+-release channel confer susceptibility to malignant hyperthermia, which may be triggered by inhalational anesthetics such as halothane. Using immunoblotting, we show here that the ryanodine receptor, calmodulin, junctin, calsequestrin, sarcalumenin, calreticulin, annexin-VI, sarco(endo)plasmic reticulum Ca2+-ATPase, and the dihydropyridine receptor exhibit no major changes in their expression level between normal human skeletal muscle and biopsies from individuals susceptible to malignant hyperthermia. In contrast, protein gel-shift studies with halothane-treated sarcoplasmic reticulum vesicles from normal and susceptible specimens showed a clear difference. Although the alpha2-dihydropyridine receptor and calsequestrin were not affected, clustering of the Ca2+-ATPase was induced at comparable halothane concentrations. In the concentration range of 0.014-0.35 mM halothane, anesthetic-induced oligomerization of the RyR1 complex was observed at a lower threshold concentration in the sarcoplasmic reticulum from patients with malignant hyperthermia. Thus the previously described decreased Ca2+-loading ability of the sarcoplasmic reticulum from susceptible muscle fibers is probably not due to a modified expression of Ca2+-handling elements, but more likely a feature of altered quaternary receptor structure or modified functional dynamics within the Ca2+-regulatory apparatus. Possibly increased RyR1 complex formation, in conjunction with decreased Ca2+ uptake, is of central importance to the development of a metabolic crisis in malignant hyperthermia.     

Changes in cytoplasmic free calcium caused by halothane. Role of the plasma membrane and intracellular Ca2+ stores

Malignant hyperthermia is a muscle disease characterized by an abnormal response to anaesthetics, stress, and exercise. It is typified by muscle contracture and a dramatic elevation in body temperature. A defect in the regulation of the concentration of cytoplasmic free calcium, [Ca2]i, is thought to underlie this disease, but the actual [Ca2+]i was not measurable until recently. We have shown that the anaesthetic halothane increases [Ca2+]i in isolated lymphocytes from malignant hyperthermia-susceptible humans and pigs but not in the normal counterparts. In this report we extend these observations to a larger number of cases and analyze the molecular mechanisms responsible for the increase. The halothane-mediated rise in [Ca2+]i required external Ca2+ and was prevented by nifedipine, an inhibitor of the voltage-sensitive Ca2+ channels of the cell membrane. In addition, the effect of halothane on the releasable Ca2+ from intracellular stores was determined by measuring the size of the releasable pool before and after addition of the anaesthetic. After addition of halothane, about 73% of this Ca2+ pool was still available for release by the Ca2+ ionophore ionomycin in cells from normal humans and pigs. In contrast, only about 45% of the free Ca2+ in intracellular stores was left after treatment with halothane in cells from malignant hyperthermia-susceptible humans and swine. These results indicate that halothane acts both at the cell membrane and at intracellular organelles, and that this action results in a net increase in [Ca2+]i in malignant hyperthermia, but not in normal cells. The action at the cell membrane appears to be on the voltage-sensitive Ca2+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipid peroxidation, antioxidant concentrations, and fatty acid contents of muscle tissue from malignant hyperthermia-susceptible swine.

Homogenates of semitendinosus muscle from malignant hyperthermia (MH)-susceptible pigs produced threefold more pentane than those from MH-resistant pigs, indicating enhanced free radical-mediated peroxidation of n-6 fatty acids. This did not reflect a deficiency in tissue antioxidants or antioxidant-enzymes but glutathione concentrations and glutathione peroxidase activities were increased in the tissue from MH-susceptible swine, consistent with an adaptive response to a sustained oxidant stress. A lower proportion of linoleic acid (18:2 n-6) in phospholipids and neutral lipids in muscle from MHS pigs indicated increased peroxidation or metabolism (desaturation and elongation). The increased oleic acid (18:1 n-9) in the MHS muscle indicated that desaturase activity was elevated in all lipid classes. The results are consistent with the hypothesis that enhanced free radical activity and lipid peroxidation contributes to the abnormalities in Ca2+ homeostasis and polyunsaturated fatty acid metabolism in MH.     

Comparative effects of the ionophore A23187 on the mechanical responses of muscle in normal Pietrain pigs and pigs with malignant hyperthermia

Since increased intracellular Ca2+ is believed to be the main factor causing skeletal muscle contracture in human and porcine malignant hyperthermia, the potential effects of the ionophore A23187, which enhances intracytoplasmic Ca2+, were investigated in Pietrain pig muscles. These effects were compared with those of caffeine, known to induce dose-dependent contracture in vitro in isolated muscle from human subjects with malignant hyperthermia. For this purpose, the mechanical and biochemical actions of caffeine and A23187 were tested in intercostal muscle biopsies from 10 normal pigs and 10 with malignant hyperthermia. The results show that A23187 allowed very clear differentiation between the muscles of normal and pathological animals. In view of the wide spectrum of drug sensitivity characterizing subjects with malignant hyperthermia, it is suggested that exposure to A23187 be added to the halothane and caffeine tests currently used to detect this disease.     

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.     

Voltage-dependent calcium release in human malignant hyperthermia muscle fibers.

Malignant hyperthermia (MH) results from a defect of calcium release control in skeletal muscle that is often caused by point mutations in the ryanodine receptor gene (RYR1). In malignant hyperthermia-susceptible (MHS) muscle, calcium release responds more sensitively to drugs such as halothane and caffeine. In addition, experiments on the porcine homolog of malignant hyperthermia (mutation Arg615Cys in RYR1) indicated a higher sensitivity to membrane depolarization. Here, we investigated depolarization-dependent calcium release under voltage clamp conditions in human MHS muscle. Segments of muscle fibers dissected from biopsies of the vastus lateralis muscle of MHN (malignant hyperthermia negative) and MHS subjects were voltage-clamped in a double vaseline gap system. Free calcium was determined with the fluorescent indicator fura-2 and converted to an estimate of the rate of SR calcium release. Both MHN and MHS fibers showed an initial peak of the release rate, a subsequent decline, and rapid turn-off after repolarization. Neither the kinetics nor the voltage dependence of calcium release showed significant deviations from controls, but the average maximal peak rate of release was about threefold larger in MHS fibers.     

Skeletal muscle mitochondrial respiration of malignant hyperthermia-susceptible patients. Ca2+-induced uncoupling and free fatty acids

1. Skeletal muscle mitochondria of malignant hyperthermia (MH)-susceptible patients showed normal oxidative phosphorylation but were more easily uncoupled than normal by exogenous Ca2+. 2. Fatty acids, in stimulating the mitochondrial ATPase activity, are responsible for the enhanced State 4 respiration in MH-susceptible patients. 3. These results imply that skeletal muscle mitochondria and free fatty acids are associated with the development of MH syndrome.     

Defective Mg2+ regulation of RyR1 as a causal factor in malignant hyperthermia

In skeletal muscle, Mg(2+) exerts a dual inhibitory effect on RyR1, by competing with Ca(2+) at the activation site and binding to a low affinity Ca(2+)/Mg(2+) inhibitory site. Pharmacological activators of RyR1 must overcome the inhibitory action of Mg(2+) before Ca(2+) efflux can occur. In normal muscle, where the free [Mg(2+)](i) is approximately 1mM, even prolonged exposure to millimolar levels of volatile anesthetics does not initiate SR Ca(2+) release. However, when the cytosolic [Mg(2+)] is reduced below the physiological range, low levels of volatile anesthetic within the clinically relevant range (1mM) can initiate SR Ca(2+) release, in the form of a propagating Ca(2+) wave. In human muscle fibers from malignant hyperthermia susceptible patients, such Ca(2+) waves occur when 1mM halothane is applied at physiological [Mg(2+)](i). There is increasing evidence to suggest that defective Mg(2+) regulation of RyR1 confers susceptibility to malignant hyperthermia. At the molecular level, interactions between critical RyR1 subdomains may explain the clustering of RyR1 mutations and associated effects on Mg(2+) regulation.     

Abnormal membrane properties of the sarcoplasmic reticulum of pigs susceptible to malignant hyperthermia: modes of action of halothane, caffeine, dantrolene, and two other drugs

The role of sarcoplasmic reticulum (SR) in malignant hyperthermia (MH) was studied using the heavy microsomal fraction prepared from semitendinosus muscles of both normal and genetically MH-susceptible pigs. In the presence of ATP, SR was loaded with 70 nmol Ca2+/mg SR protein. Under these conditions, MH-SR demonstrated Ca2+-induced Ca2+ release (Ca-ICaR) and halothane-induced Ca2+ release (halothane-ICaR; halothane concentrations as low as 10 microM). Normal SR did not demonstrate these release phenomena. Dantrolene inhibited the halothane-ICaR, but did not inhibit the Ca-ICaR. Ruthenium red and tetracaine inhibited both types of Ca2+ release. From the measurement of passive Ca2+ efflux, it was shown that dantrolene did not affect the Ca2+ permeability of the SR itself, but suppressed only the halothane-induced increment of the permeability. The membrane order parameter of the SR, as measured by the spin-probe EPR technique, indicated that halothane disordered the lipid bilayer of MH-SR to a greater extent than it did of normal SR. This halothane disordering effect on MH-SR was antagonized by dantrolene. Ruthenium red and tetracaine did not antagonize the halothane disordering effect. These results raise the possibility that halothane could disturb the structure of the lipoprotein complex in MH-SR in such a way that it could open the Ca2+-release channels. The Ca2+ thus released further opens the channel through the Ca-ICaR mechanism in a positive feedback fashion, thus triggering the MH syndrome. The efficacy of dantrolene in ameliorating the MH syndrome might be related to the inhibition of this halothane effect.     

Ryanodine receptor channelopathies

Ryanodine receptors (RyR) are the Ca2+ release channels of sarcoplasmic reticulum that provide the majority of the [Ca2+] necessary to induce contraction of cardiac and skeletal muscle cells. In their cellular environment, RyRs are exquisitely regulated by a variety of cytosolic factors and accessory proteins so that their output signal (Ca2+) induces cell contraction without igniting signaling pathways that eventually lead to contractile dysfunction or pathological cellular remodeling. Here we review how dysfunction of RyRs, most commonly expressed as enhanced Ca2+ release at rest (skeletal muscle) or during diastole (cardiac muscle), appears to be the fundamental mechanism underlying several genetic or acquired syndromes. In skeletal muscle, malignant hyperthermia and central core disease result from point mutations in RYR1, the skeletal isoform of RyRs. In cardiac muscle, RYR2 mutations lead to catecholaminergic polymorphic ventricular tachycardia and other cardiac arrhythmias. Lastly, an altered phosphorylation of the RyR2 protein may be involved in some forms of congestive heart failure.     

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

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

Azumolene inhibits a component of store-operated calcium entry coupled to the skeletal muscle ryanodine receptor

Dantrolene reduces the elevated myoplasmic Ca(2+) generated during malignant hyperthermia, a pharmacogenetic crisis triggered by volatile anesthetics. Although specific binding of dantrolene to the type 1 ryanodine receptor (RyR1), the Ca(2+) release channel of skeletal muscle sarcoplasmic reticulum, has been demonstrated, there is little evidence for direct dantrolene inhibition of RyR1 channel function. Recent studies suggest store-operated Ca(2+) entry (SOCE) contributes to skeletal muscle function, but the effect of dantrolene on this pathway has not been examined. Here we show that azumolene, an equipotent dantrolene analog, inhibits a component of SOCE coupled to activation of RyR1 by caffeine and ryanodine, whereas the SOCE component induced by thapsigargin is not affected. Our data suggest that azumolene distinguishes between two mechanisms of cellular signaling to SOCE in skeletal muscle, one that is coupled to and one independent from RyR1.     

Reduced threshold for luminal Ca2+ activation of RyR1 underlies a causal mechanism of porcine malignant hyperthermia

Naturally occurring mutations in the skeletal muscle Ca(2+) release channel/ryanodine receptor RyR1 are linked to malignant hyperthermia (MH), a life-threatening complication of general anesthesia. Although it has long been recognized that MH results from uncontrolled or spontaneous Ca(2+) release from the sarcoplasmic reticulum, how MH RyR1 mutations render the sarcoplasmic reticulum susceptible to volatile anesthetic-induced spontaneous Ca(2+) release is unclear. Here we investigated the impact of the porcine MH mutation, R615C, the human equivalent of which also causes MH, on the intrinsic properties of the RyR1 channel and the propensity for spontaneous Ca(2+) release during store Ca(2+) overload, a process we refer to as store overload-induced Ca(2+) release (SOICR). Single channel analyses revealed that the R615C mutation markedly enhanced the luminal Ca(2+) activation of RyR1. Moreover, HEK293 cells expressing the R615C mutant displayed a reduced threshold for SOICR compared with cells expressing wild type RyR1. Furthermore, the MH-triggering agent, halothane, potentiated the response of RyR1 to luminal Ca(2+) and SOICR. Conversely, dantrolene, an effective treatment for MH, suppressed SOICR in HEK293 cells expressing the R615C mutant, but not in cells expressing an RyR2 mutant. These data suggest that the R615C mutation confers MH susceptibility by reducing the threshold for luminal Ca(2+) activation and SOICR, whereas volatile anesthetics trigger MH by further reducing the threshold, and dantrolene suppresses MH by increasing the SOICR threshold. Together, our data support a view in which altered luminal Ca(2+) regulation of RyR1 represents a primary causal mechanism of MH.     

The use of Fura-2 to estimate myoplasmic [Ca2+] in human skeletal muscle

Fura-2 was used to estimate myoplasmic [Ca2+] in intact fibers and fiber segments from normal and diseased human muscles. Small muscle bundles (20-50 fibers) were loaded with the membrane-permeant form of the dye (Fura-2 AM). High-performance liquid chromatography was utilized to study the ability of these cells to hydrolyze Fura-2 AM. Immediately after the 30 min loading period, Fura-2 (the Ca2+ indicator) was the predominant form of the dye in all preparations and the concentration within these fibers remained stable for over 4 1/2 hours. In addition, the resting myoplasmic [Ca2+] in fiber segments from normal subjects and those susceptible to malignant hyperthermia were the same. However, halothane administration (1.5%) induced correlated increases in myoplasmic [Ca2+] and force only in fibers from the susceptible patients. In contrast, caffeine administration causes correlated increases in myoplasmic [Ca2+] and force in both types of muscle, but lower concentrations were needed to do so in the fibers from the susceptible patients. The effects of halothane and caffeine were reversible. We conclude that Fura-2 can be used successfully to estimate resting levels and changes in myoplasmic [Ca2+] in human skeletal muscle.     

Effects of BAY K 8644, nifedipine, and low Ca2+ on halothane and caffeine potentiation.

The purpose of this investigation was to examine the effects of the Ca2+ agonist BAY K 8644 and the Ca2+ antagonist nifedipine on halothane- and caffeine-induced twitch potentiation of mammalian skeletal muscle. Muscle fiber bundles were taken from normal Landrace pigs and exposed to BAY K 8644 (10 microM), nifedipine (1 microM), and low Ca2+ media administered alone and in combination with halothane (3%) or with increasing concentrations of caffeine (0.5-8.0 mM). Both BAY K 8644 and halothane potentiated twitches by approximately 80%; when they were administered in combination, twitch potentiation was nearly double that caused by either drug alone. In the presence of nifedipine, halothane increased twitches by less than 30%. Low Ca2+ significantly depressed twitches by approximately 25% but also inhibited halothane's inotropic effect. BAY K 8644 augmented caffeine potentiation but only at low caffeine concentrations (0.5-2.0 mM). Nifedipine and low Ca2+ failed to inhibit caffeine's inotropic effects. These results suggest that halothane potentiates twitches via a mechanism that involves or is influenced by extracellular Ca2+.     

Studies of the halothane-cooling contractures of skeletal muscle

The characteristics of transient contractures elicited by rapid cooling of frog or mouse muscles perfused in vitro with solutions equilibrated with 0.5-2.0% halothane are reviewed. The data indicate that these halothane-cooling contractures are dose dependent and reproducible, and their amplitude is larger in muscles containing predominantly slow-twitch type fibers, such as the mouse soleus, than in muscles in which fast-twitch fibers predominate, such as the mouse extensor digitorum longus. The halothane-cooling contractures are potentiated in muscles exposed to succinylcholine. The effects of Ca2+-free solutions, of the local anesthetics procaine, procainamide, and lidocaine, and of the muscle relaxant dantrolene on the halothane-cooling contractures are consistent with the proposal that the halothane-cooling contractures result from synergistic effects of halothane and low temperature on Ca sequestration by the sarcoplasmic reticulum. Preliminary results from skinned rabbit muscle fibers support this proposal. The halothane concentrations required for the halothane-cooling contractures of isolated frog or mouse muscles are comparable with those observed in serum of patients during general anesthesia. Accordingly, fascicles dissected from muscle biopsies of patients under halothane anesthesia for programmed surgery develop large contractures when rapidly cooled. The amplitude of these halothane-cooling contractures declined with the time of perfusion of the muscle fascicles in vitro with halothane-free physiological solutions. It is suggested that the halothane-cooling contractures could be used as a simple experimental model for the investigation of the effects of halothane on Ca homeostasis and contractility in skeletal muscle and for study of drugs of potential use in the management of the contractures associated with the halothane-induced malignant hyperthermia syndrome.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+ release in the endoplasmic reticulum of guinea pig peritoneal macrophages

Passive permeability of the endoplasmic reticulum of saponin-treated macrophages to Ca2+ was studied by the filtration method using 45Ca. The Ca2+ release from the endoplasmic reticulum of macrophages was enhanced by the presence of submicromolar concentrations of Ca2+ in the medium. The Ca2+ release was enhanced by caffeine, and suppressed by MgCl2. These phenomena are similar to the Ca2+-induced Ca2+ release reported for the sarcoplasmic reticulum of skeletal muscle. On the other hand, adenine suppressed the Ca2+ release from the endoplasmic reticulum, while it reportedly enhanced the Ca2+-induced Ca2+ release of the skeletal muscle. The threshold concentration of Ca2+ for the Ca2+-induced Ca2+ release was approximately 10(-8) M in the presence of 0.95 mM MgCl2 in macrophages. The spontaneous spreading of macrophages and spontaneous migration of macrophages were inhibited by adenine, and also by caffeine in spite of the enhancement of the Ca2+-induced Ca2+ release.     

Toward a molecular understanding of the structure-function of ryanodine receptor Ca2+ release channels

Identification of the genetic basis of human diseases linked to dysfunctional free calcium (Ca2+) signaling has triggered an explosion of interest in the functional characterization of the molecular components regulating intracellular Ca2+ homeostasis. There is a growing appreciation of the central role of intracellular ryanodine-sensitive Ca2+ release channel (RyR) regulation in skeletal and cardiac muscle pathologies, including malignant hyperthermia, heart failure, and sudden cardiac death. The use of cloned RyR isoforms and recombinant expression techniques has greatly facilitated the elucidation of the molecular basis of RyR Ca2+ release functionality. This review will focus on the recombinant techniques used in the functional characterization of recombinant RyR isoforms and the insights that these approaches have yielded in unraveling the mechanistic basis of RyR channel functionality.     

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.