Effects of modulators of sarcoplasmic Ca2+ release on the development of skeletal muscle fatigue.

The reduced release of Ca2+ from sarcoplasmic reticulum (SR) is considered a major determinant of muscle fatigue. In the present study, we investigated whether the presence of dantrolene, an established inhibitor of SR Ca2+ release, or caffeine, a drug facilitating SR Ca2+ release, modifies muscle fatigue development. Accordingly, the effects of Ca2+ release modulators were analyzed in vitro in mouse fast-twitch [extensor digitorum longus (EDL)] and slow-twitch (soleus) muscles, fatigued by repeated short tetani (40 Hz for 300 ms, 0.5 s(-1) in soleus and 60 Hz for 300 ms, 0.3 s(-1) in EDL, for 6 min). Caffeine produced a substantial increase of tetanic tension of both EDL and soleus muscles, whereas dantrolene decreased tetanic tension only in EDL muscle. In both EDL and soleus muscles, 5 microM dantrolene did not affect fatigue development, whereas 20 microM dantrolene produced a positive staircase during the first 3 min of stimulation in EDL muscle and a slowing of fatigue development in soleus muscle. The development of the positive staircase was abolished by the addition of 15 microM ML-7, a selective inhibitor of myosin light chain kinase. On the other hand, caffeine caused a larger and faster loss of tension in both EDL and soleus muscles. The results seem to indicate that the changes in fatigue profile induced by caffeine or dantrolene are mainly due to the changes in the initial tetanic tension caused by the drugs, with the resulting changes in the level of contraction-dependent factors of fatigue, rather than to changes in the SR Ca2+ release during fatigue development.     

Ca2+ Modulation of sarcoplasmic reticulum Ca2+ release in rat skeletal muscle fibers

Ca²⁺ transients and the rate of Ca²⁺ release (dCa_REL/dt) from the sarcoplasmic reticulum (SR) in voltage-clamped, fast-twitch skeletal muscle fibers from the rat were studied with the double Vaseline gap technique and using mag-fura-2 and fura-2 as Ca²⁺ indicators. Single pulse experiments with different returning potentials showed that Ca²⁺ removal from the myoplasm is voltage independent. Thus, the myoplasmic Ca²⁺ removal (dCa_REM/dt) was studied by fitting the decaying phase of the Ca²⁺ transient (Melzer, Ríos & Schneider, 1986) and dCa_REL/dt was calculated as the difference between dCa/dt and dCa_REM/dt. The fast Ca²⁺ release decayed as a consequence of Ca²⁺ inactivation of Ca²⁺ release. Double pulse experiments showed inactivation of the fast Ca²⁺ release depending on the prepulse duration. At constant interpulse interval, long prepulses (200 msec) induced greater inactivation of the fast Ca²⁺ release than shorter depolarizations (20 msec). The correlation (r) between the myoplasmic [Ca²⁺]_i and the inhibited amount of Ca²⁺ release was 0.98. The [Ca²⁺]_i for 50% inactivation of dCa_REL/dt was 0.25 m, and the minimum number of sites occupied by Ca²⁺ to inactivate the Ca²⁺ release channel was 3.0. These data support Ca²⁺ binding and inactivation of SR Ca²⁺ release.This work was supported by Grant-in-Aid from the American Heart Association (National) and Muscular Dystrophy Association (USA). Part of this work was developed in Dr. Stefani's laboratory at Baylor College of Medicine.     

Modulation of ryanodine-induced Ca2+ release in amphibian skeletal muscle

We examined effects of ryanodine on tension in intact and skinned amphibian skeletal muscle. 100 microM ryanodine (RY) alone in the frog Ringer's solution (FR) produced tension in the intact muscle reaching its peak by 1 h; 10 min treatment with RY augmented depolarization-induced tension and prevented a subsequent caffeine-induced contraction. In contrast, RY in Ca2+-free FR was unable to produce tension, after which caffeine produced irreversible tension. In skinned fibers, RY at pCa 6.5 produced tension and abolished a subsequent caffeine-induced contraction; while Ry in 2 mM EGTA did not produce tension. These data indicate that RY, in the presence of CA2+, releases CA2+ from the SR resulting in subsequent depletion of CA in the SR.     

Intraluminal Ca2+ dependence of Ca2+ and ryanodine-mediated regulation of skeletal muscle sarcoplasmic reticulum Ca2+ release.

The action of ryanodine upon sarcoplasmic reticulum (SR) Ca2+ handling is controversial with evidence for both activation and inhibition of SR Ca2+ release. In this study, the role of the intraluminal SR Ca2+ load was probed as a potential regulator of ryanodine-mediated effects upon SR Ca2+ release. Through dual-wavelength spectroscopy of Ca2+:antipyrylazo III difference absorbance, the intraluminal Ca2+ dependence of ryanodine and Ca(2+)-induced Ca2+ release (CICR) from skeletal SR vesicles was examined. Ryanodine addition after initiation of Ca2+ uptake (a) increased the intraluminal Ca2+ sensitivity of CICR and (b) stimulated spontaneous Ca2+ release with a delayed onset. These ryanodine effects were inversely proportional to the intraluminal Ca2+ load. Ryanodine also inhibited subsequent CICR after reaccumulation of Ca2+ released from the initial CICR. These results provide evidence that ryanodine inhibits transitions between low and high affinity Ca2+ binding states of an intraluminal Ca2+ compartment, possibly calsequestrin. Conformational transitions of calsequestrin may be reciprocally coupled to transitions between open and closed states of the Ca2+ release channel.     

Calcium phosphate precipitation in the sarcoplasmic reticulum reduces action potential-mediated Ca2+ release in mammalian skeletal muscle

During vigorous exercise, P_i concentration levels within the cytoplasm of fast-twitch muscle fibers may reach 30 mM. Cytoplasmic P_i may enter the sarcoplasmic reticulum (SR) and bind to Ca²⁺ to form a precipitate (CaP_i), thus reducing the amount of releasable Ca²⁺. Using mechanically skinned rat fast-twitch muscle fibers, which retain the normal action potential-mediated Ca²⁺ release mechanism, we investigated the consequences of P_i exposure on normal excitation-contraction coupling. The total amount of Ca²⁺ released from the SR by a combined caffeine/low-Mg²⁺ concentration stimulus was reduced by 20%, and the initial rate of force development slowed after 2-min exposure to 30 mM P_i (with or without the presence creatine phosphate). Peak (50 Hz) tetanic force was also reduced (by 25% and 45% after 10 and 30 mM P_i exposure, respectively). Tetanic force responses produced after 30 mM P_i exposure were nearly identical to those observed in the same fiber after depletion of total SR Ca²⁺ by 35%. Ca²⁺ content assays revealed that the total amount of Ca²⁺ in the SR was not detectably changed by exposure to 30 mM P_i, indicating that Ca²⁺ had not leaked from the SR but instead formed a precipitate with the P_i, reducing the amount of available Ca²⁺ for rapid release. These results suggest that CaP_i precipitation that occurs within the SR could contribute to the failure of Ca²⁺ release observed in the later stages of metabolic muscle fatigue. They also demonstrate that the total amount of Ca²⁺ stored in the SR cannot drop substantially below the normal endogenous level without reducing tetanic force responses. muscle fatigue; excitation-contraction coupling     

Apocalmodulin and Ca2+ calmodulin bind to the same region on the skeletal muscle Ca2+ release channel.

The skeletal muscle Ca2+ release channel (RYR1) is regulated by calmodulin in both its Ca2+-free (apocalmodulin) and Ca2+-bound (Ca2+ calmodulin) states. Apocalmodulin is an activator of the channel, and Ca2+ calmodulin is an inhibitor of the channel. Both apocalmodulin and Ca2+ calmodulin binding sites on RYR1 are destroyed by a mild tryptic digestion of the sarcoplasmic reticulum membranes, but calmodulin (either form), bound to RYR1 prior to tryptic digestion, protects both the apocalmodulin and Ca2+ calmodulin sites from tryptic destruction. The protected sites are after arginines 3630 and 3637 on RYR1. These studies suggest that both Ca2+ calmodulin and apocalmodulin bind to the same or overlapping regions on RYR1 and block access of trypsin to sites at amino acids 3630 and 3637. This sequence is part of a predicted Ca2+ CaM binding site of amino acids 3614-3642 [Takeshima, H., et al. (1989) Nature 339, 439-445].     

Comparison of the effects of inorganic phosphate on caffeine-induced Ca2+ release in fast- and slow-twitch mammalian skeletal muscle

We compared the effects of 50 mM P(i) on caffeine-induced Ca(2+) release in mechanically skinned fast-twitch (FT) and slow-twitch (ST) skeletal muscle fibers of the rat. The time integral (area) of the caffeine response was reduced by approximately 57% (FT) and approximately 27% (ST) after 30 s of exposure to 50 mM P(i) in either the presence or absence of creatine phosphate (to buffer ADP). Differences in the sarcoplasmic reticulum (SR) Ca(2+) content between FT and ST fibers [ approximately 40% vs. 100% SR Ca(2+) content (pCa 6.7), respectively] did not contribute to the different effects of P(i) observed; underloading the SR of ST fibers so that the SR Ca(2+) content approximated that of FT fibers resulted in an even smaller ( approximately 21%), but not significant, reduction in caffeine-induced Ca(2+) release by P(i). These observed differences between FT and ST fibers could arise from fiber-type differences in the ability of the SR to accumulate Ca(2+)-P(i) precipitate. To test this, fibers were Ca(2+) loaded in the presence of 50 mM P(i). In FT fibers, the maximum SR Ca(2+) content (pCa 6.7) was subsequently increased by up to 13 times of that achieved when loading for 2 min in the absence of P(i). In ST fibers, the SR Ca(2+) content was only doubled. These data show that Ca(2+) release in ST fibers was less affected by P(i) than FT fibers, and this may be due to a reduced capacity of ST SR to accumulate Ca(2+)-P(i) precipitate. This may account, in part, for the fatigue-resistant nature of ST fibers.     

Calcium release flux underlying Ca2+ sparks of frog skeletal muscle.

An algorithm for the calculation of Ca2+ release flux underlying Ca2+ sparks (Blatter, L.A., J. Hüser, and E. Ríos. 1997. Proc. Natl. Acad. Sci. USA. 94:4176-4181) was modified and applied to sparks obtained by confocal microscopy in single frog skeletal muscle fibers, which were voltage clamped in a two-Vaseline gap chamber or permeabilized and immersed in fluo-3-containing internal solution. The performance of the algorithm was characterized on sparks obtained by simulation of fluorescence due to release of Ca2+ from a spherical source, in a homogeneous three-dimensional space that contained components representing cytoplasmic molecules and Ca2+ removal processes. Total release current, as well as source diameter and noise level, was varied in the simulations. Derived release flux or current, calculated by volume integration of the derived flux density, estimated quite closely the current used in the simulation, while full width at half magnitude of the derived release flux was a good monitor of source size only at diameters >0. 7 micrometers. On an average of 157 sparks of amplitude >2 U resting fluorescence, located automatically in a representative voltage clamp experiment, the algorithm reported a release current of 16.9 pA, coming from a source of 0.5 micrometer, with an open time of 6.3 ms. Fewer sparks were obtained in permeabilized fibers, so that the algorithm had to be applied to individual sparks or averages of few events, which degraded its performance in comparable tests. The average current reported for 19 large sparks obtained in permeabilized fibers was 14.4 pA. A minimum estimate, derived from the rate of change of dye-bound Ca2+ concentration, was 8 pA. Such a current would require simultaneous opening of between 8 and 60 release channels with unitary Ca2+ currents of the level recorded in bilayer experiments. Real sparks differ from simulated ones mainly in having greater width. Correspondingly, the algorithm reported greater spatial extent of the source for real sparks. This may again indicate a multichannel origin of sparks, or could reflect limitations in spatial resolution.     

Ca2+ release by inositol-trisphosphorothioate in isolated triads of rabbit skeletal muscle.

The effectiveness of the nonmetabolizable second messenger analogue DL-myo-inositol 1,4,5-trisphosphorothioate (IPS3) described by Cooke, A. M., R. Gigg, and B. V. L. Potter, (1987b. Jour. Chem. Soc. Chem. Commun. 1525-1526.) was examined in triads purified from rabbit skeletal muscle. A Ca2+ electrode uptake-release assay was used to determine the size and sensitivity of the IPS3-releasable pool of Ca2+ in isolated triads. Uptake was initiated by 1 mM MgATP, pCa 5.8, pH 7.5 Release was initiated when the free Ca2+ had lowered to pCa approximately 7. We found that 5-25 microM myo-inositol 1,4,5-trisphosphate (IP3), and separately IPS3, consistently released 5-20% of the Ca2+ pool actively loaded into triads. Single channel recording was used to determine if ryanodine receptor Ca2+ release channels were affected by IPS3 at the same myoplasmic Ca2+ and IPS3 concentrations. Open probability of ryanodine receptor Ca2+ release channels was monitored in triads fused to bilayers over long periods (200 s) in the absence and following addition of 30 microM IPS3 to the same channel. At myoplasmic pCa approximately 7, IPS3 had no effect in the absence of MgATP (Po = 0.0094 +/- 0.001 in control and Po = 0.01 +/- 0.006 after IPS3) and slightly increased activity in the presence of 1 mM MgATP (Po = 0.024 +/- 0.03 in control and Po = 0.05 +/- 0.03 after IPS3). Equally small effects were observed at higher myoplasmic Ca2+. The onset of channel activation by IPS3 or IP3 was slow, on the time scale 20-60 s. We suggest that in isolated triads of rabbit skeletal muscle, IP3-induced release of stored Ca2+ is probably not mediated by the opening of Ca2+ release channels.     

Ca2+ release through ryanodine receptors regulates skeletal muscle L-type Ca2+ channel expression

Skeletal muscle obtained from mice that lack the type 1 ryanodine receptor (RyR-1), termed dyspedic mice, exhibit a 2-fold reduction in the number of dihydropyridine binding sites (DHPRs) compared with skeletal muscle obtained from wild-type mice (Buck, E. D., Nguyen, H. T., Pessah, I. N., and Allen, P. D. (1997) J. Biol. Chem. 272, 7360-7367 and Fleig, A., Takeshima, H., and Penner, R. (1996) J. Physiol. (Lond.) 496, 339-345). To probe the role of RyR-1 in influencing L-type Ca(2+) channel (L-channel) expression, we have monitored functional L-channel expression in the sarcolemma using the whole-cell patch clamp technique in normal, dyspedic, and RyR-1-expressing dyspedic myotubes. Our results indicate that dyspedic myotubes exhibit a 45% reduction in maximum immobilization-resistant charge movement (Q(max)) and a 90% reduction in peak Ca(2+) current density. Calcium current density was significantly increased in dyspedic myotubes 3 days after injection of cDNA encoding either wild-type RyR-1 or E4032A, a mutant RyR-1 that is unable to restore robust voltage-activated release of Ca(2+) from the sarcoplasmic reticulum (SR) following expression in dyspedic myotubes (O'Brien, J. J., Allen, P. D., Beam, K., and Chen, S. R. W. (1999) Biophys. J. 76, A302 (abstr.)). The increase in L-current density 3 days after expression of either RyR-1 or E4032A occurred in the absence of a change in Q(max). However, Q(max) was increased 85% 6 days after injection of dyspedic myotubes with cDNA encoding the wild-type RyR-1 but not E4032A. Because normal and dyspedic myotubes exhibited a similar density of T-type Ca(2+) current (T-current), the presence of RyR-1 does not appear to cause a general overall increase in protein synthesis. Thus, long-term expression of L-channels in skeletal myotubes is promoted by Ca(2+) released through RyRs occurring either spontaneously or during excitation-contraction coupling.     

Effects of azumolene on doxorubicin-induced Ca2+ release from skeletal and cardiac muscle sarcoplasmic reticulum

The mechanism of doxorubicin-induced Ca2+ release from skeletal and cardiac muscle sarcoplasmic reticulum (SR) was studied by examining the effects of azumolene (a water soluble dantrolene analog) on doxorubicin-mediated Ca2+ release and ryanodine binding. Doxorubicin induced a rapid Ca2+ release from both skeletal and cardiac SR in a similar concentration range (EC50 = 5-10 microM). Maximal doxorubicin-induced Ca2+ release was seen at 2 and 0.2 microM Ca2+ for skeletal and cardiac SR, respectively. Addition of 400 microM azumolene caused approx. 30% inhibition of doxorubicin-induced Ca2+ release from both skeletal and cardiac SR; skeletal SR had significantly higher sensitivity to azumolene than cardiac SR. In the presence of Ca2+, doxorubicin increased [3H]ryanodine binding to both skeletal and cardiac SR; whereas in the absence of Ca2+, doxorubicin led to significant ryanodine binding to skeletal SR, but not to cardiac SR. In both types of SR, doxorubicin-activated, but not Ca2+ activated ryanodine binding was inhibited by azumolene. Azumolene sensitivity for inhibition of doxorubicin-activated ryanodine binding was much higher in skeletal SR than cardiac SR, consistent with the results for effects of azumolene on Ca2+ release. Our results are consistent with the possibility that azumolene inhibits doxorubicin binding by direct competition for the drug receptor(s).     

Effects of a domain peptide of the ryanodine receptor on Ca2+ release in skinned skeletal muscle fibers

Mutations in the central domain of the skeletal muscle ryanodinereceptor (RyR) cause malignant hyperthermia (MH). A synthetic peptide(DP4) in this domain (Leu-2442-Pro-2477) produces enhanced ryanodine binding and sensitized Ca²⁺ release in isolatedsarcoplasmic reticulum, similar to the properties in MH, possiblybecause the peptide disrupts the normal interdomain interactions thatstabilize the closed state of the RyR (Yamamoto T, El-Hayek R, andIkemoto N. J Biol Chem 275: 11618-11625, 2000). Here, DP4 was applied to mechanically skinned fibers of rat muscle thathad the normal excitation-contraction coupling mechanism stillfunctional to determine whether muscle fiber responsiveness wasenhanced. DP4 (100 µM) substantially potentiated the Ca²⁺release and force response to caffeine (8 mM) and to low[Mg²⁺] (0.2 mM) in every fiber examined, with nosignificant effect on the properties of the contractile apparatus. DP4also potentiated the response to submaximal depolarization of thetransverse tubular system by ionic substitution. Importantly, DP4 didnot significantly alter the size of the twitch response elicited byaction potential stimulation. These results support the proposal thatDP4 causes an MH-like aberration in RyR function and are consistentwith the voltage sensor triggering Ca²⁺ release bydestabilizing the closed state of the RyRs.

     

Oxidation of the skeletal muscle Ca2+ release channel alters calmodulin binding

This study presents evidence for a close relationship betweenthe oxidation state of the skeletal muscleCa²⁺ release channel (RyR1) andits ability to bind calmodulin (CaM). CaM enhances the activity of RyR1in low Ca²⁺ and inhibits itsactivity in high Ca²⁺. Oxidation,which activates the channel, blocks the binding of ¹²⁵I-labeled CaM at bothmicromolar and nanomolar Ca²⁺concentrations. Conversely, bound CaM slows oxidation-induced cross-linking between subunits of the RyR1 tetramer. Alkylation ofhyperreactive sulfhydryls (<3% of the total sulfhydryls) on RyR1with N-ethylmaleimide completelyblocks oxidant-induced intersubunit cross-linking and inhibitsCa²⁺-free¹²⁵I-CaM but notCa²⁺/¹²⁵I-CaMbinding. These studies suggest that1) the sites on RyR1 for bindingapocalmodulin have features distinct from those of theCa²⁺/CaM site,2) oxidation may alter the activityof RyR1 in part by altering its interaction with CaM, and3) CaM may protect RyR1 fromoxidative modifications during periods of oxidative stress.

     

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.     

Ca2+ sparks in skeletal muscle

The study of Ca²⁺ sparks has led to extensive new information regarding the gating of the Ca²⁺ release channels underlying these events in skeletal, cardiac and smooth muscle cells, as well as the possible roles of these local Ca²⁺ release events in muscle function. Here we review basic procedures for studying Ca²⁺sparks in skeletal muscle, primarily from frog, as well as the basic results concerning the properties of these events, their pattern and frequency of occurrence during fiber depolarization and the mechanisms underlying their termination. Finally, we also consider the contribution of different ryanodine receptor (RyR) isoforms to Ca²⁺ sparks and the number of RyR Ca²⁺ release channels that may contribute to the generation of a Ca²⁺ spark. Over the decade since their discovery, Ca²⁺ sparks have provided a wealth of information concerning the function of Ca²⁺ release channels within their intracellular environment.     

Abnormal human sarcoplasmic reticulum Ca2+ release channels in malignant hyperthermic skeletal muscle.

Single sarcoplasmic reticulum (SR) Ca2+ release channels were reconstituted from normal and malignant hyperthermic (MH) human skeletal muscle biopsies (2-5 g samples). Conduction, gating properties, and myoplasmic Ca2+ dependence of human SR Ca2+ release channels were similar to those in other species (rabbit, pig). The MH diagnostic procedure distinguishes three phenotypes (normal, MH-equivocal, and MH-susceptible) on the basis of muscle contracture sensitivity to caffeine and/or halothane. Single channel studies reveal that human MH muscles (both MH phenotypes) contain SR Ca2+ release channels with abnormally greater caffeine sensitivity. Muscles from MH-equivocal and MH-susceptible patients appear to contain channels with the same abnormality. Further, our data (n = 115, 21 channels, 11 patients) reveals that human MH muscles (both phenotypes) may contain two populations of SR Ca2+ release channels, possibly corresponding to normal and abnormal isoforms. Thus, whole cell phenotypic variation (MH-equivocal vs. MH-susceptible) arises in muscles containing channels with similar caffeine sensitivity suggesting that human MH does not arise from a single defect. These results have important ramifications concerning (a) correlation of functional and genetic MH studies, (b) identification of other, yet to be determined, factors which may influence MH expression, and (c) characterization of normal SR Ca2+ release channel function by exploring genetic channel defects.     

Mechanism of chloride-dependent release of Ca2+ in the sarcoplasmic reticulum of rabbit skeletal muscle.

We investigated the effect of Cl- on the Ca2+ permeability of rabbit skeletal muscle junctional sarcoplasmic reticulum (SR) using 45Ca2+ fluxes and single channel recordings. In 45Ca2+ efflux experiments, the lumen of the SR was passively loaded with solutions of 150 mM univalent salt containing 5 mM 45Ca2+. Release of 45Ca2+ was measured by rapid filtration in the presence of extravesicular 0.4-0.8 microM free Ca2+ and 150 mM of the same univalent salt loaded into the SR lumen. The rate of release was 5-10 times higher when the univalent salt equilibrated across the SR-contained Cl- (Tris-Cl, choline-Cl, KCl) instead of an organic anion or other halides (gluconate-, methanesulfonate-, acetate-, HEPES-, Br-, I-). Cations (K+, Tris+) could be interchanged without a significant effect on the release rate. To determine whether Cl- stimulated ryanodine receptors, we measured the stimulation of release by ATP (5 mM total) and caffeine (20 mM total) and the inhibition by Mg2+ (0.8 mM estimated free) in Cl(-)-free and Cl(-)-containing solutions. The effects of ATP, caffeine, and Mg2+ were the largest in K-gluconate and Tris-gluconate, intermediate in KCl, and notably poor or absent in choline-Cl and Tris-Cl. Procaine (10 mM) inhibited the caffeine-stimulated release measured in K-gluconate, whereas the Cl- channel blocker clofibric acid (10 mM) but not procaine inhibited the caffeine-insensitive release measured in choline-Cl. Ruthenium red (20 microM) inhibited release in all solutions. In SR fused to planar bilayers we identified a nonselective Cl- channel (PCl: PTris: PCa = 1:0.5:0.3) blocked by ruthenium red and clofibric acid but not by procaine. These conductive and pharmacological properties suggested the channel was likely to mediate Cl(-)-dependent SR Ca2+ release. The absence of a contribution of ryanodine receptors to the Cl(-)-dependent release were indicated by the lack of an effect of Cl- on the open probability of this channel, a complete block by procaine, and a stimulation rather than inhibition by clofibric acid. A plug model of Cl(-)-dependent release, whereby Cl- removed the inhibition of the nonselective channel by large anions, was formulated under the assumption that nonselective channels and ryanodine receptor channels operated separately from each other in the terminal cisternae. The remarkably large contribution of Cl- to the SR Ca2+ permeability suggested that nonselective Cl- channels may control the Ca2+ permeability of the SR in the resting muscle cell.     

Inactivation of a Ca2+-induced Ca2+ release channel from skeletal muscle sarcoplasmic reticulum during active Ca2+ transport

ATP-dependent Ca2+ uptake by subfractions of skeletal muscle sarcoplasmic reticulum (SR) was studied with the Ca2+ indicator dye, antipyrylazo III. Ca2+ uptake by heavy SR showed two phases, a slow uptake phase and a fast uptake phase. By contrast, Ca2+ uptake by light SR exhibited a monophasic time course. In both fractions a steady state of Ca2+ uptake was observed when the concentration of free Ca2+ outside the vesicles was reduced to less than 0.1 microM. In the steady state, the addition of 5 microM Ca2+ to the external medium triggered rapid Ca2+ release from heavy SR but not from light SR, indicating that the heavy fraction contains a Ca2+-induced Ca2+ release channel. During Ca2+ uptake, heavy SR showed a constant Ca2+-dependent ATPase activity (1 mumol/mg protein X min) which was about 150 times higher than the rate of Ca2+ uptake in the slow uptake phase. Ruthenium red, an inhibitor of Ca2+-induced Ca2+ release, enhanced the rate of Ca2+ uptake during the slow phase without affecting Ca2+-dependent ATPase activity. Adenine nucleotides, activators of Ca2+ release, reduced the Ca2+ uptake rate. These results suggest that the rate of Ca2+ accumulation by heavy SR is not proportional to ATPase activity during the slow uptake phase due to the activation of the channel for Ca2+-induced Ca2+ release. In addition, they suggest that the release channel is inactivated during the fast Ca2+ uptake phase.     

Structure of the skeletal muscle calcium release channel activated with Ca2+ and AMP-PCP.

The functional state of the skeletal muscle Ca2+ release channel is modulated by a number of endogenous molecules during excitation-contraction. Using electron cryomicroscopy and angular reconstitution techniques, we determined the three-dimensional (3D) structure of the skeletal muscle Ca2+ release channel activated by a nonhydrolyzable analog of ATP in the presence of Ca2+. These ligands together produce almost maximum activation of the channel and drive the channel population toward a predominately open state. The resulting 30-A 3D reconstruction reveals long-range conformational changes in the cytoplasmic region that might affect the interaction of the Ca2+ release channel with the t-tubule voltage sensor. In addition, a central opening and mass movements, detected in the transmembrane domain of both the Ca(2+)- and the Ca2+/nucleotide-activated channels, suggest a mechanism for channel opening similar to opening-closing of the iris in a camera diaphragm.