Activation of the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum by caffeine and related compounds

The caffeine-sensitive Ca2+ release pathway in skeletal muscle was identified and characterized by studying the release of 45Ca2+ from heavy sarcoplasmic reticulum (SR) vesicles and by incorporating the vesicles or the purified Ca2+ release channel protein complex into planar lipid bilayers. First-order rate constants for 45Ca2+ efflux of 1 s-1 were obtained in the presence of 1-10 microM free Ca2+ or 2 X 10(-9) M free Ca2+ plus 20 mM caffeine. Caffeine- and Ca2+-induced 45Ca2+ release were potentiated by ATP and Mg.ATP, and were both inhibited by Mg2+. Dimethylxanthines were similarly (3,9-dimethylxanthine) or more (1,7-, 1,3-, and 3,7-dimethylxanthine) effective than caffeine in increasing the 45Ca2+ efflux rate. 1,9-Dimethylxanthine and 1,3-dimethyluracil (which lacks the imidazole ring) did not appreciably stimulate 45Ca2+ efflux. Recordings of calcium ion currents through single channels showed that the Ca2+- and ATP-gated SR Ca2+ release channel is activated by addition of caffeine to the cis (cytoplasmic) and not the trans (lumenal) side of the channel in the bilayer. The single channel measurements further revealed that caffeine activated Ca2+ release by increasing the number and duration of open channel events without a change of unit conductance (107 pS in 50 mM Ca2+ trans). These results suggest that caffeine exerts its Ca2+ releasing effects in muscle by activating the high-conductance, ligand-gated Ca2+ release channel of sarcoplasmic reticulum.     

Conformational change of the foot protein of sarcoplasmic reticulum as an initial event of calcium release

Heavy sarcoplasmic reticulum vesicles were labeled with the thiol-reacting fluorescent probe N-(7-dimethylamino-4-methyl-4-coumarinyl)maleimide (DACM), and the DACM-labeled foot protein moiety was purified. The fluorescence intensity of the DACM attached to the foot protein decreased by the addition of low (activating) concentrations of ryanodine, while it increased at higher (inhibitory) concentrations, suggesting that the lower fluorescence represents the active state of the foot protein, while the higher fluorescence, its inactive state. Under conditions that induce Ca2+ release from SR (Ca2+ jump, addition of Ca2+ release inducing reagents such as caffeine and polylysine), the fluorescence intensity of the protein-attached DACM decreased rapidly (e.g. k congruent to 70 s-1 under optimum conditions). The initial rate of Ca2+ release from the DACM-labeled SR showed a close correlation with the amplitude of the fluorescence change of the foot protein-attached DACM under variety of conditions; e.g. in the presence of Ca2+, polylysine, ATP, and ruthenium red, etc. The fluorescence change of the foot protein was much faster than Ca2+ release from SR under a variety of conditions of Ca2+ release. We propose that the binding of release triggering reagents to the foot protein induces a rapid conformational change, which in turn regulates Ca2+ release.     

Effects of silver ion on the calcium-induced calcium release channel in isolated sarcoplasmic reticulum

Ag+-induced Ca2+ release in isolated sarcoplasmic reticulum (SR) was studied by the stopped flow method monitoring chlortetracycline fluorescence change. After improving the experimental procedure, the initial rate of Ca2+ release could be determined more precisely than before. Micromolar concentrations of Ag+ specifically enhanced Ca2+ efflux from heavy fraction of SR vesicles (HSR). This specific effect was referred to as Ag+-induced calcium release. The Ag+-induced Ca2+ efflux was activated by caffeine and ATP, but was inhibited by Mg2+ and procaine. Further, Ag+ enhanced the Ca2+-induced Ca2+ release over the whole range of Ca2+ concentrations, similarly to ATP. Parallel to Ca2+ efflux, Mg2+ efflux, measured by the same method, was also activated by Ag+. Choline permeability determined by the light scattering method was also activated by Ag+. The results suggest that Ag+ binds to the activation site of the Ca2+-induced Ca2+ release channel and opens the channel. The Ag+ binding site is different from the Ca2+ binding site but similar to the ATP binding site.     

Ca2+-induced Ca2+ release from fragmented sarcoplasmic reticulum: Ca2+-dependent passive Ca2+ efflux

Characterization of the putative Ca2+-gated Ca2+ channel of sarcoplasmic reticulum, which is thought to mediate Ca2+-induced Ca2+ release, was carried out in order to elucidate the mechanism of Ca2+-induced Ca2+ release. Heavy and light fractions of fragmented sarcoplasmic reticulum isolated from rabbit skeletal muscle were loaded passively with Ca2+, and then passive Ca2+ efflux was measured under various conditions. The fast phase of the Ca2+ efflux depended on the extravesicular free Ca2+ concentration and was assigned to the Ca2+ efflux through the Ca2+-gated Ca2+ channel. Vesicles with the Ca2+-gated Ca2+ channels comprised about 85% of the heavy fraction and about 40% of the light fraction. The amount of Ca2+ loaded in FSR was found to be much larger than that estimated on the basis of vesicle inner volume and the equilibration of intravesicular with extravesicular Ca2+, indicating Ca2+ binding inside FSR. Taking this fact into account, the Ca2+ efflux curve was quantitatively analyzed and the dependence of the Ca2+ efflux rate constant on the extravesicular free Ca2+ concentration was determined. The Ca2+ efflux was maximal, with the rate constant of 0.75 s-1, when the extravesicular free Ca2+ was at 3 microM. Caffeine increased the affinity for Ca2+ of Ca2+-binding sites for opening the channel with only a slight change in the maximum rate of Ca2+ efflux. Mg2+ inhibited the Ca2+ binding to the sites for opening the channel while procaine seemed to inhibit the Ca2+ efflux by blocking the ionophore moiety of the channel.     

Postulated role of calsequestrin in the regulation of calcium release from sarcoplasmic reticulum

Ca2+ release from heavy sarcoplasmic reticulum (SR) vesicles was induced by 2 mM caffeine, and the amount (A) and the rate constant (k) of Ca2+ release were investigated as a function of the extent of Ca2+ loading. Under both passive and active loading conditions, the A value increased monotonically in parallel to Ca2+ loading. On the other hand, k sharply increased at partial Ca2+ loading, and upon further loading, it decreased to a lower level. Since most of the intravesicular calcium appears to be bound to calsequestrin both under passive and under active loading conditions, these results suggest that the kinetic properties of induced Ca2+ release show significant variation depending upon how much calcium has been bound to calsequestrin at the time of the induction of Ca2+ release. An SR membrane segment consisting of the junctional face membrane (jfm) and attached calsequestrin (jfm-calsequestrin complex) was prepared. The covalently reacting thiol-specific conformational probe N-[7-(dimethylamino)-4-methyl-3-coumarinyl]maleimide (DACM) was incorporated into several proteins of the jfm, but not into calsequestrin. The fluorescence intensity of DACM increased with Ca2+. Upon dissociation of calsequestrin from the jfm by salt treatment, the DACM fluorescence change was abolished, while upon reassociation of calsequestrin by dilution of the salt it was partially restored. These results suggest that the events occurring in the jfm proteins are mediated via the attached calsequestrin rather than by a direct effect of Ca2+ on the jfm proteins. We propose that the [Ca2+]-dependent conformational changes of calsequestrin affect the jfm proteins and in turn regulate the Ca2+ channel functions.     

Rapid calcium release from cardiac sarcoplasmic reticulum vesicles is dependent on Ca2+ and is modulated by Mg2+, adenine nucleotide, and calmodulin

A subpopulation of canine cardiac sarcoplasmic reticulum vesicles has been found to contain a "Ca2+ release channel" which mediates the release of intravesicular Ca2+ stores with rates sufficiently rapid to contribute to excitation-contraction coupling in cardiac muscle. 45Ca2+ release behavior of passively and actively loaded vesicles was determined by Millipore filtration and with the use of a rapid quench apparatus using the two Ca2+ channel inhibitors, Mg2+ and ruthenium red. At pH 7.0 and 5-20 microM external Ca2+, cardiac vesicles released half of their 45Ca2+ stores within 20 ms. Ca2+-induced Ca2+ release was inhibited by raising and lowering external Ca2+ concentration, by the addition of Mg2+, and by decreasing the pH. Calmodulin reduced the Ca2+-induced Ca2+ release rate 3-6-fold in a reaction that did not appear to involve a calmodulin-dependent protein kinase. Under various experimental conditions, ATP or the nonhydrolyzable ATP analog, adenosine 5'-(beta, gamma-methylene)triphosphate (AMP-PCP), and caffeine stimulated 45Ca2+ release 2-500-fold. Maximal release rates (t1/2 = 10 ms) were observed in media containing 10 microM Ca2+ and 5 mM AMP-PCP or 10 mM caffeine. An increased external Ca2+ concentration (greater than or equal to 1 mM) was required to optimize the 45Ca2+ efflux rate in the presence of 8 mM Mg2+ and 5 mM AMP-PCP. These results suggest that cardiac sarcoplasmic reticulum contains a ligand-gated Ca2+ channel which is activated by Ca2+, adenine nucleotide, and caffeine, and inhibited by Mg2+, H+, and calmodulin.     

Distinct immunopeptide maps of the sarcoplasmic reticulum Ca2+ release channel in malignant hyperthermia

Sarcoplasmic reticulum isolated from malignant hyperthermia-susceptible (MHS) muscle exhibits abnormalities in the regulation of calcium release. To identify the molecular basis of this abnormality, the Ca2+ release channel from both normal and MHS sarcoplasmic reticulum was examined using proteolytic digestion followed by immunoblot staining with a polyclonal antibody against the rabbit Ca2+ release channel protein. Under appropriate conditions, trypsin digestion of isolated sarcoplasmic reticulum vesicles from the two types of pigs revealed a distinct difference in the immunostaining pattern of the Ca2+ release channel-derived peptides. An approximate 86-kDa peptide was the predominant fragment in normal sarcoplasmic reticulum while an approximate 99-kDa peptide fragment was the major peptide detected in MHS sarcoplasmic reticulum. Digestion of sarcoplasmic reticulum vesicles isolated from four normal and four MHS pigs showed that the differences were highly reproducible. Trypsin digestion of sarcoplasmic reticulum isolated from heterozygous pigs, which contain one normal and one MHS allele, showed an antibody staining pattern that was intermediate between MHS and normal sarcoplasmic reticulum. These results can be explained by a primary amino acid sequence difference between the normal and MHS Ca2+ release channels and support the hypothesis that a mutation in the gene coding for the sarcoplasmic reticulum Ca2+ release channel is responsible for malignant hyperthermia.     

Release of Ca2+ ions from the sarcoplasmic reticulum of skeletal muscle induced by heparin. Relation between the Ca2+ release caused by Ca2+ ions and caffeine

Using a Ca2+-selective electrode and Quin 2 and chlortetracycline fluorescence, a Ca2+ release from terminal cysterns of skeletal muscle sarcoplasmic reticulum under effects of heparin, caffeine and Ca2+ has been studied. It was shown that Ca2+ release induced by heparin is insensitive to the blockers of Mg2+-dependent system of Ca2+-induced Ca2+ release, i.e., Mg2+, tetracaine and dimethylsulfoxide. Preliminary release of Ca2+ in the presence of caffeine, which activates Mg2+-dependent Ca2+ release, does not prevent the heparin-induced Ca2+ release. At the same time, after Ca2+ release caused by Ca2+ in a Mg2+-independent system, heparin cannot cause additional efflux of Ca2+. It has been shown that the heparin-induced release of Ca2+ diminishes with a decrease in a decrease in Ca2+ concentration. This effect is less pronounced in the presence of Na+ than with K+. The data obtained suggest that sarcoplasmic reticulum terminal cysterns contain two systems of Ca2+-induced release of Ca2+, i.e., a Mg2+-dependent, caffeine-sensitive and a Mg2+-independent heparin-sensitive ones. The mechanism of activation of both systems by caffeine and heparin consists, in all probability, in their increased affinity for Ca2+.     

Effects of sulfhydryl reagents and other inhibitors on Ca2+ transport and inositol trisphosphate-induced Ca2+ release from human platelet membranes

The effects of modifiers of Ca2+ uptake and release in sarcoplasmic reticulum were studied in human platelet membranes. AgNO3,p-chloromercuribenzoate (pClHgBzO), N-ethylmaleimide (MalNEt), quercetin, vanadate, A23187, and caffeine all had the same effects on Ca2+ uptake in platelet membranes as had been observed for sarcoplasmic reticulum. These results strengthen our earlier conclusion that the Ca2+-pump proteins from internal human platelet membranes and muscle sarcoplasmic reticulum are very similar in functional properties. The sulfhydryl reagents Ag+ and pClHgBzO elicited rapid release of Ca2+ from platelet membranes in the presence of ATP, whereas MalNEt induced slow release. Quercetin also caused slow release of Ca2+ from platelet membranes in the presence of ATP. The effects of all three sulfhydryl reagents could be reversed by dithiothreitol, and Ag+-induced release was also reversed by ruthenium red. These effects are similar to those observed in sarcoplasmic reticulum, but in contrast caffeine did not induce Ca2+ release. In the absence of ATP, passively loaded platelet membranes did not release Ca2+ when exposed to sulfhydryl reagents. However, AgCl and pClHgBzO inhibited inositol trisphosphate (InsP3)-induced Ca2+ release from platelet membranes and this effect was reversed by dithiothreitol. Ruthenium red also inhibited InsP3-induced release, but ATP was found not to be required for InsP3-mediated release. LiCl enhanced Ca2+ release from platelet membranes. These results demonstrate that the InsP3-gated Ca2+ release channel is a separate entity from the Ca2+-pump and that essential protein sulfhydryls are involved in the release process.     

Modulation of Ca2+ release channel activity from sarcoplasmic reticulum by annexin VI (67-kDa calcimedin)

The effect of annexin VI (67-kDa calcimedin) on the activity of the Ca2+ release channel was studied using heavy sarcoplasmic reticulum membranes reconstituted into planar bilayers. Annexin VI, in a range of 5-40 nM, modified the gating behavior of the Ca2+ release channel by increasing the probability of opening by 2.7-fold and the mean open time by 82-fold relative to controls. Annexin VI caused no change in the slope conductance of the channel. The modulatory effect of annexin VI on the activity of Ca2+ release channels was Ca2+ dependent, and the annexin VI-modified channel was sensitive to both ruthenium red and ryanodine. The effect of annexin VI was observed when this protein was added specifically to the trans chamber, which corresponds to the luminal side of sarcoplasmic reticulum as determined by the ATP activation of the channel. In addition, differential extraction studies demonstrated that some annexin VI is localized within the lumen of the isolated heavy sarcoplasmic reticulum vesicles prepared by several different procedures. Annexin VI did not modify, from either the cis or trans chambers, the activity of K+ or Cl- channels from sarcoplasmic reticulum or the dihydropyridine sensitive Ca2+ channel from transverse tubules. In addition, the 38-kDa core proteolytic fragments of annexin VI had no effect on the Ca2+ release channel activity. Annexin VI is therefore a candidate for a physiological modulator of the Ca2+ release channel and as such, may play an important role in the excitation-contraction coupling.     

Release of Ca2+ ions from the sarcoplasmic reticulum of skeletal muscles after treatment with caffeine

Using a Ca2+-selective electrode and Quin 2 and chlortetracycline fluorescence spectra, a comparative study of caffeine- and Ca2+-induced release of Ca2+ from the terminal cisterns of rabbit fast skeletal muscle sarcoplasmic reticulum was carried out. It was shown that the caffeine-induced release of Ca2+ depends on Ca2+ and Mg2+ concentration in the medium; Mg2+ inhibit, while Ca2+ stimulate this process. The caffeine-induced transport of Ca2+ is blocked by ruthenium red, tetracaine and dimethylsulfoxide. The Ca2+ release induced by Ca2+ was shown to occur in two ways, i. e., via Mg2+-dependent (inhibited by Mg2+ and caffeine blockers) and Mg2+-independent (insensitive to caffeine inhibitors, including Mg2+) routes. It was assumed that caffeine stimulates the Mg2+-dependent, Ca2+-induced release of Ca2+. The sensitivity of Ca2+ transport to caffeine testifies to the fact that about 80% of the total Ca2+ transport activity of fast skeletal muscle homogenates belongs to terminal cisterns. The total amount of sarcoplasmic reticulum membranes in the muscle makes up to 15-20 mg of protein/g of tissue.     

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.     

Rapid filtration studies of Ca2+-induced Ca2+ release from skeletal sarcoplasmic reticulum. Role of monovalent ions

We have developed a rapid filtration technique for the measurement of Ca2+ release from isolated sarcoplasmic reticulum vesicles. Using this technique, we have studied the Ca2+-induced Ca2+ release of sarcoplasmic reticulum vesicles from rabbit skeletal muscle passively loaded with 5 mM Ca2+. The effect of known effectors (adenine nucleotides and caffeine) and inhibitors (Mg2+ and ruthenium red) of this release were investigated. In a medium composed of 100 mM KCl buffered at pH 6.8 with 20 mM K/3-(N-morpholino)propanesulfonic acid the Ca2+ release rate was maximal (500 nmol of Ca2+ released.(mg of protein)-1.s-1) at 1 micron external Ca2+ and 5 mM ATP. We also observed a rapid Ca2+ release induced by micromolar Ag+ in the presence of ATP (at 1 nM Ca2+). The Ag+-induced Ca2+ release was totally inhibited by 5 micron ruthenium red. We have also investigated the effect of monovalent ions on the Ca2+ release elicited by Ca2+ or Ag+. We show that the Ca2+ release rate: 1) was dependent upon the presence of K+ or Na+ in the release medium and 2) was influenced by a K+ gradient created across the sarcoplasmic reticulum membrane. These results directly support the idea of the involvement of an influx of K+ (through K+ channels) during the Ca2+ release and allow to reconsider a possible influence of the membrane potential of the sarcoplasmic reticulum on the Ca2+ release.     

Effects of alkaline pH on sarcoplasmic reticulum Ca2+ release and Ca2+ uptake.

Alkalinization-induced Ca2+ release from isolated frog or rabbit sarcoplasmic reticulum vesicles appears to consist of two distinct components: 1) a direct activation of ruthenium red-sensitive Ca2+ release channels in terminal cisternae and 2) an increased ruthenium red-insensitive Ca2+ efflux through some other efflux pathway distributed throughout the sarcoplasmic reticulum. The first of these releases exhibits an alkalinization-induced inactivation process and does not depend on the ruthenium red-insensitive form of Ca2+ release as a triggering agent for secondary Ca(2+)-induced Ca2+ release. Both releases are inhibited when the extravesicular (i.e. cytoplasmic) free [Ca2+] is reduced. This may reflect an increased sensitivity of the Ca2+ release channels to Ca2+ at alkaline pH. The pH sensitivity of the ruthenium red-sensitive Ca2+ release channels could be of significance during excitation-contraction coupling. The ruthenium red-insensitive form of Ca2+ release is less likely to be physiologically relevant, but it probably has contributed greatly to reports of alkalinization-induced decreases in net sarcoplasmic reticulum Ca2+ uptake, particularly under conditions where oxalate supported Ca2+ uptake is much less affected, as here.     

Adenine nucleotide stimulation of Ca2+-induced Ca2+ release in sarcoplasmic reticulum

Rabbit skeletal muscle sarcoplasmic reticulum was fractionated into a "Ca2+-release" and "control" fraction by differential and sucrose gradient centrifugation. External Ca2+ (2-20 microM) caused the release of 40 nmol of 45Ca2+/mg of protein/s from Ca2+-release vesicles passively loaded at pH 6.8 with an internal half-saturation Ca2+ concentration of 10-20 mM. Ca2+-induced Ca2+ release had an approximate pK value of 6.6 and was half-maximally inhibited at an external Ca2+ concentration of 2 X 10(-4) M and Mg2+ concentration of 7 X 10(-5) M. 45Ca2+ efflux from control vesicles was slightly inhibited at external Ca2+ concentrations that stimulated the rapid release of Ca2+ from Ca2+-release vesicles. Adenine, adenosine, and derived nucleotides caused stimulation of Ca2+-induced Ca2+ release in media containing a "physiological" free Mg2+ concentration of 0.6 mM. At a concentration of 1 mM, the order of effectiveness was AMP-PCP greater than cAMP approximately AMP approximately ADP greater than adenine greater than adenosine. Other nucleoside triphosphates and caffeine were minimally effective in increasing 45Ca2+ efflux from passively loaded Ca2+-release vesicles. La3+, ruthenium red, and procaine inhibited Ca2+-induced Ca2+ release. Ca2+ flux studies with actively loaded vesicles also indicated that a subpopulation of sarcoplasmic reticulum vesicles contains a Ca2+ permeation system that is activated by adenine nucleotides.     

Bromo-eudistomin D, a novel inducer of calcium release from fragmented sarcoplasmic reticulum that causes contractions of skinned muscle fibers

Bromo-eudistomin D induced a contraction of the chemically skinned fibers from skeletal muscle at concentrations of 10 microM or more. This contractile response to bromo-eudistomin D was completely blocked by 10 mM procaine. The extravascular Ca2+ concentrations of the heavy fractions of the fragmented sarcoplasmic reticulum (HSR) were measured directly by a Ca2+ electrode to examine the effect of bromo-eudistomin D on the sarcoplasmic reticulum. After the HSR was loaded with Ca2+ by the ATP-dependent Ca2+ pump, the addition of 10 microM bromo-eudistomin D caused Ca2+ release that was followed by spontaneous Ca2+ reuptake. In the presence of 2 microM ruthenium red or 4 mM MgCl2, no Ca2+ release was induced by 20 microM bromo-eudistomin D. The rate of 45Ca2+ efflux from HSR, which had been passively preloaded with 45Ca2+, was accelerated 7 times by 10 microM bromo-eudistomin D. The concentration of bromo-eudistomin D for half-maximum effect on the apparent efflux rate was 1.5 microM, while that of caffeine was 0.6 mM. The bromo-eudistomin D-evoked efflux of 45Ca2+ was abolished by 2 microM ruthenium red or 0.5 mM MgCl2. Bromo-eudistomin D was found to be 400 times more potent than caffeine in its Ca2+-releasing action but was similar in its action in other respects. These results indicate that bromo-eudistomin D may induce Ca2+ release from the sarcoplasmic reticulum through physiologically relevant Ca2+ channels.     

Activation of the skeletal muscle Ca2+ release channel by the triazine dyes cibacron blue F3A-G and reactive red 120

Vesicle-45Ca2+ ion flux and planar lipid bilayer single-channel measurements have shown that the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (SR) is activated by micromolar concentrations of Cibacron Blue F3A-G (Reactive Blue 2) and Reactive Red 120. Cibacron Blue increased the 45Ca2+ efflux rate from heavy SR vesicles by apparently interacting with both the adenine nucleotide and caffeine activating sites of the channel. Dye-induced 45Ca2+ release was inhibited by Mg2+ and ruthenium red. In single channel recordings with the purified channel protein complex, Cibacron Blue increased the open time of the Ca2+ release channel without an apparent change in the conductance of the main and subconductance states of the channel.     

Fast release of calcium from sarcoplasmic reticulum vesicles monitored by chlortetracycline fluorescence

Rapid Ca2+ release rate from sarcoplasmic reticulum vesicles was determined by the stopped flow method in terms of chlortetracycline fluorescence. Intensity of chlortetracycline fluorescence was proportional to the intravesicular free Ca2+ concentration. Ca2+ efflux was activated by extravesicular Ca2+ with an apparent dissociation constant of 25 microM and was inhibited with an inhibition constant of 120 microM in the absence of Mg2+. Caffeine enhanced the Ca2+ release rate by increasing only the affinity of Ca2+ for the activation site. Mg2+ reduced the Ca2+ release rate by competitive binding to the activation site. ATP increased the Ca2+ release rate very much without changing the affinities of Ca2+ for the activation and inhibition sites, i.e., ATP seems to increase the pore radius or number of the Ca2+ channels without affecting the gating mechanism of the channel. These results are consistent with those reported in skinned muscle sarcoplasmic reticulum. The maximum rate of Ca2+ release in the presence of ATP reached 80 s-1. This value is considered to be sufficient to cause muscular contraction.     

Effect of caffeine on kinetics of accumulation and release of Ca2+ by vesicles of the sarcoplasmic reticulum of skeletal muscle

The action of caffeine was studied on the heavy sarcoplasmic reticulum fraction enriched by vesicles derived from terminal cisterns. Caffeine lowers the ATP-dependent accumulation of Ca2+ by vesicles and enhances the first rapid phase of the Ci2+ release from vesicles. The action of caffeine was transient, reversed, Ca2+-dependent. The data obtained suggest that the reduction of ATP-dependent calcium accumulation and enhancement of calcium release by caffeine are mediated by the mechanism of Ca2+-induced Ca2+ release and support the view that caffeine may regulate the equilibrium between open and closed states of Ca2+-channel by increasing the affinity of Ca2+-receptor site of the channel.     

Calcium-activated neutral protease effects upon skeletal muscle sarcoplasmic reticulum protein structure and calcium release.

In this study, the effects of Ca(2+)-activated neutral protease (CANP) upon skeletal muscle heavy sarcoplasmic reticulum (HSR) structure and function were investigated. CANP was immunolocalized to the 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid detergent-insoluble fraction of purified HSR membranes. Ca2+ activation of the endogenous membrane-bound CANP produced a characteristic partial fragmentation of the HSR 565-kDa Ca2+ release channel. Similarly, the major substrate for both micromolar and millimolar Ca(2+)-sensitive isoforms of exogenous CANP was the Ca2+ release channel with proteolysis of a 88-kDa HSR protein also observed. Ca2+ release channel proteolysis was initiated at a single cleavage site with coincidental production of 410- and 150-kDa peptide fragments. Appearance of 160- and 137-kDa limiting peptides accompanied secondary proteolysis of the primary 410- and 150-kDa fragments, respectively. Despite extensive proteolysis of the Ca2+ release channel, CANP did not dramatically alter the Ca2+ handling and ryanodine binding properties of HSR membranes. The association of CANP with isolated HSR membranes suggests that, in vivo, this protease may modify an additional property of the Ca2+ release channel. This may be related to the CANP-susceptible structural association of the Ca2+ release channel with dihydropyridine receptors at T-tubule/sarcoplasmic reticulum junctions.