Mechanism of calcium release from skeletal sarcoplasmic reticulum

Summary Ca²⁺-induced Ca²⁺ release at the terminal cisternae of skeletal sarcoplasmic reticulum was demonstrated using heavy sarcoplasmic reticulum vesicles. Ca²⁺ release was observed at 10 m Ca²⁺ in the presence of 1.25mm free Mg²⁺ and was sensitive to low concentrations of ruthenium red and was partially inhibited by valinomycin. These results suggest that the Ca²⁺-induced Ca²⁺ release is electrogenic and that an inside negative membrane potential created by the Ca²⁺ flux opens a second channel that releases Ca²⁺. Results in support of this formulation were obtained by applying a Cl^– gradient or K⁺ gradient to sarcoplasmic reticulum vesicles to initiate Ca²⁺ release. Based on experiments the following hypothesis for the excitation-contraction coupling of skeletal muscle was formulated. On excitation, small amounts of Ca²⁺ enter from the transverse tubule and interact with a Ca²⁺ receptor at the terminal cisternae and cause Ca²⁺ release (Ca²⁺-induced Ca²⁺ release). This Ca²⁺ flux generates an inside negative membrane potential which opens voltage-gated Ca²⁺ channels (membrane potential-dependent Ca²⁺ release) in amounts sufficient for contraction.     

Drug-induced calcium release from heavy sarcoplasmic reticulum of skeletal muscle

Calcium release from isolated heavy sarcoplasmic reticulum of rabbit skeletal muscle by several calmodulin antagonistic drugs was measured spectrophotometrically with arsenazo III and compared with the properties of the caffeine-induced calcium release. Trifluoperazine and W7 (about 500 microM) released all actively accumulated calcium (half-maximum release at 129 microM and 98 microM, respectively) in the presence 0.5 mM MgCl2 and 1 mg/ml sarcoplasmic reticulum protein; calmidazolium (100 microM) and compound 48/80 (70 micrograms/ml) released maximally 30-40% calcium, whilst bepridil (100 microM) and felodipin (50 microM) with calmodulin antagonistic strength similar to trifluoperazine (determined by inhibition of the calcium, calmodulin-dependent protein kinase of cardiac sarcoplasmic reticulum) did not cause a detectable calcium release, indicating that this drug-induced calcium release is not due to the calmodulin antagonistic properties of the tested drugs. Calcium release of trifluoperazine, W7 and compound 48/80 and that of caffeine was inhibited by similar concentrations of magnesium (half-inhibition 1.4-4.2 mM compared with 0.97 mM for caffeine) and ruthenium red (half-inhibition for trifluoperazine, W7 and compound 48/80 was 0.22 microM, 0.08 microM and 0.63 micrograms/ml, respectively, compared with 0.13 microM for caffeine), suggesting that this drug-induced calcium release occurs via the calcium-gated calcium channel of sarcoplasmic reticulum stimulated by caffeine or channels with similar properties.     

Quercetin stimulation of calcium release from rabbit skeletal muscle sarcoplasmic reticulum

To elucidate the mechnism by which quercetin enhances the rate of tension development in skinned muscle fibers, effects on calcium release from longitudinal tubule-derived SR (LSR) after phosphate-supported calcium uptake were examined. In all studies, 100 μM quercetin (which inhibits initial calcium uptake velocity 85%) was added at or shortly after the time calcium content reached a maximum at various extravesicular Ca²⁺ concentrations (Ca_o). At moderate Ca_o (0.2–1.0 μM). where spontaneous calcium release rate depended on Ca_o, quercetin caused a marked stimulation of calcium release. This was accompanied by a 60% reduction in calcium influx and a 30-fold increase in calcium efflux. Thus, the previously reported quercetin-induced increase in the rate of tension development by skinned muscle fibers may result, at least in part, from sensitization of Ca²⁺-triggered calcium release to lower Ca_o.     

G-protein dependent potentiation of calcium release from sarcoplasmic reticulum of skeletal muscle

Skinned fibre experiments were conducted to determine if guanine nucleotide-binding proteins play a role in excitation-contraction coupling of skeletal muscle. By itself, the GTP-gamma S, a non hydrolysable GTP analogue was unable to induce calcium release from the sarcoplasmic reticulum, even at concentrations as high as 500 microM. However, calcium- or caffeine-induced calcium releases were enhanced by GTP-gamma S in micromolar concentrations. This response was blocked by GDP-beta S or Pertussis toxin. 32P-ADP-ribosylation catalysed by Pertussis toxin, radiolabelled G-protein alpha subunits in the range of 40 kDa on membrane subcellular fractions of rat skeletal muscle. Using Western blot analysis with antibodies raised against the bovine transducin, G-proteins were identified in frog and rat skeletal muscle subcellular fractions. In most of the muscle fractions (plasma membrane, T-tubules, triads, sarcoplasmic reticulum), the anti-beta subunit antibodies recognized a 36 kDa protein which comigrated with transducin beta subunit. It appears therefore that some of the G-proteins identified by ADP-ribosylation or immunostaining in several subcellular fractions from skeletal muscle, are implicated in the modulation of calcium release from sarcoplasmic reticulum. These results suggest that a Pertussis toxin sensitive G-protein is present at the loci of E-C coupling, and that it serves to regulate the calcium release.     

Phosphatidylinositol 4,5-bisphosphate enhances calcium release from sarcoplasmic reticulum of skeletal muscle

In the course of our study on the function of sarcoplasmic reticulum (SR) in skeletal muscle, the stimulatory action of phosphatidylinositol 4,5-bisphosphate (PIP2) on the Ca2+ release from SR was demonstrated by using chemically skinned fibers and fragmented SR vesicles. PIP2 induced a tension spike followed by sustained contraction in skinned fibers. PIP2 enhanced the caffeine-induced Ca2+ release from SR vesicles at low concentrations and triggered Ca2+ release by itself at high concentrations. PIP2 also enhanced 45Ca2+ efflux from SR vesicles. However, inositol 1,4,5-triphosphate never produced these effects. The Ca2+-releasing action of PIP2 was only weakly affected by ruthenium red or procaine. These observations suggest that PIP2 activates an SR Ca2+ release channel whose properties are different from those of the Ca2+-induced Ca2+ release channel.     

Pathways of calcium release from heavy sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle

The active uptake and efflux of Ca2+ from suspensions of vesicles from heavy rabbit muscle sarcoplasmic reticulum have been examined using the antipyrylazo III dye method in the presence of various nucleotide triphosphate substrates to support active Ca2+ accumulation. On addition of ATP, Ca2+ is rapidly accumulated and maintained at high internal concentrations until the substrate for pump protein is exhausted. Ca2+-induced Ca2+ release which is inhibited by ruthenium red can be demonstrated. The kinetics of Ca2+ release via these channels is different from the Ca2+ efflux observed after substrate exhaustion. This rate was found to be dependent on the type of nucleotide triphosphate, decreasing in the order ATP greater than GTP greater than CTP greater than ITP UTP. It is suggested that different conformations of the Ca2+ pump protein induced by the different substrates may result in the creation of pathways for the facilitated diffusion of Ca2+.     

Photooxidation of skeletal muscle sarcoplasmic reticulum induces rapid calcium release.

The photooxidizing xanthene dye rose bengal is shown to induce rapid Ca2+ release from skeletal muscle sarcoplasmic reticulum (SR) vesicles. In the presence of light, nanomolar concentrations of rose bengal increase the Ca2+ permeability of the SR and stimulate the production of singlet oxygen (1O2). In the absence of light, no 1O2 production is measured. Under these conditions, higher concentrations of rose bengal (micromolar) are required to stimulate Ca2+ release. Furthermore, removal of oxygen from the release medium results in marked inhibition of the light-dependent reaction rate. Rose bengal-induced Ca2+ release is relatively insensitive to Mg2+. At nanomolar concentrations, rose bengal inhibits [3H]ryanodine binding to its receptor. beta,gamma-Methyleneadenosine 5'-triphosphate, a nonhydrolyzable analog of ATP, inhibits rose bengal-induced Ca2+ release and prevents rose bengal inhibition of [3H]ryanodine binding. Ethoxyformic anhydride, a histidine modifying reagent, at millimolar concentrations induces Ca2+ release from SR vesicles in a manner similar to that of rose bengal. The molecular mechanism underlying rose bengal modification of the Ca2+ release system of the SR appears to involve a modification of a histidyl residue associated with the Ca2+ release protein from SR. The light-dependent reaction appears to be mediated by singlet oxygen.     

Hypochlorous acid modifies calcium release channel function from skeletal muscle sarcoplasmic reticulum.

We have previously demonstrated that H2O2 at millimolar concentrations induces Ca(2+) release from actively loaded sarcoplasmic reticulum (SR) vesicles and induces biphasic [(3)H]ryanodine binding behavior. Considering that hypochlorous acid (HOCl) is a related free radical and has been demonstrated to be a more effective oxidant of proteins, we evaluated the effects of HOCl on sarcoplasmic reticulum Ca(2+)-channel release mechanism. In a concentration-dependent manner, HOCl activates the SR Ca(2+) release channel and induces rapid release of Ca from actively loaded vesicles. HOCl-induced Ca(2+) release is inhibited in the presence of millimolar concentrations of DMSO. High-affinity [(3)H]ryanodine binding is also enhanced at concentrations from 10 to 100 microM. At HOCl concentrations of >100 microM, equilibrium binding is inhibited. HOCl stimulation of binding is inhibited by the addition of dithiothreitol. The direct interaction between HOCl and the Ca(2+) release mechanism was further demonstrated in single-channel reconstitution experiments. HOCl, at 20 microM, activated the Ca(2+) release channel after fusion of a SR vesicle to a bilayer lipid membrane. At 40 microM, Ca(2+)-channel activity was inhibited. Pretreatment of SR vesicles with HOCl inhibited the fluorescence development of a fluorogenic probe specific to thiol groups critical to channel function. These results suggest that HOCl at micromolar concentrations can modify SR Ca(2+) handling.     

Time course of activation of calcium release from sarcoplasmic reticulum in skeletal muscle.

Myoplasmic free calcium transients were measured with antipyrylazo III in voltage clamped segments of frog skeletal muscle fibers and were used to calculate the rate of release (Rrel) of calcium from the sarcoplasmic reticulum. Intramembrane charge movement was measured for the same pulses in the same fibers. During a depolarizing pulse Rrel rose to an early peak and then decayed relatively rapidly but incompletely due to calcium-dependent inactivation (Schneider M.F., and B.J. Simon. 1988. J. Physiol. (Lond.). 405:727-745). Two approaches were used to determine release activation independent of the effects of inactivation: (a) a mathematical correction based on the assumption that inactivation was a process occurring in parallel with and independently of activation; (b) an experimental procedure in which release was maximally inactivated by a large short prepulse and then the remaining noninactivatable component of release was monitored during a subsequent test pulse. Both procedures gave the same time course of activation of release. Release activation paralleled the time course of intramembrane charge movement but was delayed by a few milliseconds.     

Characterization of the tetraphenylboron-induced calcium release from skeletal sarcoplasmic reticulum

Release of Ca2+ from skeletal sarcoplasmic reticulum vesicles was studied by the spectrophotometric stopped-flow technique using tetraphenylboron as a releasing agent. The extent of Ca2+ release shows a sigmoidal response, with respect to the tetraphenylboron concentration, being dependent on Ca2+ preloading and Ca2+-ATPase activity, since these experiments were performed on actively loaded vesicles. The release process has a rapid component with an apparent rate constant of 6-8 s-1, showing a linear relationship between the rapid rate of Ca2+ release and the Ca2+ content of the vesicles. The release is not mediated by the reversal of the Ca2+ pump. Since the amphipathic anion tetraphenylboron was unable to elicit a Ca2+-release response when added to a preparation of sarcoplasmic reticulum phospholipid vesicles, it is suggested that there may be an interaction with some membrane protein(s) at the hydrophobic/hydrophilic interface leading to the opening of some specific Ca2+-release pathway.     

Calcium dependence of inactivation of calcium release from the sarcoplasmic reticulum in skeletal muscle fibers

The steady-state calcium dependence of inactivation of calcium release from the sarcoplasmic reticulum was studied in voltage-clamped, cut segments of frog skeletal muscle fibers containing two calcium indicators, fura-2 and anti-pyrylazo III (AP III). Fura-2 fluorescence was used to monitor resting calcium and relatively small calcium transients during small depolarizations. AP III absorbance signals were used to monitor larger calcium transients during larger depolarizations. The rate of release (Rrel) of calcium from the sarcoplasmic reticulum was calculated from the calcium transients. The equilibrium calcium dependence of inactivation of calcium release was determined using 200-ms prepulses of various amplitudes to elevate [Ca2+] to various steady levels. Each prepulse was followed by a constant test pulse. The suppression of peak Rrel during the test pulse provided a measure of the extent of inactivation of release at the end of the prepulse. The [Ca2+] dependence of inactivation indicated that binding of more than one calcium ion was required to inactivate each release channel. Half-maximal inactivation was produced at a [Ca2+] of approximately 0.3 microM. Variation of the prepulse duration and amplitude showed that the suppression of peak release was consistent with calcium-dependent inactivation of calcium release but not with calcium depletion. The same calcium dependence of inactivation was obtained using different amplitude test pulses to determine the degree of inactivation. Prepulses that produced near maximal inactivation of release during the following test pulse produced no suppression of intramembrane charge movement during the test pulse, indicating that inactivation occurred at a step beyond the voltage sensor for calcium release. Three alternative set of properties that were assumed for the rapidly equilibrating calcium-binding sites intrinsic to the fibers gave somewhat different Rrel records, but gave very similar calcium dependence of inactivation. Thus, equilibrium inactivation of calcium release appears to be produced by rather modest increases in [Ca2+] above the resting level and in a steeply calcium-dependent manner. However, the inactivation develops relatively slowly even during marked elevation of [Ca2+], indicating that a calcium-independent transition appears to occur after the initial calcium-binding step.     

Target size of calcium pump protein from skeletal muscle sarcoplasmic reticulum

The oligomeric size of calcium pump protein (CPP) in fast skeletal muscle sarcoplasmic reticulum membrane was determined using target theory analysis of radiation inactivation data. There was a parallel decrease of Ca2+-ATPase and calcium pumping activities with increasing radiation dose. The loss of staining intensity of the CPP band, observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, also correlated directly with the loss of activity. The target size molecular weight of the CPP in the normal sarcoplasmic reticulum membrane ranged between 210,000 and 250,000, which is consistent with a dimeric structure. Essentially the same size is obtained for the non-phosphorylated CPP or for the phosphoenzyme form generated from either ATP (E1 state) or inorganic phosphate (E2 state). Hence, the oligomeric state of the pump does not appear to change during the catalytic cycle. Similar results were obtained with reconstituted sarcoplasmic reticulum membrane vesicles with different lipid to protein ratios. We conclude that the CPP is a dimer in both native and reconstituted sarcoplasmic reticulum membranes. The target size of the calcium-binding protein (calsequestrin) was found to be 50,000 daltons, approximating a monomer.     

Calcium release from two fractions of sarcoplasmic reticulum from rabbit skeletal muscle

Calcium release from sarcoplasmic reticulum vesicles presumably derived from longitudinal tubules (LSR) and terminal cisternae (HSR) of rabbit skeletal muscle was investigated by dual wavelength spectrophotometry using the calcium-indicator antipyrylazo III. In 120 mM KCl, 5 mM MgCl2, 30 microM, CaCl2, 50 microM MgATP, 100 microM antipyrylazo III, 40 mM histidine (pH 6.8, 25 degrees C), LSR and HSR sequestered approx. 115 nmol calcium/mg, and then spontaneously released calcium. Analysis of ATP hydrolysis and phosphoenzyme level during LSR and HSR calcium sequestration indicated that this calcium release process was passive, occurring in the virtual absence of ATP and phosphoenzyme. Moreover, subsequent addition of ATP reinitiated the calcium sequestration-release sequence. Calcium release by HSR was more than 4-times faster than that by LSR. Analysis of the calcium release phase demonstrated a biexponential decay for both LSR (0.10 and 0.63 min-1) and HSR (0.26 and 1.65 min-1), suggestive of heterogeneity within each fraction. Replacement of 120 mM KCl with either 120 mM choline chloride, 240 mM sucrose, or H2O reduced maximal calcium sequestration by LSR, but had less effect on LSR calcium release rate constants. In the case of HSR, these changes in the ionic composition of the medium drastically reduced calcium release rate constants with little effect on calcium content. These marked differences between LSR and HSR are consistent with the hypothesis that the calcium permeability of the terminal cisternae is greater and more sensitive to the ionic environment than is that of the longitudinal tubules of sarcoplasmic reticulum.     

Chemical modification of calcium release from the sarcoplasmic reticulum of mechanically skinned skeletal bullfrog muscle fibers

Several types of reagents that react with amino acid side chains induced repetitive phasic contracture of skinned skeletal muscle from frogs. The presence of 10 mM procaine or 5 mM magnesium in the medium or disruption of the sarcoplasmic reticulum (SR) eliminated this contracture, indicating that the calcium-induced calcium-release mechanism of SR is involved in the contraction. Dithiothreitol inhibited the contracture induced by chloramine T, N-acetylimidazole, or p-chloromercuriphenylsulfonic acid (pCMPS) but not in the case of carbodiimide, phenylglyoxal, trinitrobenzenesulfonic acid, diethylpyrocarbonate (DEP), or N-chlorosuccinimide (NCS). Therefore, modification of groups other than the sulfhydryl ones seems to induce contractures under such conditions. The amplitude of the caffeine-induced contracture decreased after treatment with pCMPS, DEP, or NCS. NCS shifted the pCa-tension curve toward low pCa in the SR-disrupted fibers. This shift would explain the decrease in the caffeine contracture. It is tentatively concluded that pCMPS and DEP release a large amount of calcium from SR.     

Phosphorylation modulates the function of the calcium release channel of sarcoplasmic reticulum from skeletal muscle.

The modulation of the calcium release channel (CRC) by protein kinases and phosphatases was studied. For this purpose, we have developed a microsyringe applicator to achieve sequential and multiple treatments with highly purified kinases and phosphatases applied directly at the bilayer surface. Terminal cisternae vesicles of sarcoplasmic reticulum from rabbit fast twitch skeletal muscle were fused to planar lipid bilayers, and single-channel currents were measured at zero holding potential, at 0.15 microM free Ca2+, +/- 0.5 mM ATP and +/- 2.6 mM free Mg2+. Sequential dephosphorylation and rephosphorylation rendered the CRC sensitive and insensitive to block by Mg2+, respectively. Channel recovery from Mg2+ block was obtained by exogenous protein kinase A (PKA) or by Ca2+/calmodulin-dependent protein kinase II (CalPK II). Somewhat different characteristics were observed with the two kinases, suggesting two different states of phosphorylation. Channel block by Mg2+ was restored by dephosphorylation using protein phosphatase 1 (PPT1). Before application of protein kinases or phosphatases, channels were found to be "dephosphorylated" (inactive) in 60% and "phosphorylated" (active) in 40% of 51 single-channel experiments based on the criterion of sensitivity to block by Mg2+. Thus, these two states were interconvertable by treatment with exogenously added protein kinases and phosphatases. Endogenous Ca2+/calmodulin-dependent protein kinase (end CalPK) had an opposite action to exogenous CalPK II. Previously, dephosphorylated channels using PPT (Mg2+ absent) were blocked in the closed state by action of endogenous CalPK. This block was removed to normal activity by the action of either PPT or by exogenous CalPK II. Our findings are consistent with a physiological role for phosphorylation/dephosphorylation in the modulation of the calcium release channel of sarcoplasmic reticulum from skeletal muscle. A corollary of our studies is that only the phosphorylated channel is active under physiological conditions (mM Mg2+). Our studies suggest that phosphorylation can be at more than one site and, depending on the site, can have different functional consequences on the CRC.     

Effects of diltiazem or verapamil on calcium uptake and release from chicken skeletal muscle sarcoplasmic reticulum

The purpose of this study was to determine the effects of 2 Ca2+ channel blockers, verapamil and diltiazem, on calcium loading (active Ca2+ uptake) and the following Ca2+ release induced by silver ion (Ag+) and Ca2+ from the membrane of heavy sarcoplasmic reticulum (SR) of chicken skeletal muscle. A fluorescent probe technique was employed to determine the calcium movement through the SR. Pretreatment of the medium with diltiazem and verapamil resulted in a significant decrease in the active Ca2+ uptake, with IC50 of about 290 micromol/L for verapamil and 260 micromol/L for diltiazem. Inhibition of Ca2+ uptake was not due to the development of a substantial drug-dependent leak of Ca2+ from the SR. It might, in part, have been mediated by a direct inhibitory effect of these drugs on the Ca2+ ATPase activity of the SR Ca2+ pump. We confirmed that Ca2+ channel blockers, administered after SR Ca2+ loading and before induction of Ca2+ release, caused a dose-dependent inhibition of both Ca2+- and Ag+-induced Ca2+ release rate. Moreover, if Ca2+ channel blockers were administered prior to SR Ca2+ loading, in spite of Ca2+ uptake inhibition the same reduction in Ca2+- and Ag+-induced Ca2+ release rate was seen. We showed that the inhibition of Ag+-induced Ca2+ release by L-channel blockers is more sensitive than Ca2+-induced Ca2+ release inhibition, so the IC50 for Ag+- and Ca2+-induced Ca2+ release was about 100 and 310 micromol/L for verapamil and 79 and 330 micromol/L for diltiazem, respectively. Our results support the evidence that Ca2+ channel blockers affect muscle microsome of chicken skeletal muscle by 2 independent mechanisms: first, reduction of Ca2+ uptake rate and Ca2+-ATPase activity inhibition, and second, inhibition of both Ag+- and Ca2+-induced Ca2+ release by Ca2+ release channels. These findings confirm the direct effect of Ca2+ channel blockers on calcium release channels. Our results suggest that even if the SR is incompletely preloaded with Ca2+ because of inhibition of Ca2+ uptake by verapamil and diltiazem, no impairment in Ca2+ release occurs.     

Pharmacology of calcium release from sarcoplasmic reticulum

Calcium release from sarcoplasmic reticulum (SR) has been elicited in response to additions of many different agents. Activators of Ca²⁺ release are here tentatively classified as activators of a Ca²⁺-induced Ca²⁺ release channel preferentially localized in SR terminal or as likely activators of other Ca²⁺ efflux pathways. Some of these pathways may be associated with several different mechanisms for SR Ca²⁺ release that have been postulated previously. Studies of various inhibitors of excitation-contraction coupling and of certain forms of SR Ca²⁺ release are summarized. The sensitivity of isolated SR to certain agents is unusually affected by experimental conditions. These effects can seriously undermine attempts to anticipate effects of the same pharmacological agentsin situ. Finally, mention is made of a new preparation (sarcoballs) designed to make the pharmacological study of SR Ca²⁺ release more accessible to electrophysiologists, and some concluding speculations on the future of SR pharmacology are offered.     

Evidence of a role for calmodulin in the regulation of calcium release from skeletal muscle sarcoplasmic reticulum

The effect of calmodulin and calmodulin inhibitors on the "Ca2+ release channel" of "heavy" skeletal muscle sarcoplasmic reticulum (SR) vesicles was investigated. SR vesicles were passively loaded with 45Ca2+ in the presence of calmodulin and its inhibitors, followed by measurement of 45Ca2+ release rates by means of a rapid-quench-Millipore filtration method. Calmodulin at a concentration of 2-10 microM reduced 45Ca2+ efflux rates from passively loaded vesicles by a factor of 2-3 in media containing 10(-6)-10(-3) M Ca2+. At 10(-9) M Ca2+, calmodulin was without effect. 45Ca2+ release rates were varied 1000-fold (k1 approximately equal to 0.1-100 s-1) by using 10(-5) M Ca2+ with either Mg2+ or the ATP analogue adenosine 5'-(beta,gamma-methylenetriphosphate) in the release medium. In all instances, a similar 2-3-fold reduction in release rates was observed. At 10(-5) M Ca2+, 45Ca2+ release was half-maximally inhibited by about 2 X 10(-7) M calmodulin, and this inhibition was reversible. Heavy SR vesicle fractions contained 0.1-02 micrograms of endogenous calmodulin/mg of vesicle protein. However, the calmodulin inhibitors trifluoperazine, calmidazolium, and compound 48/80 were without significant effect on 45Ca2+ release at concentrations which inhibit calmodulin-mediated reactions in other systems. Studies with actively loaded vesicles also suggested that heavy SR vesicles contain a Ca2+ permeation system that is inhibited by calmodulin.