Fragmented Sarcoplasmic Reticulum Research Articles

Soluble calcium-binding proteins (SCBPs) of the earthworm Lumbricus terrestris: possible role as relaxation factors in muscle.

The soluble Ca2+-binding protein (SCBP) from the earthworm Lumbricus terrestris was analyzed with regard to its role as a soluble muscle relaxation factor. The actomyosin ATPase activity was inhibited by the addition of decalcified SCBP as it binds Ca2+ stronger than the regulatory proteins associated with the actomyosin. Competitive 45Ca2+-binding assays with decalcified actomyosin and SCBP showed that 45Ca2+ is first bound to actomyosin and is subsequently taken over by SCBP with increasing incubation time. Ca2+ competition experiments carried out with 45Ca2+ loaded SCBP and fragmented sarcoplasmic reticulum vesicles revealed that 45Ca2+ bound to SCBP can be deprived by the ATP-dependent Ca2+ uptake of the sarcoplasmic reticulum. Furthermore, experiments in a diffusion chamber showed that the addition of SCBP significantly enhances the 45Ca2+ flux in a concentration dependent manner. The amount of the Ca2+ flux increasetends to reach a maximum value of about 70%. With all protein components isolated from the obliquely striated muscle, our in vitro experiments consistently show that SCBP may accelerate muscle relaxation similar as assumed for vertebrate parvalbumin.

Local Ca2+ coupling between mitochondria and sarcoplasmic reticulum following depolarization in guinea pig urinary bladder smooth muscle cells.

Spatiotemporal changes in cytosolic Ca2+ concentration ([Ca2+]c) trigger a number of physiological functions in smooth muscle cells (SMCs). We previously imaged Ca2+-induced Ca2+ release following membrane depolarization as local Ca2+ transients, Ca2+ hotspots, in subplasmalemmal regions. In this study, the physiological significance of mitochondria on local Ca2+ signaling was examined. Cytosolic and mitochondrial Ca2+ images following depolarization or action potentials were recorded in single SMCs from the guinea pig urinary bladder using a fast-scanning confocal fluorescent microscope. Depolarization- and action potential-induced [Ca2+]c transients occurred at several discrete sites in subplasmalemmal regions, peaked within 30 ms, and then spread throughout the whole-cell. In contrast, Ca2+ concentration in the mitochondria matrix ([Ca2+]m) increased after a delay of ~50 ms from the start of depolarization, and then peaked within 500 ms. Following repolarization, [Ca2+]c returned to the resting level with a half-decay time of ~500 ms, while [Ca2+]m recovered more slowly (∼1.5 s). Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, a mitochondrial uncoupler, abolished depolarization-induced [Ca2+]m elevations and slowed [Ca2+]c changes. Importantly, short depolarization-induced changes in [Ca2+]m and transmembrane potential in mitochondria coupled to Ca2+ hotspots were significantly larger than those in other mitochondria. Total internal reflection fluorescence imaging revealed that a subset of mitochondria closely localized with ryanodine receptors and voltage-dependent Ca2+ channels. These results indicate that particular mitochondria are functionally coupled to ion channels and sarcoplasmic reticulum fragments within the local Ca2+ microdomain, and thus, strongly contribute to [Ca2+]c regulation in SMCs.

Calcium-Induced Calcium Release in Skeletal Muscle

Calcium-induced calcium release (CICR) was first discovered in skeletal muscle. CICR is defined as Ca2+ release by the action of Ca2+ alone without the simultaneous action of other activating processes. CICR is biphasically dependent on Ca2+ concentration; is inhibited by Mg2+, procaine, and tetracaine; and is potentiated by ATP, other adenine compounds, and caffeine. With depolarization of the sarcoplasmic reticulum (SR), a potential change of the SR membrane in which the luminal side becomes more negative, CICR is activated for several seconds and is then inactivated. All three types of ryanodine receptors (RyRs) show CICR activity. At least one RyR, RyR1, also shows non-CICR Ca2+ release, such as that triggered by the t-tubule voltage sensor, by clofibric acid, and by SR depolarization. Maximum rates of CICR, at the optimal Ca2+ concentration in the presence of physiological levels of ATP and Mg2+ determined in skinned fibers and fragmented SR, are much lower than the rate of physiological Ca2+ release. The primary event of physiological Ca2+ release, the Ca2+ spark, is the simultaneous opening of multiple channels, the coordinating mechanism of which does not appear to be CICR because of the low probability of CICR opening under physiological conditions. The coordination may require Ca2+, but in that case, some other stimulus or stimuli must be provided simultaneously, which is not CICR by definition. Thus CICR does not appear to contribute significantly to physiological Ca2+ release. On the other hand, CICR appears to play a key role in caffeine contracture and malignant hyperthermia. The potentiation of voltage-activated Ca2+ release by caffeine, however, does not seem to occur through secondary CICR, although the site where caffeine potentiates voltage-activated Ca2+ release might be the same site where caffeine potentiates CICR.

Stabilization of membranes upon interaction of amphipathic polymers with membrane proteins.

Amphipathic polymers derived from polysaccharides, namely hydrophobically modified pullulans, were previously suggested to be useful as polymeric substitutes of ordinary surfactants for efficient and structure-conserving solubilization of membrane proteins, and one such polymer, 18C(10), was optimized for solubilization of proteins derived from bacterial outer membranes (Duval-Terrie et al. 2003). We asked whether a similar ability to solubilize proteins could also be demonstrated in eukaryotic membranes, namely sarcoplasmic reticulum (SR) fragments, the major protein of which is SERCA1a, an integral membrane protein with Ca(2+)-dependent ATPase and Ca(2+)-pumping activity. We found that 18C(10)-mediated solubilization of these SR membranes did not occur. Simultaneously, however, we found that low amounts of this hydrophobically modified pullulan were very efficient at preventing long-term aggregation of these SR membranes. This presumably occurred because the negatively charged polymer coated the membranous vesicles with a hydrophilic corona (a property shared by many other amphipathic polymers), and thus minimized their flocculation. Reminiscent of the old Arabic gum, which stabilizes Indian ink by coating charcoal particles, the newly designed amphipathic polymers might therefore unintentionally prove useful also for stabilization of membrane suspensions.

Local Ca(2+) transients and distribution of BK channels and ryanodine receptors in smooth muscle cells of guinea-pig vas deferens and urinary bladder.

1. The relationship between Ca(2+) sparks spontaneously occurring at rest and local Ca(2+) transients elicited by depolarization was analysed using two-dimensional confocal Ca(2+) images of single smooth muscle cells isolated from guinea-pig vas deferens and urinary bladder. The current activation by these Ca(2+) events was also recorded simultaneously under whole-cell voltage clamp. 2. Spontaneous transient outward currents (STOCs) and Ca(2+) sparks were simultaneously detected at -40 mV in approximately 50 % of myocytes of either type. Ca(2+) sparks and corresponding STOCs occurred repetitively in several discrete sites in the subplasmalemmal area. Large conductance Ca(2+)-dependent K(+) (BK) channel density in the plasmalemma near the Ca(2+) spark sites generating STOCs was calculated to be 21 channels microm(-2). 3. When myocytes were depolarized from -60 to 0 mV, several local Ca(2+) transients were elicited within 20 ms in exactly the same peripheral sites where sparks occurred at rest. The local Ca(2+) transients often lasted over 300 ms and spread into other areas. The appearance of local Ca(2+) transients occurred synchronously with the activation of Ca(2+)-dependent K(+) current (I(K,Ca)). 4. Immunofluorescence staining of the BK channel alpha-subunit (BKalpha) revealed a spot-like pattern on the plasmalemma, in contrast to the uniform staining of voltage-dependent Ca(2+) channel alpha1C subunits along the plasmalemma. Ryanodine receptor (RyR) immunostaining also suggested punctate localization predominantly in the periphery. Double staining of BKalpha and RyRs revealed spot-like co-localization on/beneath the plasmalemma. 5. Using pipettes of relatively low resistance, inside-out patches that included both clustered BK channels at a density of over 20 channels microm(-2) and functional Ca(2+) storage sites were obtained at a low probability of approximately 5%. The averaged BK channel density was 3-4 channels microm(-2) in both types of myocyte. 6. These results support the idea that a limited number of discrete sarcoplasmic reticulum (SR) fragments in the subplasmalemmal area play key roles in the control of BK channel activity in two ways: (i) by generating Ca(2+) sparks at rest to activate STOCs and (ii) by generating Ca(2+) transients presumably triggered by sparks during an action potential to activate a large I(K,Ca) and also induce a contraction. BK channels and RyRs may co-localize densely at the junctional areas of plasmalemma and SR fragments, where Ca(2+) sparks occur to elicit STOCs.

Different effects of two gold compounds on muscle contraction, membrane potential and ryanodine receptor

Effects of gold sodium thiomalate and NaAuCl 4 on skeletal muscle function were studied using intact single fibres of frog skeletal muscle and fragmented sarcoplasmic reticulum prepared from frog and rabbit skeletal muscles. Gold sodium thiomalate at a concentration of 500 μM decreased tension amplitude by 27% and resting membrane potential by 5.3% after 30 and 22 min, respectively. The duration of tetanus tension was markedly shortened by 500 μM gold sodium thiomalate. When 10 μM NaAuCl 4 was applied to gold sodium thiomalate-pretreated fibres, the fibres lost the ability to contract upon electrical stimulation, similar to the effects of 10 μM NaAuCl 4 alone. In the presence of thiomalic acid, on the other hand, NaAuCl 4 did not completely block tetanus tension even at 50 μM. Thiomalic acid also inhibited NaAuCl 4-induced membrane depolarization. These findings suggest that thiomalate masks the effects of gold ion on muscle function. When sarcoplasmic reticulum vesicles were incorporated into lipid bilayers, exposure of the cis side of the Ca 2+-release channel to 100 μM gold sodium thiomalate rapidly increased the open probability of the channel 3.3-fold, from 0.032 in controls to 0.105, with an increase in number of open events and a decrease in mean closed time. The ability of NaAuCl 4 to activate the Ca 2+-release channel was much stronger than that of gold sodium thiomalate. Only 1 μM NaAuCl 4 was enough to activate the channel and this gold was effective from either side of the channel. These results suggest that gold sodium thiomalate could be used as an antirheumatic drug without considering severe side-effects on skeletal muscle. Coexistent thiomalate probably contributes to protection of muscle function from side-effects of gold ion.

Properties of amentoflavone, a potent caffeine-like Ca 2+ releaser in skeletal muscle sarcoplasmic reticulum

Amentoflavone, like caffeine, caused a concentration-dependent increase in Ca 2+ release from the heavy fraction of fragmented sarcoplasmic reticulum of rabbit skeletal muscle. The Ca 2+-releasing activity of amentoflavone was approximately 20 times more potent than that of caffeine. The bell-shaped profile of Ca 2+ dependence for amentoflavone was almost the same as that for caffeine. Typical blockers of Ca 2+-induced Ca 2+ release channels, such as Mg 2+, procaine and ruthenium red, inhibited markedly amentoflavone- and caffeine-induced 45 Ca 2+ release. The maximum 45 Ca 2+ release in response to amentoflavone was not changed by caffeine, but was further increased by adenosine-5′-(β,γ-methylene) triphosphate. This compound enhanced [ 3 H ]ryanodine binding to the heavy fraction of fragmented sarcoplasmic reticulum with a decrease in K D but without a change in B max. These results suggest that amentoflavone, which does not contain a nitrogen atom, probably binds to the caffeine-binding site in Ca 2+ channels and thus potentiates Ca 2+-induced Ca 2+ release from the heavy fraction of fragmented sarcoplasmic reticulum.

Two novel types of calcium release from skeletal sarcoplasmic reticulum by phosphatidylinositol 4,5-bisphosphate

In both the heavy and light fractions of fragmented sarcoplasmic reticulum (SR) vesicles from the fast skeletal muscle, about 27 min after beginning the active Ca2+ uptake, the extravesicular Ca2+ concentration suddenly increased to reach a steady level (delayed Ca2+ release). Phosphatidylinositol 4,5-bisphosphate (PIP2) not only shortened the time to delayed Ca2+ release but also induced prompt Ca2+ release from the heavy fraction of SR. Delayed Ca2+ release and prompt Ca2+ release stimulated by 100 microM PIP2 were not modified by ruthenium red. PIP2 (>0.1 microM) markedly accelerated the rate of 45Ca2+ efflux from SR vesicles in a concentration-dependent manner. The PIP(2)-induced 45Ca2+ efflux was potentiated by ruthenium red but profoundly inhibited by La3+. The concentration-response curve for Ca2+ or Mg2+ in PIP2-induced 45Ca2+ release was clearly different from that in the Ca(2+)-induced Ca2+ release. PIP2 caused a concentration-dependent increase in Ca2+ release from SR of chemically skinned fibers from skeletal muscle. Furthermore, [3H]ryanodine or [3H]methyl-7-bromoeudistomin D (MBED) binding to SR was increased by PIP2 in a concentration-dependent manner. These observations present the first evidence that PIP2 most likely activates two types of SR Ca2+ release channels whose properties are entirely different from those of Ca(2+)-induced Ca2+ release channels (the ryanodine receptor 1).

Two novel types of calcium release from skeletal sarcoplasmic reticulum by phosphatidylinositol 4,5-bisphosphate

In both the heavy and light fractions of fragmented sarcoplasmic reticulum (SR) vesicles from the fast skeletal muscle, about 27 min after beginning the active Ca2+ uptake, the extravesicular Ca2+ concentration suddenly increased to reach a steady level (delayed Ca2+ release). Phosphatidylinositol 4,5-bisphosphate (PIP2) not only shortened the time to delayed Ca2+ release but also induced prompt Ca2+ release from the heavy fraction of SR. Delayed Ca2+ release and prompt Ca2+ release stimulated by 100 µM PIP2 were not modified by ruthenium red. PIP2 (>0.1 µM) markedly accelerated the rate of 45Ca2+ efflux from SR vesicles in a concentration-dependent manner. The PIP2-induced 45Ca2+ efflux was potentiated by ruthenium red but profoundly inhibited by La3+. The concentration-response curve for Ca2+ or Mg2+ in PIP2-induced 45Ca2+ release was clearly different from that in the Ca2+-induced Ca2+ release. PIP2 caused a concentration-dependent increase in Ca2+ release from SR of chemically skinned fibers from skeletal muscle. Furthermore, [3H]ryanodine or [3H]methyl-7-bromoeudistomin D (MBED) binding to SR was increased by PIP2 in a concentration-dependent manner. These observations present the first evidence that PIP2 most likely activates two types of SR Ca2+ release channels whose properties are entirely different from those of Ca2+-induced Ca2+ release channels (the ryanodine receptor 1).Key words: phosphatidylinositol 4,5-bisphosphate, sarcoplasmic reticulum, calcium release, ryanodine receptor, ryanodine.

Time-resolved charge translocation by the Ca-ATPase from sarcoplasmic reticulum after an ATP concentration jump

Time-resolved measurements of currents generated by Ca-ATPase from fragmented sarcoplasmic reticulum (SR) are described. SR vesicles spontaneously adsorb to a black lipid membrane acting as a capacitive electrode. Charge translocation by the enzyme is initiated by an ATP concentration jump performed by the light-induced conversion of an inactive precursor (caged ATP) to ATP with a time constant of 2.0 ms at pH 6.2 and 24 degrees C. The shape of the current signal is triphasic, an initial current flow into the vesicle lumen is followed by an outward current and a second slow inward current. The time course of the current signal can be described by five relaxation rate constants, lambda1 to lambda5 plus a fixed delay D approximately 1-3 ms. The electrical signal shows that 1) the reaction cycle of the Ca-ATPase contains two electrogenic steps; 2) positive charge is moved toward the luminal side in the first rapid step and toward the cytoplasmic side in the second slow step; 3) at least one electroneutral reaction precedes the electrogenic steps. Relaxation rate constant lambda3 reflects ATP binding, with lambda(3,max) approximately 100 s(-1). This step is electroneutral. Comparison with the kinetics of the reaction cycle shows that the first electrogenic step (inward current) occurs before the decay of E2P. Candidates are the formation of phosphoenzyme from E1ATP (lambda2 approximately 200 s[-1]) and the E1P --> E2P transition (D approximately 1 ms or lambda1 approximately 300 s[-1]). The second electrogenic transition (outward current) follows the formation of E2P (lambda4 approximately 3 s[-1]) and is tentatively assigned to H+ countertransport after the dissociation of Ca2+. Quenched flow experiments performed under the conditions of the electrical measurements 1) demonstrate competition by caged ATP for ATP-dependent phosphoenzyme formation and 2) yield a rate constant for phosphoenzyme formation of 200 s(-1). These results indicate that ATP and caged ATP compete for the substrate binding site, as suggested by the ATP dependence of lambda3 and favor correlation of lambda2 with phosphoenzyme formation.

Apoptosis precedes necrosis of dystrophin-deficient muscle.

The current view that death of dystrophin-deficient muscle fibers is a necrotic process relies primarily upon the histological appearance of the tissue after the degenerative process is well advanced. Here, we tested this view by examining the possibility that apoptosis is a component of dystrophin-deficient muscle cell death. Three assays for apoptosis were employed in analyzing prenecrotic, peak necrotic and regenerated hindlimb muscle of mdx mice: (1) terminal deoxynucleotidyl transferase (TdT) mediated end-labeling of DNA in nuclei in tissue sections; (2) assays for DNA ladders; and (3) electron microscopic assays for the presence of organelles undergoing structural changes characteristic of apoptosis. At all ages sampled, mdx muscle contained apoptotic nuclei, according to TdT-mediated dUTP labeling of tissue sections. Nuclei in regenerated mdx muscle fibers did not display apoptosis. dUTP-labeled nuclei in control C57 muscles were rare or absent at all ages sampled. DNA from 4-week-old mdx mice was found to be cleaved into fragments indicative of preferential cleavage at internucleosomal sites. Electron microscopic analysis showed that organelle structural changes indicating apoptosis appear before pathological changes diagnostic of necrosis. For example, condensed mitochondria, fragmented sarcoplasmic reticulum and nuclei with chromatin condensations resembling apoptosis appear in fibers that otherwise possess normal morphology. Together, the findings show that apoptosis precedes any detectable necrotic change in mdx muscle, and that apoptotic events continue into the stage of dystrophic pathology that is currently viewed as necrosis. Thus, apoptosis characterizes the onset of pathology in dystrophin-deficient muscle which is followed secondarily by necrotic processes.

Ultrastructural and biochemical analysis of the sarcoplasmic reticulum from crayfish fast and slow striated muscles

AbstractUltrastructural and biochemical properties were compared between two types of the sarcoplasmic reticulum (SR), one from the deep extensor abdominal muscle medialis (fast muscle) and the other from the flexor muscle of carpopodite in meropodite (slow muscle) of crayfish Procambarus clarkii. Electron microscopic observations revealed that terminal cisternae of the SR of crayfish fast muscle was well‐developed and contained feet‐like structures. In transverse sections, the SR of slow muscle was tubule‐like in shape and the terminal cisternae of the SR occupied smaller area than those of the fast muscle SR. The fragmented SR obtained from the homogenized fast muscle was poorly separated by ultracentrifugation with a sucrose gradient (25–50%): both vesicles from light and heavy fractions contained terminal cisternae and were connected to transverse tubules via the feet‐like structures. In contrast, the slow muscle fragmented SR could be separated in to the light and heavy fractions, which contained elliptical vesicles and vesicles with the feet‐like structures, respectively. Sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis and subsequent 45Ca ligand overlay analyses revealed that a 105,000 ( =105 k) Da protein was the major protein in both SRs, which lacked Ca2+‐binding proteins corresponding to vertebrate calsequestrins. On the other hand, peptide‐mapping of the 105 kDa protein in SDS‐gels suggested no difference in the primary structure between fast and slow SR. Ca2+‐ATPase and Ca2+ uptake activities of the fast muscle SR at 20°C were 0.39 μmol Pi/min·mg and 0.57 μmol Ca/min·mg, respectively, which were clearly higher than those of the slow muscle SR, 0.27 μmol Pi/min·mg, and 0.37 μmol Ca/min·mg, respectively.These results suggest that the SR of crayfish fast and slow muscles were morphologically and enzymatically different, which might partly cause differences in the contractile properties of fast and slow muscles. © 1993Wiley‐Liss, Inc.

Sulfhydryl oxidation induces calcium release from fragmented sarcoplasmic reticulum even in the presence of glutathione.

Alcian blue and plumbagin induced transient Ca2+ release from fragmented sarcoplasmic reticulum. Dithiothreitol (DTT) and glutathione (GSH) partially blocked Ca2+ release induced by these oxidizing compounds. Pretreatment of alcian blue and plumbagin with DTT or GSH for more than 1 min was required to abolish the ability of the oxidizing compounds to release Ca2+. Mg2+ and ruthenium red completely blocked alcian blue-and plumbagin-induced Ca2+ release. These results suggest that oxidation of sulfhydryls on Ca2+ release channels induces Ca2+ release even in the presence of GSH in situ.

Effect of the biochemical state of the Ca-ATPase protein of scallop sarcoplasmic reticulum on its interaction with trans-parinaric acid

The polyene flourescent probe trans-parinaric acid (tPA) was used to compare lipid-protein interactions in the scallop fragmented sarcoplasmic reticulum (FSR) between biochemical states where the Ca-ATPase molecules were arranged differently in the membrane and had different tertiary comformations. The state of the bulk lipid phase was examined over the temperature range −3 to + 32°C by exciting the tPA directly at 320 nm. The state of the system close to the Ca-ATPase protein was followed over the same temperature range by indirectly exciting the tPA through resonance energy transfer from the Ca-ATPase protein, with approximately one twentyfifth the quantum yield of the directly excited probe. Raising the tPA/lipid ration in the membrane to high levels (approx. 1 : 9), caused the quantum yield of indirectly excited tPA to reach a maximum, which may reflect saturation of the annular lipid phase with the probe, or contribution to the fluorescence from indirectly excited tPA bound directly to the protein. In the presence of 0.1 M KCl, a thermal perturbation was observed at approx. 7°C using indirect excitation when the Ca 2+-binding sites on the Ca-ATPase were occupied, and the subunits were disorganized. This transition was not detected in the presence of 0.1 M KCl and EGTA, when the Ca 2+-binding sites were empty, and the Ca-ATPase subunits were organized in dimeric arrays. The transition seen with the E 1(Ca 2+) 2 form of the membrane may involve an event at the protein/lipid interface, or a change in the environment of tPA bound to the Ca-ATPase. The temperature at which the perturbation occurs is close to that of a discontinuity in the Arrhenius plot of the Ca-ATPase enzyme activity determined in the presence of 0.1 M KCl (Kalabokis, V.N. and Hardwicke, P.M.D. (1988) J. Biol. Chem. 263, 15184–15188). No perturbation was observed in the bulk properties of the lipid component of the membrane in either the E 1(Ca 2+) 2 or E 2 states.

Inhibitory action of 4-aminopyridine on Ca(2+)-ATPase of the mammalian sarcoplasmic reticulum.

In the isolated guinea pig diaphragm muscle, 4-aminopyridine (4-AP) elicited a marked potentiation of twitch contraction evoked by direct electrical stimuli. Although tetraethylammonium (TEA) and charybdotoxin only slightly potentiated twitch contraction, 4-AP, but not TEA, also augmented a contractile response to caffeine. These effects of 4-AP on muscle contraction could not be interpreted by a simple inhibition of potassium channels on the plasma membrane. In the fragmented sarcoplasmic reticulum (SR) prepared from the guinea pig psoas muscle, 4-AP inhibited the ATP-driven Ca2+ uptake from the extravesicular medium. Furthermore, 4-AP at concentrations less than 10 mM elicited a selective inhibition of Ca(2+)-activated SR ATPase in a competitive manner against the Ca2+ concentration of the medium and 10 mM 4-AP showed the unsurmountable inhibition. 4-AP at 30 mM apparently inhibited activities of other ATPases such as Na+,K+- and myosin ATPases. In contrast, other potassium channel blockers such as TEA, apamin, charybdotoxin, and glibenclamide did not inhibit the SR function. These results suggest that, although the specific concentration range is rather small, 4-AP elicits an inhibition of SR Ca(2+)-pumping activity, leading to the marked potentiation of muscle contractile responses to electrical stimuli and caffeine.

Ca2+ release from isolated sarcoplasmic reticulum of guinea-pig psoas muscle induced by K(+)-channel blockers.

A Ca(2+)-sensitive electrode was used to measure the Ca2+ concentration of the medium containing the heavy fraction of the fragmented sarcoplasmic reticulum (SR) prepared from guinea-pig psoas muscle. Among K(+)-channel blockers tested, 4-aminopyridine (4-AP), tetraethylammonium (TEA) and charybdotoxin elicited Ca2+ release from the SR, but apamin and glibenclamide did not. These results suggest that a reduction of SR K+ conductance leads to Ca2+ release from the SR.

Characterization of the inhibition of intracellular Ca2+ transport ATPases by thapsigargin.

The effects of thapsigargin (TG), a specific inhibitor of intracellular Ca(2+)-ATPases, were studied on vesicular fragments of sarcoplasmic reticulum (SR) membranes. Inhibition of Ca2+ transport and ATPase activity was observed following stoichiometric titration of the membrane bound enzyme with TG. When Ca2+ binding to the enzyme was measured in the absence of ATP, or when one cycle of Ca(2+)-dependent enzyme phosphorylation by ATP was measured under conditions preventing turnover, protection against TG by Ca2+ was observed. The protection by Ca2+ disappeared if the phosphoenzyme was allowed to undergo turnover, indicating that a state reactive to TG is produced during enzyme turnover, whereby a dead end complex with TG is formed. Enzyme phosphorylation with Pi, ATP synthesis, and Ca2+ efflux by the ATPase in its reverse cycling were also inhibited by TG. However, under selected conditions (millimolar Ca2+ in the lumen of the vesicles, and 20% dimethyl sulfoxide in the medium) TG permitted very low rates of enzyme phosphorylation with Pi and ATP synthesis in the presence of ADP. It is concluded that the mechanism of ATPase inhibition by TG involves mutual exclusion of TG and high affinity binding of external Ca2+, as well as strong (but not total) inhibition of other partial reactions of the ATPase cycle. TG reacts selectively with the state acquired by the ATPase in the absence of Ca2+. This state is obtained either by enzyme exposure to EGTA, or by utilization of ATP and consequent displacement of bound Ca2+ during catalytic turnover.

Vanadate inhibition of ATP and p-nitrophenyl phosphate hydrolysis in the fragmented sarcoplasmic reticulum.

The vanadate inhibition of the Ca(2+)-ATPase activity was analysed both in intact sarcoplasmic reticulum vesicles and in the presence of low concentrations of Tween 20, using ATP and p-nitrophenyl phosphate as substrates. The saturation of the internal low-affinity calcium-binding sites protects the enzyme against vanadate inhibition, because: (1) p-nitrophenyl phosphate hydrolysis is not inhibited by vanadate in intact vesicles, but inhibition developed after solubilization with detergents; (2) the vanadate inhibition of the p-nitrophenyl phosphate hydrolysis in solubilized preparations is prevented by free Ca2+ concentrations higher than 10(-3) M and vanadate competes with calcium (10(-5)-10(-3) M); and (3) the vanadate inhibition of ATP hydrolysis is decreased with an increase in vesicular Ca2+ concentration. The presence of magnesium ions is indispensable for the vanadate effect. The vanadate inhibition is non-competitive with respect to Mg-p-nitrophenyl phosphate and uncompetitive with respect to Mg-ATP. However, in the presence of dimethyl sulfoxide, which facilitates phosphorylation of the enzyme, the inhibition is converted to a competitive one with respect to a substrate. The results suggest, that in the process of enzyme operation vanadate interacts with the unliganded E form of Ca(2+)-ATPase, occupying probably an intermediate position between the E2 and E1 forms, with the formation of an E2 Van complex, that imposes the inhibition on the Ca(2+)-ATPase activity.

Functional interactions of catalytic site and transmembrane channel in the sarcoplasmic reticulum ATPase.

Vesicular fragments of longitudinal sarcoplasmic reticulum were loaded with calcium by active transport, sedimented by centrifugation, and resuspended in neutral buffer and [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA). Under these conditions, calcium efflux from the loaded vesicles occurred at rates varying from 100 to 700 nmol/mg/min, depending on the calcium load. If either Ca2+ (microM), Mg2+ (mM), K+ or Na+ (greater than 10 mM) were added to the resuspension medium, the rate of efflux was reduced. In the presence of Mg2+ and EGTA, a large inhibition of calcium efflux was produced by formation of phosphoenzyme intermediate with Pi. In this case, addition of ADP again started calcium efflux, coupled with ATP synthesis. The rates of uncoupled or coupled efflux were approximately the same. The observed calcium fluxes are attributed to a slow channel formed by ATPase transmembrane helices (MacLennan, D. H., Brandl, C. J., Korczak, B., and Green, N. M. (1985) Nature 316, 686-700) and are capable of long range interaction with the catalytic site. Coupling of transport and catalytic activities is thereby produced by phosphorylation and ligand binding. The channel includes negatively charged residues that are likely to influence calcium fluxes through cation binding. It is proposed that this channel is the mechanistic device for active transport of calcium across the sarcoplasmic reticulum membrane, and for its reversal.

Guanosine Triphosphate Utilization by Canine Cardiac Muscle Sarcoplasmic Reticulum1

We investigated the reaction mechanism for GTP-dependent Ca2+ uptake by canine cardiac microsomes enriched in fragmented sarcoplasmic reticulum (SR), because previous studies reported that GTP utilization in cardiac SR occurs via a pathway very different from that for ATP utilization (for a review, see "Entman, M.L., Bick, R., Chu, A., Van Winkle, W.B., & Tate, C.A. (1986) J. Mol. Cell. Cardiol. 18, 781-792"). In cardiac microsomes, we detected slow but distinct oxalate-dependent Ca2+ accumulation, which reached 550 nmol/mg protein in 10 min, and similarly slow Ca2+-dependent GTP hydrolysis. In 50 microM [gamma-32P]-GTP at 0 degrees C, we detected Ca2+-dependent formation of phosphoprotein whose level in the steady state was about a half of the maximum obtained with [gamma-32P]ATP. Kinetic properties of the phosphoprotein, its molecular weight and its chemical stability after the acid treatment are consistent with the conclusion that the phosphoprotein is an acylphosphate intermediate for Ca2+-dependent GTP hydrolysis catalyzed by the Ca2+-pump ATPase. Analysis of the kinetics of the turnover of phosphoprotein revealed that slow GTP hydrolysis is due to slow phosphoprotein formation; at 25 degrees C, the latter arises mainly from slow binding of Ca2+ to the dephosphorylated enzyme. These results indicate that, contrary to the previous data, the reaction pathway for GTP-dependent Ca2+ transport in cardiac SR is basically the same as that for ATP-dependent transport.