Optical probe responses on sarcoplasmic reticulum: oxacarbocyanines as probes of membrane potential.

The relationship between Ca2+ fluxes and the ion diffusion potential was analyzed on sarcoplasmic reticulum membranes using oxacarbocyanine dyes as optical probes for membrane potential. 3.3'-Diethyloxodicarbocyanine responds to ATP-induced Ca2+ uptake by isolated sarcoplasmic reticulum vesicles with a decrease in absorbance at 600 nm. The optical change is reversed during Ca2+ release from sarcoplasmic reticulum induced by KCl or by ADP and inorganic phosphate. The absorbance changes are largely attributable to the binding of accumulated Ca2+ to the membrane. There is no indication that sustained changes in membrane diffusion potential would accompany pump-mediated Ca2+ fluxes. A large change in the absorbance of 3,3'-diethyloxodicarbocyanine was observed on sarcoplasmic reticulum vesicles under the influence of membrane potential generated by valinomycin in the presence of a K+ gradient or by ionophore A23187 in the presence of a Ca2+ gradient. The maximum of the potential-dependent absorbance change is at 575--580 nm. The potentials generated by valinomycin or ionophore A23187 are short-lived due to the high permeability of sarcoplasmic reticulum membranes for cations and anions. There is no correlation between the direction and magnitude of the artifically imposed membrane potential and the rate of Ca2+ uptake or release by isolated sarcoplasmic reticulum vesicles.     

Membrane crystals of Ca2+-ATPase in sarcoplasmic reticulum of fast and slow skeletal and cardiac muscles

Crystalline arrays of Ca2+ transport ATPase develop in sarcoplasmic reticulum membranes after treatment with Na3VO4 in a calcium-free medium [ Dux , L. and Martonosi , A. (1983) J. Biol. Chem. 258, 2599-2603]. The proportion of vesicles containing Ca2+-ATPase crystals in microsome preparations isolated from rat muscle of different fiber types (semimembranosus, levator ani, extensor digitorum longus, diaphragm, soleus, and heart) correlates well with the Ca2+-ATPase content and Ca2+-modulated ATPase activity. This implies that the concentration of Ca2+-ATPase in sarcoplasmic reticulum membranes of fast and slow skeletal or cardiac muscles differs only slightly, and the low Ca2+ transport activity of 'sarcoplasmic reticulum' preparations isolated from slow-twitch skeletal and cardiac muscles is due to the presence of large amount of non-sarcoplasmic-reticulum membrane elements. This is in accord with the relatively small differences in the density of 8.5-nm intramembranous particles seen by freeze-etch electron microscopy in sarcoplasmic reticulum of red and white muscles. The dimensions of the Ca2+-ATPase crystal lattice are similar in sarcoplasmic reticulum membranes of different fiber types; therefore if structural differences exist between 'isoenzymes' of Ca2+-ATPase, these are not reflected in the crystal-lattice.     

High-affinity Ca2+-binding site inhibiting Ca2+ release from sarcoplasmic reticulum

Ca2+ release from sarcoplasmic reticulum membranes, activated by alkaline pH occurs only when EGTA is present in the release medium. Addition of very low concentrations of Ca2+ to the medium inhibits Ca2+ release. The concentration of free Ca2+ required for 50% inhibition ranges from between 5 and 20 nM in different experiments and/or membrane preparations, irrespective of whether the free Ca2+ concentration is controlled by EGTA or CDTA. Other divalent cations such as Mn2+, Ba2+, Cu2+, Cd2+ and Mg2+ also exert an inhibitory effect on Ca2+ release, with higher or lower potency than that of Ca2+. The inactivation of Ca2+ release by Ca2+ is reversible. We suggest the involvement of high-affinity Ca2+-binding sites in the control of Ca2+ release.     

Ca 2+ outflow from sarcoplasmic reticulum vesicles during changes in membrane potential, Ca2+ concentration and pH

The concentration gradient Ca2+ outflux from the vesicles of the fragmented sarcoplasmic reticulum of rabbit skeletal muscles has been studied under conditions of the induced membrane potential, the concentrations of Ca2+ and H+ in the medium washing over the vesicles being different. The Ca2+ outflux from vesicles is shown to be the same with a decrease of the membrane potential from--80 down to -10 mV and gets higher with the zero and subsequent positive values of the latter. A significant intensification of the Ca2+ outflux from vesicles under the effect of external-vesicular Ca2+ has been observed at its concentration of 10(-5) M. Against this background of external-vesicular Ca2+ and zero value of the membrane potential either exogenous AMP or the pH increase from 6.5 up to 7.8 favour a release of more than 70% of passively accumulated Ca2+. The pH effect grows with a decrease in the external-vesicular concentration of Ca2+. A conclusion is drawn on the significance of protons in the regulation of the Ca2+ release from the sarcoplasmic reticulum.     

Transient kinetics of a Ca2+-induced fluorescence change from membrane-associated and solubilized sarcoplasmic reticulum Ca2+-ATPase

The kinetics and extent of the fluorescence change induced by Ca2+ interaction with the Ca2+-ATPase from sarcoplasmic reticulum have been compared by stopped flow fluorimetry for three preparations: sarcoplasmic reticulum; purified ATPase in membrane vesicles; and solubilized, delipidated ATPase. The kinetics of Ca2+ release and binding for both purified preparations could be described by a single exponential as has been observed for sarcoplasmic reticulum. The rate and extent of the fluorescence change for the solubilized and membrane-associated preparations are shown to be quite similar to those of the sarcoplasmic reticulum. From these results, it is concluded that all of the Ca2+-induced fluoescence change in sarcoplasmic reticulum originates from the Ca2+-ATPase. In addition, since the change in fluorescence is probably result of a conformational change in the ATPase during the Ca2+ pumping cycle, the results provide additional evidence that monomeric Ca2+-ATPase may be capable of Ca2+ transport since the delipidated preparation is monomeric under the conditions used for these experiments. Finally, it is concluded that phospholipid bilayer is not essential for this conformational change.     

Ca2+-accumulating capacity of mitochondria, sarcolemma and sarcoplasmic reticulum of rat heart

Using the isotope exchange technique including 45Ca, the Ca2+-binding and Ca2+-accumulating capacity of mitochondria, sarcolemma and sarcoplasmic reticulum of rat heart was studied. The ATP-independent binding of Ca2+ to isolated membrane fractions is by 1--2 orders of magnitude less than the ATP-dependent Ca2+-accumulating capacity of the fractions. The Ca2+-accumulating capacity of mitochondria is increased 6--8 fold after addition of physiological concentrations of succinate and Pi to the incubation medium. Under these conditions the ratio of Ca2+-accumulating capacity of mitochondria, sarcolemma and sarcoplasmic reticulum of the heart is 100:3,12:2,9. The initial rate of Ca2+-uptake by the sarcoplasmic reticulum is much higher in comparison with sarcolemma and mitochondria. A high Ca2+-accumulating capacity of heart mitochondria probably determines a long-term regulation of the concentration of "troponin-accessible" Ca2+ in the sarcoplasm, whereas the high initial rate of Ca2+ accumulation by the sarcoplasmic reticulum provides for a rapid decrease of Ca2+ concentration during rhythmic contractions of the heart.     

Functional interaction of the cytoplasmic domain of triadin with the skeletal ryanodine receptor

Triadin has been shown to co-localize with the ryanodine receptor in the sarcoplasmic reticulum membrane. We show that immunoprecipitation of solubilized sarcoplasmic reticulum membrane with antibodies directed against triadin or ryanodine receptor, leads to the co-immunoprecipitation of ryanodine receptor and triadin. We then investigated the functional importance of the cytoplasmic domain of triadin (residues 1-47) in the control of Ca2+ release from sarcoplasmic reticulum. We show that antibodies directed against a synthetic peptide encompassing residues 2-17, induce a decrease in the rate of Ca2+ release from sarcoplasmic reticulum vesicles as well as a decrease in the open probability of the ryanodine receptor Ca2+ channel incorporated in lipid bilayers. Using surface plasmon resonance spectroscopy, we defined a discrete domain (residues 18-46) of the cytoplasmic part of triadin interacting with the purified ryanodine receptor. This interaction is optimal at low Ca2+ concentration (up to pCa 5) and inhibited by increasing calcium concentration (IC50 of 300 microM). The direct molecular interaction of this triadin domain with the ryanodine receptor was confirmed by overlay assay and shown to induce the inhibition of the Ca2+ channel activity of purified RyR in bilayer. We propose that this interaction plays a critical role in the control, by triadin, of the Ca2+ channel behavior of the ryanodine receptor and therefore may represent an important step in the regulation process of excitation-contraction coupling in skeletal muscle.     

Functional characteristics of reconstituted sarcoplasmic reticulum membranes as a function of the lipid-to-protein ratio

The ATP-induced Ca2+ accumulation efficiency and rates of Ca2+ uptake of the reconstituted sarcoplasmic reticulum (RSR) model membrane system were measured over an extended range of lipid-to-protein (L/P) molar ratios and were compared to those of isolated light sarcoplasmic reticulum (LSR). Highly purified sarcoplasmic reticulum (SR), dissociated in the presence of deoxycholate, was reconstituted for several L/P ratios, according to the same procedure, forming closed membranes vesicles composed of greater than 95% Ca2+ pump protein and SR lipids which were capable of ATP-induced Ca2+ accumulation in the absence of oxalate or other Ca2+ precipitating agents. This suggests that dissociation of SR and reconstitution to form RSR does not significantly affect the ability of the Ca2+ pump protein incorporated into the SR lipid bilayer to establish Ca2+ gradients. Electron micrographs of fixed and stained dispersions of RSR revealed a structural organization of the membrane that was dependent upon the L/P molar ratio. RSR with L/P greater than 88 were composed of closed vesicles whose membranes stained asymmetrically, similar to that observed for LSR. Closed vesicles of RSR with L/P less than 88 were composed of membrane that stained symmetrically. In addition, reconstituted SR preparations with well-defined L/P molar ratios greater than 88 possess a functional behavior similar to that of LSR (in the absence of oxalate, energy efficiencies are 60-70% and apparent initial uptake rates are 80% that of isolated LSR controls); RSR preparations with L/P less than 88 are characterized by significantly depressed values of the energy efficiencies and apparent initial uptake rates especially at low L/P ratios. Thus, we are the first to report a reconstituted SR model membrane system capable of attaining rates of Ca2+ uptake comparable to isolated LSR controls at comparable L/P ratios in the absence of oxalate or other Ca2+ precipitating agents.     

Stimulation of Ca2+ uptake by cyclic AMP and protein kinase in sarcoplasmic reticulum-rich and sarcolemma-rich microsomal fractions from rabbit heart.

The effect of cyclic AMP on Ca2+ uptake by rabbit heart microsomal vesicular fractions representing mainly fragments of either sarcoplasmic reticulum or sarcolemma was investigated in the presence and absence of soluble cardiac protein kinase and with microsomes prephosphorylated by cyclic AMP-dependent protein kinase. The acceleration of oxalate-promoted Ca2+ uptake by fragmented sarcoplasmic reticulum following cyclic AMP-dependent membrane protein phosphorylation, observed by other authors, was confirmed. In addition it was found that the acceleration was greatest at pH 7.2 and almost negligible at pH 6.0 and pH 7.8. A very marked increase in Ca2+ uptake by cyclic AMP-dependent membrane protein phosphorylation was observed in the presence of boric acid, a reversible inhibitor of Ca2+ uptake. In addition to the microsomal fraction thought to represent mainly fragments of the sarcoplasmic reticulum, the effect of protein kinase and cyclic AMP on Ca2+ uptake was investigated in a cardiac sarcolemma-enriched membrane fraction. Ca2+ uptake by sarcolemmal vesicles, unlike Ca2+ uptake by sarcoplasmic reticulum vesicles, was inhibited by low doses of digitoxin. The acceleration of oxalate-promoted Ca2+ uptake by cyclic AMP and soluble cardiac protein kinase, however, was quite similar to what was seen in preparations of fragmented sarcoplasmic reticulum, which suggests that it may reflect an acceleration of active Ca2+ transport across the myocardial cell surface membrane.     

Characterization of ATP-dependent Ca2+ uptake by canine brain microsomes with saponin

ATP-dependent Ca2+ uptake by brain microsomes was classified into two fractions according to the sensitivity to saponin. Properties of each fraction of Ca2+ uptake were examined and compared with those of inside-out membrane vesicles of erythrocyte and cardiac sarcoplasmic reticulum. The concentration of saponin for 50% inhibition (IC50) of major saponin-sensitive Ca2+ uptake was 11 micrograms/ml, and this uptake was enhanced by calmodulin. The minor saponin-insensitive Ca2+ uptake fraction (IC50; 90 micrograms/ml) was not affected by calmodulin but was enhanced by oxalate or 0.1 M KCl. The IC 50 of saponin for inside-out membrane vesicles of erythrocyte and cardiac sarcoplasmic reticulum was 11.3 and 114.8 micrograms/ml, respectively. A characteristic ring-like saponin-cholesterol micellar structure was observed electron microscopically in most membrane vesicles of brain microsomes and erythrocyte membrane vesicles but not in the cardiac sarcoplasmic reticulum. These observations indicate that saponin-sensitive and insensitive Ca2+ uptake was derived from plasma membranes and endoplasmic reticulum, respectively. Saponin proved useful for distinguishing the Ca2+ transport activity of plasma membrane from the Ca2+ uptake of other cellular organelles in the membrane preparations.     

Rapid kinetics of calcium ion transport and ATPase activity in the sarcoplasmic reticulum of dystrophic muscle.

Vesicular fragments of sarcoplasmic reticulum were isolated from pectoralis muscle of normal and dystrophic chicken. Purification of both preparations was equally satisfactory, as shown by a prominent ATPase band in electrophoresis gels. Measurements of ATPase phosphorylation, Ca2+ transport and Pi cleavage by rapid quench methods revealed a lower specific activity of the dystrophic vesicles with respect to all of these functions. On the other hand, Ca2+-independent ATPase activity was found to be increased in dystrophic vesicles. It is suggested that a fraction of ATPase units of dystrophic sarcoplasmic reticulum is not activated by Ca2+, owing to an altered protein assembly within the membrane bilayer. In fact, when the membrane structure is perturbed by detergents normal and dystropic preparations acquire an equally high Ca2+-dependent ATPase.     

Cause of increase in the efficiency of Ca2+ transport by fragments of sarcoplasmic reticulum from fast skeletal muscles induced by protein kinase

The effect of cAMP-dependent protein kinases from rabbit skeletal muscles on Ca2+ uptake by fragments of skeletal muscle sarcoplasmic reticulum was studied. It was shown that incubation of the sarcoplasmic reticulum fragments with protein kinase increases the rate of Ca2+ uptake without changing the activity of Ca2+-dependent ATPase. This phenomenon is not accompanied by phosphorus incorporation into the protein components of the reticulum membranes. The protein kinase preparation subjected to "self-phosphorylation" is also capable to increase the rate of Ca2+ uptake. Using (14C) -oleic acid, it was shown that the increase of the rate of Ca2+ transport under effects of the "self-phosphorylated" protein kinase occurs due to the binding of free fatty acids present in the sarcoplasmic reticulum membranes. It was found that the effect observed is due to phosphofructokinase (ATP : D-fructose-6-phosphate-1-phosphotransferase) present in the protein kinase preparation.     

The distribution and properties of the Mg2(+)-ATPase in the membranes of functionally different muscles in the hen]

From striated (m. pectoralis and myocardium) and smooth (myometrium) muscle tissues of hen, by means of differential centrifugation with Ca-oxalate loading, membrane preparations were obtained with high activity of Mg(2+)-ATPase, i.e. a marker enzyme of tubular membranes of T-system of skeletal muscles. Some properties (pH and temperature optima) of this enzyme were investigated and compared to those of Ca(2+)-ATPase from membranes of the sarcoplasmic reticulum. It was shown that in all the investigated muscles, Mg(2+)-ATPase is associated with membrane fraction which in its density corresponds to tubular membranes of T-system. Activation of this enzyme is characterized by similar optimal levels of pH (7.2) and temperature (25 degrees C). The activity of Ca(2+)-ATPase in the membranes of the sarcoplasmic reticulum, in contrast to that of Mg(2+)-ATPase, is observed in more narrow bands of pH and temperature, exhibiting tissue specificity. The data obtained, indicating a possibility of chromatographic separation of these enzymes, confirm their biochemical individuality.     

The base-exchange enzyme activities of sarcolemma and sarcoplasmic reticulum from rat heart

The Ca2+ dependent incorporation of [14C]ethanolamine, L-[14C]serine and [14C]choline into phosphatidylethanolamine, phosphatidylserine and phosphatidylcholine, respectively, were investigated in membrane preparations from rat heart. The ethanolamine and serine base-exchange enzyme-catalyzed reactions were associated with the sarcolemma and sarcoplasmic reticulum. There was a 17.2-fold and 6.8-fold enrichment, respectively, of the serine and the ethanolamine base-exchange enzyme activities in the sarcolemma compared to the starting whole homogenate. The sarcoplasmic reticulum was enriched in the ethanolamine and serine base-exchange enzyme activities. The choline base-exchange enzyme activity of all membranes fractions was negligible compared to the ethanolamine or serine base-exchange enzyme activities. The apparent Km for the ethanolamine and serine base-exchange enzyme in sarcolemma was 14 microM and 25 microM, respectively. The pH optimum for these base-exchange activities was 7.5-8.0. There was a dependence upon Ca2+ for these reactions with a 1 or 4 mM concentration required for maximal activity. The properties of the sarcoplasmic reticulum base-exchange enzymes were similar to the sarcolemmal base-exchange enzymes.     

Damage to the Ca2+-transport function and the lipid component of the sarcoplasmic reticulum membrane in total ischemia of the rat myocardium

The Ca2+-transporting activity, lipoperoxide chemiluminescence and phospholipid spectrum of sarcoplasmic reticular membranes were studied in ischemic rats. It was shown that a substantial reduction in Ca2+ uptake rate by the sarcoplasmic reticulum occurred within the first 30 minutes and correlated with the increase in chemiluminescence intensity and accumulation of lysophosphatidylcholine. It has been suggested that free radical lipid peroxidation and phospholipase activation are directly related to the reduction of Ca2+-transporting rate by sarcoplasmic reticulum in myocardial ischemia.     

Isolation of Ca 2+-activated photoprotein obelin from Obelia longissima and its use for the recording the outflow of Ca 2+ from fragmented sarcoplasmic reticulum of skeletal muscles

A high-active stable preparation of obelin has been obtained from the luminescent hydroid Obelia longissima. The preparation is appropriate for determining free Ca2+ in the physiological range of its concentrations Obelin is shown possible to be used to record the processes of Ca2+ release from vesicles of the sarcoplasmic reticulum. In this case a rapid initial phase of Ca2+ outflux replaced by a slower one has been registered. A sharp increase of luminescence caused by the appearance of free Ca2+ in the medium has been registered under the effect of agents either increasing permeability of sarcoplasmic reticulum membranes for Ca2+ (A23187) or destroying the membrane (ethanol, triton X-100). The observed effects are confirmed, a radioactive label being used.     

Interaction between free fatty acids and Ca2+-dependent ATPase from sarcoplasmic reticulum membranes

The interaction between free fatty acids and Ca2+-dependent ATPase, an intrinsic protein of sarcoplasmic reticulum membranes, was studied with relevance to the changes in membrane permeability induced by free fatty acids. It was found that only unsaturated fatty acids increase the permeability of reticulum membranes for Ca2+, this effect being completely reversible. The increase in the membrane permeability by fatty acids is coupled to a generation of a channel for Ca2+ efflux under effect of Ca2+-dependent ATPase. The interaction between fatty acids and Ca2+-dependent ATPase was demonstrated by the protein fluorescence and electron paramagnetic resonance methods, using spin-labelled fatty acid derivatives. A model demonstrating the increase of sarcoplasmic reticulum membrane permeability for Ca2+ in the presence of the fatty acid-Ca2+-dependent ATPase complex is proposed.     

Effect of the purified (Mg2+ + Ca2+)-activated ATPase of sarcoplasmic reticulum upon the passive Ca2+ permeability and ultrastructure of phospholipid vesicles.

The passive Ca2+ permeability of fragmented sarcoplasmic reticulum membranes is 10(4) to 10(61 times greater than that of liposomes prepared from natural or synthetic phospholipids. The contribution of membrane proteins to the Ca2+ permeability was studied by incorporating the purified [Ca2+ + Mg2+]-activated ATPase into bilayer membranes prepared from different phospholipids. The incorporation of the Ca2+ transport ATPase into the lipid phase increased its Ca2+ permeability to levels approaching that of sarcoplasmic reticulum membranes. The permeability change may arise from a reordering of the structure of the lipid phase in the environment of the protein or could represent a specific property of the protein itself. The calcium-binding protein of sarcoplasmic reticulum did not produce a similar effect. The increased rate of Ca2+ release from reconstituted ATPase vesicles is not a carrier-mediated process as indicated by the linear dependence of the Ca2+ efflux upon the gradient of Ca2+ concentration and by the absence of competition and countertransport between Ca2+ and other divalent metal ions. The increased Ca2+ permeability upon incorporation of the transport ATPase into the lipid phase is accompanied by similar increase in the permeability of the vesicles for sucrose, Na+, choline, and SO42- indicating that the transport ATPase does not act as a specific Ca2+ channel. Native sarcoplasmic reticulum membranes are asymmetric structures and the 75-A particles seen by freeze-etch electron microscopy are located primarily in the outer fracture face. In reconstituted ATPase vesicles the distribution of the particles between the two fracture faces is even, indicating that complete structural reconstitution was not achieved. The Ca2+ transport activity of reconstituted ATPase vesicles is also much less than that of fragmented sarcoplasmic reticulum. The density of the 40-A surface particles visible after negative staining of native or reconstituted vesicles is greater than that of the intramembranous particles and the relationship between these two structures remains to be established.     

Electrogenic activity of Ca2+-ATPase fragments of the sarcoplasmatic reticulum during the early stages of radiation injury

It is established that local X-ray irradiation of the rabbit hind limb produces a decrease in Ca2+, Mg2+-ATP-dependent formation of electric potentials difference on the membrane of the sarcoplasmic reticulum (SR). These results agree with the observed decrease in the Ca2+-ATPase activity of SR membranes and increase in their electric conduction after irradiation.     

The functional coupling between Ca2+-ATPase and creatine phosphokinase in heart muscle sarcoplasmic reticulum]

The functional role of creatine phosphokinase (CPK) in the process of energy supply for the Ca2+-ATPase reaction and ion transport across the membrane of heart sarcoplasmic reticulum (SR) has been studied. It has been shown that isolated and purified preparations of heart SR contain significant activity of CPK. The localization of CPK on the membrane of SR has been revealed also by an electron microscopic histochemical method. Under conditions of the Ca+-ATPase reaction in the presence of creatine phosphate the release of creatine into the reaction medium is observed, the rate of the latter process being dependent upon the MgATP concentration in accordance with the kinetic parameters of the Ca2+-ATPase reaction. CPK localized on the SR membrane is able to maintain higher rate of calcium uptake by SR vesicles, as compared to that with added ATP-regenerating system. The results obtained demonstrate the close functional coupling between CPK and Ca2+-ATPase in the membrane of SR.