Calcium efflux from sarcoplasmic reticulum vesicles

Ca²⁺ efflux was studied in sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle. In experimental conditions in which the Ca²⁺ pump is reversed, the rate of Ca^2? efflux varies with the ADP, orthophosphate and Mg²⁺ concentrations of the assay medium and is inhibited by Na⁺.     

Channel-mediated monovalent cation fluxes in isolated sarcoplasmic reticulum vesicles

The permeability of isolated sarcoplasmic reticulum (SR) vesicles to monovalent cations was studied using a stopped-flow fluorescence quenching technique that permits the measurement of ion fluxes on a millisecond time scale. Approximately 70% of the SR vesicles carry a cation conductance pathway mediating fluxes of Tl+, K+, Na+, and Li+, but not of choline. Both K+ and Na+ equilibrate faster than the 3-ms dead time of the apparatus and Li+ equilibrates in approximately 50 ms. These cation fluxes are reduced by a bis-guanidinium blocker of the SR K+ channel previously studied in planar bilayers. The remaining 30% of the vesicles are permeable to these cations on a time scale of seconds. We conclude that the SR K+ channel is present in a major fraction of vesicles and that its properties in the native membrane are similar to those found in planar bilayers. Moreover, the ion fluxes in fractionated SR vesicles suggest that the channels are distributed along the entire surface of the SR membrane, but in higher concentration in vesicles derived from the terminal cisternae region. From the measured rates of K+ movement, we calculate a conductance on the order of 10(-1) S/cm2 for the SR membrane in situ, which implies that this membrane cannot develop a potential of more than a few millivolts under physiological conditions.     

Calcium control of passive permeability to calcium in sarcoplasmic reticulum vesicles

Calcium efflux from sarcoplasmic reticulum vesicles that have been equilibrated with 1-100 mM CaCl2 in the absence of ATP has two apparently first order components. The initial calcium content of each component increases with the total Ca content of the sarcoplasmic reticulum, which reaches 5, 24, and 80 nmol/mg of protein after equilibration with 1, 10, and 100 mM CaCl2, respectively. Initial rates of Ca efflux into a medium containing 10 mM EGTA increase in proportion to Ca in the loading medium up to 20 mM. Above 20 mM, efflux from the slow component clearly saturates, whereas efflux from the fast component continues to increase. The rate constant for the smaller, faster component to efflux (k congruent to 0.5 min-1) is not affected by changing the concentration of Ca either inside or outside the vesicles. The rate constant of the larger, slower component (k congruent to 0.05 min-1) is also unaffected by changes in internal Ca concentration. However, external [Ca2+] diminishes the rate constant of the slow component 6-10-fold. Inhibition by external [Ca2+] is characterized by cooperative interaction between two sites with an apparent Kd of 5.3 X 10(-6) M. The two components may represent two populations of sarcoplasmic reticulum vesicles that differ 10-fold in passive permeability to Ca when external [Ca2+] is less than 10(-6) M, and 60-100-fold when external [Ca2+] is greater than 10(-5) M. The passive permeability in one of these populations seems to be regulated by external, high affinity Ca binding sites.     

The effects of ryanodine on passive calcium fluxes across sarcoplasmic reticulum membranes

Ryanodine at concentrations of 0.01-10 microM increased, while greater concentrations of 10-300 microM decreased the calcium permeability of both rabbit fast twitch skeletal muscle junctional and canine cardiac sarcoplasmic reticulum membranes. Ryanodine did not alter calcium binding by either sarcoplasmic reticulum membranes or the calcium binding protein, calsequestrin. Therefore, the effects by this agent appear to involve only changes in membrane permeability, and the characteristics of the calcium permeability pathway affected by ryanodine were those of the calcium release channel. Consistent with this, the actions by ryanodine were localized to junctional sarcoplasmic reticulum membranes and were not observed with either longitudinal sarcoplasmic reticulum or transverse tubular membranes. In addition, passage of the junctional sarcoplasmic reticulum membranes through a French press did not diminish the effects of ryanodine indicating that intact triads were not required. Under the conditions used for the permeability studies, the binding of [3H]ryanodine to skeletal junctional sarcoplasmic reticulum membranes was specific and saturable, and Scatchard analyses indicated the presence of a single binding site with a Kd of 150-200 nM and a maximum capacity of 10.1-18.9 pmol/mg protein. [3H]ryanodine binding to this site and the increase in membrane calcium permeability caused by low concentrations of ryanodine had similar characteristics suggesting that actions at this site produce this effect. Depending on the assay conditions used, ryanodine (100-300 microM) could either increase or decrease ATP-dependent calcium accumulation by skeletal muscle junctional sarcoplasmic reticulum membranes indicating that the alterations of sarcoplasmic reticulum membrane calcium permeability caused by this agent can be determined in part by the experimental environment.     

Calcium uptake by sarcoplasmic reticulum vesicles from the leg muscles of Blaberus fuscus

By measuring Ca²⁺-uptake and Ca²⁺-dependent phosphoprotein formation sarcoplasmic reticulum vesicles could be detected in all subcellular fractions obtained from the leg muscles of Blaberus fuscus. The highest Ca²⁺-uptake and Ca²⁺-dependent phosphoprotein formation were found in the ‘microsomal’ fraction. The sarcoplasmic reticulum vesicles from this fraction were further purified by step gradient centrifugation. Ca²⁺-uptake by purified sarcoplasmic reticulum vesicles was found to be strongly dependent on pH, temperature and the concentration of ATP and Mg²⁺. Monovalent cations especially Na⁺ considerably inhibited Ca²⁺-uptake.     

Calcium release from vesicles of heavy sarcoplasmic reticulum of rabbit skeletal muscles

The release of Ca2+ from vesicles of heavy sarcoplasmic reticulum after its accumulation due to hydrolysis of ATP, GTP, CTP, UTP or ITP has been studied using Antipyrylazo III, a metal-chromic Ca-indicator. All the studied substrates of the Ca-pump provide Ca2+ accumulation inside the heavy sarcoplasmic reticulum vesicles, the spontaneous Ca2+ outflux rate being different for different nucleoside triphosphates. It is only ATP that provides Ca-(caffeine)-induced Ca2+ release, however AMP, ADP, beta, gamma-methylene-ATP induce Ca2+ ejection in the presence of nonadenylic nucleotides. The ruthenium red (10(-7M) inhibits the induced ejection of Ca2+ from vesicles of the heavy sarcoplasmic reticulum, but does not prevent the spontaneous release of Ca2+ in the same concentrations. A conclusion is drawn that besides Ca-channels sensitive to Ca2+ and caffeine in the presence of ATP (or to AMP, ADP, beta, gamma-methylene-ATP in the presence of nonadenylic nucleotides) and possessing high sensitivity to the ruthenium red there is another pathway for Ca2+ in the heavy reticulum membranes along which its spontaneous release occurs after the substrate exhaustion. It is supposed that this release is provided by the presence of the Ca-ATPase protein.     

The modification of the unidirectional calcium fluxes of sarcoplasmic reticulum vesicles by monovalent cation ionophores

Calcium uptake by rabbit skeletal muscle sarcoplasmic reticulum vesicles in phosphate-containing media exhibits time-dependent changes that arise from changing rates of calcium influx and efflux. The monovalent cation ionophore gramicidin, added before the start of the calcium uptake reaction, delayed the spontaneous calcium release that normally occurred after approx. 6 min in such reactions; the rate of calcium efflux was inhibited while calcium influx was little affected. Under these conditions, Ca²⁺-activated ATPase activity could remain unaltered.Gramicidin stimulated calcium uptake irrespective of the presence of a K⁺ gradient across the vesicle membrane. Valinomycin stimulated calcium uptake in a manner similar to that for gramicidin even in an NaCl-containing medium lacking potassium. Thus, dissipation of a transmembrane K⁺ gradient is unlikely to account for the effects of these ionophores on the spontaneous changes in calcium flux rates.Addition of gramicidin to partially calcium-filled vesicles inhibited the phase of spontaneous calcium reuptake because both calcium influx and efflux were inhibited. Addition of gramicidin to partially calcium-filled vesicles in the presence of a water-soluble protein, such as bovine serum albumin, creatine kinase or pyruvate kinase, markedly stimulated calcium uptake. This stimulatory effect was due primarily to inhibition of calcium efflux, calcium influx being minimally influenced by the ionophore.After cleavage of the 100 000 dalton ATPase to 50 000 dalton fragments, which was not associated with changes in Ca²⁺-activated ATPase activity or initial calcium uptake rate, gramicidin increased rather than decreased calcium content when added to vesicles after the initial maximum in calcium content. Thus, the ability of monovalent cation ionophores to block calcium efflux from calcium-filled vesicles may reflect their interaction with a portion of the Ca²⁺-activated ATPase protein.     

The modification of the unidirectional calcium fluxes of sarcoplasmic reticulum vesicles by monovlent cation ionophroes

Calcium uptake by rabbit skeletal muscle sarcoplasmic reticulum vesicles in phosphate-containing media exhibits time-dependent changes that arise from changing rates of calcium influx and efflux. The monovalent cation ionophore gramicidin, added before the start of the calcium uptake reaction, delayed the spontaneous calcium release that normally occurred after approx. 6 min in such reactions; the rate of calcium efflux was inhibited while calcium influx was little affected. Under these conditions, Ca2+-activated ATPase activity could remain unaltered. Gramicidin stimulated calcium uptake irrespective of the presence of a K+ gradient across the vesicle membrane. Valinomycin stimulated calcium uptake in a manner similar to that for gramicidin even in an NaCl-containing medium lacking potassium. Thus, dissipation of a transmembrane K+ gradient is unlikely to account for the effects of these ionophores on the spontaneous changes in calcium flux rates. Addition of gramicidin to partially calcium-filled vesicles inhibited the phase of spontaneous calcium reuptake because both calcium influx and efflux wre inhibited. Addition of gramicidin to partially calcium-filled vesicles in the presence of a water-soluble protein, such as bovine serum albumin, creatine kinase or pyruvate kinase, markedly stimulated calcium uptake. This stimulatory effect was due primarily to inhibition of calcium efflux, calcium influx being minimally influenced by the ionophore. After cleavage of the 100,000 dalton ATPase to 50,000 dalton fragments, which was not associated with changes in Ca2+-activated ATPase activity or initial calcium uptake rate, gramicidin increased rather than decreased calcium content when added to vesicles after the initial maximum in calcium content. Thus, the ability of monovalent cation ionophores to block calcium efflux from calcium-filled vesicles may reflect their interaction with a portion of the Ca2+-activated ATPase protein.     

Ca2+ uptake and membrane potential in sarcoplasmic reticulum vesicles

The rate of calcium uptake by sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle was stimulated by inside-negative membrane potential generated by K+ gradients in the presence of valinomycin. The increase in the calcium transport rate was accompanied by a proportional increase in the rate of calcium-dependent ATP hydrolysis, without significant change in the steady state level of the phosphorylated enzyme intermediate. Changes in the sarcoplasmic reticulum membrane potential during calcium transport were monitored with the optical probe, 3,3'-diethylthiadicarbocyanine. The decrease in the absorbance of 3,3'-diethylthiadicarbocyanine at 660 nm following generation of inside-negative membrane potential was reversed during ATP-induced calcium uptake. These observations support an electrogenic mechanism for the transport of calcium by the sarcoplasmic reticulum.     

The effect of ionomycin on calcium fluxes in sarcoplasmic reticulum vesicles and liposomes.

Ionomycin, a recently discovered calcium ionophore, inhibits the ATP-dependent active Ca2+ transport of rabbit sarcoplasmic reticulum vesicles at concentrations as low as 10(-8) to 10(-6) M. The effect is due to an increase in the Ca2+ permeability of the membrane which is also observed on liposomes. The inhibition of Ca2+ uptake is accompanied by an increase in the Ca2+-sensitive ATPase activity of sarcoplasmic reticulum vesicles.     

Functional characterization of reconstituted sarcoplasmic reticulum vesicles

A detailed functional characterization of reconstituted sarcoplasmic reticulum (SR) vesicles with similar lipid content as normal SR was obtained by studies of ATPase activity and calcium transport in transient state, steady state, and equilibrium conditions. For this purpose, enzyme phosphorylation with ATP, hydrolytic activity, calcium transport, phosphorylation with Pi, and ATP synthesis by reversal of the pump were measured, and utilized to demonstrate function and orientation of catalytic sites. The preparations used in these studies displayed the highest activity reported for reconstituted sarcoplasmic reticulum systems. The rates of phosphoenzyme formation from ATP and hydrolysis as well as steady state levels matched the values obtained with normal SR vesicles. Calcium transport and repeated cycles of ATP synthesis by reversal of the pump were also obtained. However, the efficiency of transport and ATP synthesis from a Ca2+ gradient was approximately three times lower than in native vesicles. This deficiency could not be attributed to passive calcium leak from the reconstituted vesicles but, in part, can be explained by the bidirectional alignment of the calcium pump in reconstituted SR. It is suggested that vectorial transport requires a more complex level of protein structure than that for sustaining simple ATPase activity. Time resolution of the phosphorylation reaction by rapid quench methods can be used to estimate the orientation of the calcium pump in the membrane. Such studies indicate that the calcium pump protein is largely bidirectionally oriented in reconstituted SR vesicles.     

Trypsin digestion of junctional sarcoplasmic reticulum vesicles

A putative constituent of the junctional processes, connecting the terminal cisternae of sarcoplasmic reticulum and the transverse tubules of skeletal muscle fibers, is a greater than or equal to 350,000-dalton (Da) protein that displays ryanodine binding and Ca2+ channel properties. Ryanodine modulation of Ca2+ fluxes suggests that the ryanodine receptor and calcium channel are integral parts of one functional unit corresponding to the greater than or equal to 350,000-Da protein [Inui, M., Saito, E., & Fleischer, S. (1987) J. Biol. Chem. 262, 1740-1747; Campbell, K. P., Knudson, C. M., Imagawa, T., Leung, A. L., Sutko, J. L., Kahl, S. D., Raab, C. R., & Madson, L. (1987) J. Biol. Chem. 262, 6460-6463]. We subjected vesicular fragments of junctional-cisternal membrane to stepwise trypsin digestion. The greater than or equal to 350,000-Da protein is selectively cleaved in the early stage of digestion, with consequent disappearance of the corresponding band in electrophoretic gels. The Ca2+-ATPase is cleaved at a later stage, while calsequestrin is not digested under the same experimental conditions. While the Ca2+-ATPase yields two complementary fragments that are relatively resistant to further digestion, the greater than or equal to 350,000-Da protein yields fragments that are rapidly broken down to small peptides. Under conditions producing extensive digestion of the greater than or equal to 350,000-Da protein, the junctional processes are still visualized by electron microscopy, with no discernible alterations of their ultrastructure. The functional properties of the Ca2+ release channel are also maintained following trypsin digestion, including blockage by Mg2+ and ruthenium red and activation by Ca2+ and nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium transport by sarcoplasmic reticulum of skeletal muscle is inhibited by antibodies against the 53-kilodalton glycoprotein of the sarcoplasmic reticulum membrane

The effects of an antiserum against the 53-kDa glycoprotein (GP-53) of the sarcoplasmic reticulum (SR) and of monoclonal antibodies against GP-53 on Ca2+ transport and ATP hydrolysis by SR of rabbit skeletal muscle have been investigated. Preincubation of SR with an antiserum against GP-53 resulted in decreased ATP-driven Ca2+ transport by the SR but had no effect on Ca2+-stimulated ATP hydrolysis. Preincubation of SR with preimmune serum had no significant effect on either Ca2+ transport or Ca2+-ATPase activity. The effect of anti-GP-53 serum was time and concentration dependent. Preincubation of SR with two monoclonal antibodies against GP-53 had no effect on Ca2+ transport or on Ca2+-stimulated ATP hydrolysis. However, preincubation of SR with either monoclonal antibody against GP-53 together with a monoclonal antibody against the Ca2+-ATPase (at levels which had little effect alone) resulted in markedly decreased rates of Ca2+ uptake and ATP hydrolysis. Preincubation of SR with anti-GP-53-serum or with monoclonal antibodies, under the same conditions that inhibited Ca2+ uptake, did not increase the passive permeability of the SR membrane to Ca2+, did not decrease the permeability of the SR to oxalate, and did not cause significant proteolysis of the Ca2+-ATPase. Our results are consistent with the interpretation that GP-53 may modulate the function of the Ca2+-ATPase of the SR membrane.     

Studies on the heterogeneity of sarcoplasmic reticulum vesicles.

Isolated sarcoplasmic reticulum vesicles from rabbit white muscle were separated into a light (15--20% of total microsomes) and a heavy (80--85%) fraction by density gradient centifugation. The ultrastructure, chemical composition, enzymic activities and localization of membrane components in the vesicles of both fractions were investigated. From the following results it was concluded that both fractions are derived from the membranes of the sarcoplasmic reticulum system of the muscle: (i) The protein pattern of both fractions is essentially the same, except for different ratios of acidic, Ca2+-binding proteins. (ii) The 105000 dalton protein of the light fraction cross-reacts immunologically with the Ca2+-dependent ATPase of the heavy fraction. (iii) Ca2+-dependent ATPase, although of different specific activity, is found in both fractions. After rendering the vesicles leaky, specific activities in both fractions reach the same value. The light fraction was found to consist of "inside-out" vesicles by the following criteria: (i) No Ca2+ accumulation can be measured and the Ca2+-dependent ATPase activity is low and variable. (ii) The rate of trypsin digestion is lower and, compared to the heavy microsomes, a different ratio of degradation products is obtained. (iii) The sarcoplasmic reticulum membrane has a highly asymmetrical lipid distribution. This distribution of aminophospholipids is opposite to that in vesicles of heavy fraction. The light sarcoplasmic reticulum fraction has a higher phospholipid to protein ratio than the heavy one. This is consistent with the possibility that the two fractions derive from different parts of the sarcoplasmic reticulum system.