Thermogenic activity of Ca2+-ATPase from skeletal muscle heavy sarcoplasmic reticulum: the role of ryanodine Ca2+ channel

The sarcoplasmic reticulum Ca(2+) ATPase 1 (SERCA 1) is able to handle the energy derived from ATP hydrolysis in such a way as to determine the parcel of energy that is used for Ca(2+) transport and the fraction that is converted into heat. In this work we measured the heat production by SERCA 1 in the two sarcoplasmic reticulum (SR) fractions: the light fraction (LSR), which is enriched in SERCA and the heavy fraction (HSR), which contains both the SERCA and the ryanodine Ca(2+) channel. We verified that although HSR cleaved ATP at faster rate than LSR, the amount of heat released during ATP hydrolysis by HSR was smaller than that measured by LSR. Consequently, the amount of heat released per mol of ATP cleaved (DeltaH(cal)) by HSR was lower compared to LSR. In HSR, the addition of 5 mM Mg(2+) or ruthenium red, conditions that close the ryanodine Ca(2+) channel, promoted a decrease in the ATPase activity, but the amount of heat released during ATP hydrolysis remained practically the same. In this condition, the DeltaH(cal) values of ATP hydrolysis increased significantly. Neither Mg(2+) nor ruthenium red had effect on LSR. Thus, we conclude that heat production by SERCA 1 depends on the region of SR in which the enzyme is inserted and that in HSR, the DeltaH(cal) of ATP hydrolysis by SERCA 1 depends on whether the ryanodine Ca(2+) channel is opened or closed.     

Uncoupled ATP hydrolysis and thermogenic activity of the sarcoplasmic reticulum Ca2+-ATPase: coupling effects of dimethyl sulfoxide and low temperature

The sarcoplasmic reticulum Ca(2+)-ATPase transports Ca(2+) using the energy derived from ATP hydrolysis. During catalysis, part of the energy is used to translocate Ca(2+) across the membrane, and part is dissipated as heat. At 35 degrees C the heat released during the hydrolysis of each ATP molecule varies depending on the formation of a Ca(2+) gradient across the membrane. With leaky vesicles (no gradient) the heat released varies between 9 and 12 kcal/mol of ATP cleaved, and with intact vesicles (gradient), the heat released increases to 20-24 kcal/mol of ATP. After Ca(2+) accumulation, 82% of the Ca(2+)-ATPase activity is not coupled to Ca(2+) transport, and the ratio between Ca(2+) transported and ATP cleaved is 0.3. The addition of 20% dimethyl sulfoxide (v/v) to the medium or decreasing the temperature from 35 to 20 degrees C abolishes the difference of heat produced during ATP hydrolysis in the presence and absence of a gradient. This is accompanied by a simultaneous inhibition of the uncoupled ATPase activity and an increase of the Ca(2+)/ATP ratio from 0.3 to 1.3-1.4. It is concluded that the uncoupled Ca(2+)-ATPase is responsible for both the low Ca(2+)/ATP ratio measured during transport and the difference of heat produced during ATP hydrolysis in the presence and absence of a gradient.     

Uncoupled ATPase activity and heat production by the sarcoplasmic reticulum Ca2+-ATPase. Regulation by ADP

Sarcoplasmic reticulum vesicles of rabbit skeletal muscle accumulate Ca2+ at the expense of ATP hydrolysis. The heat released during the hydrolysis of each ATP molecule varies depending on whether or not a Ca2+ gradient is formed across the vesicle membrane. After Ca2+ accumulation, a part of the Ca2+-ATPase activity is not coupled with Ca2+ transport (Yu, X., and Inesi, G. (1995) J. Biol. Chem. 270, 4361-4367). I now show that both the heat produced during substrate hydrolysis and the uncoupled ATPase activity vary depending on the ADP/ATP ratio in the medium. With a low ratio, the Ca2+ transport is exothermic, and the formation of the gradient increases the amount of heat produced during the hydrolysis of each ATP molecule cleaved. With a high ADP/ATP ratio, the Ca2+ transport is endothermic, and formation of a gradient increased the amount of heat absorbed from the medium. Heat is absorbed from the medium when the Ca2+ efflux is coupled with the synthesis of ATP (5.7 kcal/mol of ATP). When there is no ATP synthesis, the Ca2+ efflux is exothermic (14-16 kcal/Ca2+ mol). It is concluded that in the presence of a low ADP concentration the uncoupled ATPase activity is the dominant route of heat production. With a high ADP/ATP ratio, the uncoupled ATPase activity is abolished, and the Ca2+ transport is endothermic. The possible correlation of these findings with thermogenesis and anoxia is discussed.     

Correlation between uncoupled ATP hydrolysis and heat production by the sarcoplasmic reticulum Ca2+-ATPase: coupling effect of fluoride.

The sarcoplasmic reticulum Ca(2+)-ATPase transports Ca(2+) using the chemical energy derived from ATP hydrolysis. Part of the chemical energy is used to translocate Ca(2+) through the membrane (work) and part is dissipated as heat. The amount of heat produced during catalysis increases after formation of the Ca(2+) gradient across the vesicle membrane. In the absence of gradient (leaky vesicles) the amount of heat produced/mol of ATP cleaved is half of that measured in the presence of the gradient. After formation of the gradient, part of the ATPase activity is not coupled to Ca(2+) transport. We now show that NaF can impair the uncoupled ATPase activity with discrete effect on the ATPase activity coupled to Ca(2+) transport. For the control vesicles not treated with NaF, after formation of the gradient only 20% of the ATP cleaved is coupled to Ca(2+) transport, and the caloric yield of the total ATPase activity (coupled plus uncoupled) is 22.8 kcal released/mol of ATP cleaved. In contrast, the vesicles treated with NaF consume only the ATP needed to maintain the gradient, and the caloric yield of ATP hydrolysis is 3.1 kcal/mol of ATP. The slow ATPase activity measured in vesicles treated with NaF has the same Ca(2+) dependence as the control vesicles. This demonstrates unambiguously that the uncoupled activity is an actual pathway of the Ca(2+)-ATPase rather than a contaminating phosphatase. We conclude that when ATP hydrolysis occurs without coupled biological work most of the chemical energy is dissipated as heat. Thus, uncoupled ATPase activity appears to be the mechanistic feature underlying the ability of the Ca(2+)-ATPase to modulated heat production.     

Brown adipose tissue Ca2+-ATPase: uncoupled ATP hydrolysis and thermogenic activity

In this report a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) was identified in rats brown adipose tissue. Electrophoretic analysis of brown fat microssomal protein yields a 110-kDa band that is reactive to SERCA 1 antibody but is not reactive to SERCA 2 antibodies. Nevertheless, the kinetics properties of the brown fat SERCA differ from the skeletal muscle SERCA 1 inasmuch they manifest a different Ca2+ affinity and a much higher degree of ATPase/Ca2+ uncoupling. A SERCA enzyme is not found in white fat. Fatty acids promoted Ca2+ leakage from brown fat vesicles. The heat released during ATP hydrolysis was -24.7 kcal/mol when a Ca2+ gradient was formed across the vesicles membrane and -14.4 kcal/mol in the absence of a gradient. The data reported suggest that in addition to storing Ca2+ inside the endoplasmic reticulum, the Ca2+-ATPase may represent a source of heat production contributing to the thermogenic function of brown adipose tissue.     

The presence of sarcolipin results in increased heat production by Ca(2+)-ATPase

Skeletal muscle sarcoplasmic reticulum of large mammals such as rabbit contains sarcolipin (SLN), a small peptide with a single transmembrane alpha-helix. When reconstituted with the Ca(2+)-ATPase from skeletal muscle sarcoplasmic reticulum into sealed vesicles, the presence of SLN leads to a reduced level of accumulation of Ca(2+). Heats of reaction of the reconstituted Ca(2+)-ATPase with ATP were measured using isothermal calorimetry. The heat released increased linearly with time over 30 min and increased with increasing SLN content. Rates ATP hydrolysis by the reconstituted Ca(2+)-ATPase were constant over a 30-min time period and were the same when measured in the presence or absence of an ATP-regenerating system. The calculated values of heat released per mol of ATP hydrolyzed increased with increasing SLN content and fitted to a simple binding equation with a dissociation constant for the SLN.ATPase complex of 6.9 x 10(-4) +/- 2.9 x 10(-4) in units of mol fraction per monolayer. It is suggested that the interaction between Ca(2+)-ATPase and SLN in the sarcoplasmic reticulum could be important in thermogenesis by the sarcoplasmic reticulum.     

Identification of Ca(2+)-activated Mg(2+)-dependent ATP-hydrolase activity of membrane microsomal fraction of loach embryo (Misgurnus fossilis L.)

The functional confirmation of availability of Ca2+ transport initially-active systems in the embryo cells of loach Misgurnus fossilis L. has been obtained. Using thapsigargin, the specific inhibitor of endoplasmic reticulum of Ca2+, Mg(2+)-ATPase, this enzyme activity was divided into thapsigargin-sensitive (actually endoplasmic reticulum Ca2+, Mg(2+)-ATPase) and thapsigargin-insensitive (plasma membrane Ca2+, Mg(2+)-ATPase) constituents. The Ca(2+)-independent Mg(2+)-dependent ATPase activity makes above 39.7% of the common Ca2+, Mg(2+)-ATPase activity of embryo loach. The periodic changes of Ca2+, Mg(2+)-ATPase activity (except for the changes of plasma membrane Ca2+, Mg(2+)-ATPase activity) were found out, which coincide with periodic [Ca2+]i oscillations during the synchronous divisions of loach blastomers embryos.     

Role of Sarco/Endoplasmic Reticulum Ca2+-ATPase in Thermogenesis

Enzymes are able to handle the energy derived from the hydrolysis of phosphate compounds in such a way as to determine the parcel that is used for work and the fraction that is converted into heat. The sarco/endoplasmic reticulum Ca²⁺-ATPases (SERCA) is a family of membrane-bound ATPases that are able to transport Ca²⁺ ion across the membrane using the chemical energy derived from ATP hydrolysis. The heat released during ATP hydrolysis by SERCA may vary from 10 up to 30 kcal/mol depending on the SERCA isoform used and on whether or not a Ca²⁺ gradient is formed across the membrane. Drugs such as heparin, dimethyl sulfoxide and the platelet-activating factor (PAF) are able to modify the fraction of the chemical energy released during ATP hydrolysis that is used for Ca²⁺ transport and the fraction that is dissipated in the surrounding medium as heat. The thyroid hormone 3,5,3′-triiodo L-thyronine (T₃) regulates the expression and function of the thermogenic SERCA isoforms. Modulation of heat production by SERCA might be one of the mechanisms involved in the increased thermogenesis found in hyperthyroidism.     

Quercetin interaction with the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum

In sarcoplasmic reticulum vesicles or in the (Ca2+ + Mg2+)-ATPase purified from sarcoplasmic reticulum, quercetin inhibited ATP hydrolysis, Ca2+ uptake, ATP-Pi exchange, ATP synthesis coupled to Ca2+ efflux, ATP-ADP exchange, and steady state phosphorylation of the ATPase by inorganic phosphate. Steady state phosphorylation of the ATPase by ATP was not inhibited. Quercetin also inhibited ATP and ADP binding but not the binding of Ca2+. The inhibition of ATP-dependent Ca2+ transport by quercetin was reversible, and ATP, Ca2+, and dithiothreitol did not affect the inhibitory action of quercetin.     

Neuronal-specific endoplasmic reticulum Mg(2+)/Ca(2+) ATPase Ca(2+) sequestration in mixed primary hippocampal culture homogenates

Endoplasmic reticulum Mg(2+)/Ca(2+) ATPase Ca(2+) sequestration is crucial for maintenance of neuronal Ca(2+) homeostasis. The use of cell culture in conjunction with modern Ca(2+) imaging techniques has been invaluable in elucidating these mechanisms. While imaging protocols evaluate endoplasmic reticulum Ca(2+) loads, measurement of Mg(2+)/Ca(2+) ATPase activity is indirect, comparing cytosolic Ca(2+) levels in the presence or absence of the Mg(2+)/Ca(2+) ATPase inhibitor thapsigargin. Direct measurement of Mg(2+)/Ca(2+) ATPase by isolation of microsomes is impossible due to the minuscule amounts of protein yielded from cultures used for imaging. In the current study, endoplasmic reticulum Mg(2+)/Ca(2+) ATPase Ca(2+) sequestration was measured in mixed homogenates of neurons and glia from primary hippocampal cultures. It was demonstrated that Ca(2+) uptake was mediated by the endoplasmic reticulum Mg(2+)/Ca(2+) ATPase due to its dependence on ATP and Mg(2+), enhancement by oxalate, and inhibition by thapsigargin. It was also shown that neuronal Ca(2+) uptake, mediated by the type 2 sarco(endo)plasmic reticulum Ca(2+) ATPase isoform, could be distinguished from glial Ca(2+) uptake in homogenates composed of neurons and glia. Finally, it was revealed that Ca(2+) uptake was sensitive to incubation on ice, extremely labile in the absence of protease inhibitors, and significantly more stable under storage conditions at -80 degrees C.     

Differentiation induces up-regulation of plasma membrane Ca(2+)-ATPase and concomitant increase in Ca(2+) efflux in human neuroblastoma cell line IMR-32

Precise regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) is achieved by the coordinated function of Ca(2+) channels and Ca(2+) buffers. Neuronal differentiation induces up-regulation of Ca(2+) channels. However, little is known about the effects of differentiation on the expression of the plasma membrane Ca(2+)-ATPase (PMCA), the principal Ca(2+) extrusion mechanism in neurons. In this study, we examined the regulation of PMCA expression during differentiation of the human neuroblastoma cell line IMR-32. [Ca(2+)](i) was monitored in single cells using indo-1 microfluorimetry. When the Ca(2+)-ATPase of the endoplasmic reticulum was blocked by cyclopiazonic acid, [Ca(2+)](i) recovery after small depolarization-induced Ca(2+) loads was governed primarily by PMCAs. [Ca(2+)](i) returned to baseline by a process described by a monoexponential function in undifferentiated cells (tau = 52 +/- 4 s; n = 25). After differentiation for 12-16 days, the [Ca(2+)](i) recovery rate increased by more than threefold (tau = 17 +/- 1 s; n = 31). Western blots showed a pronounced increase in expression of three major PMCA isoforms in IMR-32 cells during differentiation, including PMCA2, PMCA3 and PMCA4. These results demonstrate up-regulation of PMCAs on the functional and protein level during neuronal differentiation in vitro. Parallel amplification of Ca(2+) influx and efflux pathways may enable differentiated neurons to precisely localize Ca(2+) signals in time and space.     

Inhibition of sarcoplasmic reticulum Ca(2+)-ATPase by 2,5-di(tert-butyl)-1,4-benzohydroquinone.

We characterized the interaction of 2,5-di(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) with the sarcoplasmic reticulum (SR) Ca(2+)-ATPase from rabbit fast-twitch skeletal and canine cardiac muscles by examining the effect of this agent on the ATPase reaction. tBuBHQ at less than 10 microM inhibited ATP hydrolysis by both isoforms of Ca(2+)-ATPase by up to 80 and 90%, respectively. The half maximal inhibition of these enzymes was observed at about 1.5 microM tBuBHQ. Thus, this agent potently inhibits the fast-twitch skeletal and slow-twitch skeletal/cardiac isoforms of SR Ca(2+)-ATPase. tBuBHQ at 5-10 microM inhibited the rate of decomposition of the phosphoenzyme intermediate (EP), measured as a ratio between ATPase activity and the EP level in the steady state, by 35-40%. It also inhibited formation of EP by decreasing the rate of Ca2+ binding to the Ca(2+)-deficient, nonphosphorylated enzyme to about 1/8 of the control value. These results indicate that tBuBHQ has at least two sites of action in the reaction sequence for the SR Ca(2+)-ATPase.     

Role of regulator of G protein signaling 2 (RGS2) in Ca(2+) oscillations and adaptation of Ca(2+) signaling to reduce excitability of RGS2-/- cells

Regulators of G protein signaling (RGS) proteins accelerate the GTPase activity of Galpha subunits to determine the duration of the stimulated state and control G protein-coupled receptor-mediated cell signaling. RGS2 is an RGS protein that shows preference toward Galpha(q).To better understand the role of RGS2 in Ca(2+) signaling and Ca(2+) oscillations, we characterized Ca(2+) signaling in cells derived from RGS2(-/-) mice. Deletion of RGS2 modified the kinetic of inositol 1,4,5-trisphosphate (IP(3)) production without affecting the peak level of IP(3), but rather increased the steady-state level of IP(3) at all agonist concentrations. The increased steady-state level of IP(3) led to an increased frequency of [Ca(2+)](i) oscillations. The cells were adapted to deletion of RGS2 by reducing Ca(2+) signaling excitability. Reduced excitability was achieved by adaptation of all transporters to reduce Ca(2+) influx into the cytosol. Thus, IP(3) receptor 1 was down-regulated and IP(3) receptor 3 was up-regulated in RGS2(-/-) cells to reduce the sensitivity for IP(3) to release Ca(2+) from the endoplasmic reticulum to the cytosol. Sarco/endoplasmic reticulum Ca(2+) ATPase 2b was up-regulated to more rapidly remove Ca(2+) from the cytosol of RGS2(-/-) cells. Agonist-stimulated Ca(2+) influx was reduced, and Ca(2+) efflux by plasma membrane Ca(2+) was up-regulated in RGS2(-/-) cells. The result of these adaptive mechanisms was the reduced excitability of Ca(2+) signaling, as reflected by the markedly reduced response of RGS2(-/-) cells to changes in the endoplasmic reticulum Ca(2+) load and to an increase in extracellular Ca(2+). These findings highlight the central role of RGS proteins in [Ca(2+)](i) oscillations and reveal a prominent plasticity and adaptability of the Ca(2+) signaling apparatus.     

Energetics of Ca(2+)-EDTA interactions: calorimetric study

The interaction between Ca(2+) and EDTA has been studied using isothermal titration calorimetry to elucidate the detailed mechanism of complex formation and to relate the apparent thermodynamic parameters of calcium binding to the intrinsic effects of ionization. It has been shown that Ca(2+) binding to EDTA is an exothermic process in the temperature range 5-48 degrees C and is highly dependent on the buffer in which the reaction occurs. Calorimetric measurements along with pH-titration of EDTA under different solvent conditions shows that the apparent enthalpy effect of the binding is predominantly from the protonation of buffer. Subtraction of the ionization effect of buffer from the total enthalpy shows that the enthalpy of binding Ca(2+) to EDTA is -0.56 kcal mol(-1) at pH 7.5. The DeltaH value strongly depends on solvent conditions as a result of the degree of ionization of the two amino groups in the EDTA molecule, but depends little on temperature, indicating that the heat capacity increment for metal binding is close to zero. At physiological pH values where the amino groups of EDTA with pK(a)=6.16 and pK(a)=10.26 are differently ionized, the coordination of the Ca(2+) ion into the complex leads to release of one proton due to deprotonation of the amino group having pK(a)=10.26. Increasing the pH up to 11.2, where little or no ionization occurs, leads to elimination of the enthalpy component due to ionization, while its decrease to pH 2 leads to its increase, due to protonation of the two amino groups. The heat effect of Ca(2+)/EDTA interactions, excluding the deprotonation enthalpy of the amino groups, i.e. that associated with the intrinsic enthalpy of binding, is higher in value (Delta(b)H(o)=-5.4 kcal mol(-1)) than the apparent enthalpy of binding. Thus, the large DeltaG value for Ca(2+) binding to EDTA arises not only from favorable entropic but also enthalpic changes, depending on the ionization state of the amino groups involved in coordination of the calcium. This explains the great variability in DeltaH obtained in previous studies. The ionization enthalpy is always unfavorable, and therefore dramatically decreases Ca(2+) affinity by reduction of the enthalpy term of the stability function. The origin of the enthalpy and entropy terms in the stability of the Ca(2+)-EDTA complex is discussed.     

Inhibition of plasma membrane Ca(2+)-ATPase by CrATP. LaATP but not CrATP stabilizes the Ca(2+)-occluded state

The bidentate complex of ATP with Cr(3+), CrATP, is a nucleotide analog that is known to inhibit the sarcoplasmic reticulum Ca(2+)-ATPase and the Na(+),K(+)-ATPase, so that these enzymes accumulate in a conformation with the transported ion (Ca(2+) and Na(+), respectively) occluded from the medium. Here, it is shown that CrATP is also an effective and irreversible inhibitor of the plasma membrane Ca(2+)-ATPase. The complex inhibited with similar efficiency the Ca(2+)-dependent ATPase and the phosphatase activities as well as the enzyme phosphorylation by ATP. The inhibition proceeded slowly (T(1/2)=30 min at 37 degrees C) with a K(i)=28+/-9 microM. The inclusion of ATP, ADP or AMPPNP in the inhibition medium effectively protected the enzyme against the inhibition, whereas ITP, which is not a PMCA substrate, did not. The rate of inhibition was strongly dependent on the presence of Mg(2+) but unaltered when Ca(2+) was replaced by EGTA. In spite of the similarities with the inhibition of other P-ATPases, no apparent Ca(2+) occlusion was detected concurrent with the inhibition by CrATP. In contrast, inhibition by the complex of La(3+) with ATP, LaATP, induced the accumulation of phosphoenzyme with a simultaneous occlusion of Ca(2+) at a ratio close to 1.5 mol/mol of phosphoenzyme. The results suggest that the transport of Ca(2+) promoted by the plasma membrane Ca(2+)-ATPase goes through an enzymatic phospho-intermediate that maintains Ca(2+) ions occluded from the media. This intermediate is stabilized by LaATP but not by CrATP.     

A mathematical model predicts that calreticulin interacts with the endoplasmic reticulum Ca(2+)-ATPase.

A robust mathematical model developed from single cell calcium (Ca(2+)) dynamics has enabled us to predict the consequences of over-expression of endoplasmic reticulum-located chaperones. Model predictions concluded that calreticulin interacts with the lumenal domain of the sarcoplasmic and endoplasmic reticulum Ca(2+)-activated ATPase (SERCA) pump, altering pump affinity for Ca(2+) (K(1/2) switches from 247 to 431 nM) and hence generating Ca(2+) oscillations. Expression of calreticulin in the ER generated an average of six transient-decline oscillations during the Ca(2+) recovery phase, upon exposure to maximal levels of the agonist ATP. In contrast, normal cells produced a single Ca(2+) transient with few or no oscillations. By conditioning the model to experimental data, parameters for generation and decay of IP(3) and SERCA pump kinetics were determined. To elucidate the possible source of the oscillatory behavior three possible oscillators, 1) IP(3), 2) IP(3)R, and 3) SERCA pump, were investigated and parameters constrained by experimental data to produce the best candidate. Each of the three oscillators generated very good fits with experimental data. However, converting a normal exponential recovery to a transient-decline oscillator predicted that the SERCA pump is the most likely candidate for calreticulin-mediated Ca(2+) release, highlighting the role of this chaperone as a signal protein within the endoplasmic reticulum.     

Ca(2+) signals in Pmr1-GFP-expressing COS-1 cells with functional endoplasmic reticulum

We studied the role of the Pmr1-containing Ca(2+) store in COS-1 cells endowed with a functional endoplasmic reticulum. Transfected cells could be recognized by using a green-fluorescent-protein (GFP)-tagged form of Pmr1. Pmr1-GFP fluorescence showed a typical juxtanuclear Golgi-like distribution. Pmr1-GFP-containing cells with functional endoplasmic reticulum responded to 100 microM ATP with baseline Ca(2+) spiking, while non-transfected cells produced an initial Ca(2+) peak followed by a long-lasting plateau. The Ca(2+) signal often appeared after a long latency in Pmr1-GFP-expressing cells. ATP-stimulated Pmr1-GFP-expressing cells with functional endoplasmic reticulum responded after a latency period to extracellular Ca(2+) with a regenerative Ca(2+) signal, while non-transfected control cells responded with an immediate slow rise in free cytosolic Ca(2+) concentration. These results demonstrate the importance of the Pmr1-containing Ca(2+) store in generating or modifying cellular Ca(2+) signals.     

Role of Mg2+ in the Ca2+-Ca2+ exchange mediated by the membrane-bound (Ca2+, Mg2+)-ATPase of sarcoplasmic reticulum vesicles

Sarcoplasmic reticulum vesicles were preloaded with either 45Ca2+ or unlabeled Ca2+. The unidirectional Ca2+ efflux and influx, together with Ca2+-dependent ATP hydrolysis and phosphorylation of the membrane-bound (Ca2+, Mg2+)-ATPase, were determined in the presence of ATP and ADP. The Ca2+ efflux depended on ATP (or ADP or both). It also required the external Ca2+. The Ca2+ concentration dependence of the efflux was similar to the Ca2+ concentration dependences of Ca2+ influx, Ca2+-dependent ATP hydrolysis, and phosphoenzyme formation. The rate of the efflux was approximately in proportion to the concentration of the phosphoenzyme up to 10 microM Ca2+. These results and other findings indicate that the Ca2+ efflux represents the Ca2+-Ca2+ exchange (between the external medium and the internal medium) mediated by the phosphoenzyme. In the range of 0.6-5.2 microM Mg2+, no appreciable Ca2+-Ca2+ exchange was detected although phosphoenzyme formation occurred to a large extent. Elevation of Mg2+ in the range 5.2 microM-4.8 mM caused a remarkable activation of the exchange, whereas the amount of the phosphoenzyme only approximately doubled. The kinetic analysis shows that this activation results largely from the Mg2+-induced acceleration of an exchange between the bound Ca2+ of the phosphoenzyme and the free Ca2+ in the internal medium. It is concluded that Mg2+ is essential for the exposure of the bound Ca2+ of the phosphoenzyme to the internal medium.