©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Lumenal Ca Dissociation from the Phosphorylated Ca-ATPase of the Sarcoplasmic Reticulum Is Sequential (*)

(Received for publication, March 21, 1995)

Vincent Forge Elisabeth Mintz Denis Canet Florent Guillain

From the Commissariat l'Energie Atomique and Unit de Recherche 1290 Associe au Centre National de la Recherche Scientifique, Section de Biophysique des Protines et des Membranes, Dpartement de Biologie Cellulaire et Molculaire, Centre d'Etudes de Saclay, 91191 Gif-sur-Yvette Cedex, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Once two radioactive Ca coming from the cytoplasm are bound to the transport sites of the nonphosphorylated ATPase, excess EGTA induces rapid dissociation of both ions, whereas excess nonradioactive Ca only reaches one of the two bound Ca. This difference has been explained assuming that the two Ca sites are in a single file channel in which the superficial Ca is freely exchangeable from the cytoplasm, whereas the deeper Ca is exchangeable only when the superficial site is vacant. The same experiment was done using phosphorylated ATPase to determine whether Ca dissociation toward the lumen is sequential as well. Under conditions that allow ADP-sensitive phosphoenzyme to accumulate (leaky vesicles, 5 °C, pH 8, 300 mM KCl), we found the same two pools of Ca. Excess EGTA induced dissociation of both ions together with dephosphorylation. Excess nonradioactive Ca induced the exchange of half the radioactive Ca without any effect on the phosphoenzyme level. Our results show a close similarity between the transport sites of the nonphosphorylated and the phosphorylated enzymes, although the orientation, affinities, and dissociation rate constants are different.


INTRODUCTION

Sarcoplasmic reticulum Ca-ATPase is a membranous enzyme that pumps Ca from the cytoplasm of muscle cells into the reticulum lumen, requiring ATP hydrolysis. The ATPase cycle transports 2Ca per molecule of ATP and per monomer of ATPase, as described in Fig. S1. During the cycle, the transport sites change their orientation and affinity, depending on whether the ATPase is phosphorylated. The high-affinity transport sites of the nonphosphorylated ATPase are accessible from the cytoplasm, whereas once the ATPase has been phosphorylated the transport sites have lower affinity and are accessible from the lumen. This allows Ca release into the SR (^1)lumen and is followed by dephosphorylation of the phosphoenzyme.


Figure S1: Scheme 1



Ca binding to E, the Ca-deprived nonphosphorylated ATPase, has been well characterized. Two ions bind sequentially with a high affinity and a positive cooperativity which both depend on the experimental conditions, Ca = 0.1-10 µM and n(H) = 1.3-2, for pH 8-6, and 0-3 mM Mg at 20 °C (1) . The two Ca ions are known to be kinetically distinguishable, since Dupont (2) showed that the dissociation of half the Ca bound to E was impaired by the presence of excess Ca in the medium. This has been confirmed by several authors under various experimental conditions, different temperatures, pH, Mg concentrations . . .(3, 4, 5, 6, 7, 8) . A simple example of two sites being sequentially accessible from the cytoplasm by the two Ca ions is a channel with a deep site and a superficial site (see the sketch in Fig. 2). The first ion must reach the deep site to leave the superficial site vacant for the second ion. The Ca bound to the superficial site is freely exchangeable with the outer medium, whereas the Ca bound to the deep site is not.


Figure 2: Ca dissociation from the nonphosphorylated ATPase (leaky vesicles). Cytoplasmic sites were saturated with Ca by manual perfusion with 10 µM [Ca]Ca as described and then perfused with either 1 mM EGTA () or 1 mMCa (bullet) for various times. Cartoons illustrating the experiment: A, initial state; B, final state for EGTA; C, final state for Ca.



Such a description of a channel-like structure for the transport sites was first proposed by Inesi(5) . Inesi (5) took advantage of the possibility to selectively place one Ca on top of one Ca, to determine whether their dissociation toward the lumen is sequential. By monitoring the internalization of the Ca ions after phosphorylation, Inesi (5) concluded that their dissociation toward the lumen is sequential and that the first Ca bound to E is the first to be internalized.

The question whether the dissociation of the Ca ions toward the lumen is sequential or not has been reinvestigated more recently by Hanel and Jencks (9) and Orlowski and Champeil(10) . In observing the dissociation of Ca from the phosphorylated ATPase, Orlowski and Champeil (10) found no difference between the dissociation kinetics induced by EGTA or cold Ca and no difference in the dissociation rates of each individual ion. Hanel and Jencks (9) found no difference between the Ca internalization rates observed using empty vesicles, or vesicles loaded with 20 mM Ca, and also no difference between the internalization rates of each individual ion. In both papers, the authors concluded that the two ions cannot be kinetically distinguished. The explanation given by Hanel and Jencks (9) is that a slow conformational change corresponding to deocclusion of Ca precedes fast dissociation of Ca from the phosphoenzyme, therefore making the measurement of the individual Ca dissociation steps impossible.

The discrepancy between the results of Inesi (5) on the one hand and those of Hanel and Jencks (9) and Orlowski and Champeil (10) on the other hand is difficult to understand, because they all used similar experimental conditions, typically pH 6.8-7.5, room temperature, 80-100 mM KCl. Here we show that with 300 mM KCl, at pH 8 and 5 °C, the lumenal dissociation of the two Ca ions from the phosphorylated ATPase is sequential, as is the cytoplasmic dissociation of the two Ca ions from the nonphosphorylated ATPase.


MATERIALS AND METHODS

SR vesicles were prepared and tested as described in (1) from rabbits subjected to a 48-h starvation diet to lower the contamination by phosphorylase(11) . All experiments were carried out at 5 °C in a cold room, and the buffer was always 100 mM Tes-Tris, pH 8, 300 mM KCl. It was prepared with water filtered through a Milli-Q Water Purification System (Millipore). All salts were added as chlorides. Vesicles were made leaky by an incubation of at least 1 h at 2 mg/ml in 50 mM Tris, 10 mM KCl, 2 mM EDTA, at room temperature.

[Ca]Caand [-P]ATP Measurements

Kinetic measurements involving [Ca]Ca or [-P]ATP all started with the same incubation and rinsing steps. Vesicles (0.2 mg/ml) were first incubated in the pH 8 buffer, plus Mg, as specified. 1 ml of this suspension was deposited on a filter (Millipore HA 0.45), and the adsorbed vesicles were rinsed with 1 ml of 100 µM EGTA to deprive the enzyme of contaminating Ca. For cytoplasmic Ca dissociation experiments, ATPase was converted to the Ca(2)E state by manually perfusing the filters for 5 s with 1 ml of 10 µM [Ca]Ca. For lumenal Ca dissociation experiments, ATPase was converted to the Ca(2)E-P state by manually perfusing the filters for 5 s with 2 ml of 100 µM [Ca]Ca or Ca, 100 µM [-P]ATP or ATP, and Mg as specified. The kinetic measurements were started immediately after this step using a rapid filtration apparatus (Biologic, Claix, France). They were done by perfusing 1 mM EGTA, or 1 mMCa, plus Mg as specified, for various times.

All solutions containing [Ca]Ca or [-P]ATP also contained 1 mM [^3H]glucose, which allows evaluation of the filter wet volume, usually about 30 µl. ^3H and Ca or P retained on the filter were simultaneously measured by scintillation. [-P]ATP or [Ca]Ca contained in the wet volume was subtracted from the total P or Ca counts to evaluate the phosphoenzyme and the Ca bound to the ATPase.

[Ca]Ca accumulation in the vesicles was measured in the presence of [Ca]Ca, [^3H]glucose, ATP, and Mg as specified. The reaction was started by addition of vesicles at 0.2 mg/ml. After various periods of time, 1 ml of the reaction mixture was deposited on a filter (Millipore HA 0.45), and the radioactivity retained on the filter was counted.

Steady-state ATPase Activity

Steady-state ATPase activity was measured spectrophotometrically by coupling ATP hydrolysis to NADH oxidation in the presence of 0.4 mg/ml pyruvate kinase, 0.2 mg/ml lactate dehydrogenase, 1 mM phosphoenolpyruvate, 0.45 mM NADH, 1-3 mM Mg, 0.1-1 mM ATP, 300 mM KCl, at pH 8 and 6-10 °C. NADH absorbance variations were followed at 350 nm by an HP 8452A diode array spectrophotometer.


RESULTS

If it is assumed that the dissociation of the two Ca ions from the phosphorylated ATPase is intrinsically sequential, the fact that Inesi (5) found that the two ions dissociate sequentially, whereas Hanel and Jencks (9) and Orlowski and Champeil (10) found that the two ions could not be distinguished can simply indicate that this assumption was difficult to prove under their approximately similar conditions. Keeping this in mind, we looked for experimental conditions that would allow measuring an effect of cold Ca on the lumenal dissociation of radioactive Ca bound to the phosphorylated ATPase. Alkaline pH has been shown to increase the affinity of the phosphorylated ATPase for Ca(12) and to be even more effective at low temperatures(13) . Also high KCl concentrations are known to favor Ca(2)E-P, the ADP-sensitive phosphoenzyme(14) . Thus, 300 mM KCl, pH 8 and 5 °C, were the conditions chosen for these experiments, as we expected to have all the ATPase in its Ca(2)E-P form, with a low ATPase activity and a high enough affinity for lumenal Ca to see an effect of cold Ca on the dissociation of radioactive Ca.

CaAccumulation into the Vesicles and Steady-state ATPase Activity

Ca accumulation in the vesicles and steady-state ATPase activity were measured under the conditions of the Ca dissociation experiments, i.e. 300 mM KCl, pH 8 at 5 °C, to check the activity of the enzyme under such conditions.

Fig. 1shows Ca accumulation into the vesicles during 30-min incubation in the presence of 100 µM ATP. With 3 mM Mg, tight vesicles accumulated 70 nmol/mg, a value that is comparable with the maximum amount of Ca accumulated into the vesicles under standard conditions (i.e. 80 nmol/mg at pH 6 and 20 °C), and the so-called leaky vesicles did not accumulate Ca, as was intended. In the absence of Mg, accumulation was lower, as expected from slower turnover in the presence of CaATP(15) .


Figure 1: Ca accumulation into the vesicles. Vesicles were incubated for various times with 100 µM [Ca]Ca and 100 µM ATP and no added Mg (tight vesicles, ) or 3 mM Mg (tight vesicles, , leaky vesicles, ▪).



Steady-state ATPase activity was measured as described above, using tight vesicles permeabilized by 4% (w/w) A23187 or leaky vesicles. The steady-state activity measured in the presence of 3 mM Mg and 100 µM ATP was 240 nmol/mg/min at 8 °C.

CaDissociation from the Nonphosphorylated ATPase

Once two radioactive Ca coming from the cytoplasm have been bound to the transport sites of the nonphosphorylated ATPase, excess EGTA induces rapid dissociation of both ions, whereas excess nonradioactive Ca induces rapid dissociation of only one of the two bound Ca(2) . This rapidly exchangeable Ca has been identified as the last Ca bound to ATPase(5, 6) . As the dissociation of the other Ca is impaired by the binding of cold Ca at the exchangeable site, it has been identified as the first Ca bound to ATPase(6) .

The two sites are sketched in Fig. 2, which also reports the results of cytoplasmic Ca dissociation experiments under the present conditions, namely using leaky vesicles at pH 8 and 5 °C, in the presence of 300 mM KCl. Ca(2)E was formed by perfusing the vesicles with 10 µM [Ca]Ca plus 3 mM Mg, and dissociation was initiated by perfusing 1 mM EGTA or 1 mMCa, plus 3 mM Mg. EGTA induced biphasic dissociation of Ca ions with rates of 20 and 1 s, whereas Ca induced dissociation of only half the radioactive Ca with the rate of 9 s. This experiment was repeated using tight vesicles that yielded similar rates for cytoplasmic Ca dissociation (data not shown), indicating that the Ca transport sites are not modified by the incubation in EDTA and Tris.

CaOcclusion by the Phosphorylated ATPase (Tight Vesicles)

Phosphorylated ATPase was formed by perfusing tight vesicles with 100 µM [Ca]Ca, 100 µM [-P]ATP, 3 mM Mg, 300 mM KCl for 5 s. It was then perfused with 1 mM EGTA plus 3 mM Mg (Fig. 3, open symbols). The amounts of bound [Ca]Ca and phosphoenzyme were unchanged even after 10 s, indicating that Ca was no longer accessible from the cytoplasmic side.


Figure 3: Ca occlusion by the phosphorylated ATPase (tight vesicles). ATPase was first phosphorylated by manual perfusion with 100 µM [Ca]Ca, 100 µM [-P]ATP, 3 mM Mg as described and then perfused with 1 mM EGTA plus 3 mM Mg (, ) or 1 mM EGTA plus 300 µM ADP (bullet, ▪) for various times. Bound [Ca]Ca (, bullet), phosphoenzyme (, ▪). Cartoons illustrating the experiment: A, initial state; B, final state for EGTA plus Mg (, ); C, final state for EGTA plus ADP (bullet, ▪).



This so-called occluded Ca can be either occluded in the membrane in the Ca(2)E-P form of ATPase as the Ca bound to this form is not accessible from the cytoplasmic side or it can be occluded inside the vesicles, if ATPase turnover is such that some Ca has been transported during the phosphorylation step(16, 17) . The existence of these two types of occluded Ca is demonstrated by the effect of ADP, as shown in Fig. 3(closed symbols).

Almost all the occluded Ca was trapped in the membrane in the Ca(2)E-P form of ATPase. Phosphorylated ATPase, prepared as described above, was perfused with a mixture of 300 µM ADP and 1 mM EGTA. At variance with the perfusion with 1 mM EGTA alone, with ADP present there was fast dephosphorylation together with Ca dissociation. This experiment showed that all the phosphoenzyme was ADP sensitive, as expected from the use of 300 mM KCl at low temperature, and that there was 8 nmol/mg calcium accumulated in the vesicle lumen during the phosphorylation step.

CaDissociation from the Phosphorylated ATPase (Leaky Vesicles)

Phosphorylated ATPase was formed as above, but using leaky vesicles. It was then perfused with 1 mM EGTA plus 3 mM Mg (Fig. 4) or 1 mMCa plus 3 mM Mg (Fig. 5). The perfusion with EGTA induced biphasic dissociation of all the bound [Ca]Ca, together with dephosphorylation of ATPase at rates of 0.6 and 0.06 s, whereas the perfusion with Ca induced biphasic dissociation of [Ca]Ca at 0.4 and 0.03 s and slow dephosphorylation at 0.03 s. To determine whether the phosphoenzyme measured during these perfusions was still the Ca(2)E-P form of ATPase, its ADP sensitivity was tested at time 0 and after 19 s of perfusion. That is the perfusions with EGTA or Ca were followed by manual perfusion of a mixture of 300 µM ADP and 1 mM EGTA for 5 s. These measurements are represented by filled symbols in Fig. 4and Fig. 5. They confirm that after perfusion with EGTA or Ca, the phosphoenzyme was ADP-sensitive, as it was before perfusion, and that all the Ca bound to the vesicles was bound to the ADP-sensitive phosphoenzyme, as expected from leaky vesicles.


Figure 4: Ca dissociation from the phosphorylated ATPase (leaky vesicles). ATPase was first phosphorylated as in Fig. 3and then perfused with 1 mM EGTA plus 3 mM Mg for various times (, ). ADP sensitivity of the phosphoenzyme was evaluated by perfusing a mixture of 300 µM ADP and 1 mM EGTA (bullet, ▪). , bullet, Bound [Ca]Ca; , ▪, phosphoenzyme. Cartoons illustrating the experiment: A, initial state; B, final state for EGTA (, ).




Figure 5: Ca exchange from the phosphorylated ATPase (leaky vesicles). ATPase was first phosphorylated as in Fig. 3and then perfused with 1 mMCa plus 3 mM Mg for various times (, ). ADP sensitivity of the phosphoenzyme was evaluated by perfusing a mixture of 300 µM ADP and 1 mM EGTA (bullet, ▪). , bullet, Bound [Ca]Ca; , ▪, phosphoenzyme. Cartoons illustrating the experiment: A, initial state; B, final state for Ca (, ).



The experiments reported in Fig. 3and Fig. 4were identical, except that the vesicles in Fig. 4were leaky. Thus, although in the phosphorylated ATPase the Ca sites are not accessible from the cytoplasm (Fig. 3), they are accessible from the lumen (Fig. 4). The use of leaky vesicles enabled EGTA (Fig. 4) and Ca (Fig. 5) to induce Ca dissociation, and moreover, Ca impaired dephosphorylation. The stability of the ADP-sensitive phosphoenzyme during the perfusion with Ca suggests that half the bound [Ca]Ca was replaced by Ca on the Ca(2)E-P form of ATPase. This appears clearly in Fig. 6which shows that the [Ca]Ca/E-P ratio varies from 1.6 to 1 during the perfusion by Ca, whereas it remains around 1.8 during the dephosphorylation induced by EGTA.


Figure 6: Bound [Ca]Ca to phosphoenzyme ratio calculated from Fig. 4and Fig. 5. The ratio was calculated after correction for the small amount of Ca and phosphoenzyme remaining after the perfusion with a mixture of ADP and EGTA. , 1 mM EGTA; ▴, 1 mMCa.



Sequential Dissociation or Ca/MgExchange at the Catalytic Site?

In the experiment reported in Fig. 4and Fig. 5, 3 mM Mg were present, together with EGTA or Ca, in the perfusion buffers. Recalling that the phosphorylation step was done in the presence of 3 mM Mgversus 100 µM Ca, ATPase was phosphorylated by MgATP, so that we can assume the presence of Mg at the catalytic site of the phosphoenzyme. Nevertheless, during the perfusion with 1 mMCa and 3 mM Mg, partial substitution of Ca for Mg at the catalytic site cannot be excluded. ATPase turnover with CaATP as substrate is known to be much slower than with MgATP, possibly because of slower dissociation of Ca from the Ca(2)E-P form when it has Ca at its catalytic site(15) . Such a substitution at the catalytic site during the perfusion could induce a slow phase in the Ca dissociation kinetics that would be difficult to distinguish from a slow phase due to sequential dissociation from the transport sites. This possibility was tested by repeating the experiment described in Fig. 5with various Mg concentrations.

Phosphorylated ATPase was formed as described in the legend to Fig. 5and perfused with 1 mMCa and either no added Mg or 1, 3, or 5 mM Mg (Fig. 7). The concentration of Mg did not significantly modify the Ca dissociation kinetics, as Ca impaired dissociation of half the bound [Ca]Ca and dephosphorylation. The rate of the fast phase was 0.2-0.5 s, whereas the rate of the slow phase was the same as that of the phosphoenzyme, 0.02-0.03 s. If a Mg/Ca exchange was to take place at the catalytic site during the perfusion, one would expect the amplitudes and the rates of the fast and slow phases to be modified by the Mg/Ca ratio in the perfusion buffer. It is therefore more likely that the observed slow phase corresponds to a non-exchangeable Ca.


Figure 7: Effect of Mg on Ca dissociation from the phosphorylated ATPase (leaky vesicles). ATPase was first phosphorylated as in Fig. 3and then perfused with 1 mMCa and no added Mg (, bullet), 1 mM Mg (, ▪), 3 mM Mg (, ▴), 5 mM Mg (, ▾) for various times. , , , , bound [Ca]Ca; bullet, ▪, ▴, ▾, phosphoenzyme.




DISCUSSION

Understanding how Ca is transported from the cytoplasm to the SR lumen requires some knowledge of the Ca binding sites on the different forms of ATPase. In the absence of ATP, when there is no turnover, the cytoplasmic Ca binding reaction (step 4 in Fig. S1) has been well characterized, as it can be studied both kinetically and at equilibrium (1, 8, 18, 19, 20, 21) . The situation is different for the Ca binding sites on the phosphorylated ATPase (step 2 in Fig. S1), which should be studied during turnover. Knowledge of the lumenal affinity for Ca can be obtained with tight vesicles loaded with Ca. For instance, the study of phosphoenzyme formation from P(i) in the presence of various lumenal Ca concentrations has yielded information on the lumenal affinity for Ca, which was found in the millimolar range at pH 7 and 20 °C(22) . Direct access to the Ca binding sites on E-P requires working with leaky vesicles. In this case, as the affinity of the lumenal sites is lower than that of the cytoplasmic sites, addition of Ca to E-P induces dephosphorylation and Ca binding to E(23) . Nevertheless, de Meis et al. (13) have shown that working on leaky vesicles ATP could be synthesized in the absence of a Ca gradient, provided that a mixture of Ca and ADP was added to E-P. In this paper, ATP synthesis was studied as a function of the Ca concentration under various conditions that yielded other estimations of the luminal affinity for Ca. Of particular interest is that at pH 8 and 0 °C, half the maximal amount of ATP synthesized was obtained with 10 µM Ca, instead of 300 µM at pH 8 and 30 °C or pH 7 and 0 °C. Although these numbers should not be taken as absolute values for Ca affinity, it is likely that alkaline pH and low temperature are conditions that increase the lumenal affinity for Ca.

Lumenal CaDissociation from Phosphorylated ATPase

In the absence of any known possibility to measure Ca binding to the phosphoenzyme directly, we have studied Ca dissociation from phosphorylated ATPase toward the vesicle lumen using the rapid filtration technique. The experimental conditions were chosen to yield (i) as high as possible lumenal affinity for Ca, i.e. an affinity high enough to use reasonable concentrations of Ca to compete with bound [Ca]Ca, (ii) as much as possible Ca-bound phosphoenzyme, (iii) as low as possible ATPase activity, in order to have enough ATP in the 30-µl wet volume of the filter to maintain the ATPase phosphorylated during the few seconds necessary to start the rapid filtration experiment. All three requirements were achieved while working at pH 8, 5 °C, 300 mM KCl and using 100 µM ATP for phosphorylation.

Under these conditions, the ATPase activity was effectively low, and the vesicles were able to accumulate Ca ( Fig. 1and Fig. 3) showing that the ATPase cycled normally. After phosphorylation, the phosphoenzyme was ADP-sensitive, and there were two Ca bound per phosphoenzyme ( Fig. 3and Fig. 4). Thus, the vast majority of ATPase was in the Ca(2)E-P form, in which Ca is said to be occluded, because it cannot be released on the cytoplasmic side unless the ATPase has bound ADP. When this occluded Ca was formed in leaky vesicles, perfusion with EGTA simultaneously induced dissociation of the two Ca ions and dephosphorylation, as expected from the fact that hydrolysis of E-P, the Ca-deprived phosphoenzyme, is fast in the presence of KCl(14) .

The luminal Ca dissociation, i.e. the EGTA-induced dissociation of Ca from Ca(2)E-P (Fig. 4) was slow compared with the cytoplasmic dissociation of Ca from Ca(2)E (Fig. 2). It is likely that this slow rate illustrates Ca deocclusion from the Ca-bound phosphoenzyme. Keeping in mind that hydrolysis of E-P is fast with KCl present and that the ADP sensitivity is lost together with Ca dissociation, the fact that both the dephosphorylation and the Ca dissociation are biphasic suggests that there is an equilibrium between two forms of Ca(2)E-P. One form, denoted as [Ca(2)]E-P, would bear occluded Ca, and the other form would be able to exchange Ca with the lumen. Thus, biphasic dissociation of Ca and biphasic dephosphorylation would be due to a fast dissociation of Ca from the non-occluded form and the slow conversion of the occluded form into the non-occluded form. At this point, more details on the luminal dissociation of Ca come from the Ca exchange experiments.

Sequential Dissociation from the Phosphorylated ATPase

Comparison of the Ca exchange experiments performed on the phosphorylated ATPase (Fig. 5) and on the nonphosphorylated ATPase (Fig. 2) shows that cytoplasmic Ca exchange was faster than lumenal Ca exchange. On the cytoplasmic side, this exchange is explained by the following sequence,

where the departure of one Ca is followed by the binding of one Ca. The same sequence on the phosphorylated ATPase suggests the existence of an intermediate phosphoenzyme having bound only one Ca and able to bind one Ca. Taking into account the occluded state, Ca exchange on the phosphorylated ATPase can be described by the following sequence.

Such a sequence would provide a stable phosphoenzyme in the presence of cold Ca. In the absence of lumenal Ca, sequential dissociation of both Ca ions would thus occur following.

Our data show a close similarity between the transport sites of the nonphosphorylated ATPase that are accessible from the cytoplasm and those of the phosphorylated ATPase that are accessible from the vesicle lumen. In both states, the transport sites interact with each other as bulk Ca impairs the dissociation of one of the two Ca bound.

Our results agree with the conclusion of Inesi (5) whose model includes luminal sequential dissociation, but they differ from those of Hanel and Jencks (9) and Orlowski and Champeil (10) who found that the two Ca ions could not be distinguished once the ATPase was phosphorylated. As stated above, this discrepancy is probably due to the difference in the experimental conditions. For instance, at room temperature the luminal affinity for Ca is low and the ATPase activity is high, so that it is necessary to phosphorylate ATPase and to exchange cold Ca for radioactive Ca simultaneously, i.e. to perfuse at least 10 mMCa and ATP together. In this case, an ATP/ADP-mediated Mg/Ca exchange can occur in the filter, leading to various populations of phosphoenzymes displaying different rates at step 2 in Fig. S1, as discussed by Orlowski and Champeil (10) . Under our conditions, there was only 1 mMCa perfused, and ATP and ADP were washed out of the filter after the first 25 ms of perfusion; thus, there was very little possibility to have an ATP/ADP-mediated Mg/Ca exchange at the catalytic site. There still remains the possibility that the effect of cold Ca is due to a direct substitution of Ca for Mg at the catalytic site. Fig. 7shows that this was not the case, because varying the Mg concentration during the Ca exchange did not change its kinetics.

Finally, as Hanel and Jencks (9) found the same rate of internalization of Ca whether they monitored internalization of both ions or each individual ion into empty or Ca-loaded vesicles, they suggested that they could only measure a slow deocclusion rate preceding the Ca dissociation steps. This slow deocclusion step was also seen in our experiments, but at variance with Hanel and Jencks(9) , we observed different effects when EGTA or Ca was used. Thus, the comparison between their results and ours suggests that the experimental conditions modify the equilibrium between the occluded and deoccluded states of Ca on the phosphoenzyme. Under our conditions, there would be enough Ca in the deoccluded state to observe the dissociation of the first ion and thus the impairment of the dissociation of the second ion in the presence of cold Ca.

Our finding that Ca dissociation from the phosphorylated enzyme is sequential, as is Ca binding to the nonphosphorylated enzyme, suggests at first sight that during the transport cycle, the Ca ions cross the membrane sequentially via a channel-like structure such as the one sketched here. Nevertheless, a few points should be emphasized. First, a channel-like structure is obviously the simplest way to describe the interactions between the two Ca sites, but it is not the only one. Second, a channel-like structure also suggests that the first ion bound to the cytoplasmic sites is the first ion to dissociate toward the lumen. Such a first-in-first-out model has been suggested by Inesi(5) . Third, there is a controversy about the possibility to have a channel-like structure crossing the membrane and including the putative Ca sites. Site-directed mutagenesis has yielded information on the location of the Ca sites, as six charged residues from the membrane helices M4, M5, M6, and M8 (Glu, Glu, Asn, Thr, Asp, and Glu) were shown to be crucial for Ca transport(24) , and among them, the five residues belonging to M4, M5, and M6 were shown to be crucial for Ca occlusion(25, 26) . According to Inesi (27) , M4, M5, M6, and M8 could form a channel providing the residues coordinating the two Ca ions at its inner surface. According to Andersen(28) , the three residues belonging to M6 cannot at the same time coordinate the two Ca ions and be part of the same alpha-helix.

We have shown here that the lumenal dissociation of the Ca ions is compatible with a channel-like structure, because it is sequential. However, we did not try to obtain any information about what happens in the occluded state, as we did not follow a specified Ca ion, i.e. the first or the second, from the cytoplasmic side to the lumenal side.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^1
The abbreviations used are: SR, sarcoplasmic reticulum; Tes, N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid; A23187, calcimycin.


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