Calcium Occlusion in Plasma Membrane Ca2+-ATPase*

In this work, we set out to identify and characterize the calcium occluded intermediate(s) of the plasma membrane Ca2+-ATPase (PMCA) to study the mechanism of calcium transport. To this end, we developed a procedure for measuring the occlusion of Ca2+ in microsomes containing PMCA. This involves a system for overexpression of the PMCA and the use of a rapid mixing device combined with a filtration chamber, allowing the isolation of the enzyme and quantification of retained calcium. Measurements of retained calcium as a function of the Ca2+ concentration in steady state showed a hyperbolic dependence with an apparent dissociation constant of 12 ± 2.2 μm, which agrees with the value found through measurements of PMCA activity in the absence of calmodulin. When enzyme phosphorylation and the retained calcium were studied as a function of time in the presence of LaIII (inducing accumulation of phosphoenzyme in the E1P state), we obtained apparent rate constants not significantly different from each other. Quantification of EP and retained calcium in steady state yield a stoichiometry of one mole of occluded calcium per mole of phosphoenzyme. These results demonstrate for the first time that one calcium ion becomes occluded in the E1P-phosphorylated intermediate of the PMCA.

The plasma membrane calcium ATPase (PMCA) 3 is a calmodulin-modulated P-type ATPase responsible for the maintenance of low intracellular concentrations of Ca 2ϩ in most eukaryotic cells. It couples the transport of Ca 2ϩ out of cells with the hydrolysis of ATP into ADP and inorganic phosphate. PMCAs consist of a single polypeptide chain of 127,000 to 137,000 Da. Mammalian PMCAs are encoded by four separate genes (PMCA1-4), and additional isoforms are generated via alternative RNA splicing, which augments the number of variants to Ͼ20 (1). The current kinetic model for PMCA function proposes that the enzyme exists in two main conformations, E 1 and E 2 . E 1 has a high affinity for Ca 2ϩ and is readily phosphorylated by ATP, whereas E 2 has a low affinity for Ca 2ϩ and can be phosphorylated by P i . After binding of intracellular Ca 2ϩ to high affinity sites, E 1 can be phosphorylated by ATP with formation of the intermediate E 1 P. After a conformational transition to E 2 P, Ca 2ϩ would be released to the extracellular medium from low affinity sites, followed by the hydrolysis of the phosphoenzyme to E 2 and a new conformational transition to E 1 ( Fig. 1) (2). During some stages of the reaction cycle, Ca 2ϩ becomes occluded, i.e. trapped in the enzyme machinery while it is transported from one side to the other side of the membrane. The principal aim of this study is to identify and kinetically characterize the intermediate(s) of the PMCA containing occluded calcium. Evidence for occlusion in Na,K-ATPase and sarcoplasmic reticulum Ca 2ϩ -ATPase (SERCA) has been well established (3), and a good deal of information exists about the occlusion and deocclusion steps of the transported cations in these pumps. Na ϩ and K ϩ are occluded in the E 1 P and E 2 intermediates of the Na ϩ /K ϩ -ATPase (4), respectively, and Ca 2ϩ becomes occluded in the E 1 P intermediate of the SERCA (5).
By analogy with SERCA, one would expect (5) that Ca 2ϩ occlusion occurs in the E 1 P state of the PMCA. However, it has not been possible until now to obtain definitive experimental evidence of such a phenomenon, mainly because PMCA in microsomal preparations obtained from natural sources is not sufficiently abundant and pure. To overcome this difficulty, we have used a procedure to overexpress PMCA in Sf9 insect cells using a baculovirus system and also to isolate the microsomal membranes. Binding of Ca 2ϩ to these membranes was measured as a function of time using a method (6) that combines a quench-flow apparatus with a rapid filtration device, with a time resolution of 3.5 ms. The procedure included inhibition of endogenous SERCA and the permeabilization of microsomal vesicles using the pore-forming peptide alamethicin, which prevents 45 Ca 2ϩ accumulation (7). Our results suggest that a single calcium ion is occluded in E 1 P of the PMCA and that the formation of this phosphoenzyme and the occlusion of calcium are simultaneous events. Amplification of Recombinant Baculovirus-The viral stocks were amplified using a multiplicity of infection of 0.1-0.2 following standard procedures, and the titer of the amplified stock was determined. The viral stock was kept at 4°C in the dark.

EXPERIMENTAL PROCEDURES
Microsomal Preparation-Crude microsomal membranes from Sf9 cells were prepared essentially as described for COS cells with some minor modifications (9). After washing the cells with phosphate-buffered saline containing increased amounts of protease inhibitors, 10 g/ml aprotinin, and 4 g/ml leupeptin, the cell pellets were immediately frozen in aliquots of 250⅐10 6 cells until processing. The volumes of buffers used for homogenizing the relatively large cell pellets were also increased two or three times to maintain the concentrations required to promote formation of inside-out vesicles. Per 250⅐10 6 cells, 7 ml of hypotonic and 7 ml of homogenization buffers were used. Aliquots of microsomes were stored in liquid nitrogen.
Reagents and Reaction Conditions-( 45 Ca)CaCl 2 , [␥-32 P]-ATP, and 125 I were obtained from Perkin-Elmer Life Sciences. All other reagents were of analytical grade.
All experiments were performed at 25°C in medium containing 30 mM MOPS (pH 7.4 at 25°C), 120 mM KCl, 3 mM MgCl 2 , 200 nM thapsigargin, and enough CaCl 2 to obtain the concentration of free Ca 2ϩ indicated in the figures. The concentrations of other components varied according to the experiments and are indicated in the figure legends. For measuring time courses of phosphoenzyme and Ca 2ϩ retained in the millisecond-second timescale, we used a rapid mixing apparatus SFM4 from Bio-Logic.
Measurement of Free Ca 2ϩ Concentrations-The Ca 2ϩ concentration in the incubation medium was measured using a selective Ca 2ϩ electrode (93-20, Orion Research, Inc.), as described by Kratje et al. (10).
Thapsigargin Treatment-Thapsigargin (octanoic acid derivative of azulene [4,5-b]furan) was obtained from Sigma (catalog no. T9033). Thapsigargin was dissolved in dimethyl sulfoxide to a concentration of 153.6 M. Dilutions of this solution were added to a suspension containing the protein. The final concentration of dimethyl sulfoxide never exceeded 0.1% in volume. Controls of Ca 2ϩ -ATPase activity with and without dimethyl sulfoxide showed no significant differences. In agreement with Sagara et al. (11), we found that inhibition of SERCA activity was achieved after a 15-min incubation of the microsomal preparation with 200 nM thapsigargin at 25°C. This inhibition lasted for at least 3 min after addition of Ca 2ϩ . Therefore, all experiments were performed within this time frame.
Measurements of ATPase Activity-This was measured as the amount of [ 32 P]P i released from [␥-32 P]ATP, according to a slight modification of the method described by Schwarzbaum et al. (12). Incubation time was short enough to prevent the hydrolysis of Ͼ10% of the ATP present and to ensure initial rate conditions. Enzyme concentration was 60 g of total protein/ ml, and blanks were included in an assay in the same medium without Ca 2ϩ in the presence of 1 mM EGTA. The medium contained 5 mM NaN 3 and 0.5 mM ouabain.
Measurements of Calcium Retained-Ca 2ϩ retained was measured using the method of Rossi et al. (6) where the rapid mixing apparatus is connected to a quenching-and-washing chamber (supplemental Fig. S1), through a suitable polyethylene tubing. In a typical experiment, one volume of a microsomal preparation suspended in a solution with 30 mM MOPS (pH 7.4 at 25°C), 120 mM KCl, and 400 nM thapsigargin was mixed with the same volume of a solution containing the same concentrations of MOPS and KCl, plus 6 mM MgCl 2 and enough ATP and ( 45 Ca 2ϩ )CaCl 2 to obtain the concentrations of the nucleotide and of free Ca 2ϩ indicated in the figures. For some experiments, 100 M La III was also included in the latter solution. Measurements were carried out at 25°C. Reactions were quenched after the appropriate time by injecting the reaction mixture into the quenching-and-washing chamber at a flow rate of 1-5 ml. s Ϫ1 . During the injection process, the fluid was mixed with an ice-cold washing solution flowing at a rate of 30 -40 ml/s and then filtered through a Millipore filter (AA, 0.8-m pore size) placed in the quenching-and-washing chamber to retain the microsomal suspension that includes the enzyme.   SEPTEMBER 16, 2011 • VOLUME 286 • NUMBER 37 labeled enzyme was recovered irrespective of the filter pore size.

Calcium Occlusion in PMCA
To ensure that the initial temperature in the quenching-andwashing chamber was 1-2°C and that the flow was constant, ϳ50 ml of washing solution was allowed to run through the filter prior to the injection of the reaction mixture, and 240 ml of washing solution was applied to the filter from that moment. The composition of the washing solution was 10 mM Tris, 10 mM EDTA, pH 7.4, at 2°C. Control experiments show that the washing procedure effectively removes the unbound 45 Ca 2ϩ . After the washing solution was drained, the filter was removed, dried under a lamp, and counted for 45 Ca 2ϩ radioactivity in a scintillation counter. This was converted into nanomoles of Ca 2ϩ using the specific activity value of the 45 Ca 2ϩ in the reaction mixture. Retained Ca 2ϩ was considered equal to the 45 Ca 2ϩ radioactivity retained by the enzyme after subtracting the blank values. These were estimated from the amount of 45 Ca 2ϩ retained by the filters in the presence of enzyme that was heat-inactivated for 2 h at 50°C (see supplemental Figs. S2 and S3).
Determination of Phosphorylated Intermediates-The phosphorylated intermediates (EP), were measured as the amount of acid-stable 32 P incorporated in the enzyme from [␥-32 P]ATP after stopping the reaction with an ice-cold solution containing 10% trichloroacetic acid. The isolation of the intermediate was performed according to two methodologies.
In the first methodology, the suspension was transferred to Millipore filters (Type GS, 0.22 m pore size) where the phosphoenzyme was washed with 20 ml of a solution of 10% trichloroacetic acid and 50 mM H 3 PO 4 . For the blanks, similar experiments were done with heat-inactivated enzyme.
In the second methodology, when the microsomes were phosphorylated with high concentration of ATP, we used the method described by Echarte et al. (14). The phosphorylation reaction was stopped, and the tubes were spun down at 7000 rpm for 3.5 min at 4°C. The samples were then washed once with 7% TCA, 150 mM H 3 PO 4 , and once with double-distilled water and processed for SDS-PAGE. For this purpose, the pellets were dissolved in a medium containing 150 mM Tris-HCl (pH 6.5 at 14°C), 5% SDS, 5% DTT, 10% glycerol, and bromphenol blue (sample buffer). Electrophoresis was performed at pH 6.3 (14°C) in a 7.5% polyacrylamide gel. The reservoir buffer was 175 mM MOPS, pH 6.5, with 0.1% SDS. Migration of the sample components took place at 14°C, with a current of 60 mA until the tracking dye reached a distance of ϳ10 cm from the top of the gel. Gels were stained, dried, and exposed to a Storage Phosphoscreen of Molecular Dynamics (Amersham Biosciences). Unsaturated autoradiograms and stained gels were scanned with an HP Scanjet G2410 scanner. Analysis of the images was performed with GelPro Analyzer. EP quantification was achieved as described in Echarte et al. (14).
Alamethicin Treatment-Alamethicin was obtained from Sigma (catalog no. A4665). This peptide was dissolved in 60% (v/v) ethanol to a concentration of 20 mg/ml (7). This solution was added to a suspension containing the protein. Final concentrations of alamethicin added to the microsomes are expressed on a weight basis relative to microsomal protein. The final ethanol concentration of the microsomes after adding alamethicin never exceeded 0.1%, and for all of the experiments, an equivalent amount of ethanol was added to control membranes not treated with alamethicin.
Data Analysis-Theoretical equations were fitted to the results by nonlinear regression based on the Gauss-Newton algorithm using commercial programs (Excel and Sigma-Plot for Windows, the latter being able to provide not only the best fitting values of the parameters but also their S.E.). The goodness of fit of a given equation to the experimental results was evaluated by the corrected AIC criterion defined in Equation 1, where N is the number of data, P is the number of parameters plus one, and SS is the sum of weighted square residual errors (15). Unitary weights were considered in all cases, and the best equation was chosen as that giving the lower value of AIC C . The AIC criterion is based on information theory and selects an equation among several possible equations on the basis of its capacity to explain the results using a minimal number of parameters.

Calcium Retained by Microsomal Vesicles Expressing PMCA-
According to earlier findings on SERCA (16), one would expect to observe occlusion of Ca 2ϩ in PMCA as a fast phase early in the time course of 45 Ca 2ϩ uptake by microsomal vesicles.
where A 1 and A 2 are maximal amounts of Ca ret and k 1 and k 2 are rate coefficients. The best fitting values of the parameters are shown in Table 1, where it appears that A 1 and k 1 , as well as k 2 increase with the concentration of ATP. If one assumes that the fast component is due to the occlusion of Ca 2ϩ whereas the slow one reflects the accumulation of Ca 2ϩ into vesicles of the microsomal preparation, A 1 should be on the order of the amount of Ca 2ϩ -ATPase, while A 2 (but not necessarily A 1 ) should decrease upon addition of a permeabilizing agent.
Results of experiments to test these predictions are described in the following paragraphs.
Enzyme Concentration-La III is known to prevent the Mg 2ϩdependent transition E 1 P3E 2 P, acting noncompetitively with respect to Ca 2ϩ and ATP (17,18). Thus, the amount of PMCA can be evaluated by measuring the concentration of enzyme phosphorylated from [␥-32 P]ATP in the presence of this inhibitor, producing accumulation of E 1 P with the inhibition of the ATPase activity (17,18). Fig. 3A shows the amount of phosphorylated intermediates, EP (ϭ [E 1 P] ϩ [E 2 P]), as a function of [La III ]. EP increased with the concentration of La III along the following hyperbolic function, where EP 0 and EP max are the amounts of PMCA phosphorylated in the absence of lanthanum and in the presence of nonlimiting concentrations of the inhibitor, respectively, and K 0.5 is the concentration of La III at which Ep ϭ (EP max ϩ EP 0 )/2. The best fitting values for EP 0 , EP max , and K 0.5 were 0.030 Ϯ 0.002 nmol EP⅐mg Ϫ1 , 0.0765 Ϯ 0.0024 nmol EP⅐mg Ϫ1 , and 5.25 Ϯ 1.33 M, respectively. In the inset of Fig. 3A, we plotted Ca 2ϩ -ATPase activity as a function of the concentration of La III . The best fit to the experimental data were a hyperbolic decreasing function (see legend to Fig. 3), where the K i for La III was 3.5 Ϯ 0.8 M (cf. with the value of K 0.5 (La) for EP formation). The time course of Ca ret in the presence of 50 M La III was measured under similar conditions, although using a different preparation (Fig. 3B). Best fitting values using equation 2 were A 1 ϭ 0.0774 Ϯ 0.0072 nmol⅐mg Ϫ1 , A 2 ϭ 0.314 Ϯ 0.014 nmol⅐mg Ϫ1 , k 1 ϭ 2.17 Ϯ 0.38 s Ϫ1 , and k 2 ϭ 0.059 Ϯ 0.007 s Ϫ1 , respectively. Note that the value of A 1 is of the same order of magnitude as that of EP max above, which can be taken as evidence that the first fast phase of the time course of calcium retained is due to the occlusion of Ca 2ϩ in the PMCA. Moreover, although La III almost completely inhibits the ATPase activity, it does not significantly decrease the fraction of the slow phase in the time course of Ca 2ϩ retained. Effect of Alamethicin on Ca 2ϩ Accumulation by Microsomes-If the slow phase of the time course in Figs. 2 and 3B is due to the uptake of Ca 2ϩ into vesicles, addition of permeabilizing agents should facilitate the washing out of Ca 2ϩ in the quenching and washing chamber and accordingly remove a possible confounding factor for the detection of Ca 2ϩ -occluded states in the PMCA. We tested this by treating the microsomal membranes with alamethicin, a peptide that forms large pores allowing the passage of organic molecules of considerable size such as ATP. Alamethicin has been used previously in transport studies of the SERCA (7) and determinations of occlusion of Rb ϩ in the H ϩ /K ϩ -ATPase (19).
Following incubation of microsomal membranes with different concentrations of alamethicin for 30 min at 25°C, retained calcium was measured as a function of time from 1.5 to 8.5 s (Fig. 4A, inset). In Fig. 4A, we plotted both the Ca 2ϩ -ATPase activity and the slopes of the curves of retained calcium versus time as a function of the alamethicin concentration. The data show that, as [alamethicin] increases, the slope decreases, whereas the PMCA activity remains nearly constant, at least up  in our measurements of Ca ret . As a further refinement of these measurements we also took into account a slow residual linear component of bound Ca 2ϩ that was observed as well for heat inactivated enzyme and was therefore subtracted as background (supplemental Fig. S2). Fig. 4B compares the time course of calcium retained measured either in the absence of alamethicin or in the presence of 0.2 mg alamethicin per mg of total protein. Notice that La III was not present in this experiment. Although the time course with no added alamethicin shows a behavior similar to that in Fig. 2 (Fig. 5, A and B) as well as the Ca 2ϩ -ATPase activity (Fig. 5C) as a function of [Ca 2ϩ ]. Fig. 5A shows the time courses of retained calcium, after subtracting the small linear component due to nonspecific binding (see supplemental Fig. S3), measured at different [Ca 2ϩ ]. All the curves can be described by a single increasing exponential function of time, where A is the steady-state amount of the "specific" retained Ca 2ϩ , and k is an apparent rate coefficient. The best fitting values of A from Fig. 5A were plotted as a function of [Ca 2ϩ ] as shown in Fig. 5B.
Both the value of A from Equation 4 and the Ca 2ϩ -ATPase activity can be described by a rectangular hyperbola as a func- where Y max is the Ca 2ϩ -ATPase activity or the value of A when the calcium concentration tends to infinity, and K 0.5 represents the [Ca 2ϩ ] at which the half-maximum effect is achieved. The best fitting values of K 0.5 were 12.5 Ϯ 1.3 M and 12.7 Ϯ 2.2 M for activity and occluded calcium, respectively. The fact that these values are not significantly different from each other indicates that the retained calcium measured in media with alamethicin is due to an intermediate of the reaction cycle, probably that containing occluded calcium. Stoichiometry of Occluded Calcium-As shown in Fig. 3, the amount of PMCA, evaluated as the maximal concentration of EP that accumulates as E 1 P in the presence of La III , is of the same order of magnitude as the size of the fast component of the time course of Ca ret . Because the yield of PMCA expressed in Sf9 cells varies between different preparations, a strictly quantitative comparison between Ca ret and EP requires performing experiments using the same preparation under the same conditions. Results of parallel experiments in media with La III measuring steady-state levels of both EP and Ca 2ϩ occluded, and the ratio Ca occ /EP are shown in Table 2 for two different microsomal preparations. Although the values of both experimentally determined levels vary between the preparations, the ratio Ca occ /EP is nearly equal to 1, which is consistent with the stoichiometry of one Ca 2ϩ transported per molecule of ATP hydrolyzed (20 -22) and with the hypothesis that, in the presence of ATP, calcium is occluded in the E 1 P conformation of the PMCA.
Time Course of E 1 P and Occluded Calcium-To determine whether calcium occlusion in the PMCA is concomitant with the formation of the E 1 P phosphorylated enzyme intermediate, we measured the time course of EP and calcium occlusion under the same experimental conditions. Fig. 6 shows that both phosphorylation and calcium occlusion increase simultaneously and can be described by a single exponential as a function of time adjusted to Equation 4, with best fitting values of k ϭ 2.10 Ϯ 0.30 s Ϫ1 and 2.09 Ϯ 0.31 s Ϫ1 , for E 1 P and occluded calcium, respectively. Furthermore, the data reveal a stoichiometry of 1:1 for calcium occlusion and PMCA phosphorylation over time, showing that both reactions occur concomitantly.

TABLE 2
Comparison of E 1 P and occluded calcium Phosphorylated PMCA was measured in steady state at 25°C in reaction medium containing 60 g/ml total protein, 3 mM MgCl 2 , 25 M ͓␥-32 P͔ATP, 12 g/ml alamethicin, 50 M La III , and enough CaCl 2 to give 60 M of free Ca 2ϩ . The occluded calcium was measured in steady state at 25°C in reaction medium containing 60 g/ml total protein, 3 mM MgCl 2 , 25 M ATP, 12 g/ml alamethicin, 50 M La III , and enough ͓ 45 Ca͔CaCl 2 to give 60 M of free Ca 2ϩ . Note that the EP for microsomes 1 was measured by the method described by Echarte et al. (14) and the EP for microsomes 2 was measured by filtration (see "Experimental Procedures").

DISCUSSION
This work provides the first conclusive evidence that Ca 2ϩ becomes occluded in the plasma membrane Ca 2ϩ -ATPase, with a stoichiometry of one Ca 2ϩ transported per ATP hydrolyzed. The evidence is based on the following findings. First, during ATPase activity, the uptake of Ca 2ϩ by microsomes enriched in PMCA shows a time course with a fast component whose amplitude is compatible with the amount of enzyme, and a slow component, which is related to accumulation of Ca 2ϩ into vesicles. In support of this interpretation, addition of the pore-forming peptide alamethicin tends to eliminate the slow component without affecting the fast one. Second, measurements in the presence of alamethicin and La III show that the time course of the fast component of Ca 2ϩ uptake is concomitant with that of enzyme phosphorylation, with a stoichiometry of one Ca 2ϩ retained per phosphorylation site. Third, steadystate measurements of calcium retained and ATPase activity in the presence of alamethicin exhibit the same K 0.5 for Ca 2ϩ . These results indicate that Ca 2ϩ becomes occluded in the E 1 P intermediate of the PMCA, with a stoichiometry of one Ca 2ϩ per phosphorylation site, but they do not rule out the possibility that other intermediates of the reaction cycle can occlude Ca 2ϩ as well.
Steps toward Detailed Kinetic Model for PMCA-Although there is a large body of information on the kinetics of the PMCA, most of this information has not been incorporated into a detailed unified model because of the heterogeneity of the data. The main reason for this has been the lack of a reliable standard preparation of the pump. Unlike for other P-type ATPases, there are no known tissue sources rich enough in a single isoform of PMCA. Even the purified erythrocyte preparation is a mixture of PMCA4b and PMCA1b (23). Additionally, methodological challenges have made it very difficult to obtain reliable measurements of phosphorylated species at physiological (millimolar) concentrations of ATP. One of the main differences between purified enzyme preparations from erythrocytes and the insect cell-derived recombinant enzyme preparation used in this work is that in the former case the enzyme is solubilized in detergents and included in phospholipid micelles, whereas in the latter it remains embedded in the lipid bilayer of the plasma membrane. Therefore, besides the fact that the PMCA is kept in its original environment, an important advantage of the Sf9 insect cell-expressed PMCA preparation is the possibility of isolating the enzyme containing occluded ions by filtration on Millipore-type membranes. This, plus the use of alamethicin as a permeabilizing agent for efficient washing out of unbound Ca 2ϩ , provides an excellent system to measure occlusion of this cation in the PMCA.
By analogy with Ca 2ϩ occlusion in SERCA and Na ϩ occlusion in Na,K-ATPase (4, 5), Ca 2ϩ occlusion is thought to occur in the E 1 P state of the PMCA. To detect Ca 2ϩ -occluded states the strategy therefore must be to work under conditions where the expected amount of E 1 P is maximal, and its rate of breakdown is minimal. These conditions can be deduced from kinetic studies determining the ADP-sensitive phosphoenzyme (24), but an alternative approach is to perform the reactions in the presence of La III (17,18), which blocks the Mg 2ϩ -depen-dent E 1 P to E 2 P transition. In PMCA purified from erythrocytes, the steady-state amount of EP increases with the concentration of ATP along a nonhyperbolic curve, indicating a complex mechanism for nucleotide hydrolysis during calcium transport (14). ATP not only accelerates the dephosphorylation reaction, as reported by Garrahan and Rega (25), but it also modifies the rate of conformational transition E 1 P3E 2 P, accelerating this reaction at millimolar concentrations of Mg 2ϩ . However, in this study, we have shown that occluded Ca 2ϩ can be detected even in the presence of 3 mM MgCl 2 . This raises the question whether Ca 2ϩ occlusion could also take place in intermediates other than E 1 P.
Occluded Cations as Intermediates of Ca 2ϩ Transport Cycle of PMCA-In SERCA and Na,K-ATPase pumps, studies of cation occlusion gave crucial information on how the pumps couple the exergonic hydrolysis of ATP to uphill movement of the transported cations (3). According to Glynn and Karlish (3), occluded ions are defined as those that can "escape from the pump only after a change in the pump machinery that represents a step (or steps) in the working cycle." For instance, when SERCA is phosphorylated by ATP to give the ADP-sensitive form of phosphoenzyme (E 1 P), two Ca 2ϩ ions bound to high affinity, Ca 2ϩ -selective sites at the cytoplasmic surface become occluded. A change in conformation of the phosphoenzyme to E 2 P makes these sites accessible from the inner (lumenal) surface of the sarcoplasmic reticulum, and the phosphoryl group is transferred to water. Release of the occluded Ca 2ϩ ions and hydrolysis of the phosphoenzyme are followed by a spontaneous change in conformation of the dephosphoenzyme back to a form that can bind Ca 2ϩ ions at the cytoplasmic surface with high affinity. Using erythrocyte ghosts permeabilized with the ionophore A23187, Moreira et al. (26) reported evidence for occluded Ca 2ϩ in the E 1 P intermediate of PMCA. However, according to their results, ϳ1.5 mol of Ca 2ϩ are occluded per mole of enzyme. This value is in contrast with the proposed stoichiometry of 1 Ca 2ϩ transported per ATP hydrolyzed by the PMCA (20,21,22). In our hands 4 (not shown, but see Ref. 26), the preparation of erythrocyte ghosts, which is not highly enriched in PMCA, presents serious problems for filtration through Millipore-type filters, and washing out of Ca 2ϩ from the ghosts using the ionophore A23187 is slow and inefficient. Ca 2ϩ occlusion is a very rapid transient process (on the secondsubsecond scale), and passage of Ca 2ϩ ions through the ionophore can take a few minutes, producing an accumulation of the cation, which impedes reliable measurements of Ca 2ϩ occlusion. In addition, Moreira et al. (26) used reaction blanks for Ca 2ϩ occlusion with ATP but not La III , a condition that can still lead to the formation of occluded Ca 2ϩ , as we have shown in this work.
Perspectives-Like the phosphorylated states of the enzyme, which are intermediates during the hydrolysis of ATP, the occluded states of the enzyme are thought to be true intermediates during the transport of cations (27). The kinetic study of these two types of intermediates in parallel experiments will yield very important information for the construction of a detailed reaction model of the plasma membrane Ca 2ϩ -ATPase. Future experiments using the methodology employed in this paper will also include a comparative kinetic study of Ca 2ϩ occlusion in different PMCA isoforms. Of particular interest will be studies on PMCA2, which differs significantly from PMCA4 in activation kinetics and basal activity (28). These studies may then also be extended to PMCA mutants with changes in amino acids thought to be involved in the state transitions linked to Ca 2ϩ occlusion.