Ca2+ Translocation across Sarcoplasmic Reticulum ATPase Randomizes the Two Transported Ions

Cytoplasmic Ca2+ dissociation is sequential, and the Ca2+ ions bound to the nonphosphorylated ATPase are commonly represented as superimposed on each other, so that the superficial Ca2+ is freely exchangeable from the cytoplasm, whereas the deeper Ca2+ is not. Under conditions where ADP-sensitive phosphoenzyme accumulates (leaky vesicles, 5°C, pH 8, 300 mM K+), luminal Ca2+ dissociation is sequential as well, so that the representation of two superimposed Ca2+ ions still holds on the phosphoenzyme, with the superficial Ca2+ facing the lumen freely exchangeable and the deeper Ca2+ blocked by the superficial Ca2+.Under the same conditions, we have investigated whether a prebuilt Ca2+ order is maintained during membrane translocation. Starting from a prebuilt order on the cytoplasmic side, we showed that the Ca2+ ions cannot be identified after translocation to the luminal side. The same result was obtained starting from a prebuilt order on the luminal side and following the luminal to cytoplasmic translocation. We conclude that the two Ca2+ ions are mixed during ATP-induced phosphorylation as well as during ADP-induced dephosphorylation. Cytoplasmic Ca2+ dissociation is sequential, and the Ca2+ ions bound to the nonphosphorylated ATPase are commonly represented as superimposed on each other, so that the superficial Ca2+ is freely exchangeable from the cytoplasm, whereas the deeper Ca2+ is not. Under conditions where ADP-sensitive phosphoenzyme accumulates (leaky vesicles, 5°C, pH 8, 300 mM K+), luminal Ca2+ dissociation is sequential as well, so that the representation of two superimposed Ca2+ ions still holds on the phosphoenzyme, with the superficial Ca2+ facing the lumen freely exchangeable and the deeper Ca2+ blocked by the superficial Ca2+. Under the same conditions, we have investigated whether a prebuilt Ca2+ order is maintained during membrane translocation. Starting from a prebuilt order on the cytoplasmic side, we showed that the Ca2+ ions cannot be identified after translocation to the luminal side. The same result was obtained starting from a prebuilt order on the luminal side and following the luminal to cytoplasmic translocation. We conclude that the two Ca2+ ions are mixed during ATP-induced phosphorylation as well as during ADP-induced dephosphorylation. INTRODUCTIONSarcoplasmic reticulum ATPase is a membranous enzyme that pumps Ca2+ from the cytoplasm of muscle cells into the reticulum lumen, requiring ATP hydrolysis. During its cycle, each ATPase monomer transports two Ca2+ ions (Scheme 1). During transport, the Ca2+ 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 Ca2+ release into the SR 1The abbreviations used are: SRsarcoplasmic reticulumTesN-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acidBAPTA1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acidFIFOfirst-in-first-outFILOfirst-in-last-out. lumen and is followed by dephosphorylation. Scheme 1.Equation 1 Ca2+ binding to E, the Ca2+-deprived nonphosphorylated ATPase, has been well characterized. Two Ca2+ ions bind sequentially with high affinity and positive cooperativity (1Inesi G. Kurzmack M. Coan C. Lewis D.E. J. Biol. Chem. 1980; 255: 3025-3031Abstract Full Text PDF PubMed Google Scholar, 2Forge V. Mintz E. Guillain F. J. Biol. Chem. 1993; 268: 10953-10960Abstract Full Text PDF PubMed Google Scholar). In 1982, Dupont (3Dupont Y. Biochim. Biophys. Acta. 1982; 688: 75-87Crossref PubMed Scopus (88) Google Scholar) showed that the dissociation of one-half of the 45Ca2+ bound to Ca2E was impaired by the presence of excess 40Ca2+ in the medium. This was interpreted in 1987 by Inesi (4Inesi G. J. Biol. Chem. 1987; 262: 16338-16342Abstract Full Text PDF PubMed Google Scholar) as two sites being sequentially accessible from the cytoplasm by the two Ca2+ ions in a crevice with a deep site and a superficial site (Fig. 2). The first ion must reach the deep site to leave the superficial site vacant for the second ion to bind. The Ca2+ bound to the superficial site is freely exchangeable with free Ca2+ in the outer medium, whereas the Ca2+ bound to the deep site is not. Inesi took advantage of the possibility of selectively placing a 40Ca2+ on top of a 45Ca2+, to determine whether their dissociation toward the lumen is sequential. He concluded that this was the case and that the first Ca2+ bound to E was the first to be internalized by monitoring the internalization of the Ca2+ ions after phosphorylation. This would correspond to a channel-like structure with a first-in-first-out mechanism for membrane crossing.The question of whether the dissociation of the Ca2+ ions toward the lumen is sequential or not has been reinvestigated by Hanel and Jencks (5Hanel A.M. Jencks W.P. Biochemistry. 1991; 30: 11320-11330Crossref PubMed Scopus (27) Google Scholar) and Orlowski and Champeil (6Orlowski S. Champeil P. Biochemistry. 1991; 30: 11331-11342Crossref PubMed Scopus (37) Google Scholar). Both groups concluded that the two ions cannot be kinetically distinguished, probably because Ca2+ dissociation from the phosphorylated ATPase starts with a rate-limiting deocclusion step. More recently, Forge et al. (7Forge V. Mintz E. Canet D. Guillain F. J. Biol. Chem. 1995; 270: 18271-18276Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) have shown that under appropriate conditions (pH 8, 300 mM K+, 5°C), the two ions dissociate sequentially from the phosphorylated ATPase, suggesting that Ca2+ dissociation was intrinsically sequential on both sides of the membrane.As with the nonphosphorylated ATPase, the Ca2+ sites of the phosphorylated ATPase may be represented by a crevice with a deep site and a superficial site accessible from the lumen (Fig. 3). The initial suggestion arising from these sketches is that the first ion to bind to the ATPase is the first to dissociate after transport, as originally proposed by Inesi (4Inesi G. J. Biol. Chem. 1987; 262: 16338-16342Abstract Full Text PDF PubMed Google Scholar). Because the conditions chosen by Forge et al. (7Forge V. Mintz E. Canet D. Guillain F. J. Biol. Chem. 1995; 270: 18271-18276Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) have revealed the sequentiality of the luminal dissociation, they appear to be particularly appropriate for reinvestigating whether the Ca2+ ion order is kept during transport. We show that there is mixing of the two Ca2+ ions in the Ca2+-bound phosphoenzyme; i.e. the first Ca2+ bound to the nonphosphorylated ATPase cannot be identified as being the first or the second to dissociate toward the lumen. This is also shown for the reverse step, i.e. during the ADP-induced dephosphorylation of Ca2E-PMg.Fig. 3Luminal Ca2+ dissociation and exchange. ATPase was first phosphorylated by manual perfusion with 100 µM 45Ca2+ plus 90 µM EGTA, 3 mM Mg2+, 100 µM γ-32PATP and perfused with 1 mM Mg2+ in Ca2+-poor media as described under “Materials and Methods” (A) or 1 mM Mg2+ plus 10 mM 40Ca2+ (B) for various times. 300 mM K+ was present throughout the experiments. ∘, ▿, bound 45Ca2+; □, ⋄, phosphoenzyme. Diagrams: t = 0, initial state; A, final state for Ca2+-poor perfusion; B, final state for 10 mM 40Ca2+.View Large Image Figure ViewerDownload Hi-res image Download (PPT)MATERIALS AND METHODSSR vesicles were prepared and tested as described by Forge et al. (2Forge V. Mintz E. Guillain F. J. Biol. Chem. 1993; 268: 10953-10960Abstract Full Text PDF PubMed Google Scholar). All experiments were carried out at 3°C in a cold room. The buffer was 100 mM Tes-Tris, pH 8, plus 1 or 3 mM Mg2+ and 0 or 300 mM K+, as specified in the figure legends. It was prepared with water filtered through a Milli-Q Water Purification System (Millipore Corp., Milford, MA). 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 K+, 2 mM EDTA at room temperature.Ca2+ Binding and Phosphoenzyme MeasurementsCa2+ binding and phosphoenzyme levels were measured by the filtration technique. Kinetic measurements involving 45Ca2+ or γ-32PATP all started with the same incubation and rinsing steps. Vesicles (0.2 mg/ml) were first incubated in the pH 8 buffer. One ml of this suspension was deposited on a filter (Millipore HA 0.45 µm), and the adsorbed vesicles were perfused with 1 ml of 100 µM EGTA to rinse EDTA with a Ca2+-deprived buffer. For experiments starting from Ca2E (Figs. 2, 4, and 5), ATPase was converted to this state by manually perfusing the filters for 5 s with 1 ml of 100 µM 45Ca2+ (or 40Ca2+) plus 90 µM EGTA and 3 mM Mg2+. Cytoplasmic isotopic exchanges were performed in the absence of K+ by perfusing 100 µM 45Ca2+ (or 40Ca2+) plus 3 mM Mg2+. For experiments starting from Ca2E-PMg (Fig. 3, Fig. 4, Fig. 5, Fig. 6), ATPase was converted to this state by manually perfusing the filters for 5 s with 2 ml of 100 µM 45Ca2+ (or 40Ca2+) plus 90 µM EGTA, 100 µM γ-32PATP (or ATP), 3 mM Mg2+, and 300 mM K+. Unless otherwise specified, the kinetic measurements were begun immediately after this step using a rapid filtration system (Bio-Logic, Claix, France). They were carried out by perfusing media containing EGTA, or 40Ca2+, with or without ADP, for various times.Fig. 4ADP-induced Ca2+ dissociation and dephosphorylation: double filtration system experiment. ATPase was first manually perfused with 100 µM 45Ca2+, 90 µM EGTA, 3 mM Mg2+ to saturate the cytoplasmic sites. It was then perfused by the first system with 100 µM 45Ca2+, 90 µM EGTA, 100 µM γ-32PATP, 3 mM Mg2+, 300 mM K+ for 0.4 s and by the second system with 1 mM ADP, 1 mM EGTA, 300 mM K+ for various times. •, bound 45Ca2+; ▪, phosphoenzyme. The second perfusion started 2 s after the first had started.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 5Luminal Ca2+ dissociation and exchange starting from a prebuilt cytoplasmic order: double filtration system experiment. In A, the prebuilt cytoplasmic order was a 40Ca2+ on top of a 45Ca2+. It was obtained by manually saturating the cytoplasmic sites with 45Ca2+ and perfusing 100 µM 40Ca2+ for 8 s, as in Fig. 2. Phosphorylation was then induced by perfusing 100 µM 40Ca2+ plus 90 µM EGTA, 100 µM ATP, 300 mM K+ with the first filtration system. In B, the prebuilt cytoplasmic order was a 45Ca2+ on top of a 40Ca2+. Cytoplasmic order and phosphorylation were achieved as in A, except for the presence of 45Ca2+ instead of 40Ca2+. In C, cytoplasmic sites were saturated with 40Ca2+, and the cytoplasmic exchange was simulated with 40Ca2+ to reproduce the same protocol as in A and B. Phosphorylation was then achieved as in A, except for the presence of γ-32PATP. In A, B, and C, once the cytoplasmic order was built and 2 s after phosphorylation has started, the second filtration system perfused ATPase either with 10 mM EGTA (∘, □, •, ▪) or 10 mM 40Ca2+ (▿, ⋄, ▾, ♦) in the presence of 1 mM Mg2+ and 300 mM K+ for various times. Immediately after the phosphorylation perfusion or immediately after 40Ca2+ or EGTA had been perfused for 10 s, the sensitivity to ADP was tested by manually perfusing a mixture of ADP and EGTA (•, ▾, ▪, ♦). ∘, ▿, •, ▾, bound 45Ca2+; □, ⋄, ▪, ♦, phosphoenzyme. For all these curves the standard error of the mean is figured by the size of the symbols.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 6Cytoplasmic dissociation and exchange starting from two 45Ca2+ or from a prebuilt luminal order, upon cycle reversing. ATPase was first phosphorylated by manual perfusion with 100 µM 45Ca2+ plus 90 µM EGTA, 3 mM Mg2+, 100 µM ATP. In A, ATPase was perfused with 1 mM ADP plus 1 mM EGTA (•) or with 1.5 mM ADP plus 1.5 mM 40Ca2+ (▾). B, same perfusions after a luminal isotopic exchange performed for 8 s in presence of 1 mM 40Ca2+. 300 mM K+ was present throughout the experiments. Note the different scales in A and B.View Large Image Figure ViewerDownload Hi-res image Download (PPT)All solutions containing 45Ca2+ or γ-32PATP also contained 1 mM 3Hglucose, which allows evaluation of the filter wet volume, usually about 30 µl. 3H and 45Ca and/or 32P retained on the filter were simultaneously measured by scintillation. γ-32PATP or 45Ca2+ contained in the wet volume was subtracted from the total 32P or 45Ca counts to evaluate the phosphoenzyme and the Ca2+ bound to the ATPase. The values for the curves plotted in Figs. 3, 5, and 6 are the average of two to six experiments (±S.E.).Ca2+ dissociation kinetics in Fig. 3A were the same, whether measured in presence of contaminating Ca2+, 1 or 10 mM EGTA, or 1 mM BAPTA. Ca2+ dissociation kinetics proved to be insensitive to the Ca2+ ionophore calcimycin (4%, w/w). Therefore, the curve in Fig. 3A is the average of experiments conducted under these various conditions.Double Filtration SystemTwo rapid filtration systems from Bio-Logic (8Dupont Y. Anal. Biochem. 1984; 142: 504-510Crossref PubMed Scopus (59) Google Scholar) were associated to ensure that the phosphorylation reaction would last precisely for a short period of time and therefore be reproducible. Indeed, when manual phosphorylation is performed before kinetics perfusion by a single system, there can be 5-10 s between the beginning of the manual filtration of the phosphorylation medium and the beginning of the perfusion driven by the filtration system; for some experiments, this time lag is too long. To avoid this difficulty, we have coupled two rapid filtration systems face to face and linked them by on which the filter from system to the (Fig. 1). the of the filtration by the first system, the filter is manually from the first system to the second system to ensure that there are s between the of the first perfusion and the beginning of the filtration system. filter on to the first rapid filtration system; to the second rapid filtration system. the filter is in the filter the adsorbed SR can be perfused by the first system phosphorylation The filter is then toward the second system. In the filter can be perfused by the second system were obtained by perfusing for various with the second Large Image Figure ViewerDownload Hi-res image Download the experiments described the first filtration system was used to phosphorylation during 0.4 s, and the second filtration system was used to the various media precisely 2 s after the phosphorylation perfusion had This that each kinetic started from the same initial The experiments shown in Fig. 4, Fig. 5 were carried out with this system. All the of the double filtration system were as described when using a single filtration of this was to a of Ca2+ ion transport, the Inesi (4Inesi G. J. Biol. Chem. 1987; 262: 16338-16342Abstract Full Text PDF PubMed Google Scholar) showed that two different Ca2+ can be superimposed in the cytoplasmic sites and Forge et al. (7Forge V. Mintz E. Canet D. Guillain F. J. Biol. Chem. 1995; 270: 18271-18276Abstract Full Text Full Text PDF PubMed Scopus (27) Google using conditions, that the same was in the luminal sites. is some about the luminal because Inesi (4Inesi G. J. Biol. Chem. 1987; 262: 16338-16342Abstract Full Text PDF PubMed Google Scholar) that luminal dissociation was sequential, whereas Hanel and Jencks (5Hanel A.M. Jencks W.P. Biochemistry. 1991; 30: 11320-11330Crossref PubMed Scopus (27) Google Scholar) and Orlowski and Champeil (6Orlowski S. Champeil P. Biochemistry. 1991; 30: 11331-11342Crossref PubMed Scopus (37) Google Scholar) that the Ca2+ ions were not during luminal This that under the conditions the sequentiality is to and may on a of from this Forge et al. (7Forge V. Mintz E. Canet D. Guillain F. J. Biol. Chem. 1995; 270: 18271-18276Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) a of conditions that of the ADP-sensitive phosphoenzyme, this phosphoenzyme to the luminal dissociation perfusion before has and to ensure high affinity for the luminal Ca2+ sites mM K+, pH 8, and conditions were used to during its luminal dissociation, a Ca2+ ion that had been bound to or the cytoplasmic We the as from a mixing of the two Ca2+ ions after binding and during at the Inesi (4Inesi G. J. Biol. Chem. 1987; 262: 16338-16342Abstract Full Text PDF PubMed Google Scholar) proposed a mechanism for Ca2+ translocation on the of the following phosphorylation of ATPase with two bound 45Ca2+ ions induced Ca2+ suggesting that the two ions dissociate whereas phosphorylation of ATPase with a 40Ca2+ bound on top of a 45Ca2+ induced luminal Ca2+ dissociation, suggesting that the deep 45Ca2+ was the first to these experiments were conducted under conditions that probably to phosphorylation by both and Because a phosphoenzyme with Ca2+ at its site Ca2+ a phosphoenzyme with Mg2+ at its site M. S. J. Biol. Chem. Full Text PDF PubMed Google the two be to Ca2+ dissociation from Ca2E-PMg and to Ca2+ dissociation from to sequential dissociation of the two Ca2+ ions from a of this difficulty, we used the filtration which under conditions, rinsing the phosphorylation before perfusing high of Ca2+, the of In some experiments, phosphorylation was performed in the presence of 100 µM Ca2+ and without EGTA which to a of instead of when there was 90 µM We that there was in Ca2+ dissociation kinetics from with or without EGTA, suggesting that with a of not The possibility that a of Mg2+ at the site for Ca2+ during luminal perfusion of Ca2E-PMg with 40Ca2+ has been and out by luminal Ca2+ dissociation kinetics induced with various Mg2+ (7Forge V. Mintz E. Canet D. Guillain F. J. Biol. Chem. 1995; 270: 18271-18276Abstract Full Text Full Text PDF PubMed Scopus (27) Google the and Jencks (5Hanel A.M. Jencks W.P. Biochemistry. 1991; 30: 11320-11330Crossref PubMed Scopus (27) Google Scholar) and Orlowski and Champeil (6Orlowski S. Champeil P. Biochemistry. 1991; 30: 11331-11342Crossref PubMed Scopus (37) Google in the luminal sequentiality not the two Ca2+ ions and concluded that this was to a rate-limiting step Ca2+ dissociation, i.e. a change to Ca2+ conditions probably the rate-limiting step from this deocclusion step to the Ca2+ dissociation step. Because we were under these conditions to the phosphoenzyme and therefore the luminal Ca2+ we the Ca2+ order during transport from the cytoplasmic to the luminal side, as well as from the luminal to the cytoplasmic side. We followed each of the two Ca2+ ions selectively on the cytoplasmic side and that the and dissociation kinetics were different for each Therefore, mechanism as or was In the the of ion mixing during transport. The same mixing was during ADP-induced there to be an and of the phosphoenzyme in which the Ca2+ ions are they are on both cytoplasmic and luminal sides 2 and in the that there are two Ca2+ sites in a that change their affinity and orientation during phosphorylation or that the of the Ca2+ ions during these and probably during the step. this is in with the of a in the phosphoenzyme, during this step the Ca2+ ions are and to exchange as their binding to the and the structure were show six to the membrane and as for Ca2+ transport Inesi G. PubMed Scopus Google and them were for Ca2+ B. J. Biol. Chem. Full Text PDF PubMed Google Scholar, B. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The possibility that the two Ca2+ ions can be superimposed in a by and is still in the G. J. Biol. PubMed Scopus Google Scholar, C. 1993; Scopus Google Scholar, 1995; PubMed Scopus Google Scholar, G. C. Lewis D. 1995; PubMed Scopus Google and Biochemistry. PubMed Scopus Google Scholar, Biochemistry. 1993; PubMed Scopus Google Scholar) and Jencks and W.P. D. J. Biochemistry. 1993; PubMed Scopus (37) Google Scholar, J. Jencks W.P. Biochemistry. PubMed Scopus Google Scholar) proposed that the cytoplasmic and luminal sites be i.e. there are Ca2+ sites on the ATPase. this and there would be during phosphorylation and dephosphorylation a of the Ca2+ ions from of sites to the other, during which the Ca2+ ions would be (Scheme 1995; PubMed Scopus Google Scholar) proposed a for sites with two the described and an by and each Ca2+ ion in this the membrane in its not the mixing in This to be at least during phosphorylation and a different of the six i.e. in a structure the Ca2+ ions to (Scheme In this that the representation of the two Ca2+ ions as being superimposed is a of structure to the sequential The from is that the Ca2+ sites are not Scheme Because the two Ca2+ ions are to be during their to a change involving the Ca2+ sites. Starting from an state for the Ca2+ the reaction or must to state for the Ca2+ ions through a state where the Ca2+ and Because phosphorylation and dephosphorylation are this state may have not been in 1995; PubMed Scopus Google Scholar). INTRODUCTIONSarcoplasmic reticulum ATPase is a membranous enzyme that pumps Ca2+ from the cytoplasm of muscle cells into the reticulum lumen, requiring ATP hydrolysis. During its cycle, each ATPase monomer transports two Ca2+ ions (Scheme 1). During transport, the Ca2+ 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 Ca2+ release into the SR 1The abbreviations used are: SRsarcoplasmic reticulumTesN-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acidBAPTA1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acidFIFOfirst-in-first-outFILOfirst-in-last-out. lumen and is followed by dephosphorylation. Scheme 1.Equation 1 Ca2+ binding to E, the Ca2+-deprived nonphosphorylated ATPase, has been well characterized. Two Ca2+ ions bind sequentially with high affinity and positive cooperativity (1Inesi G. Kurzmack M. Coan C. Lewis D.E. J. Biol. Chem. 1980; 255: 3025-3031Abstract Full Text PDF PubMed Google Scholar, 2Forge V. Mintz E. Guillain F. J. Biol. Chem. 1993; 268: 10953-10960Abstract Full Text PDF PubMed Google Scholar). In 1982, Dupont (3Dupont Y. Biochim. Biophys. Acta. 1982; 688: 75-87Crossref PubMed Scopus (88) Google Scholar) showed that the dissociation of one-half of the 45Ca2+ bound to Ca2E was impaired by the presence of excess 40Ca2+ in the medium. This was interpreted in 1987 by Inesi (4Inesi G. J. Biol. Chem. 1987; 262: 16338-16342Abstract Full Text PDF PubMed Google Scholar) as two sites being sequentially accessible from the cytoplasm by the two Ca2+ ions in a crevice with a deep site and a superficial site (Fig. 2). The first ion must reach the deep site to leave the superficial site vacant for the second ion to bind. The Ca2+ bound to the superficial site is freely exchangeable with free Ca2+ in the outer medium, whereas the Ca2+ bound to the deep site is not. Inesi took advantage of the possibility of selectively placing a 40Ca2+ on top of a 45Ca2+, to determine whether their dissociation toward the lumen is sequential. He concluded that this was the case and that the first Ca2+ bound to E was the first to be internalized by monitoring the internalization of the Ca2+ ions after phosphorylation. This would correspond to a channel-like structure with a first-in-first-out mechanism for membrane crossing.The question of whether the dissociation of the Ca2+ ions toward the lumen is sequential or not has been reinvestigated by Hanel and Jencks (5Hanel A.M. Jencks W.P. Biochemistry. 1991; 30: 11320-11330Crossref PubMed Scopus (27) Google Scholar) and Orlowski and Champeil (6Orlowski S. Champeil P. Biochemistry. 1991; 30: 11331-11342Crossref PubMed Scopus (37) Google Scholar). Both groups concluded that the two ions cannot be kinetically distinguished, probably because Ca2+ dissociation from the phosphorylated ATPase starts with a rate-limiting deocclusion step. More recently, Forge et al. (7Forge V. Mintz E. Canet D. Guillain F. J. Biol. Chem. 1995; 270: 18271-18276Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) have shown that under appropriate conditions (pH 8, 300 mM K+, 5°C), the two ions dissociate sequentially from the phosphorylated ATPase, suggesting that Ca2+ dissociation was intrinsically sequential on both sides of the membrane.As with the nonphosphorylated ATPase, the Ca2+ sites of the phosphorylated ATPase may be represented by a crevice with a deep site and a superficial site accessible from the lumen (Fig. 3). The initial suggestion arising from these sketches is that the first ion to bind to the ATPase is the first to dissociate after transport, as originally proposed by Inesi (4Inesi G. J. Biol. Chem. 1987; 262: 16338-16342Abstract Full Text PDF PubMed Google Scholar). Because the conditions chosen by Forge et al. (7Forge V. Mintz E. Canet D. Guillain F. J. Biol. Chem. 1995; 270: 18271-18276Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) have revealed the sequentiality of the luminal dissociation, they appear to be particularly appropriate for reinvestigating whether the Ca2+ ion order is kept during transport. We show that there is mixing of the two Ca2+ ions in the Ca2+-bound phosphoenzyme; i.e. the first Ca2+ bound to the nonphosphorylated ATPase cannot be identified as being the first or the second to dissociate toward the lumen. This is also shown for the reverse step, i.e. during the ADP-induced dephosphorylation of