Ca²⁺ Release to Lumen from ADP-sensitive Phosphoenzyme E1PCa₂ without Bound K⁺ of Sarcoplasmic Reticulum Ca²⁺-ATPase

Abstract

During Ca²⁺ transport by sarcoplasmic reticulum Ca²⁺-ATPase, the conformation change of ADP-sensitive phosphoenzyme (E1PCa₂) to ADP-insensitive phosphoenzyme (E2PCa₂) is followed by rapid Ca²⁺ release into the lumen. Here, we find that in the absence of K⁺, Ca²⁺ release occurs considerably faster than E1PCa₂ to E2PCa₂ conformation change. Therefore, the lumenal Ca²⁺ release pathway is open to some extent in the K⁺-free E1PCa₂ structure. The Ca²⁺ affinity of this E1P is as high as that of the unphosphorylated ATPase (E1), indicating the Ca²⁺ binding sites are not disrupted. Thus, bound K⁺ stabilizes the E1PCa₂ structure with occluded Ca²⁺, keeping the Ca²⁺ pathway to the lumen closed. We found previously (Yamasaki, K., Wang, G., Daiho, T., Danko, S., and Suzuki, H. (2008) J. Biol. Chem. 283, 29144–29155) that the K⁺ bound in E2P reduces the Ca²⁺ affinity essential for achieving the high physiological Ca²⁺ gradient and to fully open the lumenal Ca²⁺ gate for rapid Ca²⁺ release (E2PCa₂ → E2P + 2Ca²⁺). These findings show that bound K⁺ is critical for stabilizing both E1PCa₂ and E2P structures, thereby contributing to the structural changes that efficiently couple phosphoenzyme processing and Ca²⁺ handling.

Introduction

Sarcoplasmic reticulum (SR)² Ca²⁺-ATPase (SERCA1a) catalyzes Ca²⁺ transport coupled with ATP hydrolysis against an ∼10,000-fold concentration gradient (1–9). The ATPase is first activated by the binding of two cytoplasmic Ca²⁺ ions at the transport sites with a submicromolar high affinity (E2 to E1Ca₂, see step 1 in Fig. 1) and then autophosphorylated at Asp³⁵¹ by ATP to form a phosphoenzyme intermediate (EP) (step 2). This EP is “ADP-sensitive” (E1P) because it is rapidly dephosphorylated by ADP in the reverse reaction. Upon E1P formation, the bound Ca²⁺ ions are occluded in the transport sites (E1PCa₂). Subsequently, E1PCa₂ undergoes its isomeric transition to an ADP-insensitive form (E2P), i.e. loss of ADP sensitivity, which results in a large reduction of Ca²⁺ affinity and opening of the lumenal release gate, i.e. Ca²⁺ deocclusion and release (steps 3–4). Ca²⁺ release in step 4 is very rapid, so that an E2PCa₂ intermediate state does not accumulate and in fact had never been found until we recently established its existence (10–13) and successfully trapped it for the first time (14). Finally, E2P is hydrolyzed back to the inactive E2 form (step 5).

FIGURE 1.

Reaction scheme of Ca²⁺-ATPase.

In E1PCa₂ → E2P + 2Ca²⁺, the A domain rotates parallel to the membrane plane and the P domain inclines to the A domain, thereby associating with each other to produce a compactly organized and inclined headpiece (15–27). This tight structure is stabilized by critical interaction networks between the A and P domains at three regions (10–14) (see Fig. 9 for details). The rotation and inclination of the domains result in motions and rearrangements of the transmembrane helices thereby disrupting the Ca²⁺ sites and opening the lumenal gate. In the P domain, there is a specific K⁺ binding site (28); K⁺ binding here is crucial for rapid hydrolysis of E2P (28–30). Recently, we further found (13) that the K⁺ in E2P is critical for reducing the lumenal Ca²⁺ affinity that is required to achieve the high physiological Ca²⁺ gradient and for rapid Ca²⁺ release (E2PCa₂ → E2P + 2Ca²⁺). Thus, bound K⁺ contributes to stabilization of the compactly organized and inclined E2P structure with its disrupted Ca²⁺ sites and fully opened lumenal gate, probably by cross-linking the P domain with the A domain/M3-linker (13).

FIGURE 9.

Structural change during E1PCa₂ → E2P + 2Ca²⁺ and movement of the K⁺ binding site. The structural change is modeled on the crystal structures with bound K⁺, E1PCa₂·AMPPN, and E2·AlF₄⁻ (Protein Data Bank codes 3BA6 (22) and 1XP5 (20), respectively). The two structures are aligned with the static M8–10 helices. The approximate position of the transmembrane region (TM) is shown by green lines. The area indicated by red dashed lines in the whole molecule is enlarged in the lower panel. The motions of each of N, P, and A domains during E1PCa₂·AMPPN → E2·AlF₄⁻ are indicated by curved arrows. Note that the K⁺ site with bound K⁺ on the P domain moves down to the Gln²⁴⁴ region on the A/M3-linker (blue arrow), thus likely cross-linking the P domain with the A/M3-linker. There are three critical interaction networks to realize and stabilize the compactly organized E2P structure. They are Tyr¹²² HC forming a hydrophobic interaction cluster (violet Van der Waals spheres), the Val²⁰⁰ loop (red loop), and TGES¹⁸⁴ (blue loop) (10–13). Crystal structures of E2·BeF₃⁻ (21, 22), which are analogs of the E2P ground state (25), are not used here because they were formed without K⁺ (although the above noted changes are also seen with the E2·BeF₃⁻ crystals).

Despite these findings on the Ca²⁺ release process and E2P, a possible role for K⁺ in E1PCa₂ has not been explored. The K⁺ site is situated at the bottom of the P-domain near the cytoplasmic ends of the transmembrane helices. Therefore, the lack of K⁺ binding might have a serious effect on the stability of the helices and Ca²⁺ handling in E1PCa₂. The E2-E1Ca₂ transition is markedly retarded, and its equilibrium is affected by the absence of K⁺ (31–33).

In this study, we explore a possible role of K⁺ in E1PCa₂ especially in regard to Ca²⁺ occlusion. Results reveal that K⁺-free E1PCa₂ has an open Ca²⁺ pathway to the lumen. Thus, the Ca²⁺ binding sites face the lumen, and Ca²⁺ can be released. The absence of K⁺ does not reduce the high Ca²⁺ affinity (Ca²⁺ site coordination probably unchanged), and yet the cytoplasmic gate is closed, and the lumenal gate is open. These changes probably do not involve large motions of the cytoplasmic domains and transmembrane helices. Therefore, bound K⁺ likely stabilizes the Ca²⁺ occluded structure of E1PCa₂ by simply keeping the lumenal Ca²⁺ pathway closed. The structural role of K⁺ in E1PCa₂ is discussed in detail using crystal structures of Ca²⁺-ATPase with bound K⁺.

EXPERIMENTAL PROCEDURES

Preparation of SR Vesicles

SR vesicles were prepared from rabbit skeletal muscle as described (34). The phosphorylation site content in the vesicles determined according to Barrabin et al. (35) was 4.49 ± 0.22 nmol/mg vesicle protein (n = 5).

Determination of EP

SR vesicles were phosphorylated with [γ-³²P]ATP as described in the legends to Figs. 2–6. In the experiments performed in Fig. 2, aliquots of the reaction mixture were spotted on the HAWP membrane filter (Millipore) and washed continuously with a chasing solution for the periods indicated. At the end of chase, the reaction was terminated by washing with 0.1 m HCl. To determine the amount of E2P in the phosphorylation mixture, the membrane was washed with an ADP solution for 1 s and then with 0.1 m HCl. The membrane was dried, and the radioactivity was measured by digital autoradiography. In Figs. 3 and 6, total EP was measured by quenching the phosphorylation reaction (in a test tube) with 5% (v/v) ice-cold trichloroacetic acid containing P_i, whereas for E2P determination, the reaction was chased with ADP for 1 s and quenched by addition of the trichloroacetic acid. The precipitated proteins were separated by 5% SDS-PAGE at pH 6.0 according to Weber and Osborn (36). The radioactivity associated with the separated Ca²⁺-ATPase was quantitated by digital autoradiography (37). Rapid kinetic measurements in Fig. 3 were performed with a handmade rapid mixing apparatus (38).

FIGURE 2.

Time courses of EP decay and Ca²⁺ release in the presence or absence of 0.1 m K⁺. A, the Ca²⁺-ATPase in SR vesicles (20 μg protein/ml) was phosphorylated for 10 s with 100 μm [γ-³²P]ATP in 10 μm nonradioactive CaCl₂ (closed circles and, in inset, triangles), or with 100 μm nonradioactive ATP in 10 μm ⁴⁵CaCl₂ (open circles), 0.1 m KCl, 3 μm A23187. Then, 50 μl of the reaction mixture was spotted on the membrane and washed for the time periods on the abscissa with a chasing solution containing 1 mm EGTA, 0.1 m KCl, 3 μm A23187. The amount of bound ⁴⁵Ca²⁺ (open circles) and the total amount of EP (open triangles in inset) were determined. The amounts of E2P (closed triangles in inset) were determined by a subsequent washing by a solution containing 1 mm ADP, 1 mm EGTA, and 0.1 m KCl. The amount of E1P (closed circles) was calculated by subtracting the amount of E2P from total amount of EP. B, the Ca²⁺-ATPase in SR vesicles was phosphorylated in the presence of 0.1 m KCl as in A and spotted on the filter. Then, the filter was washed with the EGTA solution (and subsequently with the ADP solution for the E2P determination) containing 0.1 m LiCl instead of KCl, otherwise as in A. Solid lines in A and B show the least squares fit to a single exponential. The rates (s⁻¹) for E1P decay and the Ca²⁺ release were 0.66 and 0.58 (A) and 0.21 and 0.81 (B), respectively.

FIGURE 3.

EP formation and decay in a single turnover. All of the solutions contained 0.1 m KCl (closed symbols) or LiCl (open symbols). SR vesicles (20 μg/ml) were incubated in 10 μm CaCl₂, and EP formation was initiated by mixing with an equal volume of a solution containing 20 μm [γ-³²P]ATP and 10 mm EGTA (triangles and squares) or 10 μm CaCl₂ (circles). The total amount of EP was determined with the addition of trichloroacetic acid (circles and triangles). To determine the amount of E2P (squares), the phosphorylated sample was mixed with an equal volume of a solution containing 2 mm ADP and 5 mm EGTA, and then the reaction was terminated by trichloroacetic acid at 1 s after the ADP addition.

FIGURE 4.

⁴⁵Ca²⁺ uptake in a single turnover of EP with and without K⁺. All of the solutions contained 0.1 m KCl (A) or LiCl (B). In the absence of the Ca²⁺ ionophore, SR vesicles (SRV; 20 μg/ml) were first incubated with 10 μm ⁴⁵CaCl₂ for ∼10 min, and then Ca²⁺ uptake in a single turnover of EP was initiated by mixing with an equal volume of a solution containing 20 μm ATP and 2 mm EGTA, as described in Fig. 3. After the indicated periods, the reaction was chased with an equal volume of a solution containing 2 mm EGTA without (closed circles) or with (open circles) 2 mm ADP. The mixture was immediately spotted on the membrane and washed for ∼10 s with 1 ml of a 2 mm EGTA solution. The amount of ⁴⁵Ca²⁺ on the membrane, i.e. transported into the vesicles and/or remained bound to the ATPase and not released to cytoplasmic side, was normalized to the maximum total amount of EP formed immediately after the addition ATP and EGTA (Fig. 3). In A, the time course obtained with the ADP chase was best described by a single exponential Ca²⁺ uptake (solid line) with a rate constant of 0.49 s⁻¹ and maximum Ca²⁺/EP value of 1.26. In B, it was best described by a double exponential (broken line) with a rate constant and maximum Ca²⁺/EP value of 5.1 s⁻¹ and 0.66 for the fast phase and 0.24 s⁻¹ and 0.98 for the slow phase (but it was not described by a single exponential increase shown by solid line with the rate constant of 1.54 s⁻¹ and maximum value of 1.31). Note also that without the ADP addition, almost of all the bound Ca²⁺ ions are transported into the vesicles during the ∼10-s EGTA wash because the single turnover of EP is nearly completed in this period (see Fig. 3).

FIGURE 5.

Uptake of site I-bound ⁴⁵Ca²⁺ in a single turnover of EP with and without K⁺. SR vesicles were incubated with 10 μm ⁴⁵Ca²⁺ as in Fig. 4 and diluted by an equal volume of a solution containing 2 mm nonradioactive CaCl₂ and 0.1 m KCl (A) or LiCl (B), and further incubated for 10 s. By this incubation, site I of the two Ca²⁺ sites (I, II) is labeled with ⁴⁵Ca²⁺ due to Ca²⁺ exchange with site II (see supplemental Fig. S1) (42–44). Then, ⁴⁵Ca²⁺ uptake assay in a single turnover was performed as in Fig. 4. In A, the time course obtained with the ADP-chase was best described by a single exponential Ca²⁺ uptake (solid line) with a rate constant of 0.45 s⁻¹ and maximum Ca²⁺/EP value of 0.75. In B, it was best described by a double exponential increase (broken line) with a rate constant and maximum value of 6.0 s⁻¹ and 0.34 for the fast phase and 0.41 s⁻¹ and 0.56 for the slow phase (but not described by a single exponential increase shown as solid line with the rate constant of 1.36 s⁻¹ and the maximum value of 0.80).

FIGURE 6.

Ca²⁺ dependence of E1P accumulation and Ca²⁺ binding in steady state. A, SR vesicles (200 μg/ml) were phosphorylated at 4 °C for 30 s with 100 μm [γ-³²P]ATP in 3 μm A23187, 0.1 m LiCl, and 20 μm CaCl₂ with various concentrations of EGTA to give the indicated free Ca²⁺ concentrations. The total amount of EP (closed circles) and amount of E1P (open circles) were determined as described in Fig. 3. Solid lines show the least squares fit to the Hill equation. The maximum, K_0.5, and Hill coefficient for the total amount of EP were 3.67 nmol/mg, 0.10 μm, and 2.6, respectively, and those for E1P were 2.12 nmol/mg, 0.11 μm, and 2.5, respectively. B, SR vesicles were phosphorylated with 100 μm ATP (open squares) or incubated without ATP (closed squares and open triangles) in 20 μm ⁴⁵CaCl₂ with various concentrations of EGTA and 0.1 m LiCl (squares) or KCl (triangles), otherwise as described in A. Then, 50 μl of the reaction mixture was spotted on the membrane, and the amount of ⁴⁵Ca²⁺ specifically bound to the ATPase was determined. Solid lines show the least squares fit to the Hill equation. The maximum, K_0.5, and Hill coefficient were 8.54 nmol/mg, 0.18 μm, and 2.1 (open triangles), 7.82 nmol/mg, 0.20 μm, and 2.3 (closed squares), and 4.14 nmol/mg, 0.15 μm, and 1.5 (open squares). C, the amount of E1P (open circles) in A and that of bound ⁴⁵Ca²⁺ under the phosphorylating condition (open squares) in B in the absence of K⁺ are replotted after normalization to the maximum total amount of EP and to the maximum ⁴⁵Ca²⁺ binding under the nonphosphorylating condition (E1) in the absence of K⁺, respectively, and shown as % values. Solid lines show the least squares fit to the Hill equation, and the maximum values were 58% for E1P and 52% for bound ⁴⁵Ca²⁺, respectively.

Determination of Bound Ca²⁺

In the experiments performed in Figs. 2 and 6, SR vesicles were incubated with ⁴⁵CaCl₂ as per the figure legends, and an aliquot of reaction mixture was spotted on the HAWP membrane filter (Millipore). Then, the membrane was perfused with a chasing solution for indicated time periods using a rapid filtration apparatus RFS-4 (Bio-Logic, Claix, France). To estimate nonspecific ⁴⁵Ca²⁺ binding, the same experiments were done in the presence of 1 μm thapsigargin. Specific ⁴⁵Ca²⁺ binding was obtained after subtracting this nonspecific binding.

Ca²⁺ Uptake into SR Vesicles in a Single Turnover of EP

In the experiments performed in Figs. 4 and 5, SR vesicles were incubated with ⁴⁵Ca²⁺, and a single turnover of EP was initiated by adding ATP and excess EGTA using the handmade rapid mixing apparatus. After chasing the reaction, the mixture was spotted on the membrane filter and washed for ∼10 s by an EGTA solution, as described in the figure legends. The background level of ⁴⁵Ca²⁺ was determined without ATP and subtracted. This background level was <3% of the maximum Ca²⁺ uptake level.

Miscellaneous

All of the reactions were performed at 4 °C in 7 mm MgCl₂ and 50 mm MOPS/Tris (pH 7.3). Protein concentrations were determined by the method of Lowry et al. (39) with bovine serum albumin as a standard. Free Ca²⁺ concentrations were calculated by the Calcon program. Data were analyzed by nonlinear regression using the program Origin (Microcal Software, Inc., Northampton, MA). Three-dimensional models of the enzyme were produced by the program VMD (40).

RESULTS

Time Courses of EP Decay and Ca²⁺ Release

The Ca²⁺-ATPase in SR vesicles was phosphorylated with MgATP in the presence of 0.1 m K⁺, 10 μm Ca²⁺, and Ca²⁺-ionophore A23187 (Fig. 2, A and B). The reaction reaches steady state within a few seconds, and almost all of the Ca²⁺-ATPase is in the ADP-sensitive form of EP (E1P) because of the rate-limiting E1P to E2P transition followed by rapid E2P hydrolysis in the presence of K⁺ (29, 30).

When the reaction was chased with excess EGTA in 0.1 m K⁺, the amount of EP decreases in a single exponential time course, and the EP during the decay is almost all ADP sensitive (Fig. 2A). The bound Ca²⁺ decreases concomitantly with E1P decay, i.e. E1PCa₂ to E2P transition. The result agrees with the established mechanism that the two Ca²⁺ ions are occluded in E1PCa₂, and Ca²⁺ release into the lumen occurs very rapidly after the rate-limiting E1PCa₂ to E2PCa₂ transition, E1PCa₂ → E2PCa₂ → E2P + 2Ca²⁺ (11–14). Thus, the EP transition and Ca²⁺ release are tightly coupled in the presence of K⁺.

Surprisingly, when E1PCa₂ formed as above in 0.1 m K⁺, and A23187 was chased with excess EGTA in the absence of K⁺, the Ca²⁺ release is considerably (∼3×) faster than the E1P decay via its transition to E2P (Fig. 2B). The result shows that in the absence of K⁺, there is an E1P species without bound Ca²⁺ and that the Ca²⁺ ions are released from E1PCa₂. We found essentially the same results in the presence of choline chloride in place of LiCl without K⁺ (data not shown).

⁴⁵Ca²⁺ Uptake in Single Turnover of E1PCa₂

We then examined whether this rapid Ca²⁺ release from E1PCa₂ in the absence of K⁺ upon the EGTA chase occurs to the lumenal side or cytoplasmic side of the membrane. For this purpose, we performed a ⁴⁵Ca²⁺ uptake assay in a single turnover of E1PCa₂ in the absence of ionophore, i.e. with sealed SR vesicles. In Fig. 3, for the single turnover of E1PCa₂, the Ca²⁺-ATPase in the vesicles in 10 μm Ca²⁺ was phosphorylated by a simultaneous addition of [γ-³²P]ATP and excess EGTA in either the presence or the absence of 0.1 m K⁺. Approximately half of the ATPase is phosphorylated rapidly to form E1PCa₂ both in the presence and absence of K⁺, and then EP decays slowly, in contrast to the full phosphorylation achieved without the removal of Ca²⁺. In sealed vesicles (without A23187), EP decays at the same rate in the presence or absence of K⁺. In the presence of K⁺, nearly all EP is E1P (ADP-sensitive), whereas in the absence of K⁺, E2P increases slowly to ∼20% at ∼2 s of the maximum amount of EP formed immediately after the ATP addition.

Then, in Fig. 4 (closed circles), the ⁴⁵Ca²⁺ uptake assay during a single turnover of E1PCa₂ was performed by membrane filtration with an EGTA chase, i.e. with extensive EGTA washing of the filter for ∼10 s under otherwise the same conditions as in the single turnover of E1PCa₂ in Fig. 3. During the ∼10 s of EGTA washing, nearly all EP was dephosphorylated (Fig. 3) as we intended; therefore, all of the bound ⁴⁵Ca²⁺ in EP was released even at the first time point (0.1 s after the start when nearly all EP is E1PCa₂) either to the cytoplasmic side or lumenal side. If released to the cytoplasmic side, the ⁴⁵Ca²⁺ will be lost from the filter by the EGTA wash, and levels will be reduced significantly from the ideal stoichiometry of two Ca²⁺ ions transported in a single turnover of E1PCa₂. However, the results (closed circles) clearly show a maximum uptake of ∼1.7 Ca²⁺ per EP in 0.1 m K⁺ and an even higher uptake of 1.8∼1.9 without K⁺, very close to the ideal stoichiometry. Therefore, during a single turnover, the bound ⁴⁵Ca²⁺ ions in E1PCa₂ formed in the absence of K⁺ are not released to the cytoplasmic side but to the lumen. It is concluded that in E1PCa₂ without K⁺, the cytoplasmic gate is closed, but a Ca²⁺ pathway to the lumen exists. Thus, the Ca²⁺ binding sites face the lumen.

ADP Chase during Single Turnover of ⁴⁵Ca²⁺ Uptake

In Fig. 4 (open circles), we assessed at each time point during the single turnover of E1PCa₂ the amount of ⁴⁵Ca²⁺ remaining on the filter with the vesicles. For this purpose, we chased the reaction with ADP and excess EGTA at each time point, i.e. dephosphorylating to E1Ca₂ very rapidly in the reverse reaction and removing ⁴⁵Ca²⁺ released to the cytoplasmic side. Both in the presence and absence of K⁺, at 0.1 s (first time point) immediately after the ATP/EGTA addition, nearly maximum EP is already formed (all E1PCa₂, Fig. 3), and all of the bound ⁴⁵Ca²⁺ is removed by the ADP chase. Then, in the presence of 0.1 m K⁺ (A), the ADP-insensitive fraction and the amount of ⁴⁵Ca²⁺ released into the lumen increased exponentially due to the forward E1PCa₂ decay via its transition to E2P with Ca²⁺ release as expected from the established transport mechanism. In fact, the time course agreed with that of EP decay via the rate-limiting E1PCa₂ to E2P transition (Fig. 3, closed triangles).

On the other hand, in the absence of K⁺ (B), ⁴⁵Ca²⁺, and the ADP-insensitive fraction increase very rapidly (within the initial ∼0.5 s) and suddenly slow, showing a clear biphasic time course. The second slow phase occurs at nearly the same rate as the EP decay via the E1P to E2P transition (Fig. 3, open triangles) and the single exponential ⁴⁵Ca²⁺ uptake in the presence of K⁺ (A), i.e. the normal transport process E1PCa₂ → E2PCa₂ → E2P + 2Ca²⁺. The initial rapid phase occurs at a significantly faster rate and to a higher extent than in E2P formation (Fig. 3) and therefore cannot be accounted for simply by formation of E2P. Actually, the initial phase is even faster than the Ca²⁺ release from K⁺-free E1PCa₂ revealed upon excess EGTA addition (without ADP) in A23187 in Fig. 2. The results suggest that, in K⁺-free E1PCa₂, different types of Ca²⁺ sites are produced in the initial rapid phase; the Ca²⁺ ions are not released to the cytoplasmic side even upon ADP-induced reverse dephosphorylation.

Behavior of ⁴⁵Ca²⁺ at Site I in E1PCa₂

We examined whether the above observed biphasic kinetics revealed by the ADP chase is related to the heterogeneity of the Ca²⁺ sites I and II in E1Ca₂. In E1Ca₂, Ca²⁺ bound at site II is rapidly exchanged with the cytoplasmic Ca²⁺, and the Ca²⁺ bound at the deeper site I can be released to the cytoplasm only when site II is vacant (15, 41–44). Therefore, we first labeled site I with ⁴⁵Ca²⁺ by exchanging the site II-bound ⁴⁵Ca²⁺ with nonradioactive Ca²⁺ (supplemental Fig. S1). In Fig. 5B, we clearly observed a biphasic ⁴⁵Ca²⁺ increase in the ADP-insensitive fraction in the absence of K⁺ as in Fig. 4B. The only difference is that, as expected, the total amount of ⁴⁵Ca²⁺ uptake (0.8–1.0 Ca²⁺ per EP) is half of that in Fig. 4 in which both sites I and II are labeled by ⁴⁵Ca²⁺. The results show that the heterogeneity of the two Ca²⁺ sites I and II in E1Ca₂ is not related to the biphasic ⁴⁵Ca²⁺ increase revealed by the ADP chase in Fig. 4B. Furthermore, we observed a nonsequential release of two Ca²⁺ ions from E1PCa₂ to the lumenal side upon removal of free Ca²⁺ in the presence of A23187 without ADP, which therefore is not related to the biphasic ⁴⁵Ca²⁺ increase in Fig. 4B (supplemental Fig. S2) (58, 59).

These results show that there are two different types of E1PCa₂, i.e. the normal Ca²⁺-occluded E1PCa₂ and another E1PCa₂ species that possesses lumen-facing Ca²⁺ binding sites (opened lumenal pathway) and a closed cytoplasmic gate. The results further indicate that in the absence of K⁺, the E1PCa₂ species with the lumen-facing Ca²⁺ binding sites is rapidly produced from normal E1PCa₂, and this process is revealed by the ADP chase as the initial rapid phase in Fig. 4B (see more in “Discussion” and a schematic model in Fig. 7).

FIGURE 7.

Schematic model for roles of K⁺ in EP processing and Ca²⁺ handling in Ca²⁺ transport. sE1PCa₂ is an E1PCa₂ species formed without K⁺ possessing a closed cytoplasmic gate and lumen-facing Ca²⁺ binding sites (an opened lumenal pathway) with high Ca²⁺ affinity (Fig. 6). sE1PCa₂ is in rapid equilibrium with the normal E1PCa₂. Here, s denotes silent because this species is apparently absent in the presence of K⁺ and also because the bound Ca²⁺ ions are not released to the cytoplasmic side even upon ADP-induced reverse dephosphorylation (to sE1Ca₂) in contrast to the normal E1PCa₂ reverse dephosphorylation. Actual active Ca²⁺ transport is achieved by a large reduction of the Ca²⁺ affinity during the normal sequence E1PCa₂ → E2PCa₂ → E2P + 2Ca²⁺ (blue arrows). The schematic is based on crystal structural models for the ADP-sensitive and -insensitive EP states and E1Ca₂, with the positions of the cytoplasmic N, P, and A domains, and membrane (orange layer) being approximate. The Ca²⁺ sites in the transmembrane domain are depicted as occluded (closed cytoplasmic and lumenal gates) in normal E1PCa₂, as lumen-facing and high Ca²⁺ affinity with the closed cytoplasmic gate in sE1PCa₂ and sE1Ca₂, and as lumenally opened with reduced Ca²⁺ affinity in E2P and E2PCa₂ (immediately before the Ca²⁺ release).

Affinity of E1P for Lumenal Ca²⁺ in Absence of K⁺

In Fig. 6, we assessed the Ca²⁺ affinity of the transport sites exposed to the lumen in K⁺-free E1PCa₂ by determining the Ca²⁺ binding to E1P in steady state in the presence of A23187. In Fig. 6A, the total amount of EP increased with increasing Ca²⁺ concentration and reached its maximum level at ∼0.5 μm Ca²⁺ due to high affinity Ca²⁺ binding at the transport sites (E2 to E1Ca₂ transition). The total amount of EP at saturating Ca²⁺ was half of the maximum Ca²⁺ binding in E1Ca₂ (B); therefore, all Ca²⁺-ATPases are phosphorylated at saturating Ca²⁺. As replotted in Fig. 6C, ∼60% of the maximum total amount of EP was E1P in steady state at saturating Ca²⁺ under these conditions.

In Fig. 6B, the amount of bound Ca²⁺ in steady state in the presence of A23187 was determined without washing the filter so as not to alter the equilibrium. As replotted in Fig. 6C with % values relative to the maximum Ca²⁺ binding in E1Ca₂, the bound Ca²⁺ under the phosphorylating condition without K⁺ increases concomitantly with an increase in E1P, and their relative values are nearly the same. Note that if the affinity of the lumen-facing Ca²⁺ sites of E1P without K⁺ is significantly lower than that of the high Ca²⁺ affinity in E1 for the phosphorylation, the Ca²⁺ binding curve would be shifted significantly to higher Ca²⁺ concentrations, and the relative value of the bound Ca²⁺ would become significantly smaller than that of E1P in the 0.1–10 μm range. However, this is obviously not the case. We conclude that the affinity of the lumen-facing Ca²⁺ sites of K⁺-free E1P is as high as the cytoplasmic Ca²⁺ affinity in E1.

DISCUSSION

Ca²⁺ Release from E1PCa₂ in Absence of K⁺

Our studies show that in the absence of K⁺, Ca²⁺ is released from E1PCa₂ to the lumenal side. This Ca²⁺ release obviously precedes the conversion of the ADP-sensitive EP (E1P) to ADP-insensitive one (E2P); thus, there is a K⁺-free E1P species without bound Ca²⁺ (Fig. 2B). Evidently, a Ca²⁺ pathway from the transport sites to the lumen is open at least to some extent in this species. K⁺, probably bound to its specific site in the ATPase (28), therefore plays a critical role in E1PCa₂ to stabilize the transport sites in an occluded state. Notable also is our finding that the Ca²⁺ affinity of the sites facing the lumen in K⁺-free E1PCa₂ is as high as the cytoplasmic Ca²⁺ affinity in the unphosphorylated E1 state (Fig. 6). Thus, the Ca²⁺ binding sites are not disrupted in this K⁺-free E1PCa₂ structure, suggesting that the opening of the lumenal Ca²⁺ pathway does not involve large structural changes such as those that occur during the EP conformation change. The observation also means that such a Ca²⁺-ATPase species cannot be involved in producing a Ca²⁺ gradient across the membrane and therefore is unlikely to contribute significantly to active Ca²⁺ transport. This is because, without a reduction in Ca²⁺ affinity, lumenal Ca²⁺ would rebind at low concentrations and inhibit the pump.

Our kinetic analysis of the lumenal Ca²⁺-induced reverse conversion E2P + 2Ca²⁺ ↔ E2PCa₂ ↔ E1PCa₂ in wild type Ca²⁺-ATPase (13) has revealed that the K⁺ in E2P is critical for lowering the lumenal Ca²⁺ affinity and for fully opening the lumenal gate, thereby accomplishing the high physiological Ca²⁺ gradient and rapid Ca²⁺ release E2PCa₂ → E2P + 2Ca²⁺. K⁺ stabilizes the E2P structure with disrupted Ca²⁺ sites and a fully open lumenal gate. In the absence of K⁺, the lumenal Ca²⁺ affinity of E2P is ∼2000 times lower than in E1P (K_0.5 values, 0.4 mm (13) and 0.15 μm (Fig. 6), respectively). Therefore, the large structural change associated with the EP conformation change is obviously required, even in the absence of K⁺, for disrupting the Ca²⁺ sites. K⁺ binding in E2PCa₂/E2P further reduces the Ca²⁺ affinity to a level (K_0.5 value, 1.5 mm (13)) appropriate for producing the high physiological Ca²⁺ gradient across the membrane.

Thus, bound K⁺ stabilizes both the Ca²⁺ occluded structure of E1PCa₂ and the Ca²⁺-released structure of E2P. Thereby, K⁺ critically contributes to the successive structural changes and ensures strict and efficient coupling for EP processing and Ca²⁺ handling in E1PCa₂ → E2PCa₂ → E2P + 2Ca²⁺, key events for Ca²⁺ transport. Also notable is the fact that the K⁺ bound in the P domain is crucial for producing a catalytic site structure in E2P appropriate for its accelerated hydrolysis (28–30).

Biphasic Ca²⁺ Release in ADP Chase of Single Turnover of E1PCa₂ without K⁺

In Fig. 7, we provide a schematic model to show the roles of K⁺ in the Ca²⁺ transport and to account for the biphasic Ca²⁺ release from K⁺-free E1PCa₂ following an ADP chase during a single turnover (Fig. 4B, open circles). The fast initial phase may be accounted for by the rapid formation of sE1PCa₂, with lumen-facing, high affinity Ca²⁺ binding sites, in rapid equilibrium with normal E1PCa₂. The bound ⁴⁵Ca²⁺ ions cannot be released to the cytoplasmic side even upon ADP-induced reverse dephosphorylation (sE1Ca₂) but only to the lumenal side (yellow arrow). Because sE1P has high affinity, Ca²⁺ rebinding occurs at low lumenal concentrations³ and inhibits flux through this pathway. The slow second phase (Fig. 4B) most probably reflects the E1PCa₂ to E2P transition as in the single exponential Ca²⁺ uptake in 0.1 m K⁺ (Fig. 4A and Fig. 7, blue arrows). The formation of sE1PCa₂ in rapid equilibrium with occluded E1PCa₂ necessarily lowers the steady state level of the latter species and hence Ca²⁺ transport through the normal route. Thus, although progression to sE1PCa₂ is relatively fast, this pathway cannot contribute to gradient formation and ultimately slows normal transport. It is concluded that K⁺ ensures the normal structural process for Ca²⁺ transport (blue arrows) by stabilizing the Ca²⁺-occluded structure of E1PCa₂ and disallowing opening of a lumenal Ca²⁺ pathway (this study), and by stabilizing the E2P structure with disrupted Ca²⁺ sites (greatly reduced affinity) and a fully opened lumenal gate (13).

Structural Role of Bound K⁺ in E1PCa₂

The crystal structures provide a likely structural role of bound K⁺ in E1PCa₂. In structures analogous to K⁺-bound E1PCa₂ (E1PCa₂·AMPPN (22) and E1Ca₂·AlF₄⁻ ·ADP as well as E1Ca₂·AMPPCP (17)), K⁺ is specifically bound at the bottom part of the P domain and coordinated by the backbone carbonyl oxygens of Leu⁷¹¹, Lys⁷¹², and Ala⁷¹⁴ on Pα6 (sixth P-domain α-helix) (near the catalytic Mg²⁺ site Asp⁷⁰³/Asp⁷⁰⁷ on Pα5 of this region) and by the Glu⁷³² side chain oxygen on Pα7 (Fig. 8). The importance of Glu⁷³² in the K⁺-induced acceleration of E2P hydrolysis was shown through mutations (28).

FIGURE 8.

Structure of E1PCa₂ with bound K⁺. The crystal structure E1PCa₂·AMPPN (Protein Data Bank code 3BA6) (22) is shown. Panel a, a space-filling model with K⁺ (blue), M3 (yellow), M4 (orange), L6–7 (lime), Pα1 (pink), Pα6/Pα7 (light purple), Glu⁷³² (red and cyan), and A/M3-linker (dark gray). b, a schematic model with the view from the same direction as in a. K⁺ and Ca²⁺ are blue and red Van der Waals spheres, respectively. M3, M4, and M5 are yellow, orange, and light purple, respectively. The lower panels in a and b are the enlarged views of the areas surrounded by red broken line. In b, the coordination of K⁺ is shown by broken green lines. c, the helices for Ca²⁺ binding (M4, M5, M6, and M8) and K⁺ binding (Pα6/Pα7) and adjacent components (M3, M7, Pα1, and A/M3-linker) are depicted. A large red arrow suggests a possible motion of M3/M4L at the lumenal end to open the Ca²⁺ pathway. The residues involved in coordination of K⁺ and Ca²⁺ are depicted in ball and stick representation. Residues possibly forming interactions at the lumenal end (Tyr²⁹⁴, Tyr²⁹⁵, Lys²⁹⁷, and Glu⁷⁸⁵) are also depicted. The lower panel in c shows the view from the lumenal side.

The K⁺ ion and these ligands are distant from and not in direct contact with the transport sites from which Ca²⁺ release occurs. On the other hand, adjacent to the K⁺ binding site on Pα6/Pα7 is Pα1, which is directly linked with the cytoplasmic end of M4 within the P domain. Pα6, Pα7, and Pα1 constitute the bottom part of one-half of the P domain and move together as a body during the transport cycle (7, 18). Furthermore, Pα1 forms a hydrogen-bonding network with L6–7 (a cytoplasmic short loop linking M6 and M7) and top parts of M3/M5. This interaction network is critical for proper arrangement of the transmembrane helices (48–50). In fact, disruption of this network by mutations causes a marked retardation of the E2-E1 transition (48, 49).

Because the bound K⁺ is deeply embedded and ligated within this part of the P domain (Fig. 8a), its absence would allow more flexibility of the structural components, such as segmental fluctuations or wobbling, which in turn would impinge on the cytoplasmic regions of the transmembrane helices and probably destabilize the interaction network Pα1/L6–7/M3/M5. The absence of K⁺ in fact markedly retards the E2 to E1 transition (31, 32), and, as noted above, disruption of the Pα1/M3/M5/L6–7 interaction network markedly retards the E1-E2 transition and also the E1P to E2P conformation change (48–50). Opening of the lumenal pathway and Ca²⁺ release from E1PCa₂ may be caused by such structural perturbations in the absence of bound K⁺.

As shown in the view from the lumen of the helices M4/M5/M6/M8 ligating Ca²⁺ in Fig. 8c, the space surrounded by these helices seems to be the only possible Ca²⁺ exit pathway. M3 is in close contact at the lumenal end with the lumenal part of M4 (M4L), and they are connected by a short lumenal loop (L3–4). During the EP conformation change and subsequent Ca²⁺ release (E1PCa₂ → E2P + 2Ca²⁺), M3 and M4L incline together and move outward, thereby opening the putative Ca²⁺ release pathway (lumenal gate) (19). The M3/M4L motion is produced by the large rotation and inclination of the A and P domains and by the consequent significant motions and rearrangements of the helices M1∼6, in which M1/M2 as a rigid body pushes M4L to open the Ca²⁺ release gate (Fig. 9) (19). The large motions concomitantly disrupt the Ca²⁺ binding sites and reduce the Ca²⁺ affinity (19). In K⁺-free E1PCa₂ (ADP sensitive), these domain motions have not yet taken place, and the Ca²⁺ sites are not disrupted and maintain a high affinity. Here, these motions are likely much less prominent and opening of the release pathway is simply the result of fluctuations and wobbling of the relevant helices, in particular M3/M4L.

The unique Ca²⁺ coordination and particular make up of the M3 and M4 helices lend themselves to creating a release pathway while maintaining a high affinity. The Ca²⁺ sites with properly positioned ligands are located at an unwound portion of the M4 helix creating intrinsic flexibility (Fig. 8). On the other hand, M3 is a continuous helix from the cytoplasmic to the lumenal end and is located at the periphery of the transmembrane domain and is not closely associated with other helices including M1/M2 (except for M4L at the lumenal end). Thus, in the crystal structures analogous to E1PCa₂, M3 seems not to have much steric restriction against possible outward movement, a shift that would open the Ca²⁺ pathway. Therefore, if the cytoplasmic region of M3 is not fixed as occurs in the absence of bound K⁺, its lumenal part and the associated M4L may become more mobile. Wobbling here could allow the Ca²⁺ pathway to fluctuate between a closed and open state. The Ca²⁺ sites are not necessarily disrupted because of the flexibility of the unwound structure of M4 and because the large motions of the A–P domains do not occur. (These are the motions that disrupt the Ca²⁺ sites by inclining the cytoplasmic region of M4/M5.) Also, M3 is not involved directly in the Ca²⁺ ligation.

Interestingly, at the lumenal end of M4L (Fig. 8c), there are bulky and hydrophobic residues (Tyr²⁹⁴/Tyr²⁹⁵/Lys²⁹⁷), which may form hydrogen bonds, e.g. Tyr²⁹⁴/Tyr²⁹⁵ with Glu⁷⁸⁵ on L5–6. Lys²⁹⁷ seems to seal the Ca²⁺ channel (51). Tyr²⁹⁵ is important for Ca²⁺ transport activity and stabilizing E2 relative to E1 (52). These residues may possibly function as the lumenal plug, and M3/M4L wobbling may destabilize their interactions helping to open the Ca²⁺ pathway in K⁺-free E1PCa₂.

Importantly, in the crystal structures of analogues of E1PCa₂, the cytoplasmic Ca²⁺ gate is closed by the Ca²⁺ ligand Glu³⁰⁹ because Leu⁶⁵ on M1 locks the Glu³⁰⁹ side chain configuration by van der Waals contact (8, 9, 18, 53). Our observation shows that this cytoplasmic gate is closed in E1PCa₂ even without bound K⁺, and therefore, the Glu³⁰⁹-gating with Leu⁶⁵ has not been affected.

Movement of K⁺ Binding Site during E1PCa₂ → E2P + 2Ca²⁺

K⁺-bound crystal structures E1PCa₂·AMPPN and E2·AlF₄⁻ may be used as a model for the overall change in E1PCa₂ → E2P + 2Ca²⁺ (Fig. 9). Hence, the P domain inclines to the A domain that also rotates and inclines (curved arrows), thus producing the A–P domain association in the most compactly organized and inclined headpiece structure, the Ca²⁺-released E2P. With this change, the cytoplasmic region of M4/M5 in the P domain inclines and disrupts the Ca²⁺ sites (19). M2 inclines with the A domain motion and consequently M1, which forms a rigid V-shaped body with M2, pushes against the lumenal part of M4, and opens the lumenal gate (19).

In these structural changes, the K⁺ site with bound K⁺ on the P domain moves down to the Gln²⁴⁴ region on the A/M3-linker (blue arrow) and brings in the Gln²⁴⁴ side chain (or neighboring residues) as an additional coordination ligand. Thus bound K⁺ likely cross-links the bottom part of the P domain and the A/M3-linker. This cross-link must contribute to the stabilization of the compactly organized and inclined E2P structure with disrupted Ca²⁺ sites and fully opened lumenal gate (13).

The A/M1′-linker of correct length has a critical function in inclining and compacting the E2P structure (14, 27). The structure is stabilized by three critical interaction networks; at the Tyr¹²² HC (hydrophobic interaction cluster involving the A and P domains and M2), at the Val²⁰⁰ loop (ionic and hydrogen bonding interactions with the P domain residues), and at the TGES¹⁸⁴ loop (hydrogen bonding interactions with the P domain residue in the catalytic site) (10–14). The TGES¹⁸⁴ loop of the rotated A domain protrudes into the catalytic site and blocks attack of ADP on the Asp³⁵¹ phosphate (causing the loss of ADP sensitivity). The Tyr¹²² HC is produced upon A–P domain inclination induced by tension on the A/M1′-linker (14, 27) and is critical for reducing the Ca²⁺ affinity and opening the lumenal gate, i.e. to deocclude/release Ca²⁺, E2PCa₂ → E2P (11–13). All of the interaction networks are essential for these changes and are also necessary for the formation of the catalytic site with hydrolytic activity (10–13). Importantly, the Val²⁰⁰ loop and Tyr¹²² HC are situated at the top and bottom of the A–P domain interface, respectively, and bound K⁺ is lower down and close to the membrane domain. Thus, these interaction networks including the K⁺ site are situated at positions most appropriate for stabilizing the compactly organized and inclined (thus strained) structure of E2P.

Ca²⁺ Release into Cytoplasm and Uncoupling

It was previously observed with SR Ca²⁺-ATPase (54–57) that Ca²⁺ in E1PCa₂ can be released to cytoplasm upon direct hydrolysis to E1Ca₂ (not via its transition to E2P) under specific conditions such as with a raised lumenal Ca²⁺ level. This causes ATP hydrolysis without Ca²⁺ transport resulting in uncoupling. de Meis and co-workers (54–56) further suggested that such uncoupled ATP hydrolysis functions as a heat-producing entity. This finding obviously differs from ours in that in K⁺-free E1PCa₂, the Ca²⁺ release pathway into the lumen is open, and the phosphoenzyme is not directly hydrolyzed.

In summary, we have found that E1PCa₂ without bound K⁺ has a perturbed structure with at least a partially open lumenal Ca²⁺ release pathway and still with the Ca²⁺ sites maintaining a high affinity. Thus, in the natural E1PCa₂ structure, bound K⁺ stabilizes the Ca²⁺ in an occluded form by not allowing the pathway to open. Bound K⁺ also stabilizes E2P following disruption of the Ca²⁺ sites and full opening of the lumenal gate (13). Thus, bound K⁺ has a crucial role in EP processing and Ca²⁺ occlusion and release to the lumen in the sequence E1PCa₂ → E2PCa₂ → E2P + 2Ca²⁺.