Ca2+ release to lumen from ADP-sensitive phosphoenzyme E1PCa2 without bound K+ of sarcoplasmic reticulum Ca2+-ATPase

Abstract

During Ca(2+) transport by sarcoplasmic reticulum Ca(2+)-ATPase, the conformation change of ADP-sensitive phosphoenzyme (E1PCa(2)) to ADP-insensitive phosphoenzyme (E2PCa(2)) is followed by rapid Ca(2+) release into the lumen. Here, we find that in the absence of K(+), Ca(2+) release occurs considerably faster than E1PCa(2) to E2PCa(2) conformation change. Therefore, the lumenal Ca(2+) release pathway is open to some extent in the K(+)-free E1PCa(2) structure. The Ca(2+) affinity of this E1P is as high as that of the unphosphorylated ATPase (E1), indicating the Ca(2+) binding sites are not disrupted. Thus, bound K(+) stabilizes the E1PCa(2) structure with occluded Ca(2+), keeping the Ca(2+) 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 Ca(2+) affinity essential for achieving the high physiological Ca(2+) gradient and to fully open the lumenal Ca(2+) gate for rapid Ca(2+) release (E2PCa(2) → E2P + 2Ca(2+)). These findings show that bound K(+) is critical for stabilizing both E1PCa(2) and E2P structures, thereby contributing to the structural changes that efficiently couple phosphoenzyme processing and Ca(2+) handling.

FIGURE 1.

Reaction scheme of Ca²⁺-ATPase.

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.

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).

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.

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).