FIGURE 8.

Ca²⁺ occlusion in E2Ca₂·BeF₃⁻ of the mutant 4Gi-46/47 formed from E1Ca₂ (A) and from E2·BeF₃⁻ (B). A, microsomes (0.2 mg/ml) expressing the mutant 4Gi-46/47 were incubated for various periods at 25 °C in 10 μl of a mixture containing 0.01 mm ⁴⁵CaCl₂, 1 mm KF, 1 μm BeSO₄, 15 mm MgCl₂, 0.1 m KCl, 50 mm MOPS/Tris (pH 7). The mixture was then diluted 200-fold at 0 °C with a washing solution containing 2 mm EGTA, 5 μm A23187, 0.1 m KCl, 7 mm MgCl₂, and 50 mm MOPS/Tris (pH 7.0), subjected to membrane filtration, and washed rapidly with 6 ml of the washing solution for 4 s at 0 °C. For determination of EP, the above BeF_x-incubation was made with ⁴⁰Ca²⁺ instead of ⁴⁵Ca²⁺ otherwise as above, and the sample was diluted 10-fold and phosphorylated with 10 μm [γ-³²P]ATP at 0 °C for 15 s as in Fig. 3C. The sample was then further diluted 20-fold at 0 °C with the washing solution, immediately filtered as above, and washed rapidly with ice-cold trichloroacetic acid containing P_i. The EP level was not changed during the above sample handling because the decay of EP (E2PCa₂) is almost completely blocked in the mutant (14). The amount of ⁴⁵Ca²⁺ specifically bound and occluded (■) and that of E³²P formed (○) in the expressed SERCA1a mutant were obtained by subtracting the background levels determined by including 1 μm TG in the BeF_x incubation mixture. The values presented are the mean ± S.D. (n = 5). Inset, the amount of EP formed was replotted versus that of occluded Ca²⁺ with the BeF_x treatment. The solid line represents the linear least squares fit. The y and x intercepts gave 4.3 and 8.4 nmol/mg of the expressed SERCA1a for the amounts of EP and of Ca²⁺ occluded, respectively. B, for formation of E2·BeF₃⁻, microsomes (1 mg/ml) expressing the mutant 4Gi-46/47 were incubated at 25 °C for 30 min with 1 mm KF and 20 μm BeSO₄ in 1 mm EGTA, 7 mm MgCl₂, 50 mm LiCl, and 50 mm MOPS/Tris (pH 7). Then the mixture was diluted 2.5-fold with a solution containing 7 mm MgCl₂, 50 mm LiCl, 50 mm MOPS/Tris (pH 7), 5 μm Ca²⁺ ionophore A23187, and various concentrations of ⁴⁵CaCl₂ to give the indicated final ⁴⁵Ca²⁺ concentrations. After incubating at 25 °C for 1 min, the mixture was further diluted with 400-fold of the washing solution containing the excess EGTA, filtered, and washed with the washing solution as above. The amount of ⁴⁵Ca²⁺ specifically bound and occluded in the SERCA1a was obtained by subtracting the nonspecific Ca²⁺ binding, which was determined without KF in the BeF_x treatment mixture. In fitting to the Hill equation (solid line), the maximum amount of occluded Ca²⁺, K_0.5 for the Ca²⁺ activation, and Hill coefficient were obtained as 7.7 nmol/mg of the expressed SERCA1a, 0.1 mm, and 1.6, respectively. The values presented are the mean ± S.D. (n = 7).