Figure 5.

Estimating the concentration of PMCA in the JS. (A) An example of the time course of Ca²⁺ accumulation in the t-system. The t-system was initially depleted of Ca²⁺, and Ca²⁺ (Ca_M, 67 nM) was returned to the fiber for the period indicated by the gray bar. Tetracaine was included in the bathing solution to inhibit RYR Ca²⁺ leak. In that case, Ca_JS, which drives PMCA, can be considered to equal Ca_M. Steady-state Ca_T, steady-state [Ca²⁺]; t_½, time to reach 50% of steady-state Ca_T. (B) Model-generated time courses of Ca²⁺ accumulation in the t-system using different combinations of PMCA concentration and $k_{\mathrm{L}\mathrm{e}\mathrm{a}\mathrm{k}}^{\mathrm{T}}.$ The example shows 15 records; the complete analysis used 5,000 records. Parameter combinations that produced records with realistic values of t_½ and steady-state Ca_T were those that passed through the red rectangle (correct t_½) and reached a final Ca_T value between the horizontal red lines. The one record in the sample meeting these criteria is shown in red. (C) Combinations of c_PMCA and $k_{\mathrm{L}\mathrm{e}\mathrm{a}\mathrm{k}}^{\mathrm{T}}$ that are consistent with observed time course of t-system filling. The analysis was performed for three $K_{\mathrm{D}}^{\mathrm{P}\mathrm{M}\mathrm{C}\mathrm{A}}$ values (150, 200, and 250 μM) that span the range of likely values; these correspond to pump Ca₅₀ values of 28, 38, and 48 nM under the conditions in the skinned fibers. Vertical dashed line, the value chosen for c_PMCA that can fulfill the requirement of producing realistic time course of t-system Ca²⁺ accumulation across the range of likely $K_{\mathrm{D}}^{\mathrm{P}\mathrm{M}\mathrm{C}\mathrm{A}}$ values. (D) Comparison of measured (solid red line) and calculated (dotted black line) time courses of Ca²⁺ accumulation in the t-system. The measured record is a digitized version of the record in A, and the calculated record was generated using the fiber model with parameter values: c_PMCA, 58 μM; $k_{\mathrm{L}\mathrm{e}\mathrm{a}\mathrm{k}}^{\mathrm{T}},$ 0.018 s⁻¹; and $K_{\mathrm{D}}^{\mathrm{P}\mathrm{M}\mathrm{C}\mathrm{A}},$ 210 μM (Ca₅₀, 40 nM).