Method used to investigate the PMCA V_maxdistribution in RBCs. (Panel A) Computer simulation of the Ca²⁺ load-extrusion protocol in an RBC population whose symmetrical distribution of PMCA V_max activity is shown in the inset. Substrate-fed RBCs are assumed to be suspended at an Hct of approximately 10% in an iso-osmotic buffer containing 80 mM K⁺ and 100 μM CaCl₂ at 37°C with magnetic stirring. A high ionophore concentration is used to rapidly induce a large and uniform RBC Ca²⁺ load. After about 2 minutes (to prevent significant reduction of cell ATP levels) Co²⁺ is added in excess of Ca²⁺ in the medium to block ionophore-mediated Ca²⁺ transport and to expose the uphill extrusion of Ca²⁺ by the pump. [Ca_T]_i was estimated in timed samples with the use of ⁴⁵Ca by dividing the measured cell-contained ⁴⁵Ca radioactivity by the specific activity of ⁴⁵Ca (there are no endogenous Ca²⁺ pools that could dilute the specific activity set by the addition of tracer). Each of the Ca²⁺ extrusion curves was obtained by solving numerically the differential equation d[Ca²⁺]_i /dt = (V_max)([Ca²⁺]_i²/(K_d² + [Ca²⁺]_i²)), using a different V_max value for each curve and applying the conversion [Ca_T]_i = [Ca²⁺]_i /α (Figure 1 legend). The thick line was computed as the weighted mean of the V_max distribution shown in the inset. Note that the Ca²⁺-extrusion curves with different V_max remain linear until [Ca_T]_i falls to about 2 to 4 μM, as expected from a Ca²⁺ desaturation kinetics with K_d values in the range of 0.2 to 0.5 μM.^2,4,36  The mean-V_max curve also renders a linear segment with a slope close to the true mean V_max of the distribution (approximately 14 mmol (340 g Hb)^-1h^-1) but with an apparent Ca²⁺ desaturation pattern starting at [Ca_T]_i approximately 100 μmol (340 g Hb)^-1 a level much higher than that of the single-V_max desaturation curves. This specious desaturation effect results from retention of Ca²⁺ within the low-V_max cells. Points A to E mark sampling times for hemolysis curves expected to have the patterns illustrated in panel B. The horizontal dashed line is at the [Ca_T]_i level above which Gardos channels are assumed to open maximally. (Panel B) Actual hemolysis curves from preliminary experiments illustrate the patterns expected with samples taken at the time points A (▴) to E (•) of the load-extrusion protocol simulated in panel A.