Generation of ⁴⁵Ca content heterogeneity during the Ca²⁺ extrusion period of the load-extrusion protocol. One experiment with protocol 1 is shown (see “Experimental protocols”) representative of 3 with similar results. (A) Measurement of the PMCA V_max by the load-extrusion extrusion protocol. RBCs were suspended at 10% Hct in medium HK supplemented with 5 mM inosine and 140 μM ⁴⁵Ca(Ca²⁺). The ionophore A21387 was added at t = 0 (final concentration 10 μM) and CoCl₂ was added at t = 2 minutes (final concentration 0.4 mM). (B) Comparison of the release of Hb and ⁴⁵Ca induced by hypotonic lysis, as a function of relative tonicity. The measurements were performed on aliquots of the same samples used for the V_max estimates in panel A, as per protocol 1. All samples were postincubated in LK medium. Solid and open symbols report percentages of ⁴⁵Ca and Hb released at each relative tonicity, respectively. One hundred percent release corresponds to the values obtained at about 0 relative tonicity. (C) Direct comparison of lysis-induced release of Hb and ⁴⁵Ca at each relative tonicity. The solid symbols and lines represent paired values from open and solid symbols of panel B. The open symbols and dashed lines are from duplicate samples from the same experiment treated as in panel A except for postincubation in HK medium to preclude volume changes.