Distribution of Pcat activity induced by Ca²⁺ loads and RBC dehydration, shown as profile migration of osmotic lysis curves. Experiments were performed at 37°C with gentle magnetic stirring of the RBC suspensions, with Hct 5% and suspension [Ca²⁺]_o of 50 μM. All media were supplemented with 5 mM inosine and 40 μg/mL gentamicin. At t = 0, A23187 was added in panels A to G and valinomycin to panel H to final concentrations of 5 and 10 μM, respectively, in the cell suspension. (A) Rehydration response in LK medium. The indicated times apply to Figure 3A-D. The rapid left shift in the osmotic fragility curve indicates full uniform dehydration of the RBCs by 10 minutes following the ionophore-induced Ca²⁺ load. This is followed by slow right shifts in the osmotic fragility curves reflecting the Pcat-mediated rehydration response. (B) At t = 11 minutes, after dehydration was complete, 1 mM EGTA was added to rapidly extract cell Ca²⁺, reducing [Ca²⁺]_i to subphysiological levels. RBCs exposed to 10 μM valinomycin at t = 0 instead of A23187 showed identical dehydration-rehydration patterns as those in this panel, and therefore Figure 1B is used to illustrate both experimental results. (C) At t = 11 minutes, after dehydration was complete, 1 mM EGTA was added to rapidly extract cell Ca²⁺, reducing [Ca²⁺]_i to subphysiological levels. The suspension was gently spun at 900g for 3 minutes, the supernatant removed, and the cells resuspended in HK medium supplemented with 1 mM EGTA to follow the rehydration pattern in HK medium with the Gardos channels inactivated by Ca²⁺ removal. (D) RBCs were suspended in LK medium in which 120 mM NaCl was replaced by 240 mM sucrose. (E) Hydration response in 90K medium. The indicated times apply to panels E to H. (F) Effect of 0.1 μM charybdotoxin (CTX) on the hydration response in 90K medium. (G) Effect of 0.1 mM ouabain on the hydration response in 90K medium. (H) Effect of valinomycin instead of A23187 in the same conditions as those of Figure 3E.