Figure 3: Cl⁻/HCO₃⁻ exchange activity across control or stomatin-deficient RBC membranes estimated from intracellular chloride concentration changes.

(a) Individual time courses of fluorescence and probe dequenching by the decrease of the chloride concentration in ghosts prepared from control and stomatin-deficient RBCs. Ghosts resealed in the presence of a chloride-sensitive dye (SPQ) were submitted to 100 mEq outwardly directed chloride and 25 mEq inwardly directed bicarbonate gradients at 20 °C in the stopped-flow spectrofluorometer. As a specificity control, ghosts were incubated with 10 μM DIDS for 30 minutes and centrifuged in order to remove unbound DIDS prior to analysis. Data from three to four time-courses were averaged. Smooth lines are monoexponential fits to the data using the simplex procedure of the Biokine (Bio-Logic), providing probe dequenching rate constants (k, s⁻¹). (b) Three independent experiments allowed the determination of an average of the rate constants k for four controls (Stom-positive) and three OHSt (Stom-deficient). From these averages, means ± SEM were calculated for Stom-positive (black bars) and Stom-deficient (grey bars) and were compared using a paired Student t test (p = 0.0013).