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Stomatin modulates the activity of the Anion Exchanger 1 (AE1, SLC4A1)

View Article: PubMed Central - PubMed

ABSTRACT

Anion Exchanger 1 (AE1) and stomatin are integral proteins of the red blood cell (RBC) membrane. Erythroid and kidney AE1 play a major role in HCO3− and Cl− exchange. Stomatins down-regulate the activity of many channels and transporters. Biochemical studies suggested an interaction of erythroid AE1 with stomatin. Moreover, we previously reported normal AE1 expression level in stomatin-deficient RBCs. Here, the ability of stomatin to modulate AE1-dependent Cl−/HCO3− exchange was evaluated using stopped-flow methods. In HEK293 cells expressing recombinant AE1 and stomatin, the permeabilities associated with AE1 activity were 30% higher in cells overexpressing stomatin, compared to cells with only endogenous stomatin expression. Ghosts from stomatin-deficient RBCs and controls were resealed in the presence of pH- or chloride-sensitive fluorescent probes and submitted to inward HCO3− and outward Cl− gradients. From alkalinization rate constants, we deduced a 47% decreased permeability to HCO3− for stomatin-deficient patients. Similarly, kinetics of Cl− efflux, followed by the probe dequenching, revealed a significant 42% decrease in patients. In situ Proximity Ligation Assays confirmed an interaction of AE1 with stomatin, in both HEK recombinant cells and RBCs. Here we show that stomatin modulates the transport activity of AE1 through a direct protein-protein interaction.

No MeSH data available.


Related in: MedlinePlus

Cl−/HCO3− exchange activity across control or stomatin-deficient RBC membranes estimated from pHi changes.(a) Individual time-courses of fluorescence and pHi changes in ghosts prepared from RBCs of a control and an OHSt patient. Ghosts resealed in the presence of a pH-sensitive dye (pyranine) were submitted to 100 mEq outwardly-directed chloride and 50 mEq inwardly-directed bicarbonate gradients at 30 °C in the stopped-flow spectrofluorometer. As a specificity control, ghosts were incubated with 20 μM DIDS for 30 minutes 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 alkalinization rate constants (k, s−1). (b) At least three experiments for each patient (Stom-positive: four controls and one Rh (black bars), Stom-deficient: four OHSt and one 7.2(−)CHC patient (grey bars)) were averaged and can be compared. (c) Means of the alkalinization rate constants (k, s−1) ± SEM for Stom-positive and Stom-deficient were reported. p value was determined using an unpaired Student t test.
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f1: Cl−/HCO3− exchange activity across control or stomatin-deficient RBC membranes estimated from pHi changes.(a) Individual time-courses of fluorescence and pHi changes in ghosts prepared from RBCs of a control and an OHSt patient. Ghosts resealed in the presence of a pH-sensitive dye (pyranine) were submitted to 100 mEq outwardly-directed chloride and 50 mEq inwardly-directed bicarbonate gradients at 30 °C in the stopped-flow spectrofluorometer. As a specificity control, ghosts were incubated with 20 μM DIDS for 30 minutes 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 alkalinization rate constants (k, s−1). (b) At least three experiments for each patient (Stom-positive: four controls and one Rh (black bars), Stom-deficient: four OHSt and one 7.2(−)CHC patient (grey bars)) were averaged and can be compared. (c) Means of the alkalinization rate constants (k, s−1) ± SEM for Stom-positive and Stom-deficient were reported. p value was determined using an unpaired Student t test.

Mentions: In order to investigate the impact of stomatin deficiency in RBCs on the activity of AE1, a stopped-flow assay based, as previously described31, on the measurements of the kinetics of pHi changes related to HCO3− entry was applied to ghosts derived from four OHSt and one 7.2(−)CHC patients. Since the pHi measurement, using a fluorescent probe, is very sensitive, inconstant residual endogenous carbonic anhydrase resulting from the ghost preparation could non-homogeneously enhance the chloride/bicarbonate exchange. However, after the addition of 2 mg/ml of bovine carbonic anhydrase, HCO3− entry could be then accurately compared between several ghost samples (discussed in ref. 31). As shown in Fig. 1a from the individual time-courses of fluorescence changes in the ghosts of one control and one OHSt (black and grey curves, respectively), an alkalinisation was obtained for both samples. However, the kinetics was slower in ghosts derived from the OHSt patient. The alkalinisation rate constants k were deduced for each patient (Stom-positive: four controls and one Rh; Stom-deficient: four OHSt and one 7.2(−)CHC patient) and compared (Fig. 1b), showing a clear decrease of these constants k in Stom-deficient, when compared to Stom-positive samples. A significant decrease of 47% could be thus calculated from the means of these constants (Fig. 1c, Stom-deficient: 2.58 ± 0.13 s−1 and Stom-positive: 4.89 ± 0.18 s−1). This decrease can be strictly correlated to a diminution of the AE1 activity, since similar expression levels of AE1 were previously demonstrated in Stom-deficient and Stom-positive RBCs12.


Stomatin modulates the activity of the Anion Exchanger 1 (AE1, SLC4A1)
Cl−/HCO3− exchange activity across control or stomatin-deficient RBC membranes estimated from pHi changes.(a) Individual time-courses of fluorescence and pHi changes in ghosts prepared from RBCs of a control and an OHSt patient. Ghosts resealed in the presence of a pH-sensitive dye (pyranine) were submitted to 100 mEq outwardly-directed chloride and 50 mEq inwardly-directed bicarbonate gradients at 30 °C in the stopped-flow spectrofluorometer. As a specificity control, ghosts were incubated with 20 μM DIDS for 30 minutes 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 alkalinization rate constants (k, s−1). (b) At least three experiments for each patient (Stom-positive: four controls and one Rh (black bars), Stom-deficient: four OHSt and one 7.2(−)CHC patient (grey bars)) were averaged and can be compared. (c) Means of the alkalinization rate constants (k, s−1) ± SEM for Stom-positive and Stom-deficient were reported. p value was determined using an unpaired Student t test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5383999&req=5

f1: Cl−/HCO3− exchange activity across control or stomatin-deficient RBC membranes estimated from pHi changes.(a) Individual time-courses of fluorescence and pHi changes in ghosts prepared from RBCs of a control and an OHSt patient. Ghosts resealed in the presence of a pH-sensitive dye (pyranine) were submitted to 100 mEq outwardly-directed chloride and 50 mEq inwardly-directed bicarbonate gradients at 30 °C in the stopped-flow spectrofluorometer. As a specificity control, ghosts were incubated with 20 μM DIDS for 30 minutes 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 alkalinization rate constants (k, s−1). (b) At least three experiments for each patient (Stom-positive: four controls and one Rh (black bars), Stom-deficient: four OHSt and one 7.2(−)CHC patient (grey bars)) were averaged and can be compared. (c) Means of the alkalinization rate constants (k, s−1) ± SEM for Stom-positive and Stom-deficient were reported. p value was determined using an unpaired Student t test.
Mentions: In order to investigate the impact of stomatin deficiency in RBCs on the activity of AE1, a stopped-flow assay based, as previously described31, on the measurements of the kinetics of pHi changes related to HCO3− entry was applied to ghosts derived from four OHSt and one 7.2(−)CHC patients. Since the pHi measurement, using a fluorescent probe, is very sensitive, inconstant residual endogenous carbonic anhydrase resulting from the ghost preparation could non-homogeneously enhance the chloride/bicarbonate exchange. However, after the addition of 2 mg/ml of bovine carbonic anhydrase, HCO3− entry could be then accurately compared between several ghost samples (discussed in ref. 31). As shown in Fig. 1a from the individual time-courses of fluorescence changes in the ghosts of one control and one OHSt (black and grey curves, respectively), an alkalinisation was obtained for both samples. However, the kinetics was slower in ghosts derived from the OHSt patient. The alkalinisation rate constants k were deduced for each patient (Stom-positive: four controls and one Rh; Stom-deficient: four OHSt and one 7.2(−)CHC patient) and compared (Fig. 1b), showing a clear decrease of these constants k in Stom-deficient, when compared to Stom-positive samples. A significant decrease of 47% could be thus calculated from the means of these constants (Fig. 1c, Stom-deficient: 2.58 ± 0.13 s−1 and Stom-positive: 4.89 ± 0.18 s−1). This decrease can be strictly correlated to a diminution of the AE1 activity, since similar expression levels of AE1 were previously demonstrated in Stom-deficient and Stom-positive RBCs12.

View Article: PubMed Central - PubMed

ABSTRACT

Anion Exchanger 1 (AE1) and stomatin are integral proteins of the red blood cell (RBC) membrane. Erythroid and kidney AE1 play a major role in HCO3− and Cl− exchange. Stomatins down-regulate the activity of many channels and transporters. Biochemical studies suggested an interaction of erythroid AE1 with stomatin. Moreover, we previously reported normal AE1 expression level in stomatin-deficient RBCs. Here, the ability of stomatin to modulate AE1-dependent Cl−/HCO3− exchange was evaluated using stopped-flow methods. In HEK293 cells expressing recombinant AE1 and stomatin, the permeabilities associated with AE1 activity were 30% higher in cells overexpressing stomatin, compared to cells with only endogenous stomatin expression. Ghosts from stomatin-deficient RBCs and controls were resealed in the presence of pH- or chloride-sensitive fluorescent probes and submitted to inward HCO3− and outward Cl− gradients. From alkalinization rate constants, we deduced a 47% decreased permeability to HCO3− for stomatin-deficient patients. Similarly, kinetics of Cl− efflux, followed by the probe dequenching, revealed a significant 42% decrease in patients. In situ Proximity Ligation Assays confirmed an interaction of AE1 with stomatin, in both HEK recombinant cells and RBCs. Here we show that stomatin modulates the transport activity of AE1 through a direct protein-protein interaction.

No MeSH data available.


Related in: MedlinePlus