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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.


Proximity ligation assays for protein interaction between AE1 and stomatin (AE1-stom).RBCs from a control (stomatin-positive) and an OHSt (stomatin-deficient) patient were treated as described in experimental procedures, using anti-AE1 along with anti-stomatin antibodies and anti-AE1 along with anti-ankyrin R antibodies. For controls, two other pairs of antibodies were used: anti-AQP1 along with anti-CAII (positive controls) and anti-AE1 along with anti-AQP1 (negative controls). After PLA, RBCs were examined by confocal microscopy using a Zeiss LSM700 inverted confocal microscope equipped with a x100 oil-immersion objective, numerical aperture 1.4. Z-stack confocal image capture was performed and analyzed using the ZEN software.
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f4: Proximity ligation assays for protein interaction between AE1 and stomatin (AE1-stom).RBCs from a control (stomatin-positive) and an OHSt (stomatin-deficient) patient were treated as described in experimental procedures, using anti-AE1 along with anti-stomatin antibodies and anti-AE1 along with anti-ankyrin R antibodies. For controls, two other pairs of antibodies were used: anti-AQP1 along with anti-CAII (positive controls) and anti-AE1 along with anti-AQP1 (negative controls). After PLA, RBCs were examined by confocal microscopy using a Zeiss LSM700 inverted confocal microscope equipped with a x100 oil-immersion objective, numerical aperture 1.4. Z-stack confocal image capture was performed and analyzed using the ZEN software.

Mentions: Stomatin-positive (control) and stomatin-deficient (OHSt) RBCs were subjected to a proximity ligation assay (PLA) for protein interaction between AE1 and stomatin, as described in experimental procedures. The pictures (Fig. 4) show the merged images of a bright-field viewing of RBCs from a control and an OHSt to a maximum intensity projection of PLA signals visualized as red spots. For the AE1-stomatin PLA, a signal was clearly visible in stomatin-positive (control) and absent in stomatin-deficient (OHSt) RBCs. This result confirms interactions between stomatin and AE1 in control RBCs, the absence of stomatin in OHSt consequently resulting in absence of PLA signal in the patient RBCs. It is noteworthy that the number of red spots does not quantitatively match with the expected number of interaction sites and is much lower than that obtained in PLA on HEK293 cells (see below). Several PLA controls were also carried out. The association of anti-AQP1 with anti-CAII gave, as expected, a positive PLA in the RBCs of both the control and the OHSt. Indeed, these results confirm, for the first time in RBCs, the observations of Vilas et al.37, that clearly showed by PLA that recombinant AQP1 and endogenous CAII were interacting in HEK293 cells. Anti-AE1 used along with anti-AnkR resulted in a positive PLA in control erythrocytes as well as in OHSt. In contrast, when anti-AE1, which gave a PLA signal with anti-stomatin or anti-AnkR, was used with anti-AQP1, which gave a PLA signal with anti-CAII, a negative PLA response was obtained in both control and OHSt RBCs. This indicates a distance >40 nm between AE1 and AQP1 and is consistent with the absence of AQP1 from the multimolecular AE1 complex.


Stomatin modulates the activity of the Anion Exchanger 1 (AE1, SLC4A1)
Proximity ligation assays for protein interaction between AE1 and stomatin (AE1-stom).RBCs from a control (stomatin-positive) and an OHSt (stomatin-deficient) patient were treated as described in experimental procedures, using anti-AE1 along with anti-stomatin antibodies and anti-AE1 along with anti-ankyrin R antibodies. For controls, two other pairs of antibodies were used: anti-AQP1 along with anti-CAII (positive controls) and anti-AE1 along with anti-AQP1 (negative controls). After PLA, RBCs were examined by confocal microscopy using a Zeiss LSM700 inverted confocal microscope equipped with a x100 oil-immersion objective, numerical aperture 1.4. Z-stack confocal image capture was performed and analyzed using the ZEN software.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Proximity ligation assays for protein interaction between AE1 and stomatin (AE1-stom).RBCs from a control (stomatin-positive) and an OHSt (stomatin-deficient) patient were treated as described in experimental procedures, using anti-AE1 along with anti-stomatin antibodies and anti-AE1 along with anti-ankyrin R antibodies. For controls, two other pairs of antibodies were used: anti-AQP1 along with anti-CAII (positive controls) and anti-AE1 along with anti-AQP1 (negative controls). After PLA, RBCs were examined by confocal microscopy using a Zeiss LSM700 inverted confocal microscope equipped with a x100 oil-immersion objective, numerical aperture 1.4. Z-stack confocal image capture was performed and analyzed using the ZEN software.
Mentions: Stomatin-positive (control) and stomatin-deficient (OHSt) RBCs were subjected to a proximity ligation assay (PLA) for protein interaction between AE1 and stomatin, as described in experimental procedures. The pictures (Fig. 4) show the merged images of a bright-field viewing of RBCs from a control and an OHSt to a maximum intensity projection of PLA signals visualized as red spots. For the AE1-stomatin PLA, a signal was clearly visible in stomatin-positive (control) and absent in stomatin-deficient (OHSt) RBCs. This result confirms interactions between stomatin and AE1 in control RBCs, the absence of stomatin in OHSt consequently resulting in absence of PLA signal in the patient RBCs. It is noteworthy that the number of red spots does not quantitatively match with the expected number of interaction sites and is much lower than that obtained in PLA on HEK293 cells (see below). Several PLA controls were also carried out. The association of anti-AQP1 with anti-CAII gave, as expected, a positive PLA in the RBCs of both the control and the OHSt. Indeed, these results confirm, for the first time in RBCs, the observations of Vilas et al.37, that clearly showed by PLA that recombinant AQP1 and endogenous CAII were interacting in HEK293 cells. Anti-AE1 used along with anti-AnkR resulted in a positive PLA in control erythrocytes as well as in OHSt. In contrast, when anti-AE1, which gave a PLA signal with anti-stomatin or anti-AnkR, was used with anti-AQP1, which gave a PLA signal with anti-CAII, a negative PLA response was obtained in both control and OHSt RBCs. This indicates a distance >40 nm between AE1 and AQP1 and is consistent with the absence of AQP1 from the multimolecular AE1 complex.

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.