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.
Highlights
Stomatin, known as protein 7.2 or as the major component of band 7, is a 31-kDa integral membrane protein expressed at the membrane of red blood cells (RBCs)[1,2]
In order to investigate the impact of stomatin deficiency in RBCs on the activity of Anion Exchanger 1 (AE1), a stopped-flow assay based, as previously described[31], on the measurements of the kinetics of pHi changes related to HCO3− entry was applied to ghosts derived from four overhydrated stomatocytosis (OHSt) and one 7.2(−)CHC patients
This decrease can be strictly correlated to a diminution of the AE1 activity, since similar expression levels of AE1 were previously demonstrated in Stomdeficient and Stom-positive RBCs12
Summary
Known as protein 7.2 or as the major component of band 7, is a 31-kDa integral membrane protein expressed at the membrane of red blood cells (RBCs)[1,2]. As regards the mdb[3] which corresponds to the physical site of the chloride/bicarbonate exchange, the recent crystal structure of this functional domain[29] allowed the identification of the anion-binding position in AE1 and showed the proximity of natural mutations that lead to diseases[30]. AE1 transport activity was tested in that paper using ghosts derived from the RBCs of a patient presenting a hereditary stomatocytosis (HSt) with a normal expression of stomatin This pathology is associated with the G796R mutation in AE132 and, in accordance with the heterozygous status of the patient, RBC exhibited a 50% reduction of the AE1 activity using our functional assay. The availability of rare stomatin-deficient RBC samples from several patients presenting RBC membrane disorders and corresponding to previously described mutations in either RhAssociated Glycoprotein (RhAG) (F65S) for overhydrated stomatocytosis (OHSt)[33] or GLUT1 (G286D) for a cryohydrocytosis (CHC)[34], presented a unique opportunity to investigate the influence of stomatin in the AE1 activity
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