Rapid Cl⁻/HCO₃⁻ exchange kinetics of AE1 in HEK293 cells and hereditary stomatocytosis red blood cells

Abstract

Anion exchanger 1 (AE1) or band 3 is a membrane protein responsible for the rapid exchange of chloride for bicarbonate across the red blood cell membrane. Nine mutations leading to single amino-acid substitutions in the transmembrane domain of AE1 are associated with dominant hereditary stomatocytosis, monovalent cation leaks, and reduced anion exchange activity. We set up a stopped-flow spectrofluorometry assay coupled with flow cytometry to investigate the anion transport and membrane expression characteristics of wild-type recombinant AE1 in HEK293 cells, using an inducible expression system. Likewise, study of three stomatocytosis-associated mutations (R730C, E758K, and G796R), allowed the validation of our method. Measurement of the rapid and specific chloride/bicarbonate exchange by surface expressed AE1 showed that E758K mutant was fully active compared with wild-type (WT) AE1, whereas R730C and G796R mutants were inactive, reinforcing previously reported data on other experimental models. Stopped-flow analysis of AE1 transport activity in red blood cell ghost preparations revealed a 50% reduction of G796R compared with WT AE1 corresponding to a loss of function of the G796R mutated protein, in accordance with the heterozygous status of the AE1 variant patients. In conclusion, stopped-flow led to measurement of rapid transport kinetics using the natural substrate for AE1 and, conjugated with flow cytometry, allowed a reliable correlation of chloride/bicarbonate exchange to surface expression of AE1, both in recombinant cells and ghosts and therefore a fine comparison of function between different stomatocytosis samples. This technical approach thus provides significant improvements in anion exchange analysis in red blood cells.

Evidence of a Structural and Functional Ammonium Transporter RhBG·Anion Exchanger 1·Ankyrin-G Complex in Kidney Epithelial Cells

“…T-REx (Invitrogen) is a Tet-regulated mammalian expression system based on the binding of tetracycline to a Tet repressor and derepression of the promoter controlling the expression of the gene of interest. The establishment of a HEK293 cell line inducible for expression of recombinant kAE1 protein was achieved as described previously for recombinant eAE1 expression (19). In kAE1-RhBG coexpression experiments, HEK293 stable transfectants (pcDNA6/TR ϩ pcDNA4/TO-kAE1) were cotransfected with pcDNA3-RhBG vector (1) and selected using 0.8 mg/ml neomycin (Geneticin; Invitrogen).…”

“…For immunofluorescence experiments, HEK293 stable transfectants were cultured on poly-L-lysine coverslips (BD Biosciences). Cells were induced or not for kAE1 expression, fixed, permeabilized or left untreated, immunostained with appropriate primary and Alexa Fluor secondary antibodies as described previously (19), and examined by confocal microscopy using a Zeiss LSM700 inverted confocal microscope equipped with a ϫ100 oil immersion objective with a numerical aperture of 1.4. MDCK cells grown to subconfluency on poly-L-lysine coverslips were transiently transfected with pcDNA4/TO-kAE1 vector using TurboFect transfection reagent (Thermo Scientific, St. Leon-Rot, Germany), cultured for 4 days to allow polarization, immunostained with appropriate primary and Alexa Fluor secondary antibodies, and analyzed by confocal microscopy.…”

“…Stopped Flow Analysis-Ammonium transport (RhBG) and Cl Ϫ /HCO 3 Ϫ exchange (kAE1) functions were determined by stopped flow spectrofluorometry analysis using the fluorescent pH-sensitive probe BCECF-AM (10 M) at pH 7.2, essentially as described previously (10,19). Briefly, parental and transfected HEK293 cells were exposed either to an outwardly directed 20 mEq NH 4 ϩ at 15°C (ammonium transport) or to inwardly directed 10 meq HCO 3 Ϫ /CO 2 and outwardly directed 67.5 meq Cl Ϫ gradients at 30°C (Cl Ϫ /HCO 3 Ϫ exchange).…”

“…HEK293 cells, lacking endogenous expression of kAE1 (22), were stably transfected with wild-type or mutant kAE1 cDNA using the tetracycline-inducible expression system previously described (19) to circumvent the loss of long term expression of AE1 protein (23,24). Flow cytometry experiments using BRIC6 antibody revealed wild-type kAE1 at the surface of tetracyclineinduced transfected cells ( Fig.…”

“…Cl Ϫ /HCO 3 Ϫ Exchange Activity of Recombinant kAE1 in HEK293 Cells-Recently, we described the measurement of the rapid and specific Cl Ϫ /HCO 3 Ϫ exchange by stably expressed eAE1 in HEK293 cells, using stopped flow spectrofluorometry (19). Likewise, we determined here the Cl Ϫ /HCO 3 Ϫ exchange capacity of recombinant wild-type kAE1 or delABS mutant by this methodology, using BCECF-AM and in the presence of inwardly directed 10 meq HCO 3 Ϫ /CO 2 and outwardly directed 67.5 meq Cl Ϫ gradients at 30°C and pH 7.2.…”