title: Rapid Cl⁻/HCO⁻₃exchange 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.
authors.affiliation: Inserm U665, Paris, France;
authors.forename: Etienne
authors.initials: E
authors.lastname: Frumence
authors.forename: Sandrine
authors.initials: S
authors.lastname: Genetet
authors.forename: Pierre
authors.initials: P
authors.lastname: Ripoche
authors.forename: Achille
authors.initials: A
authors.lastname: Iolascon
authors.forename: Immacolata
authors.initials: I
authors.lastname: Andolfo
authors.forename: Caroline
authors.initials: C
authors.lastname: Le Van Kim
authors.forename: Yves
authors.initials: Y
authors.lastname: Colin
authors.forename: Isabelle
authors.initials: I
authors.lastname: Mouro-Chanteloup
authors.forename: Claude
authors.initials: C
authors.lastname: Lopez
mesh_terms: D019943:Amino Acid Substitution; D000745:Anemia, Hemolytic, Congenital / Q000097:blood* / Q000235:genetics / Q000473:pathology; D001457:Anion Exchange Protein 1, Erythrocyte / Q000235:genetics / Q000378:metabolism*; D000838:Anions / Q000378:metabolism; D001639:Bicarbonates / Q000378:metabolism*; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism* / Q000473:pathology; D057809:HEK293 Cells; D006579:Heterozygote; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D001457:Anion Exchange Protein 1, Erythrocyte; D000838:Anions; D001639:Bicarbonates; D002712:Chlorides; D008565:Membrane Proteins; D011994:Recombinant Proteins
keywords: HEK293; anion exchanger 1; hereditary stomatocytosis; red blood cells; stopped-flow spectrofluorometry