{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T21:39:58Z","timestamp":1791322798844,"version":"4.3.1"},"reference-count":33,"publisher":"American Physiological Society","issue":"6","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["American Journal of Physiology-Cell Physiology"],"published-print":{"date-parts":[[2013,9,15]]},"abstract":"<jats:p> 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. <\/jats:p>","DOI":"10.1152\/ajpcell.00142.2013","type":"journal-article","created":{"date-parts":[[2013,7,11]],"date-time":"2013-07-11T04:51:52Z","timestamp":1373518312000},"page":"C654-C662","source":"Crossref","is-referenced-by-count":13,"title":["Rapid Cl<sup>\u2212<\/sup>\/HCO<sub>3<\/sub><sup>\u2212<\/sup> exchange kinetics of AE1 in HEK293 cells and hereditary stomatocytosis red blood cells"],"prefix":"10.1152","volume":"305","author":[{"given":"Etienne","family":"Frumence","sequence":"first","affiliation":[{"name":"Inserm U665, Paris, France;"},{"name":"Universit\u00e9 Paris Diderot, Sorbonne Paris Cit\u00e9, UMR-S665, Paris, France;"},{"name":"Institut National de la Transfusion Sanguine, Paris, France;"},{"name":"Laboratoire d'Excellence GR-Ex., Paris, France;"},{"name":"Universit\u00e9 de la R\u00e9union, Saint-Denis, France; 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