Inducible expression of erythrocyte band 3 protein

Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322-3110 A permanent cell line with inducible expression of the human anion exchanger protein 1 (hAE1) was constructed in a derivative of human embryonic kidney cells (HEK-293). In the absence of the inducer, muristerone A, the new cell line had no detectable hAE1 protein by Western analysis or additional 36 Cl flux. Increasing dose and incubation time with muristerone A increased the amount of protein (both unglycosylated and glycosylated). The 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS)-inhibitable rapid Cl exchange flux was increased up to 40-fold in induced cells compared with noninduced cells. There was no DNDS-inhibitable rapid flux component in noninduced cells. This result demonstrates inducible expression of a new rapid Cl transport pathway that is DNDS sensitive. The additional transport of 36 Cl and 35 SO 4 had the characteristics of hAE1-mediated transport in erythrocytes: 1 ) inhibition by 250 µM DNDS, 2 ) activation of 36 Cl efflux by external Cl with a concentration producing half-maximal effect of 4.8 mM, 3 ) activation of 36 Cl efflux by external anions that was selective in the order NO 3 = Cl > Br > I, and 4 ) activation of 35 SO 4 influx by external protons. Under the assumption that the turnover numbers of hAE1 were the same as in erythrocytes, there was good agreement (±3-fold) between the number of copies of glycosylated hAE1 and the induced tracer fluxes. This is the first expression of hAE1 in a mammalian system to track the kinetic characteristics of the native protein. anion exchanger 1; chloride transport; ecdysone receptor; HEK-293 cells