Inducible expression of erythrocyte band 3 protein

A permanent cell line with inducible expression of the human anion exchange r protein I (hAE1) was constructed in a derivative of human embryonic kidne y cells (HEK-293). In the absence of the inducer, muristerone A, the new ce ll line had no detectable hAE1 protein by Western analysis or additional Cl -36 flux. Increasing dose and incubation time with muristerone A increased the amount of protein (both unglycosylated and glycosylated). The 4,4'-dini trostilbene-2,2'-disulfonate (DNDS)-inhibitable rapid Cl exchange flux was increased up to 40-fold in induced cells compared with noninduced cells. Th ere was no DNDS-inhibitable rapid flux component in noninduced cells. This result demonstrates inducible expression of a new rapid Cl transport pathwa y that is DNDS sensitive. The additional transport of Cl-36 and (SO4)-S-35 had the characteristics of hAE1-mediated transport in erythrocytes: I) inhi bition by 250 mu M DNDS, 2) activation of Cl-36 efflux by external Cl with a concentration producing half-maximal effect of 4.8 mM, 3) activation of C l-36 efflux by external anions that was selective in the order NO3 = CI > B r > I, and 4) activation of (SO4)-S-35 influx by external protons. Under th e assumption that the turnover numbers of hAE1 were the same as in erythroc ytes, there was good agreement (+/-3-fold) between the number of copies of glycosylated hAE1 and the induced tracer fluxes. This is the first expressi on of hAE1 in a mammalian system to track the kinetic characteristics of th e native protein.