Inducible expression of erythrocyte band 3 protein

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Abstract
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 additional36Cl 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 of36Cl and35SO4had the characteristics of hAE1-mediated transport in erythrocytes: 1) inhibition by 250 μM DNDS, 2) activation of36Cl efflux by external Cl with a concentration producing half-maximal effect of 4.8 mM, 3) activation of36Cl efflux by external anions that was selective in the order NO3= Cl > Br > I, and 4) activation of35SO4influx 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.

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