Decrease of P-glycoprotein activity in K562/ADR cells by MbetaCD and filipin and lack of effect induced by cholesterol oxidase indicate that this transporter is not located in rafts

Abstract

The effect of low-density membrane domains on function of the plasma membrane transporter P-glycoprotéine (P-gp), involved in multidrug resistance (MDR) phenotype, has been investigated in K562/ADR cells. To this end we reversibly altered the cholesterol content of K562/ADR cells by using methyl-beta-cyclodextrin as a cholesterol chelator and conversely we repleted them through incubation with cholesterol in culture medium. We also used the cholesterol-binding fluorochrome filipin and cholesterol oxidase. Our data show that either cholesterol depletion or complex formation with filipin resulted in a strong decrease of P-gp activity. However, when cells were incubated with cholesterol oxidase that are known to disrupt rafts, no modification of the P-gp activity was observed. In addition, using a free-detergent methodology to separate by ultracentrifugation, "light," "heavy," and "extra heavy" fractions we show that no P-gp is found in the "light" fraction where rafts are usually detected. Altogether, our data strongly suggest that, in this cell line, P-gp is not localized in rafts.