Relationships between rhodamine 123 transport, cell volume, and ion- channel function of P-glycoprotein

The P-glycoprotein (Pgp), a plasma membrane protein overexpressed in multidrug-resistant tumor cells, is thought to be both an ATPase that actively exports cytotoxic drugs and a Cl^- channel activated by cell swelling. The partial reversal of multidrug resistance by Cl^- transport blockers suggests a possible role for Cl^- in Pgp-mediated drug transport. We used multidrug-resistant Chinese hamster fibroblasts and human breast cancer cells expressing Pgp to study the roles of Cl^- (and also Na⁺ and HCO₃/^- /CO₂) on Pgp-mediated efflux of the fluorescent dye rhodamine 123 (R123). In Pgp-expressing Chinese hamster fibroblasts, exposed to isosmotic solutions, the unidirectional efflux of R123 was not measurably changed by a ~60-min removal of Cl^- (or by exposure to Na⁺-free, or nominally HCO₃/^-/CO₂- free medium); short term (2-3 min) ion substitutions were also ineffective. In human breast cancer cells transfected with human mdr1 cDNA, hyposmotic solutions activated a Cl^- current but had no effect on the Pgp-mediated unidirectional efflux of R123. Additionally, in human breast cancer cells, the intracellular presence of R123 did not prevent activation of the Cl^- current by hyposmotic solution. The lack of detectable effect of removal of Cl^-, Na⁺, or HCO₃/^- on Pgp-mediated R123 transport rules out direct coupling between substrate transport and transport of either of these ions by Pgp. The persistence of Pgp-mediated R123 efflux in osmotically swollen cells indicates that activation of the Pgp-associated Cl^- current does not hinder the Pgp pump function. The lack of effect of R123 on swelling-activated Cl^- current denotes that Pgp-mediated transport of organic substrates and Pgp- associated Cl^- currents can occur at the same time in a single cell. These results underscore the dissociation between Pgp-mediated active drug transport and electrodiffusive Cl^- transport.