Abstract
MDR1 (multidrug resistance) P-glycoprotein (Pgp; ABCB1) decreases intracellular concentrations of structurally diverse drugs. Although Pgp is generally thought to be an efflux transporter, the mechanism of action remains elusive. To determine whether Pgp confers drug resistance through changes in transmembrane potential (E(m)) or ion conductance, we studied electrical currents and drug transport in Pgp-negative MCF-7 cells and MCF-7/MDR1 stable transfectants that were established and maintained without chemotherapeutic drugs. Although E(m) and total membrane conductance did not differ between MCF-7 and MCF-7/MDR1 cells, Pgp reduced unidirectional influx and steady-state cellular content of Tc-Sestamibi, a substrate for MDR1 Pgp, without affecting unidirectional efflux of substrate from cells. Depolarization of membrane potentials with various concentrations of extracellular K(+) in the presence of valinomycin did not inhibit the ability of Pgp to reduce intracellular concentration of Tc-Sestamibi, strongly suggesting that the drug transport activity of MDR1 Pgp is independent of changes in E(m) or total ion conductance. Tetraphenyl borate, a lipophilic anion, enhanced unidirectional influx of Tc-Sestamibi to a greater extent in MCF-7/MDR1 cells than in control cells, suggesting that Pgp may, directly or indirectly, increase the positive dipole potential within the plasma membrane bilayer. Overall, these data demonstrate that changes in E(m) or macroscopic conductance are not coupled with function of Pgp in multidrug resistance. The dominant effect of MDR1 Pgp in this system is reduction of drug influx, possibly through an increase in intramembranous dipole potential.
Highlights
MDR1 P-glycoprotein (Pgp),1 a member of the ATP-binding cassette family of membrane transporters, decreases intracellular concentrations of structurally diverse compounds, many of which are hydrophobic and cationic
Characterization of MCF-7/MDR1 Cells—Because cell lines derived from stepwise selection with MDR drugs may have multiple mechanisms of drug resistance [22], we stably transfected MCF-7 cells with MDR1 and established clonal cell lines that express Pgp without use of MDR drugs
As determined by differences in accumulation of Tc-Sestamibi between MCF-7/MDR1 and control cells, function of Pgp was stable over at least 7 months of continuous culture. These matched cell lines provided an appropriate system for biophysical analysis of MDR1 Pgp without the confounding effects of prior exposure to MDR drugs
Summary
P-glycoprotein; MDR, multidrug resistance; TPB, tetraphenyl borate; MEBSS, modified Earle’s balanced salt solution; [Kϩ]i, intracellular Kϩ concentration. Pgp has been hypothesized to be a pump with multiple binding sites for different drugs [2], a translocase for lipids [3], or a modifier of vesicular trafficking [4] Another model suggests that MDR1 Pgp indirectly alters partitioning of substrates within cells through effects on transmembrane potential (Em), intracellular pH, and/or surface potentials and does not directly transport drugs [5]. Tc-Sestamibi has no titratable proton, making accumulation within cells independent of intracellular pH [8, 12] Using this system, we determined the effects of MDR1 Pgp on substrate content under steady-state and unidirectional influx or efflux conditions. As proposed over 25 years ago [13], we found that the dominant effect of Pgp is to establish a permeability barrier, limiting unidirectional influx of TcSestamibi without affecting Em
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