Reversing the direction of drug transport mediated by the human multidrug transporter P-glycoprotein.

P-glycoprotein (P-gp), also known as ABCB1, is a cell membrane transporter that mediates the efflux of chemically dissimilar amphipathic drugs and confers resistance to chemotherapy in most cancers. Homologous transmembrane helices (TMHs) 6 and 12 of human P-gp connect the transmembrane domains with its nucleotide-binding domains, and several residues in these TMHs contribute to the drug-binding pocket. To investigate the role of these helices in the transport function of P-gp, we substituted a group of 14 conserved residues (seven in both TMHs 6 and 12) with alanine and generated a mutant termed 14A. Although the 14A mutant lost the ability to pump most of the substrates tested out of cancer cells, surprisingly, it acquired a new function. It was able to import four substrates, including rhodamine 123 (Rh123) and the taxol derivative flutax-1. Similar to the efflux function of wild-type P-gp, we found that uptake by the 14A mutant is ATP hydrolysis-, substrate concentration-, and time-dependent. Consistent with the uptake function, the mutant P-gp also hypersensitizes HeLa cells to Rh123 by 2- to 2.5-fold. Further mutagenesis identified residues from both TMHs 6 and 12 that synergistically form a switch in the central region of the two helices that governs whether a given substrate is pumped out of or into the cell. Transforming P-gp or an ABC drug exporter from an efflux transporter into a drug uptake pump would constitute a paradigm shift in efforts to overcome cancer drug resistance.