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

Proc Natl Acad Sci U S A. 2020 Nov 24;117(47):29609-29617. doi: 10.1073/pnas.2016270117. Epub 2020 Nov 9.

Abstract

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.

Keywords: ABC transporter; P-glycoprotein; drug transport; mechanism; multidrug resistance.

Publication types

  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / metabolism*
  • ATP-Binding Cassette Transporters / metabolism
  • Amino Acid Substitution / physiology
  • Animals
  • Binding Sites / physiology
  • Biological Transport / physiology*
  • Cell Line
  • Cell Line, Tumor
  • Drug Resistance, Multiple / physiology*
  • Drug Resistance, Neoplasm / physiology
  • HeLa Cells
  • Humans
  • Insecta
  • Molecular Docking Simulation / methods
  • Pharmaceutical Preparations / metabolism*
  • Rhodamine 123 / metabolism
  • Substrate Specificity / physiology

Substances

  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • ATP-Binding Cassette Transporters
  • Pharmaceutical Preparations
  • Rhodamine 123