Dataset Information

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

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.

SUBMITTER: Sajid A

PROVIDER: S-EPMC7703596 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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

Sajid Andaleeb A Lusvarghi Sabrina S Murakami Megumi M Chufan Eduardo E EE Abel Biebele B Gottesman Michael M MM Durell Stewart R SR Ambudkar Suresh V SV

Proceedings of the National Academy of Sciences of the United States of America 20201109 47

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 ...[more]

PMID: 33168729

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Project description:P-Glycoprotein, the plasma membrane protein responsible for the multidrug resistance of some tumour cells, is an active transporter of a number of structurally unrelated hydrophobic drugs. We have characterized the modulation of its ATPase activity by a multidrug-resistance-related cytotoxic drug, vinblastine, and different multidrug-resistance-reversing agents, verapamil and the dihydropyridines nicardipine, nimodipine, nitrendipine, nifedipine and azidopine. P-Glycoprotein ATPase activity was measured by using native membrane vesicles containing large amounts of P-glycoprotein, prepared from the highly multidrug-resistant lung fibroblasts DC-3F/ADX. P-Glycoprotein ATPase is activated by verapamil and by nicardipine but not by vinblastine. Among the five dihydropyridines tested, the higher the hydrophobicity, the higher was the activation factor with respect to the basal activity and the lower was the half-maximal activating concentration. The vinblastine-specific binding on P-glycoprotein is reported by the inhibitions of the verapamil- and the nicardipine-stimulated ATPase. These inhibitions are purely competitive, which means that the bindings of vinblastine and verapamil, or vinblastine and nicardipine, on P-glycoprotein are mutually exclusive. In contrast, verapamil and nicardipine display mutually non-competitive interactions. This demonstrates the existence of two distinct specific sites for these two P-glycoprotein modulators on which they can bind simultaneously and separately to the vinblastine site. The nicardipine-stimulated ATPase activity in the presence of the other dihydropyridines shows mixed-type inhibitions. These dihydropyridines have thus different binding sites that interact mutually to decrease their respective, separately determined affinities. This could be due to steric constraints between sites close to each other. This is supported by the observation that vinblastine binding is not mutually exclusive with nifedipine or nitrendipine binding, whereas it is mutually exclusive with nicardipine. Moreover, verapamil binding also interacts with the five dihydropyridines by mixed inhibitions, with different destabilization factors. On the whole our enzymic data show that P-glycoprotein has distinct but interacting binding sites for various modulators of its ATPase function.

Dataset Information

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

Publications

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

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