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Biochemistry. 2016 Feb 23;55(7):1010-23. doi: 10.1021/acs.biochem.5b01064. Epub 2016 Feb 10.

Human-Mouse Chimeras with Normal Expression and Function Reveal That Major Domain Swapping Is Tolerated by P-Glycoprotein (ABCB1).

Author information

1
Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health , Bethesda, Maryland 20892, United States.
2
Gene Medicine Research Group, Nuffield Department of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford , Oxford OX3 9DU, U.K.
3
Laboratory of Pathology, National Cancer Institute, National Institutes of Health , Bethesda, Maryland 20892, United States.

Abstract

The efflux transporter P-glycoprotein (P-gp) plays a vital role in the transport of molecules across cell membranes and has been shown to interact with a panoply of functionally and structurally unrelated compounds. How human P-gp interacts with this large number of drugs has not been well understood, although structural flexibility has been implicated. To gain insight into this transporter's broad substrate specificity and to assess its ability to accommodate a variety of molecular and structural changes, we generated human-mouse P-gp chimeras by the exchange of homologous transmembrane and nucleotide-binding domains. High-level expression of these chimeras by BacMam- and baculovirus-mediated transduction in mammalian (HeLa) and insect cells, respectively, was achieved. There were no detectable differences between wild-type and chimeric P-gp in terms of cell surface expression, ability to efflux the P-gp substrates rhodamine 123, calcein-AM, and JC-1, or to be inhibited by the substrate cyclosporine A and the inhibitors tariquidar and elacridar. Additionally, expression of chimeric P-gp was able to confer a paclitaxel-resistant phenotype to HeLa cells characteristic of P-gp-mediated drug resistance. P-gp ATPase assays and photo-cross-linking with [(125)I]iodoarylazidoprazosin confirmed that transport and biochemical properties of P-gp chimeras were similar to those of wild-type P-gp, although differences in drug binding were detected when human and mouse transmembrane domains were combined. Overall, chimeras with one or two mouse P-gp domains were deemed functionally equivalent to human wild-type P-gp, demonstrating the ability of human P-gp to tolerate major structural changes.

PMID:
26820614
PMCID:
PMC5348299
DOI:
10.1021/acs.biochem.5b01064
[Indexed for MEDLINE]
Free PMC Article

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