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In Vitro Transport Activity and Trafficking of MRP2/ABCC2 Polymorphic Variants

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Abstract

Purpose

Multidrug resistance-associated protein 2 (MRP2/ABCC2) is an efflux pump that removes drugs and chemicals from cells. We sought to characterize the expression, trafficking and in vitro activity of seven single nucleotide polymorphisms (SNPs) in the ABCC2 gene.

Methods

ABCC2 SNPs were generated using site-directed mutagenesis and stably expressed in Flp-In HEK293 cells, which allows targeted insertion of transgenes within the genome. Total and cell surface expression of MRP2 as well as accumulation of substrates (calcein AM and 5(6)-carboxy-2′,7′-dichlorofluorescein diacetate, CDCF) were quantified in cells or inverted membrane vesicles expressing wild-type (WT) or variant forms.

Results

The cell surface expression of the C-24T-, G1249A-, G3542T-, T3563A-, C3972T- and G4544A-MRP2 variants was similar to WT-MRP2. While expression was similar, transport of calcein AM was enhanced in cells expressing the G3542T-, T3563A-, C3972T-, and G4544A-MRP2 variants. By comparison, cells expressing the C2366T-MRP2 variant had 40–50% lower surface expression, which increased the accumulation of calcein AM up to 3-fold. Accumulation of CDCF in inverted membrane vesicles expressing the C2366T-MRP2 variant was also reduced by 50%. In addition, the G1249A-MRP2 variant had decreased transport of CDCF.

Conclusions

Taken together, these data demonstrate that genetic variability in the ABCC2 gene influences the in vitro expression, trafficking, and transport activity of MRP2.

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Abbreviations

BCA:

Bicinchoninic acid

CDCF:

5(6)-carboxy-2′,7′-dichlorofluorescein diacetate

EV:

Empty vector

Flp-In 293 cells:

Flp-In human embryonic kidney 293 cells

MRP2:

Multidrug resistance-associated protein 2

MSD:

Membrance-spanning domain

NBD:

Nucleotide-binding domain

RFU:

Relative fluorescence units

SNPs:

Single nucleotide polymorphisms

WT:

Wild-type

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ACKNOWLEDGMENTS AND DISCLOSURES

The authors would like to thank Drs. Bo Kong and Shaojun Yang for technical advice. This work was supported by the National Institutes of Health Institute of Diabetes and Digestive and Kidney Diseases [Grant DK080774, DK093903] and the National Institutes of Environmental Health Sciences [Grants ES020522, ES021800, ES005022].

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Authors and Affiliations

  1. Department of Pharmacology and Toxicology, Rutgers University Ernest Mario School of Pharmacy, 170 Frelinghuysen Road, Piscataway, New Jersey, 08854, USA

    Xia Wen & Lauren M. Aleksunes

  2. Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado, Aurora, Colorado, 80045, USA

    Melanie S. Joy

  3. Environmental and Occupational Health Sciences Institute, Rutgers University, Piscataway, New Jersey, 08854, USA

    Lauren M. Aleksunes

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  1. Xia Wen
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Correspondence to Lauren M. Aleksunes.

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Wen, X., Joy, M.S. & Aleksunes, L.M. In Vitro Transport Activity and Trafficking of MRP2/ABCC2 Polymorphic Variants. Pharm Res 34, 1637–1647 (2017). https://doi.org/10.1007/s11095-017-2160-0

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