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

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.

Keywords: ABCC2; MRP2; SNP; transporter; variant.

Figure 1. Localization of variants in a predicted topology model of the human MRP2 transporter

The full-length model of the MRP2 protein was generated using the open source tool Protter (http://wlab.ethz.ch/protter/start/). The C-24T variant is found in the 5′-untranslated region of the ABCC2 promoter and not shown in the figure. MSD: membrane-spanning domain; NBD: nucleotide-binding domain.

Figure 2. Expression and activity of the MRP2 transporter using the Flp-In system

Flp-In 293 cells were stably transfected with empty vector (EV) or full-length human ABCC2 plasmids (wild-type, WT). Protein expression of MRP2 in cell lysates (A) and biotinylated membrane fractions (B) was detected by Western blot analysis. β-ACTIN and Na⁺/K⁺ ATPase were used as loading controls for total and cell surface expression, respectively. (C) EV and WT-MRP2 cells were treated with calcein AM (0.5 μM) for 30 min (uptake period), washed, and then incubated with fresh culture media for 60 min (efflux period). Intracellular accumulation of calcein AM was determined at the end of the efflux period using a fluorescent cell counter. Data are expressed as relative fluorescence units (RFU) and presented as mean ± SE (n=4). Asterisks (*) represent statistically significant differences (p < 0.05) compared to EV.

Figure 3. Transport of calcein AM in MRP2 wild-type and variant cells

Flp-In 293 cells stably expressing ABCC2 wild-type (WT) or variant plasmids were exposed to calcein AM for 30 min (uptake period), washed, and then incubated with fresh culture medium for 60 min (efflux period). Intracellular calcein AM accumulation was determined at the end of the efflux period using a fluorescent counter. (A) WT-MRP2 and variant cells were exposed to calcein AM (0.5 μM). (B) WT-MRP2 and variants were exposed to calcein AM (0.5, 2, and 10 μM). Data are expressed as relative fluorescence units (RFU) and presented as mean ± SE (n=4-6). Asterisks (*) represent statistically significant differences (p < 0.05) compared to WT.

Figure 4. Protein expression and localization of MRP2 in wild-type and variant cells

Flp-In 293 cells were stably transfected with ABCC2 wild-type (WT) or variant plasmids. Protein expression of MRP2 in cell lysates (A) and biotinylated membrane fractions (B) was detected by Western blot analysis. β-ACTIN and Na⁺/K⁺ ATPase were used as loading controls for total and cell surface expression, respectively. Data are expressed as relative protein expression and presented as mean ± SE (n=3). Asterisks (*) represent statistically significant differences (p < 0.05) compared to WT.

Figure 5. Localization of MRP2 protein in wild-type and variant cells

Flp-In 293 cells were stably transfected with ABCC2 wild-type (WT) or variant plasmids. Staining of MRP2 protein in WT, G1249A, C2366T, G3542T, T3563A, C3972T, and G4544A variant cells was performed using indirect immunofluorescence analysis. Cells were incubated with an anti-MRP2 antibody (M₂III-6) (green). Images are shown at 63× magnification.

Figure 6. Expression of MRP2 protein and CDCF transport in membrane vesicles isolated from wild-type and variant cells

Flp-In 293 cells were stably transfected with ABCC2 wild-type (WT) or variant plasmids. (A) Protein expression of MRP2 in membrane vesicles isolated from cells was detected by Western blot analysis. Na⁺/K⁺ ATPase was used as a loading control. (B) CDCF (10 μM) transport in membrane vesicles isolated from cells expressing WT-MRP2 and variants. Transport of CDCF (10 μM) in empty vector-transfected vesicles was 4.2 pmol/min/mg protein. (C) CDCF (2.5, 10 and 50 μM) transport in membrane vesicles isolated from cells expressing WT-MRP2 and variants G1249A, C2366T, G3542T. Transport was conducted using vesicles (50 μg) incubated with CDCF at 37°C for 10 min. Data are presented as mean ± SE (n=3-6). Asterisks (*) represent statistically significant differences (p < 0.05) compared to WT.