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Organic anion transporter 2 transcript variant 1 shows broad ligand selectivity when expressed in multiple cell lines.

Hotchkiss AG, Berrigan L, Pelis RM - Front Pharmacol (2015)

Bottom Line: We cloned OAT2-tv1 and OAT2-tv2, but were unsuccessful at amplifying mRNA for OAT2-tv3 from human kidney.OAT2-tv1 trafficked to the plasma membrane of all three cell types, but OAT2-tv2 did not.Not surprising given its lack of plasma membrane expression, OAT2-tv2 failed to transport any of the organic solutes examined, including penciclovir.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Dalhousie University Halifax, NS, Canada.

ABSTRACT
Organic anion transporter 2 (OAT2) is likely important for renal and hepatic drug elimination. Three variants of the OAT2 peptide sequence have been described - OAT2 transcript variant 1 (OAT2-tv1), OAT2 transcript variant 2 (OAT2-tv2), and OAT2 transcript variant 3 (OAT2-tv3). Early studies helping to define the ligand selectivity of OAT2 failed to identify the variant used, and the studies used several heterologous expression systems. In preliminary studies using OAT2-tv1, we failed to observe transport of several previously identified substrates, leading us to speculate that ligand selectivity of OAT2 differs with variant and/or heterologous expression system. The purpose was to further investigate the ligand selectivity of the OAT2 variants expressed in multiple cell types. We cloned OAT2-tv1 and OAT2-tv2, but were unsuccessful at amplifying mRNA for OAT2-tv3 from human kidney. OAT2-tv1 and OAT2-tv2 were individually expressed in human embryonic kidney (HEK), Madin-Darby canine kidney (MDCK), or Chinese hamster ovary (CHO) cells. mRNA for OAT2-tv1 and OAT2-tv2 was demonstrated in each cell type transfected with the respective construct, indicating their expression. OAT2-tv1 trafficked to the plasma membrane of all three cell types, but OAT2-tv2 did not. OAT2-tv1 transported penciclovir in all three cell types, but failed to transport para-aminohippurate, succinate, glutarate, estrone-3-sulfate, paclitaxel or dehydroepiandrosterone sulfate - previously identified substrates of OAT2-tv2. Not surprising given its lack of plasma membrane expression, OAT2-tv2 failed to transport any of the organic solutes examined, including penciclovir. Penciclovir transport by OAT2-tv1 was sensitive to large (e.g., cyclosporine A) and small (e.g., allopurinol) organic compounds, as well as organic anions, cations and neutral compounds, highlighting the multiselectivity of OAT2-tv1. The potencies with which indomethacin, furosemide, cyclosporine A and cimetidine inhibited OAT2-tv1 are in good agreement with previous studies using this variant, but inconsistent with studies using OAT2 with an unidentified sequence. This study shows that organic molecules with diverse physicochemical properties interact with OAT2-tv1, making it a likely site of drug interactions. Many previously identified substrates of OAT2 are not transported by OAT2-tv1, suggesting that variant and/or expression system may contribute. Future work should establish the expression pattern and ligand selectivity of OAT2-tv3.

No MeSH data available.


mRNA expression (A) and cell surface protein expression (B) of Organic anion transporter 2 (OAT2)-tv1 (tv1) or OAT2-tv2 (tv2) in Chinese hamster ovary (CHO), human embryonic kidney (HEK), or Madin-Darby canine kidney (MDCK) cells stably transfected with the respective construct, or in parental cells (P). mRNA expression was determined by reverse transcription polymerase chain reaction (RT-PCR) using oligonucleotide primers against OAT2. Amplified products were separated on 1% agarose gels and stained with ethidium bromide. Sulfo-NHS-SS-biotin was used for cell surface biotinylation.
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Figure 1: mRNA expression (A) and cell surface protein expression (B) of Organic anion transporter 2 (OAT2)-tv1 (tv1) or OAT2-tv2 (tv2) in Chinese hamster ovary (CHO), human embryonic kidney (HEK), or Madin-Darby canine kidney (MDCK) cells stably transfected with the respective construct, or in parental cells (P). mRNA expression was determined by reverse transcription polymerase chain reaction (RT-PCR) using oligonucleotide primers against OAT2. Amplified products were separated on 1% agarose gels and stained with ethidium bromide. Sulfo-NHS-SS-biotin was used for cell surface biotinylation.

Mentions: Organic anion transporter 2-tv1 and OAT2-tv2 were individually transfected into CHO, HEK, and MDCK cells, and placed under selection pressure in order to establish stable cell lines. RT-PCR analyses revealed expression of both OAT2-tv1 and OAT2-tv2 at the mRNA level in all three cell types, indicating successful stable expression of both constructs (Figure 1A). However, cell surface biotinylation experiments showed that only OAT2-tv1 was present at the cell surface (Figure 1B), pointing to a potential trafficking defect associated with OAT2-tv2.


Organic anion transporter 2 transcript variant 1 shows broad ligand selectivity when expressed in multiple cell lines.

Hotchkiss AG, Berrigan L, Pelis RM - Front Pharmacol (2015)

mRNA expression (A) and cell surface protein expression (B) of Organic anion transporter 2 (OAT2)-tv1 (tv1) or OAT2-tv2 (tv2) in Chinese hamster ovary (CHO), human embryonic kidney (HEK), or Madin-Darby canine kidney (MDCK) cells stably transfected with the respective construct, or in parental cells (P). mRNA expression was determined by reverse transcription polymerase chain reaction (RT-PCR) using oligonucleotide primers against OAT2. Amplified products were separated on 1% agarose gels and stained with ethidium bromide. Sulfo-NHS-SS-biotin was used for cell surface biotinylation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4594013&req=5

Figure 1: mRNA expression (A) and cell surface protein expression (B) of Organic anion transporter 2 (OAT2)-tv1 (tv1) or OAT2-tv2 (tv2) in Chinese hamster ovary (CHO), human embryonic kidney (HEK), or Madin-Darby canine kidney (MDCK) cells stably transfected with the respective construct, or in parental cells (P). mRNA expression was determined by reverse transcription polymerase chain reaction (RT-PCR) using oligonucleotide primers against OAT2. Amplified products were separated on 1% agarose gels and stained with ethidium bromide. Sulfo-NHS-SS-biotin was used for cell surface biotinylation.
Mentions: Organic anion transporter 2-tv1 and OAT2-tv2 were individually transfected into CHO, HEK, and MDCK cells, and placed under selection pressure in order to establish stable cell lines. RT-PCR analyses revealed expression of both OAT2-tv1 and OAT2-tv2 at the mRNA level in all three cell types, indicating successful stable expression of both constructs (Figure 1A). However, cell surface biotinylation experiments showed that only OAT2-tv1 was present at the cell surface (Figure 1B), pointing to a potential trafficking defect associated with OAT2-tv2.

Bottom Line: We cloned OAT2-tv1 and OAT2-tv2, but were unsuccessful at amplifying mRNA for OAT2-tv3 from human kidney.OAT2-tv1 trafficked to the plasma membrane of all three cell types, but OAT2-tv2 did not.Not surprising given its lack of plasma membrane expression, OAT2-tv2 failed to transport any of the organic solutes examined, including penciclovir.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Dalhousie University Halifax, NS, Canada.

ABSTRACT
Organic anion transporter 2 (OAT2) is likely important for renal and hepatic drug elimination. Three variants of the OAT2 peptide sequence have been described - OAT2 transcript variant 1 (OAT2-tv1), OAT2 transcript variant 2 (OAT2-tv2), and OAT2 transcript variant 3 (OAT2-tv3). Early studies helping to define the ligand selectivity of OAT2 failed to identify the variant used, and the studies used several heterologous expression systems. In preliminary studies using OAT2-tv1, we failed to observe transport of several previously identified substrates, leading us to speculate that ligand selectivity of OAT2 differs with variant and/or heterologous expression system. The purpose was to further investigate the ligand selectivity of the OAT2 variants expressed in multiple cell types. We cloned OAT2-tv1 and OAT2-tv2, but were unsuccessful at amplifying mRNA for OAT2-tv3 from human kidney. OAT2-tv1 and OAT2-tv2 were individually expressed in human embryonic kidney (HEK), Madin-Darby canine kidney (MDCK), or Chinese hamster ovary (CHO) cells. mRNA for OAT2-tv1 and OAT2-tv2 was demonstrated in each cell type transfected with the respective construct, indicating their expression. OAT2-tv1 trafficked to the plasma membrane of all three cell types, but OAT2-tv2 did not. OAT2-tv1 transported penciclovir in all three cell types, but failed to transport para-aminohippurate, succinate, glutarate, estrone-3-sulfate, paclitaxel or dehydroepiandrosterone sulfate - previously identified substrates of OAT2-tv2. Not surprising given its lack of plasma membrane expression, OAT2-tv2 failed to transport any of the organic solutes examined, including penciclovir. Penciclovir transport by OAT2-tv1 was sensitive to large (e.g., cyclosporine A) and small (e.g., allopurinol) organic compounds, as well as organic anions, cations and neutral compounds, highlighting the multiselectivity of OAT2-tv1. The potencies with which indomethacin, furosemide, cyclosporine A and cimetidine inhibited OAT2-tv1 are in good agreement with previous studies using this variant, but inconsistent with studies using OAT2 with an unidentified sequence. This study shows that organic molecules with diverse physicochemical properties interact with OAT2-tv1, making it a likely site of drug interactions. Many previously identified substrates of OAT2 are not transported by OAT2-tv1, suggesting that variant and/or expression system may contribute. Future work should establish the expression pattern and ligand selectivity of OAT2-tv3.

No MeSH data available.