The flounder organic anion transporter fOat has sequence, function, and substrate specificity similarity to both mammalian Oat1 and Oat3

Abstract

The flounder renal organic anion transporter (fOat) has substantial sequence homology to mammalian basolateral organic anion transporter orthologs (OAT1/Oat1 and OAT3/Oat3), suggesting that fOat may have functional properties of both mammalian forms. We therefore compared uptake of various substrates by rat Oat1 and Oat3 and human OAT1 and OAT3 with the fOat clone expressed in Xenopus oocytes. These data confirm that estrone sulfate is an excellent substrate for mammalian OAT3/Oat3 transporters but not for OAT1/Oat1 transporters. In contrast, 2,4-dichlorophenoxyacetic acid and adefovir are better transported by mammalian OAT1/Oat1 than by the OAT3/Oat3 clones. All three substrates were well transported by fOat-expressing Xenopus oocytes. fOat K(m) values were comparable to those obtained for mammalian OAT/Oat1/3 clones. We also characterized the ability of these substrates to inhibit uptake of the fluorescent substrate fluorescein in intact teleost proximal tubules isolated from the winter flounder (Pseudopleuronectes americanus) and killifish (Fundulus heteroclitus). The rank order of the IC(50) values for inhibition of cellular fluorescein accumulation was similar to that for the K(m) values obtained in fOat-expressing oocytes, suggesting that fOat may be the primary teleost renal basolateral Oat. Assessment of the zebrafish (Danio rerio) genome indicated the presence of a single Oat (zfOat) with similarity to both mammalian OAT1/Oat1 and OAT3/Oat3. The puffer fish (Takifugu rubripes) also has an Oat (pfOat) similar to mammalian OAT1/Oat1 and OAT3/Oat3 members. Furthermore, phylogenetic analyses argue that the teleost Oat1/3-like genes diverged from a common ancestral gene in advance of the divergence of the mammalian OAT1/Oat1, OAT3/Oat3, and, possibly, Oat6 genes.

Fig. 1

Uptake of estrone sulfate (ES), adefovir, and 2,4-dichlorophenoxyacetic acid (2,4-D) by Xenopus oocytes expressing mammalian OAT1/Oat1, OAT3/Oat3, and flounder renal organic anion transporter (fOat) cDNAs. Concentrations tested were 1 μM ES, 30 μM adefovir and 10 μM 2,4-D; h, human; r, rat. fOat 2,4-D data were generated at 50 μM, and its uptake has been divided by 5 to allow expression on the same scale as mammalian OAT/Oat data. Fold stimulation over the water controls is shown above each bar. Bars reflect the means ± SE of 5–10 replicates from a representative experiment repeated in oocytes from 3 animals. Data are presented as mediated uptake (i.e., pmol · oocyte⁻¹ · 20 min⁻¹ after subtraction of mean uptake by water-injected control oocytes).

Fig. 2

Kinetics of ES and adefovir uptake by fOat. A 20-min time period was used. Mediated uptake (□) was calculated by subtracting the mean uptake in water-injected oocytes (▴) from the mean uptake in fOat-injected oocytes (●). Points reflect the means ± SE of 7–10 replicates from a representative experiment repeated in oocytes from 4 animals. Responses in fOat-expressing oocytes were compared with those in water-injected (control) oocytes using unpaired nonparametric (Mann-Whitney) t-test (*P < 0.05).

Fig. 3

Fluorescein accumulation by fOat-expressing oocytes. A: noninjected oocytes exposed to 5 μM fluorescein showed no accumulation after at least 1.5 h of incubation. Fl, fluorescein. B: uptake of 5 μM fluorescein in fOat-injected oocytes as demonstrated by fluorescein fluorescence. C: incubation of fOat-injected oocytes with 200 μM probenecid, an organic anion transporter inhibitor, prevented the uptake of 5 μM fluorescein. Images are representative of 4 oocytes (bar, 200 μm). Pro, probenecid.

Fig. 4

Inhibition of cellular and luminal fluorescein (2 μM) accumulation in isolated winter flounder proximal tubules by ES and cimetidine. Fluorescence intensities were normalized to the untreated control cells. Bars reflect the pooled mean ± SE of 5–17 tubule replicates observed in 2–3 fish. Responses were normalized to the fluorescence intensity in cells of control tubules (100%). Responses were analyzed using unpaired nonparametric (Mann-Whitney) t-test, comparing the test concentrations to control (no inhibitor) for cell and lumen conditions (*P < 0.05).

Fig. 5

Inhibition of cellular (●) and luminal (□) fluorescein (1 μM) accumulation in isolated killifish proximal tubules by ES (A), cimetidine (B), and 2,4-D (C). Points reflect the pooled mean (includes 2 animals) ± SE of 6 –12 tubule replicates from a representative experiment repeated 2–3 times. Graphs at left were analyzed using unpaired nonparametric (Mann-Whitney) t-test, comparing the test concentrations to control (no inhibitor) for cell and lumen conditions (*P < 0.05). Graphs at right were analyzed using paired nonparametric (Wilcoxon) t-test, comparing responses at each concentration for the cellular and luminal components (*P < 0.05). RFU, relative fluorescence units.

Fig. 6

Amino acid sequence comparison of winter flounder (fOat), zebrafish (zfOat), and puffer fish (pfOat) with mammalian OAT1 and OAT3 representatives. Only the lineage-specific motifs are shown. Amino acids that differ from the motifs are in bold type.

Fig. 7

Phylogenetic analysis depicting divergence among teleost and mammalian organic anion transporters. Representatives of teleost Oat1/3-like proteins, mammalian OAT members 1– 6, and URAT1 were aligned with their teleost counterparts. Teleost Oat names are in bold type, whereas teleost Oat1/3-like proteins are designated in bold italic type. NCBI accession numbers are as follows: CAB09724 (fOat), SINFRUP00000071613 (pfOat), NP_996960 (zfOat), AAH95733 (zfOatX), NP_004781 (hOAT1), CAB62587 (rbOat1), NP_032792 (mOat1), NP_058920 (rOat1), NP_001001261 (pOat1), NP_445989 (rOat2), NP_659105 (mOat2), AAZ79452 (rbOat2), NP_006663 (hOAT2), XP_696026 (zfOat2), CAD34035 (rbOat3), NP_004245 (hOAT3), NP_999620 (pOat3), NP_112471 (mOat3), NP_112622 (rOat3), NP_060954 (hOAT4), NP_543142 (hOAT5), NP_659034 (mOat5), BAB78471 (rOat5), XP_854865 (dOat6), NP_941052 (mOat6), XP_219524 (rOat6), BAB96750 (hURAT1), and NP_033229 (mUrat1) (f, flounder; pf, puffer fish; zf, zebrafish; h, human, rb, rabbit; m, mouse; r, rat; p, pig; d, dog).