Abstract
The orphan transporter hORCTL3 (human organic cation transporter like 3; SLC22A13) is highly expressed in kidneys and to a weaker extent in brain, heart, and intestine. hORCTL3-expressing Xenopus laevis oocytes showed uptake of [(3)H]nicotinate, [(3)H]p-aminohippurate, and [(14)C]urate. Hence, hORCTL3 is an organic anion transporter, and we renamed it hOAT10. [(3)H]Nicotinate transport by hOAT10 into X. laevis oocytes and into Caco-2 cells was saturable with Michaelis constants (K(m)) of 22 and 44 microm, respectively, suggesting that hOAT10 may be the molecular equivalent of the postulated high affinity nicotinate transporter in kidneys and intestine. The pH dependence of hOAT10 suggests p-aminohippurate(-)/OH(-), urate(-)/OH(-), and nicotinate(-)/OH(-) exchange as possible transport modes. Urate inhibited [(3)H]nicotinate transport by hOAT10 with an IC(50) value of 759 microm, assuming that hOAT10 represents a low affinity urate transporter. hOAT10-mediated [(14)C]urate uptake was elevated by an exchange with l -lactate, pyrazinoate, and nicotinate. Surprisingly, we have detected urate(-)/glutathione exchange by hOAT10, consistent with an involvement of hOAT10 in the renal glutathione cycle. Uricosurics, diuretics, and cyclosporine A showed substantial interactions with hOAT10, of which cyclosporine A enhanced [(14)C]urate uptake, providing the first molecular evidence for cyclosporine A-induced hyperuricemia.
Highlights
Decade, many members of these three subfamilies were identified and functionally characterized
Some proteins, which were classified under the solute carrier family 22 (SLC22) family, e.g. ORCTL3, ORCTL4, FLIPT1, or BOCT, have still not been characterized and are called orphan transporters [4]
Protein sequence alignments of all known organic cation transporters (OCTs) and organic anion transporters (OATs), including the nonclassified orphan transporters listed in solute carrier family 22 (SLC22) revealed several highly conserved amino acids
Summary
Reagents—Materials used included fetal bovine serum, trypsin, and PBS from Invitrogen. CRNA Synthesis and Xenopus laevis Oocyte Injection—X. laevis oocytes (Nasco, Fort Atkinson, WI) of stages V and VI were isolated and defolliculated by overnight incubation at 18 °C with collagenase (Type CLSII, Biochrom KG) (0.5 mg/ml) in oocyte Ringer’s solution: 90 mM NaCl, 3 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5 mM HEPES/Tris, pH 7.6 They were injected with 23 nl of water (mock) or cRNA, which was synthesized from NotI-linearized pSPORT6-hOAT10 plasmid (mMessage mMachine-SP6 in vitro transcription kit; Ambion, Austin, TX), according to the manufacturer’s protocol. We tested whether OAT10 is expressed in the apical or basolateral membrane of tubular epithelial cells using rat BLMVs and BBMVs. Western blot analysis of protein extracts of these vesicles showed a clear cut detection
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