Characterization of a human renal organic anion transporter

by Glen Reid
Doctoral thesis
Date of Examination:2000-06-22
Date of issue:2001-05-02
Advisor:Dr. Natascha Wolff
Referee:Prof. Dr. Gerhard Burckhardt
Persistent Address: http://dx.doi.org/10.53846/goediss-74

 

 

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Abstract

English

The renal excretion of organic anions, including a variety of endogenous substances, xenobiotics and their metabolites, involves an organic anion transport system with multiple specificity. The well characterized basolateral uptake step of this process involves the organic anion transporter 1 (OAT1), recently cloned from the rat, mouse and flounder, which mediates the uptake of an organic anion in exchange for an intracellular dicarboxylate. This study represents the first reported cloning of a human OAT1 homologue, hROAT1, which was cloned via a degenerate PCR-based approach. The hROAT1 reading frame encodes a 550 amino acid protein with a calculated molecular mass of 60 kDa, and has significant homology to previously cloned OAT1 homologues. To enable heterologous expression in the Xenopus laevis oocyte system, an hROAT1 expression clone was constructed by subcloning the hROAT1 reading frame between the 5' and 3' UTRs of the flounder sodium dicarboxylate cotransporter, fNaDC-3. Expressed in Xenopus laevis oocytes, hROAT1 mediated the uptake of p-aminohippurate (PAH), the model substrate for this system. This uptake had an approximate Km of 10 µM, and was inhibited by probenecid, dicarboxylates including the probable physiological exchange partner a-ketoglutarate, and the loop diuretics bumetanide and furosemide. Uptake also showed chloride dependence, characteristic of the basolateral organic anion/dicarboxylate exchange in renal systems. Mutation of two amino acids, Lys382 and Arg466, positions positively charged in all OATs and negatively charged or neutral in the related organic cation transporters (OCTs), abrogated transport function. Deletion of exons 9 and 10 also resulted in a non-functional transporter. Deletion of exon 10 alone, which contains a consensus site for phosphorylation by casein kinase II, increased transport by approximately two-fold. A second member of the OAT family, hOAT2, was also cloned from the human kidney. The hOAT2 cDNA encodes a protein of 541 amino acids, with high homology to the previously identified rat OAT2. The sequences of hOAT2 and hNLT, a second human OAT2 homologue cloned from the liver, are identical apart from exon 10, which is completely divergent and contains alternate protein kinase consensus sites. hOAT2 and hNLT appear to represent tissue-specific transcripts from the same gene. The observed effect on hROAT-mediated transport of deleting exon 10, and the apparent tissue-specific expression of the tenth exon of hOAT2 suggest an as yet unexplored role for exon 10 in the regulation of these transporters.
 
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