Substrate Specificity in a Model of the Human Organic Anion Transporter 3

Organic anion transporters (OATs) mediate the secretion of xenobiotics and other organic anions in excretory and barrier tissues. A three‐dimensional model structure for human OAT3 (hOAT3) shows residues Y218 (TMD 5), K419 (TMD 10) and F426 (TMD 10) may contribute to substrate binding and/or translocation. Functional consequences of these residues are examined with hOAT3 mutants in a Xenopus oocyte expression system. hOAT3 substrates differing in size, charge and hydrophobicity (cimetidine, p ‐aminohippurate (PAH), estrone sulfate (ES), 2,4‐dichlorophenoxyacetic acid (2,4‐D) and benzylpenicillin) are investigated. Results indicate tritiated substrate uptake significantly decreased in the presence of Y218A, K419A or F426A mutants. In contrast, substrate uptake levels were similar or greater than wild‐type in the presence of Y218F or F426Y. Furthermore, only ES exhibited minimal uptake in the K419R mutant. Kinetic analysis distinguished whether functional consequences resulted from substrate affinity differences or from protein expression changes. These data confirm the importance of Y218 and F426 in substrate binding and/or translocation and provide insight into the substrate specificity of hOAT3. This research was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences.