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STRUCTURAL REQUIREMENTS OF ORGANIC ANION TRANSPORTING POLYPEPTIDE MEDIATED TRANSPORT

Weaver, Yi Miao
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Abstract
The organic anion transporting polypeptides (human: OATP; other: Oatp) form a mammalian transporter superfamily that mediates the transport of structurally unrelated compounds across the cell membrane. Members in this superfamily participate in the absorption, distribution and excretion of many endogenous and exogenous substances including a number of medications and environmental toxicants. Polymorphisms of OATPs have been shown clinically to give rise to inter-individual variabilities of drug efficacy and/or toxicity. Furthermore, as multi-specific transporters, they are potential sites for drug-drug interactions. Therefore, understanding the mechanism of OATP/Oatp mediated transport of endo- and xenobiotics will not only help to improve drug efficacy but also to improve the prediction and prevention of toxicity. The overall goal of this dissertation is identifying key amino acids that may play an important role in OATP/Oatp-mediated transport and investigating the spatial size of the substrate binding/translocation pocket. In this dissertation, I defended three specific aims. In the first specific aim, I evaluated the hypothesis that conserved positively charged amino acids play important roles in OATP1B1 transport function. To address this aim, site-directed mutagenesis was employed and the mutants of several conserved positively charged amino acids were studied. The two extracellular amino acids R57 and K361 were found to be important in OATP1B1 mediated transport of estradiol-17β-glucuronide, estrone-3-sulfate and BSP. In the second specific aim, I evaluated the hypothesis that quantifying transport activities of different substrates mediated by chimeras between rat Oatp1a1 and Oatp1a4 in combination with site-directed mutagenesis should allow us to identify regions and/or individual amino acids that are important for Oatp1a4-mediated substrate recognition and/or transport. The effects of chimeric proteins on transport activity were substrate dependent. Extracellular loop 4 and transmembrane domain 8 were identified to be important in transport of digoxin, taurocholate and estradiol-17β-glucuronide. The C-terminal half of Oatp1a4 and Oatp1a1 was found to be important for BSP transport and the interactions between the N-terminal half and the C-terminal half of Oatp1a4 is essential for DPDPE transport. In the third specific aim, I evaluated the hypothesis that different rat renal organic anion transporters of the Oat and Oatp families selectively transport perfluorinated carboxylates (PFCAs) depending on the chain lengths. The purpose of this study was to determine the substrate size selectivity of Oats and Oatp1a1. To address this aim, the inhibitory effects of PFCAs with chain length from C2 to C18 on transport of model substrates by rat Oat1, Oat2, Oat3, Urat1 and Oatp1a1 was quantified. Furthermore, direct uptake of the best inhibitors was characterized. The best substrates for Oats were C7 and C8, whereas Oatp1a1 transported longer PFCAs such as C9 and C10 better than C8 and C7. Altogether, this dissertation reveals that (1) some conserved positively charged amino acids and the C-terminal half are important for OATP/Oatp mediated transport of certain substrates; (2) OATP/Oatps have several binding/translocation sites for different substrates and (3) the preferred substrate size for OATP/Oatps is slightly bigger than that for Oats.
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Date
2010-04-12
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Publisher
University of Kansas
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Keywords
Health sciences, Pharmacology, Biology, Physiology, Oatp, Organic anion, Perfluoronated carboxylates, Pfca, Structure, Transporters
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