Browsing by Subject "Oatp"

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    Organic anion transporting polypeptide 1c1 structure and function.
    (2009-08) Westholm, Daniel Eric
    Organic anion transporting polypeptides (Oatps) are solute carrier family members that exhibit marked evolutionary conservation. Mammalian Oatps exhibit wide tissue expression with an emphasis on expression in barrier cells. In the brain Oatps are expressed in the blood-brain barrier (BBB) endothelial cells and blood-cerebrospinal fluid barrier (BCSFB) epithelial cells. This expression profile serves to illustrate a central role for Oatps in transporting endo- and xenobiotics across brain barrier cells. One such Oatp, Oatp1c1, is a high affinity thyroxine (T4) transporter. Among Oatps, Oatp1c1 possesses a unique expression and substrate preference profile. Outside of specialized cells in the eye and testes, Oatp1c1 is expressed solely in the BBB and BCSFB cells. In addition, Oatp1c1 appears to have a narrower substrate specificity than other Oatps and has the lowest identified Km for T4 transport of any known thyroid hormone transporter. Despite these characteristics, Oatp1c1 remains relatively uncharacterized. To better establish Oatp1c1 biology and contributions to overall brain homeostasis, my research was comprised of three components: 1) the characterization of Oatp1c1 transport mechanisms/kinetics, 2) the assessment of Oatp1c1 structure-function relationships through modeling and characterization of the role of multiple Oatp1c1 amino acid residues in substrate recognition, and 3) the identification of novel Oatp1c1 substrates and development of an Oatp1c1 pharmacophore model. To further understand Oatp transport mechanism, I began by analyzing the transport of two known Oatp1c1 substrates, T4 and E217G. Reports by other labs suggested differential Oatp1c1 recognition of these substrates. Through detailed kinetic measurements in Oatp1c1 transfected cell lines, I found that Oatp1c1 possesses multiple substrate binding sites that recognize T4 and E217G with opposite affinities. Next, topology and high resolution 3-dimensional Oatp1c1 structural models were created to evaluate substrate-transporter complimentarity. Through use of the models, we identified multiple Oatp1c1 amino acids to test for involvement in T4 transport through site-directed mutagenesis. The targeted amino acids are highly conserved amongst Oatps, are in the putative transmembrane domains, and face the putative substrate channel. Polar and charged amino acids in helix 2 (D85, E89, N92) were not expressed at the plasma membrane and thus appear required to for proper protein folding and/or trafficking. Mutations at positions R601, P609, W277W278 and G399G409 were all expressed at the plasma membrane and had varying effects on Oatp1c1 transport. Some mutants, such as R601S, diminished transport, but appeared to leave both Oatp1c1 T4 binding sites functioning. Others, such as G399V,G409V appeared to affect the low affinity Oatp1c1 T4 binding site more severely than the high affinity binding site. Next, in an effort to expand the suite of known structures Oatp1c1 interacts with, a range of known T4 transport inhibitors and multiple sterol glucuronides, structural relatives of the Oatp1c1 substrate estradiol 17--glucuronide (E217G), were surveyed to assess for Oatp1c1-specific inhibition of T4 transport. I found that the fenamate class of non-steroidal anti-inflammatory drugs (NSAIDs) were competitive inhibitors of Oatp1c1 T4 transport. In addition, sterols glucuronidated in the 17 and 21 positions also competitively inhibited Oatp1c1 T4 transport. Finally, a pharmacophore analysis was performed on Oatp1c1 inhibitors identified in Chapters 2 and 4, as well as known Oatp1c1 substrates. The Oatp1c1 pharmacophore was found to possess two distinct hydrophobic planes and a negative charge. This work on Oatp1c1 structure and function will aid the rational design of drugs that can cross the blood-brain and/or blood-tumor barriers and facilitate greatly needed treatment for a variety of neurological and metastatic diseases.