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The Blood-Brain Barrier Thyroxine Transporter Organic Anion-Transporting Polypeptide 1c1 Displays Atypical Transport Kinetics

Department of Pharmacy Practice and Pharmaceutical Sciences (D.E.W., D.R.S., K.J.V., J.N.R., G.W.A.), College of Pharmacy, University of Minnesota-Duluth, Duluth, Minnesota 55812

Address all correspondence and requests for reprints to: Grant W. Anderson, 232 Life Science, 1110 Kirby Drive, Duluth Minnesota 55812. E-mail: ander163{at}umn.edu.

Organic anion-transporting polypeptide (Oatp) 1c1 is a high-affinity T₄ transporter expressed in brain barrier cells. Oatp1c1 transports a variety of additional ligands including the conjugated sterol estradiol 17β-glucuronide (E₂17βG). Intriguingly, published data suggest that E₂17βG inhibition of Oatp1c1-mediated T₄ transport exhibits characteristics suggestive of atypical transport kinetics. To determine whether Oatp1c1 exhibits atypical transport kinetics, we first performed detailed T₄ and E₂17βG uptake assays using Oatp1c1 stably transfected HEK293 cells and a wide range of T₄ and E₂17βG concentrations (100 pM to 300 nM and 27 nM to 200 µM, respectively). Eadie-Hofstee plots derived from these detailed T₄ and E₂17βG uptake experiments display a biphasic profile consistent with atypical transport kinetics. These data along with T₄ and E₂17βG cis-inhibition dose-response measurements revealed shared high- and low-affinity Oatp1c1 binding sites for T₄ and E₂17βG. T₄ and E₂17βG recognized these Oatp1c1 binding sites with opposite preferences. In addition, sterols glucuronidated in the 17 or 21 position, exhibited preferential substrate-dependent inhibition of Oatp1c1 transport, inhibiting Oatp1c1-mediated E₂17βG transport more strongly than T₄ transport. Together these data reveal that Oatp1c1-dependent substrate transport is a complex process involving substrate interaction with multiple binding sites and competition for binding with a variety of other substrates. A thorough understanding of atypical Oatp1c1 transport processes and substrate-dependent inhibition will allow better prediction of endo- and xenobiotic interactions with the Oatp transporter.