The blood brain barrier (BBB) is impermeable to substrates that are not small lipophilic molecules. This aids in the prevention of toxins in the blood from entering the brain. However, this poses a barrier to drug delivery to the central nervous system (CNS). Transporters located on the brain barrier endothelial cells allow specific substrates to pass from the blood to the brain. One transporter located on the apical and basal side of the brain endothelial cells (BEC) is organic anion transporting polypeptide 1c1 (OATP1C1 (human), Oatp1c1 (rodent)). Oatp1c1 is a Na+-independent, bidirectional transporter of not only organic anions, but also endogenous, exogenous and amphipathic organic compounds. In particular, Oatp1c1 is a high affinity thyroxine transporter and displays atypical transport kinetics. There is little known about the structure of Oatp1c1, but it is suggested to have 12 transmembrane spanning -helices which form a barrel-like structure with a central putative pore. Because Oatp1c1 is a transmembrane spanning protein, it is difficult to obtain a crystal structure using current imaging techniques. Protein modeling has been used to create a probable 3-dimensional (3D) structure based on the previously crystallized structure of the glycerol-3-phosphate transporter, and homology modeling from other Oatps. We hypothesize that using homology modeling, and sequence conservation; we can target specific amino acids for mutagenesis that are thought to play a subtle role in thyroxine transport. I tested the hypothesis by making specific amino acid mutations allowing the study of Oatp1c1 dependent transport biochemistry. By determining amino acids that play a subtle role in Oatp1c1 mediated thyroxine transport, a probable structure can be further elucidated. Once the 3D structure of the protein is further elucidated and amino acids involved in transport are identified, drugs can be rationally designed to utilize Oatp1c1 transport from the blood to the brain. In the work presented in this thesis, twelve mutations were made to further characterize the Oatp1c1 structure. Each mutation targeted an amino acid with >80% homology amongst the Oatps, except for two mutations with less than 80% homology. The targeted amino acids are proposed to be involved in substrate transport and face toward the putative substrate channel.
Wild type (WT) Oatp1c1 was fit to a biphasic profile for thyroxine transport with a Km of 16 nM and an estimated Vmax of 2 pmol/min/mg protein. Examination of the kinetic profiles in mutant constructs F88A, I96A, and Y227L displayed a decrease in Km and Vmax for thyroxine transport, as well as a conversion from biphasic to standard Michaelis-Menten kinetics when compared to WT. Kinetic profiles of mutant constructs F370A and F374A displayed higher values for Km with similar Vmax. These results further support the hypothesized structure for Oatp1c1 and identify key amino acids involved in substrate positioning within the putative pore. Results may also indicate the presence of non-overlapping high affinity and low affinity binding sites within Oatp1c1.
University of Minnesota M.S. thesis. Major: Chemistry. Advisors:Dr. Grant W. Anderson, Dr. Jon N. Rumbley. 1 computer file (PDF); x, 60 pages.
Baldeshwiler, Gregg Anthony.
A structure function study of organic anion transporting polypeptide 1c1 (Oatp1c1).
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