Functional and Structural Relevance of Conserved Positively Charged Lysine Residues in Organic Anion Transporting Polypeptide 1B3

Abstract

The human organic anion transporting polypeptide 1B3 (OATP1B3), located in the basolateral membrane of hepatocytes, mediates the uptake of endogenous substrates such as taurocholate and drugs from blood into hepatocytes. The transport activity of OATP1B3 is influenced by positively charged amino acids, which are facing the central pore. Molecular modeling was performed to select conserved positively charged amino acids, which may influence transport activity and anchoring of OATP1B3 in the plasma membrane. The modeling revealed that Lys361 faces the pore, and Lys399 is oriented to the plasma membrane. Therefore, the mutants L361ϾA, L361ϾR, L399ϾA, and L399ϾR were generated using sitedirected mutagenesis to investigate the impact of the positive charges on transport activity and anchoring in the membrane. Transport kinetic analyses for the substrates sulfobromophthalein and taurocholate showed a loss of function for the L361ϾA mutant, whereas the transport activity was maintained by the L361ϾR mutant, indicating that the positive charge at position 361 is important for transport activity of OATP1B3. Comparative modeling with OATP1A2 and OATP2B1 revealed that the pore size around this lysine residue is larger in OATP1A2 and smaller in OATP2B1 compared with OATP1B3, which could be related to the respective substrate spectra. Cell surface expression of L399ϾA and L399ϾR was decreased to 16 and 72% compared with wild-type OATP1B3 (p Ͻ 0.001), respectively, indicating that the positive charge of lysine at position 399 is necessary for an unimpaired cell surface expression. Furthermore, we provide a summary of amino acids, which influence the transport activity of OATP1B3.

Structure-Based Identification of OATP1B1/3 Inhibitors

“…The latter two OATP isoforms were included to increase the quality of the alignment. From this alignment, amino acids previously shown to be important for OATP activity (Meier-Abt et al, 2005;Mandery et al, 2011) were selected and translated to a descriptor based on Z-scales or a feature-based ProtFP descriptor (Lapinsh et al, 2002;van Westen et al, 2011a). Supplemental Figure 1 shows the similarity of the proteins as described by the Z-scales descriptors in a twodimensional plot.…”

Hypoxia/Reoxygenation Stress Signals an Increase in Organic Anion Transporting polypeptide 1a4 (Oatp1a4) at the Blood–Brain Barrier: Relevance to CNS Drug Delivery

“…16 Although not directly studied at the BBB, OATP1A2 has been shown to transport rosuvastatin in isolated human hepatocytes and atorvastatin in human embryonic kidney cells (HEK293) stably transfected with OATP1A2. 17,18 Using our in vivo model of pain/inflammation, we have shown that Oatp1a4 is a BBB transporter that can be exploited to optimize CNS delivery of opioid peptide drugs; 19 however, Oatp1a4-mediated delivery of statins across the brain microvascular endothelium under conditions of H/R has not been clearly elucidated.…”

Interplay between the prostaglandin transporter OATP2A1 and prostaglandin E2-mediated cellular effects

“…All cell culture media supplements were obtained from Invitrogen GmbH (Karlsruhe, Germany). Transient expression of the prostaglandin transporter OATP2A1 in the HEK293-EP4 cells and the respective control cells was performed by using an OATP2A1 containing pcDNA3.1(+)-plasmid [3] and Lipofectamine 2000 (Invitrogen GmbH, Karlsruhe, Germany) as previously described [24,26].…”