Structural determination of OATP transporters utilising homology models and cell based assays

Abstract

Membrane transport proteins (transporters) are present in the lipid bilayer of cells and selectively transport compounds across the membrane. The organic anion transporting polypeptides (OATPs) are a family of membrane transporters expressed in a variety of tissues, including absorptive/excretory cells of the liver, kidney and intestine. In addition, OATPs transport an array of endogenous and xenobiotic compounds including bile salts, thyroid hormones, statins and antineoplastics. OATPs play an important role in cell homeostasis and drug absorption/excretion, and have also been implicated in disease and drug‐drug interactions. However there is still much to determine regarding the binding properties and exact structure of these proteins. The topology and binding properties of OATP1A2, OATP1B1, OATP1B3 and OATP2B1 have been investigated using a HEK293T cell model.OATP proteins have been expressed in HEK293T cells and kinetic properties determined using the OATP substrate estrone‐3‐sulfate. Bioinformatics analysis using various topology models has revealed that OATP1B1, OATP1B3 and OATP2B1 have between 11–12 transmembrane domains. However there could be as few as 8 domains, as is the predicted topology of the less well characterised OATP1A2. The topology of these proteins has been experimentally investigated using a FLAG epitope tag recognition system. Epitope tags were cloned into the putative extracellular regions of the protein, which were recognised by a FLAG antibody. The detection of the antibody was quantified by a chemiluminescence method and those detected confirmed as extracellular regions. The findings of this study will be used to further determine drug transport pathways and aid in future drug development.