The organic anion transporting polypeptides (OATPs) encompass a family of membrane transport proteins responsible for the uptake of xenobiotic compounds. Human organic anion transporting polypeptide 1B1 (OATP1B1) mediates the uptake of clinically relevant compounds such as statins and chemotherapeutic agents into hepatocytes, playing an important role in drug delivery and detoxification. The OATPs have a putative 12-transmembrane domain topology and a highly conserved signature sequence (human OATP1B1: DSRWVGAWWLNFL), spanning the extracellular loop 3/TM6 boundary. The presence of three conserved tryptophan residues at the TM interface suggests a structural role for the sequence. This was investigated by site-directed mutagenesis of selected amino acids within the sequence D251E, W254F, W258/259F, and N261A. Transport was measured using the substrate estrone-3-sulfate and surface expression detected by luminometry and confocal microscopy, facilitated by an extracellular FLAG epitope. Uptake of estrone-3-sulfate and the surface expression of D251E, W254F, and W258/259F were both significantly reduced from the wild type OATP1B1-FLAG in transfected HEK293T cells. Confocal microscopy revealed that protein was produced but was retained intracellularly. The uptake and expression of N261A were not significantly different. The reduction in surface expression and intracellular protein retention indicates a structural and/or membrane localization role for these signature sequence residues in the human drug transporter OATP1B1. 1. Introduction The organic anion transporting polypeptides (OATPs) are a family of membrane transport proteins capable of transporting an array of structurally diverse endogenous and xenobiotic compounds [1–3]. The proteins are expressed in a variety of tissues, including absorptive/excretory cells of the liver and kidney, acting as both a drug delivery and a drug detoxification system. The human organic anion transporting polypeptide 1B1 (OATP1B1) (gene symbol SLCO1B1, previously OATP2, OATP-C, and LST-1) [4] is expressed solely on the basolateral membrane of hepatocytes, facilitating the uptake of clinically relevant compounds such as antibacterial drugs, statins, and chemotherapeutic drugs [5]. As well as playing an important role in hepatic drug disposition [6], OATP1B1 also acts as an active drug delivery system for statins to the liver as a target organ [7]. The protein has been implicated in many drug-drug interactions, including those with cyclosporine A, rifampicin, and statins [8–10]. Many individual DNA nucleotide changes known
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