Putative Transmembrane Domain 6 of the Human Organic Anion Transporting Polypeptide 1A2 (OATP1A2) Influences Transporter Substrate Binding, Protein Trafficking, and Quality Control

Abstract

The human organic anion transporting polypeptides (OATPs) are a family of important membrane proteins that mediate the cellular influx of various anionic substances including clinically important drugs. Transmembrane domain 6 (TM6) is a distinctive consensus "signature" common to all OATPs. Two naturally occurring variants were previously identified in TM6 of the important transporter OATP1A2; these variants may be associated with suboptimal drug influx into cells. Because of the potential importance of TM6 in drug efficacy, this study investigated its role in substrate uptake by OATP1A2. Single amino acid replacements were introduced into TM6 of OATP1A2 (residues 245-266) by alanine-scanning mutagenesis. Uptake assays, biotinylation and immunoblotting were used to assess the function and expression of OATP1A2 and its mutants after overexpression in HEK293 cells. Uptake of the model substrates estrone-3-sulfate and methotrexate by OATP1A2 mutants carrying amino acid replacements within the TM6 subregions of 245-248 and 261-266 was impaired, while transport function was largely retained by other mutants. From kinetic, biotinylation, and immunoblot analysis the diminished function of the 245-248 and 261-266 mutants was due primarily to decreased plasma membrane and total cell expression and also to a less extent, impacted by altered substrate binding. Further experiments with proteasomal or lysosomal inhibitors were consistent with impaired maturation and impaired plasma membrane insertion of several mutants of OATP1A2 within the subregions of 245-248 and 261-266. In addition, the finding that total cellular expression, but not plasma membrane expression, was less impaired for the W245A and W246A mutants suggests that these two TM6 residues might be involved in membrane targeting of OATP1A2. These findings implicate the TM6 subregions of 245-248 and 261-266 in substrate binding, protein trafficking, and quality control of OATP1A2.

Interindividual Diversity in Expression of Organic Anion Uptake Transporters in Normal and Cirrhotic Human Liver

“…( 40 ) The protein products resulting from such polymorphisms could potentially have reduced transport activity due to factors such as stereochemical interference with ligand binding or altered trafficking to the plasma membrane from intracellular locations. ( 31,40‐43 ) Reduced trafficking to the cell surface could be due to changes in the glycosylation or phosphorylation states ( 44‐46 ) or altered interaction with chaperone proteins, such as PDZK1. ( 47 ) These parameters are important for assessment of overall transporter function and, while beyond the scope of the present report, will be the subject of future study.…”

The 5′-AMP-Activated Protein Kinase Regulates the Function and Expression of Human Organic Anion Transporting Polypeptide 1A2

“…Uptake of [ 3 H]-E3S (300 nM; 57.3 nCi/well) by OATP1A2-, OATP1B1-, and OATP2B1-expressing cells and uptake of [ 3 H]-cholecystokinin-8 (2 nM; 23.4 nCi/well) by OATP1B3-expressing cells were conducted at 37°C in phosphatebuffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na 2 HPO 4 , 1.4 mM KH 2 PO 4 , pH 7.4) containing 5 mM glucose. Eight minutes was selected for transporter uptake assays and 4 minutes for kinetic experiments, as described previously (Johnston et al, 2014;Chan et al, 2015a;Lu et al, 2018;Shams et al, 2018). Uptake was terminated by rapidly washing cells in ice-cold PBS.…”

The Altered Renal and Hepatic Expression of Solute Carrier Transporters (SLCs) in Type 1 Diabetic Mice

“…Bradford assay was used to measure protein concentration. Equivalent quantities of protein lysates from each sample were denatured at 55°C for 30 min in Lammli reducing buffer as described before [ 35 – 40 ]. Protein samples were loaded onto 7.5% polyacrylamide mini gels and electrophoresed (Bio-Rad, Gladesville, New South Wales, Australia).…”