Conserved Tryptophan Residues within Putative Transmembrane Domain 6 Affect Transport Function of Organic Anion Transporting Polypeptide 1B1

Abstract

The organic anion-transporting polypeptides (OATPs, gene symbol SLCO) are a family of transporters that play important roles in the absorption, distribution, metabolism, and excretion of various drugs. Although substrate specificity of transporter proteins is under extensive study, the underlying mechanisms for substrate binding and/or recognition remain largely unknown. Transmembrane domain 6 (TM6) is a relatively conserved region within OATP family members, and several amino acid residues on its extracellular half are part of the OATP family signatureIn the present study, two adjacent tryptophan residues (Trp258 and Trp259) within TM6 were identified as critical amino acids for the transport function of OATP1B1. Kinetic studies showed that substitution of Trp258 with alanine resulted in monophasic kinetics for estrone-3-sulfate uptake, with a significantly higher K m value (K m 5 12.0 6 2.8 mM) than the high-affinity component of wild-type OATP1B1 (K m 5 0.38 6 0.06 mM). On the other hand, W259A retained the biphasic characteristic of the transporter. K m values of the high-and lowaffinity components for estrone-3-sulfate of W259A are 1.93 6 0.76 mM and 30.8 6 4.4 mM, respectively. Further studies revealed that W258A retained transport function of another prototypic substrate, taurocholate, while W259A displayed a dramatically reduced uptake of the substrate and exhibited an 8-fold increase in the K m value compared with that of the wild-type and W258A. Our results suggest that Trp258 and Trp259 may play different roles in the uptake of different substrates by OATP1B1.

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

“…18 The relative preservation of plasma membrane and total cell expression of the TM6 mutants L250A, I251A, C252A, and N256A ( Figures 3 and 4), which are located between the two subregions of TM6 (245−248 and 261−266), was in agreement with substrate uptake data ( Figure 2).…”

Transmembrane Domain 1 of Human Organic Anion Transporting Polypeptide 2B1 Is Essential for Transporter Function and Stability

“…Cell Surface Biotinylation and Western Blotting. Cell surface expression level of OATP2B1 and mutants was examined using the membrane-impermeable biotinylation reagent NHS-SS-biotin as described before (Li et al, 2012;Huang et al, 2013). In brief, HEK293 cells expressing OATP2B1 or mutants were labeled on ice with NHS-SS-biotin in two successive 20-minute incubations and lysed with radioimmunoprecipitation assay buffer [50 mM Tris, 150 mM NaCl, 0.1% SDS, 1% NP40, and protease inhibitors phenylmethylsulfonyl fluoride (200 mg/ml) and leupeptin (3 mg/ml), pH 7.4].…”

“…Uptake Assay. HEK293 cells in a 48-well plate were used for transport measurement as described before (Li et al, 2012;Huang et al, 2013) with minor modification. In brief, cells were incubated with uptake solution that contained [ 3 H]ES (pH 7.4) or [ 3 H]taurocholic acid (pH 5.0) at 37°C for 2 minutes (1 minute for kinetic analysis), and the reaction was stopped by ice-cold phosphate-buffered saline solution.…”

Organic Anion-Transporting Polypeptides

“…According to several studies, certain amino acids in TM domains 2, 6, 8, 9 and 10 are important for the function of hOATP1B1 (Gui & Hagenbuch, 2009; J. Huang et al, 2013; N.…”

“…While wild-type hOATP1B1 has a high- and a low-affinity binding site for estrone-3-sulfate resulting in biphasic kinetics (Gui & Hagenbuch, 2009; J. Noe, et al, 2007; Tamai, et al, 2001), the W258A mutant showed only a single binding component with a K m value in between the high- and low-affinity values of wild-type hOATP1B1 (J. Huang, et al, 2013).…”