Transmembrane domain 1 of human organic anion transporting polypeptide 2B1 is essential for transporter function and stability

Zihui Fang, Jiujiu Huang, Jie Chen, Shaopeng Xu, Zhaojian Xiang, Mei Hong


Organic anion transporting polypeptides (OATPs, gene symbol SLCO) are important membrane transporter proteins that mediate the uptake of wide ranges of endogenous and exogenous compounds. OATP2B1 has been found in multiple organs and tissues including the liver, small intestine, kidney, brain, placenta, heart, skin as well as skeletal muscle and is proposed to be involved in the uptake of orally administered drugs. Quite a few reports have demonstrated that transmembrane domains (TMs) are crucial for proper functions of OATP family members. Comparative modeling proposed that TM1, along with TM2, 4 and 5 of the N-terminal half of OATP2B1 may be localized within the substrate interaction pocket and are important for uptake function of the transporter. Alanine-scanning of the putative transmembrane domain 1 of OATP2B1 revealed that substitution of L58 with alanine dramatically altered Km value, and mutation of V52, H55, Q59 and L69 resulted in significantly reduced substrate turn-over number, while A61V, Q62A and S66A exhibited significant change in both Km and Vmax values. In addition, phenylalanine at position 51 seems to play an important role in maintaining proper folding of OATP2B1 because alanine replacement of F51 caused accelerated degradation of the transporter protein. Although proteasome and lysosome inhibitors could partially recovered protein level, the mutant transporter remained non-functional. Taken together, the identification of nine essential amino acid residues within TM1 of OATP2B1 suggested that the transmembrane domain is important for maintaining proper function of the transporter.


Organic anion transporting polypeptides (OATPs, gene symbol SLCO) belong to the solute carrier family and mediate sodium-independent transport of various endogenous and exogenous compounds (Hagenbuch and Gui, 2008). Bile salts, hormones and their conjugates, toxins and a wide range of drugs have been found to be transported by OATPs. Twelve human OATP family members have been found so far: OATP1A2, 1B1, 1B3, 1B7, 1C1, 2A1, 2B1, 3A1, 4A1, 4C1, 5A1 and 6A1 (Hagenbuch et al., 2003; Nakanishi and Tamai, 2012). However, SLCO1B7 was proposed as a pseudogene because OATP1B7 is considered as non-functional (Stieger and Hagenbuch, 2014). Some OATP family members are predominantly expressed in certain organs or tissues. For example, OATP1B1 and OATP1B3 are found only in the liver; whereas others were reported to express ubiquitously (König et al., 2006). Due to their broad substrate specificity, wide tissue distribution and the involvement of drug-drug interactions, OATPs have been extensively recognized as key determinants for drug absorption, distribution and excretion (Shitara et al., 2005; Poirier et al., 2007). OATP2B1 has been found in multiple organs and tissues including the liver, small intestine, kidney, brain, placenta, heart, skin as well as skeletal muscle (Hagenbuch and Meier, 2004; Tamai et al., 2000; Kullak-Ublick et al., 2001; Nissen et al., 2009; Knauer et al., 2010; Knauer et al., 2013). In the liver, OATP2B1 is involved in hepatic uptake of a wide spectrum of xenobiotics and many organic anions, mono- and dicarboxylic acids (Kobayashi et al., 2003; Sai et al., 2006), steroid hormones and their derivatives (Grube et al., 2006) as well as drugs such as rifamycin SV (Vavricka et al., 2002), pravastatin (Kobayashi et al., 2003), cyclosporine and gemfibrozil (Tamai et al., 1997) are inhibitors of the transporter. OATP2B1 exhibits high expression in the apical membrane of human intestinal epithelial cells and may function as a pH-dependent organic anion transporter (Kis et al., 2010). Since the
physiological microclimate pH in the intestinal lumen is weakly acidic,OATP2B1 that shows higher activity at acidic pH is believed to be involved in the uptake of drugs administered orally (Kobayashi et al., 2003; Tamai, 2012). Although extensive studies have been carried
out to identify substrates of OATPs, the underlying mechanisms of substrate binding and/or recognition remain largely unclear because high resolution crystal structures of mammalian drug transporters are still not available (Niessen et al., 2009).

Transmembrane domains (TMs) have been demonstrated to be important for proper functions of various transporters. Previous studies have identified quite a few essential amino acids located within transmembrane domains of OATP members. For example, amino acid residues within TM2 (Li et al., 2012), 6 (Huang et al., 2013), 10 (Gui and Hagenbuch, 2009; Ohnishi et al., 2014), 11 (Weaver and Hagenbuch, 2010; Hong et al., 2015) were shown to be crucial for proper function of OATP1B1, and TM8 (Miyagawa et al., 2009) is believed to be important for substrate recognition of the transporter. As for OATP1B3, K41 in TM1 and R580 in TM11 are believed to be pivotal to the transporter function (Glaeser et al., 2010). In addition, G45 within TM1 along with Y537, S545 and T550 within TM10 (Gui and Hagenbuch, 2008; DeGorter et al., 2012) were demonstrated to be critical for the transport of OATP1B3-specific substrate CCK8. TM6 of OATP1A2 seems to be an important region for substrate binding, protein stability and trafficking (Chan et al., 2015). However, little information is available for the role of TMs within OATP2B1. Comparative modeling proposed that transmembrane helix 1, along with TM2, 3 and 5 of the N-terminal half of OATP2B1 may be involved in formation of the substrate interaction pore (Meier-Abt et al., 2005). In the present study, alanine-scanning of the putative TM1 of OATP2B1 identified nine amino acid residues that are essential for the transporter function. Kinetic analysis of the functional impaired mutants showed that Km and/or Vmax values of most of the mutants were affected and that F51A exhibited dramatically reduced protein expression. The identification of nine critical amino acid residues within TM1 of OATP2B1 suggested that the transmembrane domain is important for maintaining proper function of the transporter protein.

Materials and Methods

Materials – [3H]Estrone-3-sulfate (ES) and [3H]Taurocholic acid were obtained from PerkinElmer Life Sciences (Waltham, MA). Sulfosuccinimidyl 2-(biotinamido) -ethyl-1, 3-dithiopropionate (NHS-SS-biotin) and streptavidin-agarose beads were purchased from Thermo Scientific (Rockford, IL). All other reagents were from Sigma (St. Louis, MO) except otherwise stated. Site-directed Mutagenesis – Mutants were generated using QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent, Santa Clara, CA). The pReceiver M07 vector that contains the SLCO2B1 cDNA and 3-HA tags at the C-terminus was obtained from Genecopoeia (Rockville, MD) and used as template for the mutagenesis. All mutant sequences were confirmed by full length sequencing (Thermo Scientific). Cell culture and transfection of plasmid constructs into cells – HEK293 cells were grown in Dulbecco’s modified Eagle’s medium (Thermo Scientific) supplemented with 10% fetal bovine serum at 37 °C and 5% CO2. Confluent cells in 48-well or 6-well plate were transfected with DNA plasmid using LipofectAMINE 2000 reagent (Thermo Scientific) following manufacturer’s instruction and used for transport assay and cell surface biotinylation 48 hrs after transfection.
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). Briefly, HEK293 cells expressing OATP2B1 or mutants were labeled on ice with NHS-SS-biotin in two successive 20-min incubations and lysed with RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 1% NP-40, protease inhibitors phenylmethylsulfonyl fluoride, 200 g/ml, leupeptin, 3 g/ml, pH 7.4). The labeled proteins in supernatant were then pull-downed by streptavidin-agarose beads, released in 4Laemmli buffer and loaded onto a 7.5% SDS-polyacrylamide electrophoresis gel, transferred electrophoretically to a polyvinylidene difluoride membrane (Millipore, Billerica, MA) and detected with anti-HA antibody (Cell Signaling Technology, Danvers, MA).

Uptake assay —HEK293 cells in 48-well plate were used for transport measurement as described before (Li et al., 2012; Huang et al., 2013) with minor modification. Briefly, cells were incubated with uptake solution that contained [3H]ES (pH7.4) or [3H]Taurocholic acid (pH5.0) at 37°C for 2 min (1min for kinetic analysis) and the reaction was stopped by ice-cold phosphate-buffered saline (PBS) solution. Cells were then washed twice with cold PBS, solubilized in 0.2 N NaOH followed by neutralization with 0.2 N HCl and radioactivity of the cell lysate was measured using a Triathler-Hidex (Hidex, Finland) liquid scintillation counter. The uptake count was standardized by the amount of protein in each well. Statistical analysis – Data statistical analysis was carried out using one-way analysis of variance (ANOVA) with Bonferroni’s post hoc test. Differences between means are regarded as significant if p<0.05. Results Characterization of OATP2B1 function in HEK293 cells In order to see whether OATP2B1 could be properly expressed and function in HEK293 cells, we first analyzed cell surface expression and kinetic parameters of OATP2B1. As shown in Figure 1, OATP2B1 was expressed and correctly targeted to the plasma membrane and exhibited a Km value of 6.760.71 M, which is consistent with the previously reported Km of 8.091.67 M (Nozawa et al., 2004) for OATP2B1 expressed in HEK293 cells. These results indicated that OATP2B1 was properly expressed and functioned within HEK293 cells. Effect of TM1 mutants on OATP2B1 uptake function and protein expression To analyze critical amino acid residues within TM1, we performed alanine scanning. Each of the amino acid residues located within putative transmembrane domains 1 of OATP2B1 (Fig. 2) was mutated to alanine and uptake function of each mutant was measured. Interestingly, most of the alanine mutants showed statistically significant change of ES uptake and more than half of the mutants (11 out of 21) exhibited greater than 50% reduction of the transport function (Fig. 3), implicating that TM1 is important for transport activity of OATP2B1. Since OATP2B1 is a membrane protein and proper targeting of the transporter to plasma membrane is essential for its function, cell surface expression of the mutants with more than 50% decreased function was investigated. As shown in Figure 4A, with the exception of F51A, all mutants exhibited comparable cell surface protein level to wild-type OATP2B1. Total protein expression was also analyzed for the mutants and a similar trend was observed (Fig. 4B). Kinetic analysis of TM1 mutants with significantly reduced estrone-3-sulfate uptake function To evaluate whether the effect on uptake function of these mutants was due to reduced protein level on the plasma membrane, ES uptake of different mutants was normalized with their cell surface protein expression (Fig. 4C). It was shown that after protein level adjustment, ES uptake of K49A, F51A and I65A was partially recovered to more than 50% of that of wild-type OATP2B1, suggesting that these residues may affect protein expression of the transporter, which in turn led to reduced uptake function. On the other hand, the other eight mutants, i.e. V52A, H55A, L58A. Q59A, A61V, Q62A, S66A, and L69A, still exhibited more than 50% decrease of transport activity, implicating that replacement of these residues may change the interaction of the transporter with substrate. Therefore, kinetic analysis of ES uptake was performed and as shown in Table 1, A61V, Q62A and S66A affect binding affinity as well as substrate turn-over number of the transporter; while L58A only showed increased Km value; V52A, H55A, Q59A and L69A only had their Vmax altered. To see whether only ES uptake was affected by mutation at these positions, we analyzed transport activity of taurocholate by OATP2B1 and the functional impaired mutants as well. As shown in Figure 5, mutants that showed more than 50% decrease in ES uptake also exhibited significant reduction (>50%) of transport activity for taurocholate, suggesting these residues are involved in uptake of both substrates.

Alanine replacement of F51 affects protein stability of OATP2B1

To see whether the significantly reduced expression of F51A was due to an increased degradation of the transporter, the mutant was treated with proteasome inhibitor MG132 or lysosome inhibitor bafilomycin A1 (BFA1). As shown in Figure 6A, MG132 treatment resulted in partial recovery of the total protein (left panel). In addition, it was observed that only the immature form (~72kD) of the transporter was recovered after MG132 treatment, the mature form (~100kD) protein remained almost undetectable. Consistent with this result, cell surface expression of F51A remained negligible even after MG132 treatment (right panel). Accordingly, MG132 treatment showed marginal effect on uptake function of F51A (Fig. 6B). Treatment with the vacuolar proton ATPase inhibitor bafilomycin A1 resulted in significantly elevated level of F51A. Further analysis revealed that the lysosome inhibitor also increased cell surface expression of F51A (Fig. 6C). A similar result was observed with the treatment of additional lysosomal inhibitors ammonia chloride or leupeptin (data not shown). However, uptake function of the mutant transporter was not affected by BFA1 treatment (Fig. 6D).

Conservative replacement of amino acids important for OATP2B1 function

To investigate whether the side chain structures of these critical amino acids are essential for OATP2B1 function, we substituted the residues with conservative amino acids. As shown in Figure 7, replacement of hydrophobic residues (V52, L58 and L69) with an amino acid that contains similar side chain structure partially recovered transport function of the protein. In addition, substitution of F51 with tyrosine increased both protein expression and transport activity (Fig. 7A&B). On the other hand, conservative replacement of H55 with lysine significantly reduced uptake function and protein expression of the transporter; while substitution of Q62 with asparagine exhibited decreased transport activity but showed similar cell surface expression compared to Q62A. No significant change of protein expression was observed for other mutants (data not shown). Since F51Y partially recovered uptake function of ES, we performed kinetic analysis of the mutant. As shown in Table 1, after normalized with cell surface expression, Km and Vmax of F51Y are comparable to those of OATP2B1 wild-type, implicating that phenylalanine at this position may not directly interact with the substrate (Table 1).


Although quite a few reports have shown that OATP2B1 is involved in the transport of a wide range of compounds, information related to the structure-function relationship of OATP2B1 is still limited. OATP2B1 has been demonstrated to contain multiple substrate binding sites in Xenopus oocytes (Shirasaka et al., 2012) and Caco-2 cells (Kis et al., 2010). However, in our current study, the transporter only shows monophasic transport of estrone-3-sulfate. The difference may be attributable to the different systems utilized. In an earlier study of OATP2B1 expressed in HEK293 cells, it was shown that the transporter exhibited single saturation kinetics as well (Nozawa et al., 2004).
Transmembrane domains have been demonstrated to play important roles in proper function of OATP1B1, 1B3 and 1A2. In the present study, we performed alanine-scanning of putative TM1 of OATP2B1 and found that alanine substitution of eleven residues within the transmembrane domain resulted in more than 50% reduction of ES uptake by the transporter. After normalized with cell surface protein level, there were still eight mutants exhibited greatly reduced transport function (>50%, Fig. 4C). When locations of these essential residues were analyzed, it was found that from V52 on, alanine replacement of every three and/or four residues, i.e. V52, H55, L58, Q59, A61, Q62, S66, L69, resulted in a mutant that showed significantly decreased transport function. Since amino acid residues spacing 3 or 4 residues apart in protein primary sequence are spatially close to each other in an -helix (Tymoczko et al., 2015), these TM1 residues may be lined-up at the same side of the -helix, interacting with OATP2B1 substrates.

A similar phenomenon was observed in our previous study of OATP1B1. It was found that several amino acid residues along TM11 are crucial for uptake function of the transporter and they are located 3 and/or 4 residues apart from each other (Hong et al., 2015). Indeed, homology modeling (Biasini et al., 2014) of OATP2B1 (Data Supplement) with E.coli glycerol-3-phosphate transporter (PDB: 1pw4, Data Supplement) as template revealed that V52, H55, L58, Q59, A61, Q62, S66, and L69 are all localized along one side the -helix, facing the inner channel formed among different transmembrane domains (Fig. 8). These results are consistent with previous reports of putative computer models of OATP family members. It was proposed that for OATP1B3, the N-terminal half TMs 1, 2, 4 and 5 and the C-terminal half TMs 7, 8, 10 and 11 are likely facing the central pore, and that the presence of a central pore may be a conserved feature and of functional significance for OATP members (Meiet-Abt et al., 2005). A more recent structural model of OATP2B1 also revealed that amino acid residues of TMs 1, 2, 4, 5, 7, 8, 10, and 11 may be involved in formation of the putative substrate translocation pathway of the transporter (Bian et al., 2016). It should be noted that the sequence identity of glycerol-3-phosphate transporter compared to OATP2B1 is relatively low (12.87%). However, homology modeling using the transporter convers the most extended area, i.e. from TM1-TM12 of OATP2B1, among different templates with comparable identity. A similar structure was obtained using the human glucose transporter family member 1 (PDB: 4pyp), which has the highest sequence identity (15.82%) compared to OATP2B1 but exhibits a lower coverage along the sequence (data not shown) as template.
Among the identified residues, V52 are highly conserved, with 10 OATPs having a nonpolar, hydrophobic amino acid at this position (Fig. 2). At position L58, other OATPs except the two OATP1B family members have the same residue or a residue with similar structure. Similarly, the residues at position 69 are nonpolar, hydrophobic in OATP2B1 and other OATPs, with the exception of OATP1B1, 1B3 and 1A2. The conservative replacement of these residues partially recovered uptake function of the transporter, suggesting the hydrophobic property of these residues may be important for transport function of OATP members. A61 seems to be only conserved among OATP2B1 and most of the OATP1 subfamily members, while a polar residue is found in other OATPs. At position Q62, the residue is conserved in other OATPs except those of the OATP1 family, which all contain a positively charged lysine residue at this position. On the other hand, H55, Q59, and S66 seem to be unique for OATP2B1 and may be involved in OATP2B1-specific functions.

The protein expression of F51A was dramatically reduced, suggesting phenylalanine at this position may play an important role in protein stability. Proteasome inhibitor MG132 partially recovered immature form of the transporter (~72kD); while lysosomal inhibitor bafilomycin A1 increased both immature and mature form (~100 kD) of F51A and that cell surface expression of the transporter was partially recovered. However, the recovered F51A is not functional. Both proteasome and lysosome are important sites for the degradation of misfolded proteins (Ciechanover, 2005). These data suggested that alanine replacement of F51 may result in a protein that is recognized by the quality control machinery to be incorrectly folded and hence targeted for degradation by the proteasome and lysosome. However,mutation of F51 do not seem to affect maturation of the protein because blocking lysosomal pathway could partially recovered mature form of the transporter. Conservative replacement with tyrosine resulted in partial recovery of transport function as well as cell surface protein expression, which indicated that the aromatic group at F51 is important for proper protein folding. In the case of H55 and Q62, conservative replacement even reduced uptake function of the mutants, suggesting side chain structures of these residues are irreplaceable for the uptake function. Alanine replacement of L58, Q62 and S66 and valine substitution of A61 significantly decreased the binding affinity for ES, indicating that these residues may be involved in substrate binding. All mutants except L58A showed significantly reduced Vmax, which suggested that these amino acid residues are crucial for substrates turn-over by OATP2B1 as well. These results indicated that L58, A61, Q62 and S66 may be part of the substrate binding site and/or translocation pathway.

In the present study, nine critical amino acids were identified within transmembrane domain 1 of OATP2B1. Most of these residues are important for substrate interaction while F51 is crucial for correct folding of the transporter protein. Although so far there hasn’t been any report relating biological significance to non-synonymous genetic polymorphisms in OATP2B1 TMs, our search in the NCBI database found reports of missense residues within different TMs of the transporter, particularly, a V52I mutant with minor allele frequency (MAF) of 0.0002 was observed in OATP2B1. Based on our current study, such a mutation may affect uptake function of the transporter. Coordination analysis of results from biochemical studies with missense mutations reported in NBCI database may help us better understand the therapeutics significance of OATP2B1 transmembrane domains.

Authorship contributions
Participated in research design: Hong.

Conducted experiments: Fang, Huang, Chen, Xu, and Xiang.
Performed data analysis: Huang and Hong.


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