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Amino Acid Residues in the Putative Transmembrane Domain 11 of Human Organic Anion Transporting Polypeptide 1B1 Dictate Transporter Substrate Binding, Stability, and Trafficking Weifang Hong,†,§ Zhixuan Wu,†,§ Zihui Fang,† Jiujiu Huang,† Hong Huang,‡ and Mei Hong*,† †

College of Life Science, South China Agricultural University, Guangzhou, China School of Information, University of South Florida, Tampa, Florida 33620, United States



S Supporting Information *

ABSTRACT: Organic anion transporting polypeptides (OATPs, gene symbol SLCO) are membrane proteins that mediate the sodium-independent transport of a wide range of endogenous and exogenous compounds. Due to their broad substrate specificity, wide tissue distribution, and involvement in drug−drug interactions, OATPs have been considered as key players in drug absorption, distribution, and excretion. Transmembrane domains (TMs) are crucial structural features involved in proper functions of many transporters. According to computer-based modeling and previous studies of our laboratory and others, TM11 of OATP1B1 may face the substrate interaction pocket and thus play an important role in the transport function of the protein. Alanine-scanning of the transmembrane domain identified seven critical amino acid residues within the region. Further analysis revealed that alanine substitution of these residues resulted in reduced protein stability, which led to significantly decreased protein expression on the plasma membrane. In addition, all mutants exhibited an altered Km for ES uptake (either high affinity or low affinity component, or both), though Km for taurocholate transport only changed in R580A, G584A, and F591A. These results suggested that critical residues in TM11 not only affect protein stability of the transporter, but its interaction with substrates as well. The identification of seven essential residues out of 21 TM amino acids highlighted the importance of this transmembrane domain in the proper function of OATP1B1. KEYWORDS: organic anion transporting polypeptides, transmembrane domains, transporter proteins, uptake function



INTRODUCTION Organic anion transporting polypeptides (OATPs, gene symbol SLCO) belong to the solute carrier family and mediate sodiumindependent transport of many structurally independent compounds.1 Substrates of OATPs include bile salts, hormones and their conjugates, toxins, and various drugs. Besides charged compounds, OATPs also transport uncharged drugs such as glycosides digoxin2 and ouabain.3 Because of 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.4,5 So far there are 12 members of the human OATP family identified: OATP1A2, 1B1, 1B3, 1B7, 1C1, 2A1, 2B1, 3A1, 4A1, 4C1, 5A1, and 6A1,6,7 though OATP1B7 is considered as nonfunctional and SLCO1B7 was proposed as a pseudogene.8 Some OATP family members are expressed ubiquitously; while others, such as OATP1B1 and OATP1B3, are predominantly found in certain organs or tissues. OATP1B1 is the major OATP located at the basolateral membrane of human hepatocytes and plays a crucial role in drug clearance from the body.9 In recent years, more and more © 2015 American Chemical Society

drugs have been demonstrated to be transported by OATP1B1.10 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.11 As an essential structural feature of membrane proteins, transmembrane domains (TMs) have been proposed to be involved in proper functions of various transporters. Studies of transmembrane domains within OATP1B1 have shown that TM 8 and 9 in this transporter are critical for its substrate recognition.11 Several amino acid residues that are important for substrate translocation and maintaining the normal protein structure were also identified in TM10 of OATP1B1.12 Previous studies in our laboratory demonstrated that four amino acids within TM2 of OATP1B1 Received: Revised: Accepted: Published: 4270

June 16, 2015 November 5, 2015 November 12, 2015 November 12, 2015 DOI: 10.1021/acs.molpharmaceut.5b00466 Mol. Pharmaceutics 2015, 12, 4270−4276

Article

Molecular Pharmaceutics

were confirmed by full length sequencing (Invitrogen, Carlsbad, CA). Cell Culture and Transfection of Plasmid Constructs into Cells. HEK293 cells were grown at 37 °C and 5% CO2 in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum. Confluent cells in 48well or 6-well plate were transfected with DNA plasmid using LipofectAMINE 2000 reagent (Invitrogen) following manufacturer’s instruction. Transfected cells were incubated for 48 h at 37 °C and then used for transport assay or cell surface biotinylation. Uptake Assay. Cells in 48-well plate were used for transport measurement as described previously13 with minor modification. Briefly, cells were incubated with uptake solution containing [3H]ES or [3H] taurocholic acid at 37 °C for 2 min (1 min for kinetic analysis) and uptake was stopped by ice-cold phosphate-buffered saline (PBS) solution. Cells were then washed with cold PBS, solubilized in 0.2 N NaOH, and neutralized with 0.2 N HCl. Radioactivity of the cell lysate was measured with a liquid scintillation counter Triathler-Hidex (Hidex, Finland). The uptake count was standardized by the amount of protein in each well. Cell Surface Biotinylation and Western Blotting. Cell surface expression level of OATP1B1 and their mutants were examined using the membrane-impermeable biotinylation reagent NHS-SS-biotin as described previously.13 Briefly, HEK293 cells expressing OATP1B1 or mutants were labeled with NHS-SS-biotin (0.5 mg/mL in PBS). The cells were then dissolved with RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 1% NP-40, protease inhibitors phenylmethylsulfonyl fluoride, 200 μg/mL, leupeptin, 3 μg/mL, pH 7.4), and proteins in supernatant were collected after centrifugation. Streptavidin-agarose beads were added to bind the biotinlabeled membrane proteins. The streptavidin-agarose beads bound proteins were finally released in 4× Laemmli buffer and loaded onto a 7.5% SDS-polyacrylamide electrophoresis gel, then transferred electrophoretically to a polyvinylidene difluoride membrane (Millipore, Billerica, MA) and detected with anti-HA antibody (Cell Signaling Technology, Danvers, MA). Statistical Analysis. Statistical analysis of experimental data was carried out using Student’s t test. Differences between means are regarded as significant if p < 0.05.

are essential for estrone-3-sulfate (ES) uptake. D70 and F73 may be critical for substrate interaction; while E74 and G76 may play a role in maintaining the proper structure of the transporter protein.13 We also identified two adjacent tryptophan residues (W258 and W259) within the relatively conserved TM6 of OATP1B1, both of which are essential for uptake function of the transporter.14 OATP members are predicted to contain 12 transmembrane domains.6 According to computer-generated model, TM1, 2, 4, and 5 of the N-terminal half and helices 7, 8, 10, and 11 of the C-terminal half of OATP1B3, another OATP family member that has a high homology compared with OATP1B1, may face the substrate interaction pore, while TM 3, 6, 9, and 12 are largely embedded in the bilayer.15 Since our previous study on OATP1B1 TM2 had identified several amino acids critical for its transport function,13 and TM11 is located at the corresponding position of the C-terminal half of the transporter (Figure 1), these two

Figure 1. Position of TM11 relative to TM2 in putative computer model of OATP1B1. Viewed from the extracellular side (A) and the intracellular side (B). Glycerol-3-phosphate transporter (PDB 1pw4) was used as the template for homology modeling of OATP1B1. The structure of OATP1B1 was modeled with the web-based protein structure prediction service Phyre2 (http://www.imperial.ac.uk/ phyre/). Arrows indicate positions of TM11 and TM2.

TMs might coordinate with each other for OATP1B1 uptake function. In addition, R580, a positively charged amino acid that is located within TM11, has been demonstrated to be important for substrate interaction of OATP1B1.16 In the present study, we performed alanine-scanning to study the TM11 of OATP1B1. Alanine substitution of seven amino acids, including R580, showed more than 50% decreased in uptake function as compared to wild-type OATP1B1. Further studies revealed that cell surface expression of most alanine mutants decreased dramatically, but conservative replacement partially recovered expression and uptake function of F591, I595, and D596. Kinetic analysis of the functional impaired mutants was also carried out.



RESULTS Alanine-Scanning of TM11. To identify the critical amino acids within TM11 of OATP1B1, we first individually mutated amino acid residues along the predicted TM11 of the transporter protein (Figure 2) and measured their uptake function for prototypic substrate ES. The amino acids along TM11 were chosen according to the Kyte-Doolittle hydrophobicity scale. Therefore, 18 out of the 21 amino acids from position 576 to 596 of OATP1B1 were mutated to alanine. The three alanine residues (A581, 587, and 593) located in TM11 were replaced with valine to investigate whether they also play a role in proper functioning of OATP1B1. Transport activity of the mutants was analyzed using 50 nM of ES as the substrate. As shown in Figure 3, most of the mutants retained ES transport function and showed more than 50% uptake compared with wild-type OATP1B1. To rule out the possibility that the comparable uptake of these mutants was a combined result of changed cell surface expression and altered uptake function, biotin labeling and Western blotting for the mutants



EXPERIMENTAL SECTION Materials. [3H]Estrone-3-sulfate (ES) and [3H]taurocholic acid were purchased from PerkinElmer Life Sciences (Waltham, MA). Sulfosuccinimidyl 2-(biotinamido)-ethyl-1,3-dithiopropionate (NHS-SS-biotin) and streptavidin-agarose beads were from Thermo Scientific (Rockford, IL). All other reagents were purchased from Sigma except where otherwise stated. Site-Directed Mutagenesis. Mutants were generated using QuikChange Lightning Site-Directed Mutagenesis Kit from Agilent (Santa Clara, CA). The pReceiver M07 vector containing the SLCO1B1 cDNA and 3-HA tags at the Cterminus was obtained from Genecopoeia (Rockville, MD) and used as the template for the mutagenesis. All mutant sequences 4271

DOI: 10.1021/acs.molpharmaceut.5b00466 Mol. Pharmaceutics 2015, 12, 4270−4276

Article

Molecular Pharmaceutics

Figure 2. Putative transmembrane domain 11 of human organic anion transporting polypeptide family members. Amino acid residues located within TM11 were predicted according to Kyte−Doolittle hydrophobicity scale. Multiple sequence alignment of 11 OATP family members was performed with Clustal W. Only partial sequences are shown here. The corresponding sequences of TM11 are in bold.

Figure 3. Estrone-3-sulfate uptake by OATP1B1 transmembrane domain 11 mutants. Uptake of 50 nM ES by HEK293 cells expressing OATP1B1 and its alanine-substituted mutants was measured at 37 °C at a 2 min interval. Net uptake was obtained by subtracting the uptake of cells transfected with empty vector from cells expressing wild-type OATP1B1 or mutants. The results represent data from three experiments, each with duplicate measurements for OATP1B1 and mutants. The results shown are means ± SE (n = 3). The dashed line indicated 50% of ES uptake by OATP1B1 wild-type.

Figure 4. Protein expression of mutants with significantly reduced uptake function. (A) Representative blot for cell surface level analysis of OATP1B1 and mutants. (B) Cell surface expression of OATP1B1 and mutants. The intensity of protein bands relative to the wild-type was quantified with ImageJ (http://imagej.nih.gov/ij, National Institute of Health, USA). The results shown are means ± SE (n = 3). Cells were biotinylated, and the biotin-labeled cell surface proteins were precipitated with streptavidin beads, separated by SDS-PAGE, followed by Western blotting with anti-HA antibody. Same blot was probed with integrin antibody as surface protein loading control. (C) Total protein expression of OATP1B1 and mutants. Cells were lysed with RIPA buffer, separated by SDS-PAGE, followed by Western blotting with anti-HA antibody. The band intensity relative to the wildtype was quantified with ImageJ. The results shown are means ± SE (n = 3).

were performed. It was found that expression of these mutants on the plasma membrane was comparable to that of wild-type OATP1B1. When ES uptake by these mutants was corrected with cell surface expression, they exhibited transport activity ranged from ∼75%−150% of wild-type (Supplemental Figure 1), suggesting these mutants maintain most of OATP1B1 uptake function. However, seven mutants, namely, R580A, G584A, P588A, F591A, G592A, I595A, and D596A, exhibited more than 50% reduction in ES uptake. Since the involvement of R580 in transport function of OATP1B1 has been fully investigated,16 further studies would be mainly focused on the other six amino acids. Protein Expression of Mutants with Reduced Transport Activity. As a membrane protein, OATP1B1 needs to be properly targeted to the cell surface to exert its transport function. Therefore, we first investigated whether the reduced transport function is due to decreased cell surface expression of the mutants. Cell surface biotinylation was performed using a cell-impermeable reagent NHS-SS-biotin. Our results demonstrated that only G584A was abundantly (∼70%) expressed on the cell surface, while all other mutants showed a much reduced expression on the plasma membrane. Plasma membrane expression of R580A, which is around 40% of that of wildtype OATP1B1, was consistent with previous report.16 All the other five alanine mutants showed a cell surface expression of less than 30% compared to wild-type (Figure 4A,B). Total protein expression of the mutants was also analyzed. As shown in Figure 4C, total protein expression correlated well with cell surface expression of most of the mutants, though R580A and

I595A demonstrated a relatively higher level of total protein expression compared with their expression on the plasma membrane. These results suggested that replacement of these amino acid residues affected protein expression of the transporter. Treatment of Mutants with Significantly Reduced Protein Expression with Proteosomal and Lysosomal Inhibitors. To investigate the mechanisms of the significantly reduced protein expression of the mutants, we next examined the effect of proteosomal inhibitor MG132 and lysosomal inhibitor NH4Cl on these mutants since membrane proteins degraded within cells mainly through these two systems.17 As shown in Figure 5, protein expression of all the mutants was partially recovered by MG132 treatment; while the lysosomal inhibitor showed no effect. In addition, cell surface expression of P588A, F591A, G592A, and D596A was significantly increased after MG132 treatment. These results suggested that the transporter protein rescued by the inhibition of proteosomal degradation could be targeted to the cell surface. I595A on the plasma membrane, however, increased only slightly after MG132 treatment, suggesting that replacement of 4272

DOI: 10.1021/acs.molpharmaceut.5b00466 Mol. Pharmaceutics 2015, 12, 4270−4276

Article

Molecular Pharmaceutics

Moreover, cell surface expression of G584 V and G592P/V was much reduced even compared with their respective alanine mutants (Figure 6B). Substitution of P588 with glycine, however, slightly increased cell surface expression level and uptake function of the transporter protein. As for F591, I595, and D596, when they were replaced with amino acid residues that have similar side chain structures, ES uptake function was partially recovered, with the exception of D596N (Figure 7A).

Figure 5. Proteosome and lysosome inhibitor treatments of mutants with significantly reduced protein expression. (A) Total protein expression of OATP1B1 and mutants after treated with MG132 or NH4Cl. (B) Cell surface expression of OATP1B1 and mutants after treated with inhibitors. Cells expressing OATP1B1 and mutants were treated with 20 μM MG132 or 5 μM NH4Cl for 12 h, and total and cell surface proteins were isolated and detected with anti-HA antibody as described in Figure 4. The immature (72 kD) and mature form (95 kD) of OATP1B1 were indicated by arrows.

isoleucine with alanine at this position not only accelerates degradation but affects cell membrane targeting of the protein as well. ES Uptake Function of Additional Mutants. To further investigate whether the side chain structure of these critical amino acids is essential for OATP1B1 function, we substituted these residues with additional amino acids. G584 and G592 were replaced with proline, a residue that may also aid to form a “kink” structure in the protein as glycine but with much larger side group. Since glycine has a small side chain (a hydrogen atom), the replacement of these glycines with alanine, which has a methyl side group, may block substrate translocation. We therefore generated valine (with slightly larger side group than alanine) mutants of G584 and G592 as well to see if the side chain size of G584 and G592 is important for uptake function of the transporter. As shown in Figure 6A, these additional substitutions resulted in further reduction of ES uptake.

Figure 7. Additional replacement of other functional impaired mutants. (A) ES uptake of OATP1B1 and additional mutants. Uptake of 50 nM ES was measured at 37 °C at a 2 min interval. The results represent data from three experiments, each with duplicate measurements for OATP1B1 and mutants. The results shown are means ± SE (n = 3). (B) Cell surface expression of additional mutants. Biotin labeling and protein analysis were carried out as described in Figure 4A.

Protein expression on the plasma membrane was partially recovered as well. Interestingly, though D596N exhibited dramatically reduced ES uptake, its cell surface expression was comparable to that of wild-type OATP1B1 (Figure 7B). Kinetic Analysis of Two Prototypic Substrates Transported by OATP1B1. Since mutation of these residues caused reduced cell surface level of the transporter protein, we next wanted to investigate whether their replacement affected the interaction of OATP1B1 and its substrates as well. Two binding sites for ES were reported in OATP1B1;9,12,18 we therefore examined ES uptake function at concentrations ranged from 0.01 to 20 μM. The uptake was corrected for cell surface expression of the mutants relative to OATP1B1 wild-type so as to better represent their effect on the interaction of the transporter and its substrates. As shown in Table 1, all the mutants retained biphasic kinetics for ES uptake, and most of them exhibited an altered Km. R580A, G584A, and F591A demonstrated a decreased Km value for the high affinity binding but a reduced binding affinity for the low affinity component, suggesting these residues interact with both binding sites. However, Km of P588A and I595A only changed slightly compared with wild-type OATP1B1. Although Km for high affinity site of G592A was comparable to the wild-type transporter, a drastically elevated Km value was observed for the low binding affinity site. ES uptake of D596A was too low to obtain reliable results, so we examined transport activity of D596E instead. D596E exhibited significantly increased Km values for both high and low affinity binding sites of ES. Vmax of R580A and G584A was significantly reduced compared with OATP1B1 in both low and high affinity binding sites; while that of F591A and G592A showed lower Vmax for high affinity

Figure 6. Additional mutations of glycine residues. (A) ES uptake of additional mutants for glycine residues. Uptake of 50 nM ES was measured at 37 °C at a 2 min interval. The results represent data from three experiments, each with duplicate measurements for OATP1B1 and mutants. The results shown are means ± SE (n = 3). (B) Cell surface expression of additional glycine mutants. Biotin labeling and protein analysis were carried out as described in Figure 4A. 4273

DOI: 10.1021/acs.molpharmaceut.5b00466 Mol. Pharmaceutics 2015, 12, 4270−4276

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Molecular Pharmaceutics

or 4 residues apart in the primary sequence are spatially close to each other. These residues may locate at the same side of the αhelix, possibly interacting with the substrates because most of them showed an altered Km value for ES uptake. Moreover, most of these residues are highly conserved among OATP family members. R580 and P588 are conserved in all human OATPs. G592 and D596 are present in 10 out of the 11 human OATP members (Figure 2), implicating that these amino acids may play similar roles in all OATPs. The role of R580 in OATP1B1 was investigated before.16 It was demonstrated that R580A had reduced cell surface expression as well as decreased transport function.16 In the present study, it was shown that R580A had a total protein level comparable to that of wild-type OATP1B1, suggesting alanine substitution of the residue may also affect its targeting to the cell membrane. Since we speculated that by forming a saltbridge, TM11 may coordinate with TM2 in maintaining the proper structure of OATP1B1, we investigated the effect resulted from simultaneous mutation of E74 and R580 as well. However, analysis of the double mutants did not seem to suggest they interact with each other (data not shown). G584, P588, and G592 may be involved in forming a turn that maintains the α-helix structure of the transmembrane domain and/or allow structural changes during transport.19 However, replacements of these residues with proline or glycine resulted in similar effect on ES uptake as the alanine mutants. These results suggested that residues at these positions may be irreplaceable. Further, when G584 and G592 were substituted with valine, a hydrophobic amino acid with a more bulky side group than alanine, ES uptake was more dramatically reduced, implicating these two residues may be localized along the binding pocket and interact with the substrate as well. In fact, G584A showed an altered Km for both ES and taurocholate uptake; while G592A demonstrated a drastically increased (around 9-fold) Km for transport of high concentrations of ES. P588A, however, only exhibited decreased Km value for low concentrations of ES uptake. Vmax of these mutants also showed different changes for ES and taurocholate transport. These results indicated that although G584, P588, and G592 are all involved in substrate binding/transloation of OATP1B1, they may play different roles in the transport of different substrates. Replacement of F591 with alanine almost totally abolished cell surface expression of the transporter; while cell surface expression and uptake function of F591Y were partially recovered, suggesting that the aromatic group at F591 is important to its stability. Indeed, total expression as well as membrane level of F591A was partially recovered after treatment with MG132, a proteosome inhibitor. F591A exhibited altered Km for both ES and taurocholate transport, which indicated that it may interact with both substrates. In addition, F591A showed reduced Vmax for low concentrations of ES transport but significantly increased Vmax value for low affinity ES and taurocholate uptake after surface protein level correction, further implicated the interaction of this residue with both substrates. Replacement of I595 with leucine also partially recovered its uptake function and surface expression. Interestingly, although MG132 treatment led to increase in total expression of the transporter, such a treatment showed only marginal effect on its expression on the plasma membrane, suggesting that replacement of I595 with alanine affect not only its stability but also its ability to target to the cell surface. A negatively charged amino acid is found at the corresponding position of D596 of all human OATPs, with

Table 1. Kinetic Parameters of Estrone-3-Sulfate Transport by OATP1B1 Wild-Type and TM11 Mutantsa Km (μM) OATP1B1 R580A G584A P588A F591A G592A I595A D596E

0.12 0.07 0.05 0.09 0.04 0.11 0.11 0.18

± ± ± ± ± ± ± ±

0.01, 10.6 ± 0.3 0.01*, 16.2 ± 2.2* 0.01*, 21.0 ± 1.5* 0.01*, 10.3 ± 0.5 0.01*, 27.6 ± 5.7* 0.02, 91.0 ± 14.5* 0.05 16.2 ± 3.0* 0.03*, 64.8 ± 7.1*

Vmax (pmol/mg protein/min) 14.5 ± 1.1, 577 ± 15 5.84 ± 0.91*, 423 ± 51* 2.91 ± 0.61*, 352 ± 19* 19.8 ± 9.1*, 807 ± 7* 8.7 ± 0.9*, 1450 ± 350* 6.12 ± 0.76*, 23432 ± 2296* 31.3 ± 3.3*, 2333 ± 375* 17.4 ± 1.9, 3271 ± 392*

a

OATP1B1 was reported to have two binding sites for ES,9,12,18 uptake of ES was therefore measured at concentrations ranged from 0.01 to 20 μM for OATP1B1 wild-type and mutants at 37 °C at a 1 min interval and corrected for cell surface expression. All mutants retained a biphasic kinetics for ES and hence showed two Km and Vmax values. Transport kinetic parameters were calculated using the Eadie− Hofstee transformation. The results shown are means ± SE (n = 3). Asterisks indicate values significantly different (p < 0.05) from that of OATP1B1 wild-type.

binding but higher value for low affinity component. P588A and I595A, however, demonstrated significantly higher Vmax for both low and high affinity components of ES uptake. D596E exhibited an increased Vmax only for the low affinity binding site. We also examined concentration-dependent transport of taurocholate by OATP1B1 and the mutants. As shown in Table 2, Km of taurocholate uptake by R580A, G584A, and F591A Table 2. Kinetic Parameters of Taurocholate Transport by OATP1B1 Wild-Type and TM11 Mutantsa OATP1B1 R580A G584A P588A F591A G592A I595A D596A

Km (μM)

Vmax (pmol/mg,protein/min)

± ± ± ± ± ± ± ±

114 ± 4 297 ± 16* 34.4 ± 4.4* 527 ± 13* 304 ± 16* 112 ± 22 219 ± 43* 107 ± 9

8.52 17.9 13.4 10.8 12.2 6.61 7.31 8.01

1.93 6.5* 2.6* 1.9 1.8* 1.61 2.27 1.48

a

Uptake of taurocholate was measured at concentrations ranging from 0.5 to 50 μM for OATP1B1 wild-type and mutants at 37 °C at a 1 min interval and corrected for cell surface expression. Transport kinetic parameters were calculated using the Eadie−Hofstee transformation. The results shown are means ± SE (n = 3). Asterisks indicate values significantly different (p < 0.05) from that of OATP1B1 wild-type.

was significantly increased, while that of P588A, G592A, I595A, and D596A remained unchanged. Vmax of most of the mutants was greatly increased or similar to that of wild-type OATP1B1 except G584A, which exhibited a significantly decreased value of Vmax.



DISCUSSION In our present study, alanine-scanning revealed seven important residues within TM11. The first amino acid residue causing more than 50% ES uptake reduction was R580, which is located close to the cytosolic side of the TM. Interestingly, from R580 on, alanine replacement of every three and/or four residues resulted in a mutant that showed significant decrease in ES transport function, i.e., R580, G584, P588, F591, Gl592, I595, and D596. In the structure of an α-helix, amino acids spacing 3 4274

DOI: 10.1021/acs.molpharmaceut.5b00466 Mol. Pharmaceutics 2015, 12, 4270−4276

Molecular Pharmaceutics



10 out of 11 being an asparatic acid and a glutamic acid in OATP6A1. Our study demonstrated that the negative charge at this location is critical because though substitution of D596 with glutamic acid did not recover its cell surface expression, it significantly increased ES uptake by the transporter. However, when D596 was replaced by asparagine, cell surface expression was dramatically increased, while transport function was still lacking. These suggested that the charged property of D596 is essential for substrate transport, while its side group structure may be important for its stability. It is interesting that negatively charged residues shall play such a critical role in substrate interaction because OATP family members mostly transport anions. D596, as well as D70 and E74 in TM2,13 are localized at the extracellular side of the transporter and thus may play a role for the entry of substrates into the binding pocket, possibly by maintaining a proper structure of the transporter. However, further studies are needed to clarify such an issue in the transport function of OATP1B1. Another interesting finding was that D596E as well as G592A showed dramatically increased Vmax value in addition to a significantly elevated Km for the low binding component of ES transport. In addition, these two mutants exhibited higher uptake for high concentrations of ES (data not shown). The simultaneous increase of Km and Vmax was also reported in human copper transporter 1 (hCTR1), in which mutation of the positively charged H139 in TM2 to arginine caused a dramatic increase in both Km and Vmax values.20 The presence of an arginine residue at this position, which resulted in different protonation status compared with a histidine, may affect conformation of hCTR1 and facilitate faster transit of copper ions through the permeation pathway.21,22 In our study, the conversion of the negatively charged D596 to a glutamic acid that has a larger side group and substitution of G592 with alanine may similarly change the conformation of OATP1B1 and facilitate the transit of ES through the transport pore. It thus implicated that the rate-limiting interaction for high concentrations ES uptake may take place at these locations. In summary, we identified six critical amino acid residues in addition to R580 within the structure of TM11 of OATP1B1. Most of these residues are important for both substrate interaction and stability of the transporter protein. R580 and I595 may also play a role in targeting OATP1B1 to the plasma membrane (Table 3).

affect binding with ES

affect binding with tau

cell surface expression

R580

yes

yes

G584

yes

yes

P588

yes

no

F591

yes

yes

G592

yes

no

I595

yes

no

D596

yes

no

∼40% of WT ∼70% of WT

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