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Originally published In Press as doi:10.1074/jbc.M605664200 on August 18, 2006

J. Biol. Chem., Vol. 281, Issue 42, 31178-31183, October 20, 2006
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Functional Role of the C Terminus of Human Organic Anion Transporter hOAT1*

Wen Xu{ddagger}, Kunihiko Tanaka{ddagger}, An-qiang Sun§, and Guofeng You{ddagger}1

From the {ddagger}Department of Pharmaceutics, Rutgers, the State University of New Jersey and Department of Pharmacology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854 and §Department of Pediatrics, Mount Sinai School of Medicine, New York, New York 10029

Received for publication, June 13, 2006 , and in revised form, August 9, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human organic anion transporter hOAT1 plays critical roles in the body disposition of environmental toxins and clinically important drugs. In the present study, we examined the role of the C terminus of hOAT1 in its function. Combined approaches of cell surface biotinylation and transport analysis were employed for such purposes. It was found that deletion of the last 15 amino acids (residues 536–550) or the last 30 amino acids (residues 521–550) had no significant effect on transport activity. However, deletion of the entire C terminus (residues 506–550) completely abolished transport activity. Alanine scanning mutagenesis within the region of amino acids 506–520 led to the discovery of two critical amino acids: Glu-506 and Leu-512. Substitution of negatively charged Glu-506 with neutral amino acids alanine or glutamine resulted in complete loss of transport activity. However, such loss of transport activity could be rescued by substitution of Glu-506 with another negatively charged amino acid aspartic acid, suggesting the importance of negative charge at this position for maintaining the correct tertiary structure of the transporter, possibly by forming a salt bridge with a positively charged amino acid. Substitution of Leu-512 with amino acids carrying progressively smaller side chains including isoleucine, valine, and alanine resulted in mutants (L512I, L512V, and L512A) with increasingly impaired transport activity. However, the cell surface expression of these mutants was not affected. Kinetic analysis of mutant L512V revealed that the reduced transport activity of this mutant resulted mainly from a reduced maximum transport velocity Vmax without affecting the binding affinity (1/Km) of the transporter for its substrates, suggesting that the size of the side chain at position 512 critically affects transporter turnover number. Together, our results are the first to highlight the central role of the C terminus of hOAT1 in the function of this transporter.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Organic anion transporters (OAT)2 play essential roles in the body disposition of clinically important anionic drugs including anti-human immunodeficiency virus therapeutics, anti-tumor drugs, antibiotics, anti-hypertensives, and anti-inflammatories (1–3). Six OATs (OAT1–6) have been identified by different laboratories (4–15). These OATs have distinct organ and cellular localizations and different substrate specificities. OAT1 and OAT3 are predominantly expressed at the basolateral membrane of kidney proximal tubule cells and the apical membrane of brain choroid plexus. OAT4 is expressed at the apical membrane of kidney proximal tubule cells and the basolateral membrane of placental trophoblast. OAT2 is expressed at the basolateral membrane of hepatocytes and in the kidney. The cellular localization of OAT2 in the kidney is still controversial. OAT5 is expressed only in the kidney. OAT6 is expressed in the olfactory mucosa. The subcellular localization of OAT5 and OAT6 has not been defined.

In the kidney, OAT1 and OAT3 utilize a tertiary transport mechanism to move organic anions across the basolateral membrane into the proximal tubule cells for subsequent exit across the apical membrane into the urine for elimination. Through this tertiary transport mechanism, Na+K+-ATPase maintains an inwardly directed (blood-to-cell) Na+ gradient. The Na+ gradient then drives a sodium dicarboxylate cotransporter, sustaining an outwardly directed dicarboxylate gradient that is utilized by a dicarboxylate/organic anion exchanger to move the organic anion substrate into the cell. This cascade of events indirectly links organic anion transport to metabolic energy and the Na+ gradient, allowing the entry of a negatively charged substrate against both its chemical concentration gradient and the electrical potential of the cell.

Despite the clinical importance of these transporters, much remains to be understood regarding the contribution of OAT structure to their transport activity and regulation. All of the cloned OATs share several common structural features (4–15) including 12 transmembrane domains flanked by intracellular N and C termini, multiple glycosylation sites localized in the first extracellular loop between transmembrane domains 1 and 2, and multiple potential phosphorylation sites present in the intracellular loop between transmembrane domains 6 and 7 and in the C terminus (Fig. 1). Recent investigation from our laboratory on the structure-function relationship of OATs revealed that glycosylation is necessary for the targeting of these transporters to the plasma membrane (16–18). We also showed that the first transmembrane domain of hOAT1 plays an important role in both the targeting of the transporter to the cell surface and its substrate recognition (19). To explore further the involvement of different domains of hOAT1 in its functional expression, we have examined the role of its C terminus. The C terminus of hOAT1, especially its proximal region, is highly conserved among all the OATs. Our results showed that the C terminus of hOAT1 played multiple roles in the function of this transporter.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
p-[14C]Aminohippuric acid (PAH) was from PerkinElmer Life Sciences. Membrane-impermeable biotinylation reagent NHS-SS-biotin and streptavidin-agarose beads were purchased from Pierce. QuikChange site-directed mutagenesis kits were purchased from Stratagene (La Jolla, CA). COS-7 cells and LLC-PK1 cells were purchased from American Type Culture Collection (Manassas, VA). All other reagents were purchased from Sigma.

Site-directed Mutagenesis—To facilitate immunodetection of hOAT1 and its C-terminally truncated and mutated products, c-myc tag (EQKLISEEDL) was added to the N terminus of hOAT1 (hOAT1-N-myc) by site-directed mutagenesis. Mutant transporters were also generated by site-directed mutagenesis using hOAT1-N-myc or hOAT1 as a template. The mutant sequences were confirmed by the dideoxy chain termination method.

Cell Culture and Transfections—COS-7 cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and antibiotics. LLC-PK1 cells were grown in Medium 199 containing 10% fetal bovine serum. Cells were grown to 90–100% confluency and transfected with the appropriate plasmids using Lipofectamine 2000 (Invitrogen).

Transport Measurements—For each well, uptake solution was added. The uptake solution consisted of phosphate-buffered saline (PBS)/CM (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, 0.1 mM CaCl2, and 1 mM MgCl2, pH 7.3) and [14C]PAH (20 µM). At the times indicated in the figure legends, the uptake was stopped by aspirating the uptake solution off and rapidly washing the cells with ice-cold PBS solution. The cells were then solubilized in 0.2 N NaOH, neutralized in 0.2 N HCl, and aliquotted for liquid scintillation counting. The uptake count was standardized by the amount of protein in each well.

Cell Surface Biotinylation—Cell surface expression levels of hOAT1 and its mutants were examined using the membrane-impermeable biotinylation reagent, NHS-SS-biotin (Pierce). hOAT1 and its mutants were expressed in cells grown in 6-well plates using Lipofectamine 2000 as described above. After 24 h, the medium was removed and the cells were washed twice with 3 ml of ice-cold PBS/CM (pH 8.0). The plates were kept on ice, and all solutions were ice-cold for the rest of the procedure. Each well of cells was incubated with 1 ml of NHS-SS-biotin (0.5 mg/ml in PBS/CM) in two successive 20-min incubations on ice with very gentle shaking. The reagent was freshly prepared for each incubation. After biotinylation, each well was briefly rinsed with 3 ml of PBS/CM containing 100 mM glycine and then incubated with the same solution for 20 min on ice to ensure complete quenching of the unreacted NHS-SS-biotin. The cells were then dissolved on ice for 1 h in 400 µl of lysis buffer (10 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100 with 1:100 protease inhibitor mixture (Sigma)). The cell lysates were cleared by centrifugation at 16,000 x g at 4 °C. 50 µl of streptavidin-agarose beads (Pierce) was then added to the supernatant to isolate cell membrane protein. hOAT1 and its mutants were detected in the pool of surface proteins by SDS-PAGE and immunoblotting.

Electrophoresis and Immunoblotting—Protein samples (30 µg) were resolved on 7.5% SDS-PAGE minigels and electroblotted on to polyvinylidene difluoride membranes. The blots were blocked for 1 h with 5% nonfat dry milk in PBS, 0.05% Tween 20, washed, and incubated for 1 h at room temperature with appropriate primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies. The signals were detected by SuperSignal West Dura extended duration substrate kit (Pierce). Nonsaturating, immunoreactive protein bands were quantitated by scanning densitometry with the FluorChem 8000 imaging system (Alpha Innotech Corp., San Leandro, CA).

Data Analysis—Each experiment was repeated a minimum of three times. The statistical analysis given was from multiple experiments. Statistical analysis was performed using Student's paired t tests. A p value of 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Deletion of C-terminal Fragments of hOAT1—Based on hydropathy analysis (Ref. 9) and a topology prediction program (TopPred), the last 45 amino acids (506–550) of hOAT1 are modeled to be an intracellular tail (Fig. 1). To examine the role of the C terminus of hOAT1 in its function, sequential deletions were carried out to remove the last 15 amino acids ({Delta}536–550), the last 30 amino acids ({Delta}521–550), and the entire C terminus ({Delta}506–550). The functional properties of these successively truncated mutants were investigated in kidney COS-7 cells and LLC-PK1 cells. Analysis of the transport of PAH, a protypical organic anion (Fig. 2a), showed that deletion of the last 15 amino acids ({Delta}536–550) had no effect on the transport activity of hOAT1. Deletion of the last 30 amino acids ({Delta}521–550) resulted in a decrease in transport activity to about 55–60% relative to that of hOAT1 wild type. Deletion, however, of the entire C terminus ({Delta}506–550) resulted in a complete loss of transport activity. The uptake in LLC-PK1 cells showed a similar pattern (not shown). To facilitate the immunodetection of wild type hOAT1 and its mutants, epitope c-myc was tagged to the N terminus of these proteins. Functional analysis of the tagged proteins revealed a pattern of PAH uptake similar to their parental proteins (Fig. 2b), suggesting that adding the myc tag does not interfere with the functional characteristics of these proteins.

Alanine Scanning of Residues 506–520—The data obtained from above revealed a marked dependence of the transporter on the region of residues 506–520 for its function. This region is highly conserved among all the OATs. To explore this region further, we systematically mutated each residue to alanine and evaluated the impact of these mutations on activity. As shown in Fig. 3, although most of the mutants exhibited significant transport activities compared with hOAT1 wild type, mutants E506A and L512A completely lost transport activity. All the active mutants retained the characteristic of hOAT1 wild type as an organic anion exchanger. These active mutants also preserved the sensitivity of hOAT1 wild type to probenecid, an OAT1 inhibitor (data not shown). Because of the total loss in the uptake of PAH by E506A and L512A, additional studies were focused on Glu-506 and Leu-512.


Figure 1
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  		FIGURE 1.
Predicted transmembrane topology of OAT1. Twelve transmembrane domains are numbered from 1 to 12. Potential glycosylation sites are denoted by tree-like structures. Potential phosphorylation sites are labeled as P. Positions of the deletions are indicated by arrows.

 


Figure 2
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  		FIGURE 2.
Functional analysis of hOAT1 wild type (Wt) and its truncated mutants. a, PAH uptake mediated by hOAT1 wild type and its truncated mutants. b, PAH uptake mediated by hOAT1 wild type and its truncated mutants tagged with epitope c-myc. Uptake of PAH (20 µM, 3 min) was performed in COS-7 cells. Activities were normalized to those of hOAT1 wild type in a simultaneous assay. The results represent data from three experiments, with triplicate measurements for each mutant. Asterisks indicate values significantly different (p < 0.05) from that of mock control (V).

 
The Role of Glu-506—Fig. 3 showed that substitution of Glu-506 with alanine resulted in a complete loss of transport activity. To elucidate the molecular mechanisms underlying the effect of mutation at Glu-506, we mutagenized this residue to amino acids with different physicochemical properties. Negatively charged Glu-506 was replaced by glutamine carrying a side chain of a size similar to that of glutamic acid but carrying no charge or by aspartic acid carrying a slightly smaller side chain than that of Glu but carrying a negative charge. As shown in Fig. 4a, similar to the substitution by alanine, substitution by glutamine at this position resulted in a nonfunctional transporter. However, such loss of transport activity could be rescued by substitution of Glu-506 with aspartic acid, suggesting that negative charge at position 506 is critical for the function of hOAT1.

The lack of transport activities of mutant transporters could be caused by changes in the absolute number of transporters, turnover rate, substrate binding affinity, or a combination of these factors. To evaluate the possible changes, we compared the protein expression levels of wild type hOAT1 and its mutants on the cell surface and in the total cell extracts by immunoblot analysis. The cell surface expression was examined using the membrane-impermeable biotinylation reagent, NHS-SS-biotin. To ensure that NHS-SS-biotin was only labeling surface proteins, the integrity of the cell membrane during biotinylation was tested by immunoblotting with an anti-beta-actin antibody. beta-Actin immunoreactivity was detected only in cells permeabilized with 0.1% Nonidet P-40, but not in non-permeabilized cells (Fig. 4b). We then showed that although completely lacking of function, mutants E506A and E506Q had significant amounts of cell surface (Fig. 4c) and total cell expression (not shown).

The Role of Leu-512—Fig. 3 also showed that substitution of Leu-512 with alanine resulted in a complete loss of transport activity. Further studies were performed by replacing Leu-512 with amino acids carrying varying sizes of hydrophobic side chains, including valine and isoleucine. Valine has a side chain smaller than that of leucine but larger than that of alanine, whereas isoleucine has a side chain of a size similar to that of leucine. As shown in Fig. 5a, the loss of transport activity by replacing Leu-512 with alanine could be partially rescued when Leu-512 was replaced by valine and could be almost fully rescued when Leu-512 was replaced by isoleucine, suggesting that the size of the side chain at position 512 is critical for the function of hOAT1. Immunoblot analysis showed that all of the mutants of Leu-512 had a similar expression on the cell surface (Fig. 5b) as well as in the total cell extracts (not shown) as compared with that of the wild type hOAT1.


Figure 3
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  		FIGURE 3.
PAH uptake by hOAT1 wild type (Wt) and its alanine-substituted mutants. Transport of PAH (20 µM, 3 min) in COS-7 cells, expressing hOAT1 wild type and its alanine-substituted mutants, was measured. Uptake activity was expressed as a percentage of the uptake measured in wild type. The results represent data from three experiments, with triplicate measurements for each mutant. Asterisks indicate values significantly different (p < 0.05) from that of mock control (V).

 


Figure 4
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  		FIGURE 4.
Effect of mutations at Glu-506 on function and cell surface expression of hOAT1. a, PAH uptake in cells expressing mutants of Glu-506. Uptake of PAH (20 µM, 3 min) was measured in cells expressing hOAT1 wild-type (Wt), E506A, E506Q, and E506D. Uptake activity was expressed as a percentage of the uptake measured in wild type. The results represent data from three experiments, with triplicate measurements for each mutant. Asterisks indicate values significantly different (p < 0.05) from that of mock control (V). b, biotinylation of beta-actin. COS-7 cells, permeabilized with or without 0.1% Nonidet P-40 (NP-40), were biotinylated with NHS-SS-biotin, and the labeled proteins were precipitated with streptavidin beads, separated by SDS-PAGE, and visualized by immunoblot analysis using anti-actin antibody (1:200). c, top panel, Western blot analysis of cell surface expression of wild type and mutants E506A, E506Q, and E506D. COS-7 cells expressing hOAT1 wild type and its mutants were biotinylated with NHS-SS-biotin, and the labeled proteins were precipitated with streptavidin beads, separated by SDS-PAGE, and visualized by immunoblot analysis using anti-myc antibody (1:100). Bottom panel, the intensity of the cell surface labeling from the experiment shown in the top panel and other experiments was quantified relative to the wild type intensity. Each column shows the mean of three experiments, and the error bars show the range of observations.

 
To further examine the mechanisms underlying the reduced transport activity of hOAT1 by substitution of Leu-512 with valine, we determined [14C]PAH uptake at different substrate concentrations. An Eadie-Hofstee analysis of the derived data (Fig. 6) showed that a valine substitution of Leu-512 resulted in decreased Vmax (606.2 ± 3.1 pmol/mg x 3 min with wild type hOAT1, 92.5 ± 0.5 pmol/mg x 3 min with L512V) without affecting Km (53.9 ± 0.1 µM with wild type hOAT1, 54.4 ± 0.4 µM with L512V).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
OATs play essential roles in the body disposition of clinically important anionic drugs including anti-human immunodeficiency virus therapeutics, anti-tumor drugs, antibiotics, anti-hypertensives, and anti-inflammatories (1–3). OAT family and another closely related organic cation transporter, OCT, family belong to a large group of related proteins, the major facilitator superfamily (MFS). The members of MFS share common structural features, including 12 putative transmembrane-spanning domains and intracellular C and N termini. Recent elucidation of high resolution crystal structures of two other MFS members, LacY (20) and GlpT (21), suggests that all MFS members may share a common fold. Based on such an assumption, a three-dimensional structure model of rat OCT1 has been developed using the template structure of LacY (22). In such a model, transmembrane-spanning domains 1, 2, 4, 5, 7, 8, 10, and 11 form a large hydrophilic cleft for substrate binding. Because such a structural model focused mainly on the organization and alignment of residues within the 12 transmembrane-spanning domains, it offered no insight into the function of the C terminus of OATs. Our current studies are the first to highlight the central role of the C terminus of hOAT1 in the function of this transporter.

C terminus seems to be essential for maintaining the correct tertiary structure of hOAT1. We showed that substitution of amino acids Glu-506 in the C terminus with alanine led to the complete loss of transport activity. To further investigate the underlying mechanisms for such loss of transport activity, we then replaced Glu-506 with several other amino acids with different physicochemical properties. Negatively charged Glu-506 was replaced by amino acids carrying or not carrying a negative charge (Fig. 4). When Glu-506 was replaced by aspartic acid, an amino acid with a smaller side chain than that of glutamic acid yet carrying a negative charge, the resulting mutant E506D exhibited activity comparable to that of the wild type hOAT1. In contrast, when Glu-506 was replaced by glutamine, an amino acid with a similar sized side chain as that of glutamic acid but carrying no charge, the resulting mutant E506Q became nonfunctional (Fig. 4a). The lack of transport activity in mutants of Glu-506 could result from the reduced or total lack of expression of the mutant transporter protein or could result from impaired binding abilities of the mutants for their substrates. By directly measuring cell surface and total cell expression of these mutants, we observed that although they completely lacked function mutants E506A and E506Q had significant amounts of cell surface (Fig. 4c) and total cell expression (not shown). These results suggest that the negative charge at position 506 may play a critical role in maintaining the correct tertiary structure of hOAT1 possibly by forming a salt bridge with a positively charged amino acid. The correct tertiary structure is necessary for the function and possibly the stability of the transporter.


Figure 5
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  		FIGURE 5.
Effect of mutations at Leu-512 on function and cell surface expression of hOAT1. a, uptake of PAH (20 µM, 3 min) was measured in cells expressing hOAT1 wild type (Wt), L512A, L512V, and L512I. The results represent data from three experiments, with triplicate measurements for each mutant. Asterisks indicate values significantly different (p < 0.05) from that of mock control (V). b, top panel, Western blot analysis of cell surface expression of wild type and mutants L512A, L512V, and L512I. COS-7 cells expressing hOAT1 wild type and its mutants were biotinylated with NHS-SS-biotin, and the labeled proteins were precipitated with streptavidin beads, separated by SDS-PAGE, and visualized by immunoblot analysis using anti-myc antibody (1:100). Bottom panel, the intensity of the cell surface labeling from the experiment shown in the top panel and other experiments was quantified relative to the wild type intensity. Each column shows the mean of three experiments, and the error bars show the range of observations.

 


Figure 6
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  		FIGURE 6.
Kinetic analysis of PAH transport mediated by mutant L512V. Kinetic characteristics were determined at substrate concentrations ranging from 2 to 20 µM (3-min uptake) using cells expressing wild type hOAT1 and mutant L512V. Transport kinetic values were calculated using the Eadie-Hofstee transformation. V, velocity. [S], substrate concentration. Values were mean of three experiments, and the error bars show the range of observations.

 
Our studies further showed that C terminus is critical for determining the transporter turnover number. When another critical amino acid Leu-512 was replaced by amino acids with varying sizes of hydrophobic side chains, we showed that progressively smaller side chains at position 512 increasingly impaired hOAT1 function (Fig. 5a). When Leu-512 was replaced by valine, a residue with a side chain larger than that of alanine but smaller than that of leucine, the resulting mutant L512V regained 30% transport activity, which was lost by replacing Leu-512 with alanine. When Leu-512 was replaced by isoleucine, a residue with a side chain of similar size to that of leucine, the resulting mutant L512I regained ~85% transport activity. Isoleucine is an isomer of leucine. Therefore, the steric difference between isoleucine and leucine may also contribute to the functional difference between L512I and the wild-type hOAT1. However, the change in the size of the side chain at position 512 had no significant effect on the surface expression of the transporter (Fig. 5b). Our kinetic analysis of L512V showed (Fig. 6) that the reduced maximum transport velocity Vmax contributed to the reduced transport activity of this mutant without affecting the binding affinity (1/Km) for the substrates. Vmax can be affected by either the number of the transporter at the cell surface (19, 23) or the transporter turnover number (24–26). Given the fact that mutation at position 512 had no significant effect on the surface expression of the transporter, the reduced Vmax mainly reflects a reduced transporter turnover number.

The studies of other transporters have shown that the C terminus could be critical for the membrane targeting, stability, or protein processing (27–30). Our current studies are the first to highlight the central role of the C terminus of hOAT1: the negative charge at position 506 plays a essential role in maintaining the correct tertiary structure of the transporter possibly by forming a salt bridge with another positively charged amino acid residue, and the size of the side chain at position 512 has a fundamental effect on the turnover number of the transporter.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant R01-DK 60034 (to G. Y.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Pharmaceutics, Rutgers, the State University of New Jersey, 160 Frelinghuysen Rd., Piscataway, NJ 08854. Tel.: 732-445-3831 (ext. 218); E-mail: gyou{at}rci.rutgers.edu.

2 The abbreviations used are: OAT, organic anion transporters; PAH, p-aminohippuric acid; NHS-SS-biotin, biotin N-hydroxysuccinimide ester sodium salt; PBS, phosphate-buffered saline; MFS, major facilitator superfamily. Back



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 ABSTRACT
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 MATERIALS AND METHODS
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 DISCUSSION
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