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【摘要】 Human organic cation transporter 2 (hOCT2) is essential for the renal tubular secretion of many toxic organic cations. Previously, of the cysteines (C437, C451, C470, and C474) that occur within transmembrane helices that comprise the hydrophilic cleft (proposed site of substrate binding), only C474 was accessible to maleimide-PEO 2 -biotin (hydrophilic thiol-reactive reagent), and covalent modification of this residue caused lower transport rates (Pelis RM, Zhang X, Dangprapai Y, Wright SH, J Biol Chem 281: 35272-35280, 2006). Thus it was hypothesized that the environmental contaminant Hg 2+ (as HgCl 2 ) would interact with C474 to reduce hOCT2-mediated transport. Uptake of [ 3 H]tetraethylammonium (TEA) into Chinese hamster ovary cells stably expressing hOCT2 was reduced in a concentration-dependent manner by HgCl 2, with an IC 50 of 3.9 ± 0.11 µM. Treatment with 10 µM HgCl 2 caused a sixfold reduction in the maximal rate of TEA transport but did not alter the affinity of hOCT2 for TEA. To determine which cysteines interact with Hg 2+, a mutant with all four cleft cysteines converted to alanines (quadruple mutant), and four variants of this mutant, each with an individual cysteine restored, were created. The quadruple mutant was less sensitive to HgCl 2 than wild-type, whereas the C451- and C474-containing mutants were more sensitive than the quadruple mutant. Consistent with the HgCl 2 effect on transport, MTSEA-biotin only interacted with C451 and C474. These data indicate that C451 and C474 of hOCT2 reside in the aqueous milieu of the cleft and that interaction of Hg 2+ with these residues causes reduced TEA transport activity.
【关键词】 HgCl organic cation transport renal proximal tubule cysteine accessibility tetraethylammonium
IN ADDITION TO reabsorption, which facilitates the reclamation of substances from the glomerular filtrate, the renal proximal tubule performs an essential role in the secretion of potentially toxic organic compounds into the tubular lumen, including clinically important therapeutics and environmental toxins ( 19 ). Many of these compounds fall into the chemical class commonly referred to as "organic cations" (OCs), which includes a diverse array of primary, secondary, tertiary, or quaternary amines that have a net positive charge at physiological pH. Transport proteins of the renal proximal tubule epithelium mediate OC secretion, thus performing a critical role in detoxification (see reviews by Refs. 4, 9 - 11, 25, 26 ). Three homologous OC transporters (OCT1, OCT2, and OCT3) have been cloned, and each is expressed in the peritubular (i.e., basolateral) membrane of proximal tubule cells, where they mediate OC uptake ( 8, 13, 22 ), i.e., the first step in transepithelial secretion.
The OCTs are members of the SLC22A family of solute carriers [which includes the organic anion transporters (OATs) and organic cation transporters novel (OCTNs)], and structural features shared by members of this family (e.g., conservation of a 13 residue sequence found between transmembrane domains 2 and 3) further place them into the major facilitator superfamily (MFS). The elucidation of high-resolution crystal structures of two MFS transporters, the Escherichia coli lactose permease [LacY; ( 1 )] and glycerol-3-phosphate transporter [GlpT; ( 7 )], and evidence that all MFS transporters share a common structural fold ( 24 ), has permitted the application of homology modeling to develop hypothetical three-dimensional structures of several MFS transport proteins, including rat OCT1 (rOCT1) ( 18 ), rabbit OCT2 (rbOCT2) ( 29 ), and human OAT1 ( 17 ). These models provide powerful tools for generating testable hypotheses concerning the relationship between structure and function of SLC22A transport proteins.
In a previous study, we examined the accessibility of native cysteine residues in hOCT2 to an impermeant thiol-reactive reagent, maleimide-PEO 2 -biotin ( 16 ). To facilitate description of the relevant conclusions of that study, and to support the basis of the hypotheses tested in the present work, Fig. 1 shows the postulated secondary and tertiary structures of hOCT2 generated from a homology model that used the high-resolution crystal structure of GlpT as a template ( 29 ) (note: the long extra- and intracellular loops were omitted from the three-dimensional model). A large hydrophilic cleft, which is proposed to contain the substrate-binding surface ( 18, 29 ), is positioned between the NH 2 -terminal and COOH-terminal halves of the protein ( Fig. 1, A - C ). Transmembrane helices (TMHs) 1, 2, 4, 5, 7, 8, 10, and 11 of OCT2 form the hydrophilic cleft, while the other TMHs (i.e., 3, 6, 9, and 12) are peripheral. In TMH 10 and 11 (shown in blue in Fig. 1 ), E448 and D475 (red) are positioned in the hydrophilic cleft, and site-directed mutagenesis studies have shown that these residues exert a profound influence on substrate affinity and selectivity ( Fig. 1 D ) ( 5, 6, 18, 29 ). In addition to E448 and D475, four cysteine residues (yellow), at amino acid position 437, 451, 470, and 474, are distributed within TMHs 10 and 11. Of these four cleft cysteines, only C474 (note that the side-chain of C474 is highlighted using a ball and stick representation) is accessible to maleimide-PEO 2 -biotin, and covalent modification of this residue results in reduced TEA transport activity ( 16 ). It would, however, be premature to conclude that C474 is the only one of the four cleft cysteine residues that is exposed to the aqueous milieu; the size of maleimide-PEO 2 -biotin (M.W. 525) may have sterically precluded access to cysteines (C437, C451, or C470) readily accessible to smaller thiol-reactive reagents.
Fig. 1. Hypothetical 2- and 3-dimensional structures of human organic cationic transporter 2 (hOCT2) based on a homology model that used the high-resolution crystal structure of GlpT as a template ( 29 ). A : secondary structure of hOCT2 emphasizing transmembrane helices (TMHs) 10 and 11 (blue) that participates in forming the hydrophilic cleft and which contains residues (E448 and D475; red) known to influence substrate affinity and selectivity ( 5, 6, 29 ). Also present in TMHs 10 and 11 are four cysteine residues, at amino acid position 437, 451, 470, and 474 (yellow). B : side view of the OCT2 homology model, with the extracellular aspect of the transporter oriented upward. The protein is divided into homologous NH 2 - and COOH-terminal halves, which demarcate the hydrophilic cleft. C : view of the model from the cytoplasmic aspect of the protein. D : view of the COOH-terminal half of hOCT2, viewed from the aspect of the hydrophilic cleft, emphasizing the relative orientation of TMHs 10 and 11 and residues C437, C451, C470, C474, E448, and D475. C474, which has been shown to be accessible to maleimide-PEO 2 -biotin (hydrophilic thiol-reactive reagent) ( 16 ), is emphasized using a ball and stick representation (other cleft cysteines shown as sticks).
In the present study, we test the hypothesis that one or more of the other three cleft cysteine residues (C437, C451, and C470), in addition to C474, will be accessible to thiol-reactive reagents that are substantially smaller than maleimide-PEO 2 -biotin, i.e., the mercuric form of mercury and a methanethiosulfonated biotinylation reagent (MTSEA-biotin). From a toxicological perspective, we were also interested in the effects on OCT-mediated transport activity of Hg 2+, as it is the most common form of inorganic mercury in the environment that tends to accumulate in the kidneys (see extensive review by Ref. 27 ). We found that, whereas C437 and C470 remained refractory to these smaller reagents, both C451 and C474 were exposed to the aqueous milieu of the hydrophilic cleft of hOCT2.
METHODS
Chemicals. [ 3 H]tetraethylammonium (54 Ci/mmol) was synthesized by Amersham Biosciences. Ham's F12 Kaighn's modification medium, HgCl 2, and 2,3-dimercaptopropanesulfonic acid (DMPS) were from Sigma. The pcDNA5/FRT/V5-His TOPO mammalian expression vector pOG44, Platinum High Fidelity DNA polymerase, hygromycin, and Zeocin were from Invitrogen. N -biotinylaminoethyl methanethiosulfonate (MTSEA-biotin) was from Toronto Research Chemicals.
TOPO cloning of hOCT2 and site-directed mutagenesis. Generation of wild-type hOCT2 and mutants lacking selected cleft cysteine residues has been described previously ( 16 ). Briefly, the open reading frame for hOCT2 (contained in pcDNA3.1) was amplified using Platinum High Fidelity DNA polymerase and sequence-specific primers with the following PCR conditions: 35 cycles of 94°C for 1 min, 54°C for 1 min, and 72°C for 3.5 min. A final elongation step of 7 min was included after the last cycle. The PCR product was gel purified and cloned into the pcDNA5/FRT/V5-His TOPO mammalian expression vector. Mutations of the hOCT2 sequence were introduced by site-directed mutagenesis using the QuickChange system (Stratagene, La Jolla, CA). Five mutants were created: a quadruple mutant in which all four of the cleft cysteines were converted to alanines, and four variants of the quadruple mutant in which one of the cleft cysteines were restored at each individual position (C437, C451, C470, or C474). Plasmid DNA was prepared using standard methods (Genesee Scientific, San Diego, CA), and sequences were confirmed with an Applied Biosystems 3730xl DNA analyzer at the University of Arizona sequencing facility.
Cell culture and stable expression of hOCT2. Chinese hamster ovary (CHO) cells containing a single integrated Flp Recombination Target (FRT) site were acquired from Invitrogen (CHO Flp-In) and were used for stable expression of wild-type hOCT2 and the mutant constructs. Before transfection, CHO Flp-In cells were grown in Ham's F12 Kaighn's modification medium supplemented with 10% fetal calf serum and Zeocin (100 µg/ml). Cultures were split every 3 days; 5 x 10 6 cells were transfected by electroporation (BTX ECM 630, San Diego, 260 V and time constant of 25 ms) with 10 µg of salmon sperm, 18 µg of pOG44, and 2 µg of pcDNA5/FRT/V5-His TOPO containing one of the constructs of hOCT2. Cells were seeded in a T-75 flask following transfection and maintained under selection pressure with hygromycin (100 µg/ml). Cells were used for experiments 21 days after electroporation.
Transport experiments. Since TEA uptake was previously shown to be low in several of the mutants ( 16 ), cells expressing the wild-type and mutant transporters were treated with 2% dimethylsulfoxide (DMSO; diluted in culture media) for 24 h to increase the transport signal (difference in transport in the absence and presence of 2 mM unlabeled TEA). DMSO has been widely used as a chemical chaperone (e.g., 3, 14, 21), and similar treatment of CHO cells expressing rbOCT2 with DMSO was shown to increase the functional expression of transport protein ( 15 ). Two experimental protocols (preexposure vs. coexposure) were initially used to determine the effect of increasing concentrations of HgCl 2 (0-30 µM) on TEA transport mediated by hOCT2. All HgCl 2 exposures and transport experiments were conducted using buffers that were equilibrated to room temperature. For both methods, CHO cells grown to confluence in 12-well plates were rinsed twice briefly with Waymouth's buffer (WB; in mM: 135 NaCl, 13 HEPES-NaOH, pH 7.4, 28 D -glucose, 5 KCl, 1.2 MgCl 2, 2.5 CaCl 2, and 0.8 MgSO 4 ) before the cells were exposed to increasing concentrations of HgCl 2 (0-30 µM). In the preexposure method, the cells were exposed for 10 s to HgCl 2 diluted in WB, before rinsing once (15 s) with DMPS (1 mM diluted in WB) to quench any unreacted mercury, and two additional times (15 s each) with WB (without DMPS) before transport was measured. An exposure time of 10 s was used since preliminary experiments showed that maximum inhibition of TEA transport caused by preexposure to 5 µM HgCl 2 was complete at 5-10 s. For example, in a single experiment, a 5-, 10-, and 60-s preexposure to 5 µM HgCl 2 reduced [ 3 H]TEA uptake by 58, 70, and 70%, respectively. In the coexposure method, cells were only exposed to HgCl 2 during the transport period, as HgCl 2 was included in the transport buffer. TEA transport was measured by incubating the cells in transport buffer: WB containing 1 µCi/ml [ 3 H]TEA (17 nM), and in some cases, increasing concentrations of unlabeled TEA (0-2 mM). Since initial experiments showed that uptake was linear for 1 min for wild-type and each of the mutant constructs, 30-s uptakes of [ 3 H]TEA were used to approximate the initial rate. After 30 s, the transport buffer was removed, and the wells were rinsed three times with ice-cold WB to stop transport. The cells were then solubilized in 400 µl of 0.5 N NaOH with 1% SDS (vol/vol), and the resulting lysate was neutralized with 200 µl of 1 N HCl. Accumulated radioactivity was determined by liquid scintillation spectrometry (Beckman model LS3801).
TEA protection assays. To determine whether the presence of substrate within the binding region of OCT2 could protect the transporter against the effects of HgCl 2 on [ 3 H]TEA uptake, cells were preexposed to a saturating concentration of TEA (500 µM; 20-fold higher than the Michaelis constant) before treatment with 10 µM HgCl 2. In these experiments, cells were exposed to 500 µM TEA diluted in WB for 10 s, followed by incubation in WB containing 500 µM TEA and 10 µM HgCl 2 for 10 s. Before transport was measured the TEA and HgCl 2 were removed by rinsing once with WB containing 1 mM DMPS and twice with WB without DMPS.
Cell surface biotinylation with MTSEA-biotin. The method described here is a minor modification of that described by Pelis et al. ( 16 ). All solutions were kept ice-cold throughout the procedure, and long incubations were conducted on ice with gentle shaking. Cells plated to confluence in a 12-well plate were initially washed three times with 2 ml of PBS solution containing calcium and magnesium (PBS/CM; containing in mM: 137 NaCl, 2.7 KCl, 8 Na 2 HPO 4, 1.5 KH 2 PO 4, 0.1 CaCl 2, and 1 MgCl 2, pH 7.0 with HCl) followed by a single 20-min incubation in 1 mM MTSEA-biotin diluted in PBS/CM. After biotinylation, the cells were rinsed twice briefly with 3 ml of PBS/CM followed by a 20-min incubation in the same solution. The cells were lysed in 1 ml of lysis buffer (150 mM NaCl, 10 mM Tris·HCl, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS, pH 7.4) containing protease inhibitors [in µM: 200 4-(2-aminoethyl)-bezenesulfonyl-fluoride, 0.16 aprotinin, 4 leupeptin, 8 bestatin, 3 pepstatin A, 2.8 E-64; Sigma] for 1 h and centrifuged at 15,800 g (4°C) for 30 min to remove insoluble material. Fifty microliters of streptavidin-agarose beads (Pierce) were added to the lysates and incubated overnight at 4°C with constant mixing. After extensive washing with the above lysis buffer, 50 µl of Laemmli sample buffer were added, and the proteins were eluted from the beads at 100°C for 5 min. Proteins were separated on 7.5% SDS-PAGE gels, transferred to PVDF membranes, and immunoreactivity corresponding to hOCT2 was detected as previously described ( 16 ).
Statistics. All data are expressed as means ± SE, with calculations of standard errors based on the number of separate experiments conducted on cells at a different passage number. One-way ANOVA was used to test the effect of multiple treatments and was followed by the Student-Newman-Keuls test for pairwise comparisons. Paired comparison of sample means was done using unpaired Student's t -test. All statistical analyses were performed with ProStat 3.81c (Poly Software International, Pearl River, NY) and deemed significant when P < 0.05.
RESULTS
Effect of HgCl 2 on TEA transport by wild-type hOCT2. Two experimental protocols (preexposure vs. coexposure) were initially used to determine the effect of increasing concentrations of HgCl 2 on TEA transport mediated by hOCT2 stably expressed in CHO cells. The first protocol (preexposure) involved exposing the cells for 10 s to HgCl 2, followed by several rinses to remove any unreacted mercury before initiating transport (using transport buffer without HgCl 2 ). The second protocol measured transport using transport buffers that contained HgCl 2 (coexposure). Regardless of the experimental protocol used, HgCl 2 (0-30 µM) reduced TEA transport by hOCT2 in a concentration-dependent manner ( Fig. 2; P < 0.05, one-way ANOVA). At 30 µM HgCl 2, TEA transport was reduced by 85%, and higher concentrations of HgCl 2 almost completely abolished transport activity (e.g., 100 µM HgCl 2 reduced [ 3 H]TEA uptake by 96%, n = 1). The kinetics of this inhibition were determined as previously described ( 20 ). The IC 50s for inhibition of TEA transport by HgCl 2 were 3.9 ± 0.72 and 3.9 ± 0.11 µM for preexposure and coexposure, respectively, which were not significantly different from one another ( P 0.05, two-tailed t -test). In agreement with the observation that TEA transport activity is almost completely inhibited by high concentrations of HgCl 2, the asymptotes corresponding to the kinetic curves in Fig. 2 approach zero.
Fig. 2. Effect of increasing concentrations of HgCl 2 (0-30 µM) on [ 3 H]TEA (17 nM) uptake (30 s) by Chinese hamster ovary (CHO) cells stably expressing wild-type hOCT2. Preexposure and coexposure refer to the different experimental protocols used to examine the effects on transport of HgCl 2. The kinetics of the inhibition caused by increasing concentrations of HgCl 2 were determined as previously described ( 20 ). IC 50 is the concentration of HgCl 2 resulting in half-maximal transport activity. Data are expressed as a percentage of TEA uptake in the absence of HgCl 2 (% of control). The absolute rate of [ 3 H]TEA uptake in the absence of HgCl 2 ranged from 30 to 90 fmol·cm -2 ·min -1. Significant differences between the 2 methods (coexposure vs. preexposure) at each of the concentrations tested are indicated by * ( n = 3; P < 0.05, Student's t -test).
Ten micromolar HgCl 2 caused a greater reduction of TEA transport with the preexposure compared with the coexposure protocol ( Fig. 2; P < 0.05, two-tailed t -test), suggesting that, despite the similar IC 50 values, TEA transport may actually be more sensitive to HgCl 2 when the preexposure method is used. In fact, prolonged 1 min) of CHO cells to the higher-end concentration of HgCl 2 (30 µM) used in the present study appeared to be toxic to the cells, as suggested by a change in cell morphology (rounding of cells; data not shown). In the preexposure experiments, transport was conducted 1 min after the initial exposure to HgCl 2 (10-s HgCl 2 exposure, 45-s rinse period, and 30-s [ 3 H]TEA uptake period), whereas transport was initiated upon HgCl 2 exposure in the coexposure protocol (only exposed to HgCl 2 for the 30-s [ 3 H]TEA uptake period). To limit the possible confounding effects of mercury toxicity on transport, most of the subsequent experiments (except for the TEA protection assays) were conducted using coexposure to HgCl 2. Since the inhibitory constants (IC 50s ) were the same using the different methods, we considered this to be an appropriate experimental approach.
Kinetic basis for the reduction of TEA transport by HgCl 2. To determine the kinetic basis for the decrease in TEA transport caused by HgCl 2, the kinetics of TEA transport were determined in the presence and absence of 10 µM HgCl 2. This concentration caused a 60-70% reduction in TEA uptake. Increasing concentrations of unlabeled TEA in the transport medium reduced the rate of transport by a process adequately described by the Michaelis-Menten equation for competitive interaction of labeled and unlabeled substrate ( 12 )
where J is the rate of [ 3 H]TEA transport from a concentration of labeled substrate equal to [* T ]; J max is the maximum rate of transport; K t is the TEA concentration that results in half-maximal transport (Michaelis constant); is the concentration of unlabeled TEA; and C is a constant representing the component of total TEA uptake that is not saturable over the concentration range tested. This nonsaturable component likely reflects the combined influence of diffusive flux, nonspecific binding and/or incomplete rinsing of the cell layer. In four separate experiments, exposure to 10 µM HgCl 2 caused a sixfold significant reduction in the maximal rate of TEA transport ( J max of 90 ± 19.5 vs. 15 ± 2.5 pmol·cm -2 ·min -1, P < 0.05, two-tailed t -test) but had no effect on the affinity of hOCT2 for TEA ( K t of 20 ± 4.6 vs. 17 ± 6.1 µM, P 0.05, two-tailed t -test).
Influence of cleft cysteine residues on the sensitivity to HgCl 2 of TEA transport. To determine whether Hg 2+ interacts with C474 or any of the other three cleft cysteines to reduce TEA transport, a mutant of hOCT2 with all four of the cleft cysteines (see Fig. 1 ) converted to alanine ("quadruple mutant"), and four variants of this mutant, each with an individual cysteine restored (C437, C451, C470, or C474), were created. All of the mutant transporters displayed considerable TEA transport activity ( Fig. 3 ) and were previously shown by immunocytochemistry to be expressed in the plasma membrane ( 16 ). In contrast, untransfected CHO cells exhibit no mediated TEA transport activity ( 2 ). As noted previously, HgCl 2 reduced the rate of TEA transport by wild-type hOCT2 in a dose-dependent manner, with only 20% of transport activity remaining with exposure to 30 µM HgCl 2 ( Fig. 4 ). Elimination of the four cleft cysteines had a profound effect on this profile. Although increasing concentrations of HgCl 2 had a modest effect on transport by the quadruple mutant, it was not statistically significant ( P 0.05, one-way ANOVA; Fig. 4 ). Figure 5 shows the effect of HgCl 2 on [ 3 H]TEA uptake by mutants containing only a single cleft cysteine (C437, C451, C470, or C474). The mutants containing C437 and C470 exhibited profiles that were similar to the quadruple mutant, i.e., largely insensitive to HgCl 2. This is in contrast to the C451- and C474-containing mutants, which appeared more sensitive to HgCl 2 than the quadruple mutant. TEA transport by the mutants with C451 and C474 were reduced 47 and 69%, respectively, by 30 µM HgCl 2.
Fig. 3. Thirty-second uptakes of 17 nM [ 3 H]TEA by CHO cells stably expressing wild-type and mutant constructs of hOCT2. In the quadruple mutant (Quad), all 4 cleft cysteines were mutated to alanine. The quadruple mutant DNA served as a template for constructing the other mutants, with each having one of the four cleft cysteine residues (C437, C451, C470, or C474) restored. Uptake of [ 3 H]TEA was conducted in the absence and presence of 2 mM unlabeled TEA ( n = 3-4). WT, wild-type.
Fig. 4. Effect of increasing concentrations of HgCl 2 (0-30 µM) on [ 3 H]TEA (17 nM) uptake (30 s) by CHO cells stably expressing the wild-type or quadruple mutant of hOCT2. All 4 of the cleft cysteines (C437, C451, C470, and C474) were mutated to alanines in the quadruple mutant. Data are expressed as a percentage of TEA uptake in the absence of HgCl 2 (% of control). Significant differences between wild-type and the quadruple mutant at each of the concentrations tested are indicated by * ( n = 3-4; P < 0.05, Student's t -test).
Fig. 5. Effect of increasing concentrations of HgCl 2 (0-30 µM) on [ 3 H]TEA (17 nM) uptake (30 s) by CHO cells stably expressing the mutants of hOCT2 containing only a single cleft cysteine (C437, C451, C470, or C474). All 4 of the cleft cysteines were mutated to alanines in the quadruple mutant. The quadruple mutant DNA served as a template for constructing the other mutants, with each having one of the four cleft cysteine residues restored. Data are expressed as a percentage of TEA uptake in the absence of HgCl 2 (% of control). Significant differences between the mutant constructs (C437, C451, C470, or C474) and the quadruple mutant (*) were determined at each of the concentrations tested ( n = 3-5; P < 0.05, Student's t -test).
Interaction of MTSEA-biotin with C451 and C474. The aforementioned data are consistent with Hg 2+ interacting with C451 and C474, but not C437 or C470, to lower TEA transport activity. As noted earlier, interaction of the thiol-reactive reagent maleimide-PEO 2 -biotin with OCT2 was restricted to C474 ( 16 ), quite possibly because of the large size of this molecule ( 25 Å in length; Fig. 6 A ) compared with the diameter of the mercuric ion (2.2 Å; Fig. 6 A ). We hypothesized that a smaller biotinylation reagent would react with C474 and C451, but not with C437 or C470 (on the grounds that, if the mercuric ion did not access these latter residues, it was not likely that a much larger organic molecule would be able to). We tested this hypothesis by determining the accessibility of the cleft cysteines to MTSEA-biotin ( 19 Å in length; Fig. 6 A ), as it is smaller than maleimide-PEO 2 -biotin, but retains a net positive charge like Hg 2+. Similar to Hg 2+, only C451 and C474 were accessible to MTSEA-biotin from the hydrophilic cleft ( Fig. 6 B ).
Fig. 6. A : space-filling representations of mercuric ion, MTSEA-biotin, and maleimide-PEO 2 -biotin. B : Western blot showing immunoreactivity corresponding to biotinylated wild-type hOCT2 and mutant constructs expressed in CHO cells following treatment with MTSEA-biotin. All 4 of the cleft cysteines (C437, C451, C470, and C474) were mutated to alanines in the quadruple mutant (Quad). The quadruple mutant DNA served as a template for constructing the other mutants, with each having one of the 4 cleft cysteine residues (C437, C451, C470, or C474) restored.
Influence of substrate binding on the sensitivity of hOCT2 to HgCl 2. Previously, it was shown that the presence of quaternary ammonium compounds (e.g., tetrapentylammonium and TEA) within the binding surface of hOCT2 limited the interaction of maleimide-PEO 2 -biotin with C474 ( 16 ). Thus, before the cells were treated with 10 µM HgCl 2 (using the preexposure method) and transport was measured, cells expressing wild-type hOCT2 or the mutants with C451 and C474 were treated with a saturating concentration of unlabeled TEA (500 µM). As anticipated, 10 µM HgCl 2 (using the preexposure protocol) reduced TEA transport (50-80%) by each of the constructs. However, TEA occupation of the binding surface did not protect OCT2 from the inhibition of transport caused by HgCl 2 ( Fig. 7 ).
Fig. 7. [ 3 H]TEA (17 nM) uptake (at 30 s) by wild-type hOCT2 and the C451- and C474-containing mutants following treatment using the preexposure protocol with TEA (500 µM), 10 µM HgCl 2, or TEA and HgCl 2 in combination. Data are expressed as a percentage of TEA uptake in the absence of HgCl 2 and/or TEA (% of control). Different letters indicate significant differences among treatments ( n = 4; P < 0.05, Student-Newman-Keuls test).
DISCUSSION
The broad structural selectivity of the OCTs (as reviewed in Ref. 23 ) make them potential sites of harmful drug-drug interactions, and an understanding of the structural basis of the binding of substrate with OCTs moves closer to anticipating the interaction of substrates with these transport proteins. Three-dimensional homology models of several SLC22A family members were recently developed (rOCT1, rbOCT2, and hOAT1) ( 17, 18, 29 ), and these models should be amenable to generating hypotheses concerning the interrelationship between protein structure and function. Toward testing, validating, and refining the homology model of hOCT2, previous work examined the accessibility to maleimide-PEO 2 -biotin and MTSES (both hydrophilic thiol-reactive reagents) of the 13 cysteine residues contained within the hOCT2 sequence ( 16 ). Six cysteines occur in the long extracellular loop (between TMHs 1 and 2), three are in TMHs that are peripheral to the hydrophilic cleft (TMHs 3, 6, and 9), and four are in TMHs (TMHs 10 and 11) that comprise the hydrophilic cleft, the proposed region of substrate-protein interaction (see Fig. 1 ). Despite the expectation that many of these cysteines should be contiguous with the extracellular environment (e.g., the six cysteines in the long extracellular loop), and therefore accessible to hydrophilic thiol-reactive reagents, only the cleft cysteine at position 474 was accessible to maleimide-PEO 2 -biotin (it was also accessible to MTSES), and covalent modification of C474 caused diminished TEA transport activity. However, we acknowledged that the relatively large size of maleimide-PEO 2 -biotin might have prevented access to cysteine residues that would have otherwise been accessible from the cleft to smaller hydrophilic thiol-reactive reagents (e.g., Hg 2+; Fig. 6 ).
To further define OCT structure, the present study examined the sensitivity of TEA transport by hOCT2 to the most common form of inorganic mercury in the environment, Hg 2+ (as HgCl 2 ), which has a propensity for accumulating in the proximal tubule ( 28 ), the main renal site of OCT2 expression ( 8 ). It was hypothesized that Hg 2+ would reduce hOCT2-mediated transport by interacting with C474, and, depending on whether they are exposed to the solvent phase of the hydrophilic cleft, C437, C451, and/or C470. HgCl 2 reduced TEA transport by hOCT2 in a concentration-dependent manner, with high concentrations of HgCl 2 effectively eliminating all transport activity. The kinetic basis for this effect was a reduction in the maximal rate of transport, with no change in the affinity of the transport protein for TEA.
To determine whether Hg 2+ interacts with cleft cysteines, a quadruple mutant with all four cleft cysteines (C437, C451, C470, and C474) converted to alanines was created. Indeed, wild-type hOCT2 was more sensitive to HgCl 2 than the quadruple mutant, suggesting that Hg 2+ interacts with at least one of the cleft cysteines. Subsequently, the quadruple mutant was used as a template to create four additional mutants, with each having one of the cleft cysteines restored. Of these mutant transport proteins, only the C451- and C474-containing constructs were more sensitive to HgCl 2 than the quadruple mutant, indicating that the side chains of both residues reside in the solvent phase and are accessible to impermeant reagents in the extracellular medium. To obtain independent affirmation for this contention, biotinylation experiments were conducted using MTSEA-biotin. In agreement with the HgCl 2 data, MTSEA-biotin was only effective at precipitating wild-type hOCT2 and the mutants containing C451 and C474. The data reported here further support the conclusion that C474, and provide novel evidence that C451, resides in the aqueous milieu of the cleft.
Cysteine 451 of TMH10 protrudes into the cleft but is the furthest cleft cysteine from the extracellular aspect of the protein ( Fig. 1 D ). However, C451 is not accessible to maleimide-PEO 2 -biotin and does not appear to interact with MTSES ( 16 ). We previously suggested that the relative size of MTSES and maleimide-PEO 2 -biotin, and the distal location of C451, contributed to the inaccessibility of this particular residue. However, the size profile of the hydrophilic thiol-reactive reagents tested against hOCT2 (this and a previous study) was as follows: Hg 2+ The inability of maleimide-PEO 2 -biotin ( 16 ) and MTSEA-biotin to interact with the three peripheral cysteines and six loop cysteines (as well as C437 and C470) indicates that these residues are nonreactive or simply inaccessible to these reagents. The refractoriness of the peripheral cysteines supports the model of OCT2 structure, which shows the cysteines residing in TMH 3, 6, and 9 as being exposed to the lipid bilayer. The loop cysteines are clearly essential for OCT structure, as they are conserved in all orthologs of OCT1, OCT2, and OCT3 currently identified. Perhaps the loop cysteines are inaccessible because of either 1 ) their involvement in disulfide bridges or 2 ) steric hindrance, perhaps caused by the presence of N -glycosylation and/or the topology of the extracellular loop precludes access of MTSEA-biotin (and maleimide-PEO 2 -biotin) to otherwise reactive thiols. Using a mutant of hOCT2 only containing the six loop cysteines, our laboratory found that precipitation of the transport protein with maleimide-PEO 2 -biotin only occurred when the cells were pretreated with 10 mM dithiothreitol (Pelis RM and Wright SH, unpublished observations). These data demonstrate that at least two cysteine residues are paired via a disulfide bridge in the long extracellular loop of hOCT2. The interaction of maleimide-PEO 2 -biotin with C474 was almost completely prevented when substrate/inhibitor (e.g., TEA or tetrapentylammonium) was present in the hOCT2 binding surface, signifying that C474 is close to or part of a region of the protein associated with substrate interaction ( 16 ). However, occupation of the binding surface of either the wild-type or mutant transport proteins by TEA afforded no protection against HgCl 2. Together, these data suggest that although C451 and C474 may be in proximity to a binding surface, they do not interact directly with TEA. Intriguingly, C474 is adjacent to D475, a residue with a profound influence on the interaction of TEA with OCTs ( 6 ). In summary, several important aspects regarding OCT structure/function were noted in the present study. First, Hg 2+ reduced hOCT2-mediated TEA transport in a concentration-dependent manner by interacting with C451 and C474, both of which reside in the hydrophilic cleft, the purported region of substrate-protein interaction. The interaction of MTSEA-biotin with C451 and C474 provided independent confirmation that both residues are exposed to the aqueous milieu of the cleft. However, TEA occupation of the binding surface afforded no protection against the effect on transport of Hg 2+, suggesting that neither C451 nor C474 directly contributes to forming a binding surface for TEA. Consistent with this hypothesis, Hg 2+ had no effect on the affinity of hOCT2 for TEA. Perspectives Kinetic analyses suggest that many of the substrates of OCT2 (e.g., TEA and MPP) are competitive inhibitors of each other; i.e., they share a common binding site. In our hands, the inhibitory constant (IC 50 ) generated from the inhibition by unlabeled 1-methyl-4-phenylpyridinium (MPP) of [ 3 H]TEA uptake into CHO cells expressing OCT2 approximates the Michaelis constant ( K t ) derived from the inhibition by unlabeled MPP of [ 3 H]MPP uptake. The same is true for the IC 50 (inhibition of [ 3 H]MPP uptake by unlabeled TEA) and K t (inhibition of [ 3 H]TEA uptake by unlabeled TEA) generated for TEA. Despite the contention that many substrates of OCTs share a common binding site, evidence to date suggests that OCTs contain multiple spatially distinct (but overlapping) binding domains, a structural feature that likely contributes to the multispecific behavior of these transport proteins. For example, amino acids implicated in substrate-protein interaction have been identified in several different transmembrane helices (e.g., TMH 4, 10, and 11). Mutation of D475 of TMH 10 to glutamate significantly increases the affinity of OCT1 for TEA but not MPP ( 6 ), and conversion of E447 of TMH 11 to leucine in the rabbit ortholog of OCT2 resulted in virtual elimination of TEA and cimetidine transport, but near normal transport of MPP ( 29 ). Additionally, amino acids W218 and Y222 of TMH 4 have been implicated in the binding of both TEA and MPP, whereas an adjacent residue, T226, was suggested to only interact with MPP ( 18 ). Perhaps the sites (one or several amino acid residues) that interact preferentially with particular substrates are contained within larger specified areas (i.e., binding surfaces comprised of many amino acids) that overlap, resulting in interactions that appear to be competitive in nature. GRANTS R. Pelis was supported by a Ruth L. Kirschstein National Research Service Award (DK-752422) from the National Institutes of Health (NIH). This work was supported in part by NIH Grants DK-058251, HL-07249, and ES-06694. ACKNOWLEDGMENTS We thank the Faculty of Medicine Siriraj Hospital, Mahidol University, for its support of Y. Dangprapai during the course of this work. 【参考文献】 作者单位：Department of Physiology, University of Arizona, College of Medicine, Tucson, Arizona
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