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• W2021001573 abstract "Organic cation transporters (OCTs) are involved in the renal elimination of many cationic drugs and toxins. A hypothetical three-dimensional structure of OCT2 based on a homology model that used the Escherichia coli glycerol 3-phosphate transporter as a template has been described (Zhang, X., Shirahatti, N. V., Mahadevan, D., and Wright, S. H. (2005) J. Biol. Chem. 280, 34813-34822). To further define OCT structure, the accessibility to hydrophilic thiol-reactive reagents of the 13 cysteine residues contained in the human ortholog of OCT2 was examined. Maleimide-PEO2-biotin precipitated (surface biotinylation followed by Western blotting) and reduced tetraethylammonium transport by OCT2 expressed in Chinese hamster ovary cells, effects that were largely reversed by co-exposure to substrates and transport inhibitors, suggesting interaction with cysteines that are near to or part of a substrate-binding surface. Cysteines at amino acid position 437, 451, 470, and 474 were identified from the model as being located in transmembrane helices that participate in forming the hydrophilic cleft, the proposed region of substrate-protein interaction. To determine which residues are exposed to the solvent, a mutant with all four of these cysteines converted to alanine, along with four variants of this mutant each with an individual cysteine restored, were created. Maleimide-PEO2-biotin was only effective at precipitating and reducing transport by wild-type OCT2 and the mutant with cysteine 474 restored. Additionally, the smaller thiol-reactive reagent, methanethiosulfonate ethylsulfonate, reduced transport by wild-type OCT2 and the mutant with cysteine 474 restored. These data demonstrate that cysteine 474 of OCT2 is exposed to the aqueous milieu of the cleft and contributes to forming a pathway for organic cation transport. Organic cation transporters (OCTs) are involved in the renal elimination of many cationic drugs and toxins. A hypothetical three-dimensional structure of OCT2 based on a homology model that used the Escherichia coli glycerol 3-phosphate transporter as a template has been described (Zhang, X., Shirahatti, N. V., Mahadevan, D., and Wright, S. H. (2005) J. Biol. Chem. 280, 34813-34822). To further define OCT structure, the accessibility to hydrophilic thiol-reactive reagents of the 13 cysteine residues contained in the human ortholog of OCT2 was examined. Maleimide-PEO2-biotin precipitated (surface biotinylation followed by Western blotting) and reduced tetraethylammonium transport by OCT2 expressed in Chinese hamster ovary cells, effects that were largely reversed by co-exposure to substrates and transport inhibitors, suggesting interaction with cysteines that are near to or part of a substrate-binding surface. Cysteines at amino acid position 437, 451, 470, and 474 were identified from the model as being located in transmembrane helices that participate in forming the hydrophilic cleft, the proposed region of substrate-protein interaction. To determine which residues are exposed to the solvent, a mutant with all four of these cysteines converted to alanine, along with four variants of this mutant each with an individual cysteine restored, were created. Maleimide-PEO2-biotin was only effective at precipitating and reducing transport by wild-type OCT2 and the mutant with cysteine 474 restored. Additionally, the smaller thiol-reactive reagent, methanethiosulfonate ethylsulfonate, reduced transport by wild-type OCT2 and the mutant with cysteine 474 restored. These data demonstrate that cysteine 474 of OCT2 is exposed to the aqueous milieu of the cleft and contributes to forming a pathway for organic cation transport. Renal excretion, accomplished by active proximal tubular secretion, is the principal pathway for elimination of a diverse array of potentially toxic organic compounds, including clinically important therapeutics, environmental toxins, and endogenous metabolites (1Pritchard J.B. Miller D.S. Kidney Int. 1996; 49: 1649-1654Abstract Full Text PDF PubMed Scopus (121) Google Scholar). Many of these compounds fall into the chemical class commonly referred to as “organic cations” (OCs), 2The abbreviations used are: OC, organic cation; OCT, organic cation transporter; hOCT2, human OCT2; rOCT1, rat OCT1; rbOCT2, rabbit OCT2; TMH, transmembrane helix; MFS, major facilitator superfamily; TEA, tetraethylammonium; TMA, tetramethylammonium; TpropA, tetrapropylammonium; TBA, tetrabutylammonium; TPA, tetrapentylammonium; CHO, Chinese hamster ovary; maleimide-PEO2-biotin, ((+)-biotinyl-3-maleimidopropionamidyl-3,6-dioxaoctanediamine; MTSET, [2-(trimethylammonium)ethyl]methanethiosulfonate bromide; MTSES, sodium (2-sulfonatoethyl)methanethiosulfonate; GlpT, glycerol 3-phosphate transporter; PBS, phosphate-buffered saline. which includes a diverse array of primary, secondary, tertiary, or quaternary amines that have a net positive charge on the amine nitrogen at physiological pH. Transport proteins of the renal proximal tubule epithelium mediate OC secretion, thus performing a critical role in detoxification (see reviews in Refs. 2Koepsell H. Annu. Rev. Physiol. 1998; 60: 243-266Crossref PubMed Scopus (287) Google Scholar, 3Koepsell H. Gorboulev V. Arndt P. J. Membr. Biol. 1999; 167: 103-117Crossref PubMed Scopus (141) Google Scholar, 4Dresser M.J. Leabman M.K. Giacomini K.M. J. Pharm. Sci. 2001; 90: 397-421Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 5Koepsell H. Schmitt B.M. Gorboulev V. Rev. Physiol. Biochem. Pharmacol. 2003; 150: 36-90Crossref PubMed Google Scholar, 6Wright S.H. Dantzler W.H. Physiol. Rev. 2004; 84: 987-1049Crossref PubMed Scopus (349) Google Scholar, 7Wright S.H. Toxicol. Appl. Pharmacol. 2005; 204: 309-319Crossref PubMed Scopus (110) Google Scholar). Three homologous OC transporters (OCT1, OCT2, and OCT3) have been cloned and shown to be expressed in the peritubular (i.e. basolateral) membrane of proximal tubule cells where they mediate OC uptake (8Karbach U. Kricke J. Meyer-Wentrup F. Gorboulev V. Volk C. Loffing-Cueni D. Kaissling B. Bachmann S. Koepsell H. Am. J. Physiol. 2000; 279: F679-F687Crossref PubMed Google Scholar, 9Motohashi H. Sakurai Y. Saito H. Masuda S. Urakami Y. Goto M. Fukatsu A. Ogawa O. Inui K.-I. J. Am. Soc. Nephrol. 2002; 13: 866-874Crossref PubMed Google Scholar, 10Thomas M.C. Tikellis C. Kantharidis P. Burns W.C. Cooper M.E. Forbes J.M. J. Pharmacol. Exp. Ther. 2004; 311: 456-466Crossref PubMed Scopus (45) Google Scholar). The OCTs are a potential site of harmful drug-drug interactions because they are multispecific, handling OCs of diverse structure and chemistry. Consequently, understanding the structure of the OCT substrate-binding surface is a critical tool for predicting the interaction of OCs with these transport proteins. The OCTs are members of a larger family of solute carriers (SLC22A), which includes the OCTNs (OCTN1-3) and OATs (OAT1-5). SLC22A family members have common structural features, including 12 putative transmembrane-spanning helices (TMHs), intracellular C and N termini, a large extracellular loop between TMHs 1 and 2, and a large intracellular loop between TMHs 6 and 7. These and other structural features further place SLC22A transport proteins into the major facilitator superfamily (MFS). The elucidation of high-resolution crystal structures of two MFS transporters, the Escherichia coli lactose permease (LacY (11Abramson J. Smirnova I. Kasho V. Verner G. Kaback H.R. Iwata S. Science. 2003; 301: 610-615Crossref PubMed Scopus (1225) Google Scholar)) and glycerol 3-phosphate transporter (GlpT (12Huang Y. Lemieux M.J. Song J. Auer M. Wang D.N. Science. 2003; 301: 616-620Crossref PubMed Scopus (854) Google Scholar)), led to the contention that all MFS transporters share a common structural fold with similar topological organization of α-helices (13Vardy E. Arkin I.T. Gottschalk K.E. Kaback H.R. Schuldiner S. Protein Sci. 2004; 13: 1832-1840Crossref PubMed Scopus (88) Google Scholar). This realization has permitted the application of homology modeling to develop hypothetical three-dimensional structures of several MFS transport proteins, including the rat ortholog of OCT1 (rOCT1 (14Popp C. Gorboulev V. Muller T. Gorbunov D. Shatskaya N. Koepsell H. Mol. Pharmacol. 2005; 67: 1600-1611Crossref PubMed Scopus (121) Google Scholar)) and rabbit ortholog of OCT2 (rbOCT2 (15Zhang X. Shirahatti N.V. Mahadevan D. Wright S.H. J. Biol. Chem. 2005; 280: 34813-34822Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar)). Using these models others have focused on the organization and alignment of residues within the 12 TMHs, resulting in the identification of a large hydrophilic cleft centrally located within the protein that is proposed to contain the substrate-binding surface, as is the case for both LacY (11Abramson J. Smirnova I. Kasho V. Verner G. Kaback H.R. Iwata S. Science. 2003; 301: 610-615Crossref PubMed Scopus (1225) Google Scholar) and GlpT (12Huang Y. Lemieux M.J. Song J. Auer M. Wang D.N. Science. 2003; 301: 616-620Crossref PubMed Scopus (854) Google Scholar). These OCT homology models were suggested to be valid because mutation of several residues (e.g. Glu-447 and Asp-474 in rbOCT2) lining the hydrophilic cleft had effects on substrate affinity and selectivity (14Popp C. Gorboulev V. Muller T. Gorbunov D. Shatskaya N. Koepsell H. Mol. Pharmacol. 2005; 67: 1600-1611Crossref PubMed Scopus (121) Google Scholar, 15Zhang X. Shirahatti N.V. Mahadevan D. Wright S.H. J. Biol. Chem. 2005; 280: 34813-34822Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 16Gorboulev V. Shatskaya N. Volk C. Koepsell H. Mol. Pharmacol. 2005; 67: 1612-1619Crossref PubMed Scopus (79) Google Scholar, 17Gorboulev V. Volk C. Arndt P. Akhoundova A. Koepsell H. Mol. Pharmacol. 1999; 56: 1254-1261Crossref PubMed Scopus (101) Google Scholar). Despite several compelling features of the current models of OCT structure, the very low sequence identity between the OCTs and GlpT and LacY (∼15%) casts substantial doubt on the precise alignment between “target” and “template” in the homology modeling process. For example, the long extracellular loop (a structure unique to SLC22A family members) and long cytoplasmic loop (a structure shared by all MFS transporters) were omitted from the rOCT1 and rbOCT2 sequences (14Popp C. Gorboulev V. Muller T. Gorbunov D. Shatskaya N. Koepsell H. Mol. Pharmacol. 2005; 67: 1600-1611Crossref PubMed Scopus (121) Google Scholar, 15Zhang X. Shirahatti N.V. Mahadevan D. Wright S.H. J. Biol. Chem. 2005; 280: 34813-34822Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) to facilitate the modeling process, and the authors acknowledge that this approach introduced ambiguity with respect to the alignment of adjacent TMHs (15Zhang X. Shirahatti N.V. Mahadevan D. Wright S.H. J. Biol. Chem. 2005; 280: 34813-34822Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Vardy et al. (13Vardy E. Arkin I.T. Gottschalk K.E. Kaback H.R. Schuldiner S. Protein Sci. 2004; 13: 1832-1840Crossref PubMed Scopus (88) Google Scholar) present data suggesting that the accuracy of homology modeling of MFS transporters is significantly enhanced when based on a “manually optimized alignment,” i.e. when the sequence alignment is based on several criteria including experimental data. Thus, although the general organization of α-helices within the postulated three-dimensional structures of rOCT1 and rbOCT2 is probably reasonably accurate, experimental validation of the relative location of amino acid residues within these hypothetical structures is required if the models are to serve as the basis for predicting substrate-transporter interactions. In the present study, the three-dimensional model of the human ortholog of OCT2 (hOCT2) was used to make inferences about the relative positions of the thirteen cysteine residues contained within the transport protein, i.e. are they exposed to the external solvent compartment or embedded in the membrane. Based on their relative position in the model, three populations of cysteine residues were identified in hOCT2, i.e. six residues in the long extracellular loop, three residues in TMHs peripheral to the hydrophilic cleft, and four residues in TMHs that form the cleft. Of the 13 residues, only the cleft cysteine at position 474 of TMH 11 was accessible to both of the thiol-reactive reagents used. These findings are discussed in relation to the proposed three-dimensional structure of hOCT2. Chemicals—[3H]Tetraethylammonium (54 Ci/mmol) was synthesized by Amersham Biosciences. ((+)-Biotinyl-3-maleimidopropionamidyl-3,6-dioxaoctanediamine (maleimide-PEO2-biotin) was obtained from Pierce Biotechnology. [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET) and sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) were obtained from Toronto Research Chemical. Tetrapentylammonium (TPA), tetrabutylammonium (TBA), tetrapropylammonium (TpropA), tetraethylammonium (TEA), tetramethylammonium (TMA), and Ham's F12 Kaighn's modification medium were obtained from Sigma. Platinum® High Fidelity DNA polymerase, zeocin, hygromycin, Flp recombinase expression plasmid (pOG44), and the mammalian expression vector pcDNA5/FRT/V5-His TOPO were obtained from Invitrogen. TOPO Cloning of hOCT2 and Site-directed Mutagenesis— The open reading frame for hOCT2 (contained in pcDNA3.1) containing a C-terminal V5 epitope tag (amino acid sequence, GKPIPNPLLGLDST; nucleotide sequence, GGT AAG CCT ATC CCT AAC CCT CTC CTC GGT CTC GAT TCT ACG) 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 V5-tagged hOCT2 sequence were introduced by site-directed mutagenesis using the QuikChange system following the manufacturer's instructions (Stratagene, La Jolla, CA). 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—Wild-type hOCT2 contained in the pcDNA3.1 expression vector was stably transfected into CHO-K1 cells and maintained as described previously (18Suhre W.M. Ekins S. Chang C. Swaan P.W. Wright S.H. Mol. Pharmacol. 2005; 67: 1067-1077Crossref PubMed Scopus (91) Google Scholar). 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 the hOCT2 mutant constructs. Prior to 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 × 106 cells were transfected by electroporation (BTX ECM 630, San Diego, 260 volts 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 the mutant 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. Cell Surface Biotinylation with Maleimide-PEO2-biotin— The method described here is a minor modification of that described by Pelis et al. (19Pelis R.M. Suhre W.M. Wright S.H. Am. J. Physiol. 2006; 290: F1118-F1126Crossref PubMed Scopus (36) Google Scholar). 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 phosphate-buffered saline (PBS) solution containing calcium and magnesium (PBS/CM (in mm): 137 NaCl, 2.7 KCl, 8 Na2HPO4, 1.5 KH2PO4, 0.1 CaCl2, and 1 MgCl2, pH 7.0 with HCl) followed by a single incubation in maleimide-PEO2-biotin diluted in PBS/CM. The concentration and time of exposure to maleimide-PEO2-biotin is described under “Results.” In some cases, the cells were pre-exposed to the quaternary ammonium compounds TMA (10.5 mm), TEA (1 mm), TpropA (400 μm), TBA (390 μm), or TPA (210 μm) for 2 min followed by inclusion of these compounds in the biotinylation reaction. The quaternary ammonium compounds were used at concentrations 20-fold higher than their reported IC50 values for inhibition of TEA transport by hOCT2 (18Suhre W.M. Ekins S. Chang C. Swaan P.W. Wright S.H. Mol. Pharmacol. 2005; 67: 1067-1077Crossref PubMed Scopus (91) Google Scholar). 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. 50 μl 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 was added, and the proteins were eluted from the beads at 100 °C for 5 min. Proteins were separated on 7.5% SDS-PAGE and transferred to polyvinylidene difluoride membranes, and immunoreactivity corresponding to the V5-tagged hOCT2 constructs was detected as described previously (19Pelis R.M. Suhre W.M. Wright S.H. Am. J. Physiol. 2006; 290: F1118-F1126Crossref PubMed Scopus (36) Google Scholar). Immunocytochemistry—CHO cells grown on coverslips in 12-well plates were washed with PBS (137 mm NaCl, 2.7 mm KCl, 8.0 mm Na2HPO4, 1.5 mm KH2PO4, pH 7.3). All subsequent washes were performed in triplicate at room temperature in PBS. Cells were fixed in ice-cold 100% methanol for 20 min, washed, and incubated for 1 h with mouse anti-V5 antibody (Invitrogen) diluted in PBS (final concentration of 2 μg/ml). The cells were washed and incubated for 1 h in the dark with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Invitrogen) diluted to 2 μg/ml in PBS. The cells were washed before staining the nuclei with propidium iodide (5 μg/ml in PBS; Sigma) for 10 min. Cells were washed again and the coverslips mounted onto microscope slides. A confocal microscope (Nikon PCM 2000 scan head fitted to a Nikon E800 microscope) was used for detection of immunoreactivity in CHO cells. Transport Experiments—CHO cells grown to confluence in 12-well plates were rinsed twice with Waymouth's buffer (WB (in mm): 135 NaCl, 28 d-glucose, 5 KCl, 1.2 MgCl2, 2.5 CaCl2, 0.8 MgSO4, and 13 HEPES-NaOH, pH 7.4) at room temperature followed by incubation in either WB or WB containing maleimide-PEO2-biotin, MTSET, or MTSES. The concentration and time of exposure to maleimide-PEO2-biotin is described under “Results.” Cells were treated with 1 mm MTSET or 5 mm MTSES for 5 min. The dose and time of exposure to MTSET and MTSES were similar to that used to examine cysteine accessibility in the creatine transporter, CreaT (20Dodd J.R. Christie D.L. J. Biol. Chem. 2005; 280: 32649-32654Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Cells were rinsed three times with 2 ml of WB to remove any excess maleimide-PEO2-biotin, MTSET, or MTSES that had not reacted with reactive thiol groups. Control cells not treated with the thiol-reactive reagents were handled in a similar manner. To determine whether the presence of TPA in the binding surface prevented the reduction in transport elicited by maleimide-PEO2-biotin (see “Results”), the cells were pre-exposed to 210 μm TPA for 2 min followed by inclusion of TPA in the biotinylation reaction. Prior to conducting transport in these experiments, the cells were rinsed four times with 2 ml of WB over a 20-min period to remove any residual TPA and/or maleimide-PEO2-biotin. Uptake of 1 μCi/ml [3H]TEA (14 nm) diluted in WB was conducted in the presence and absence of 2 mm unlabeled TEA (∼40-fold higher than the Michaelis constant for hOCT2-mediated TEA transport) to determine the amount of [3H]TEA transport specifically mediated by hOCT2. The cells were then solubilized in 400 μl of 0.5N NaOH with 1% SDS (v/v), and the resulting lysate was neutralized with 200 μl of 1 n HCl. Accumulated radioactivity was determined by liquid scintillation spectrometry (Beckman model LS3801). Individual transport observations were performed in duplicate for each experiment, and observations were confirmed at least three times in separate experiments using cells of a different passage. Statistics—All data are expressed as means ± S.D., with calculations of standard errors based on the number of separate experiments conducted on cells at a different passage number. One-way analysis of variance 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, Inc., Pearl River, NY) and were deemed significant when p < 0.05. Interaction of Maleimide-PEO2-biotin with Wild-type hOCT2—Fig. 1 shows the secondary structure model of hOCT2 based upon the homology model of rbOCT2 (15Zhang X. Shirahatti N.V. Mahadevan D. Wright S.H. J. Biol. Chem. 2005; 280: 34813-34822Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), emphasizing the placement within the sequence of 13 cysteine residues contained within this transport protein. There are six residues in the long extracellular loop and one or two cysteines in TMHs 3, 6, 9, 10, and 11. There are no cysteine residues present in any of the predicted short extracellular loops or intracellular loops. The presence of cysteines in the hOCT2 sequence led to the initial hypothesis that the membrane-impermeable, thiol-reactive reagent maleimide-PEO2-biotin would interact with one or more of these cysteine residues. With a fixed concentration of maleimide-PEO2-biotin (0.5 mg/ml) in the biotinylation reaction, immunoreactivity on Western blots corresponding to biotinylated hOCT2 expressed at the plasma membrane of CHO cells increased with increasing time of exposure to the thiol-reactive reagent, with only a slight increase in immunoreactivity with an exposure time longer than 20 min (supplemental Fig. 1A). The relative molecular mass of hOCT2 was ∼85 kDa, a profile similar to that of rbOCT2 (19Pelis R.M. Suhre W.M. Wright S.H. Am. J. Physiol. 2006; 290: F1118-F1126Crossref PubMed Scopus (36) Google Scholar). The precipitation of hOCT2 also increased in a concentration-dependent manner, with only a modest increase in precipitated immunoreactivity produced by exposure to maleimide-PEO2-biotin above 0.1 mg/ml (supplemental Fig. 1B). The effectiveness of maleimide-PEO2-biotin to precipitate hOCT2 shows that the cysteine-modifying reagent interacts with this transport protein. To determine the effect on hOCT2-mediated transport activity of covalent modification of cysteine residues, TEA uptake was measured after exposure of CHO cells expressing hOCT2 to maleimide-PEO2-biotin. Uptake of [3H]TEA by cells expressing hOCT2 increased with time in a near linear manner for 30 s, with more than 95% of transport blocked by the addition of 2 mm unlabeled TEA to the transport reaction (supplemental Fig. 2, inset). At a concentration of 0.5 mg/ml, maleimide-PEO2-biotin reduced TEA transport in a time-dependent manner with a 70% reduction in transport at 20 min (supplemental Fig. 2A). Using a fixed exposure time of 20 min, there was a concentration-dependent decrease in TEA transport with a maximum effect at 0.5 mg/ml (80% reduction in TEA uptake) (supplemental Fig. 2B). Subsequent biotinylation and transport experiments were conducted by exposing the cells for 20 min to 0.5 mg/ml maleimide-PEO2-biotin. Protection of Biotinylation and Transport by Quaternary Ammonium Compounds—The quaternary ammonium compounds, TMA, TEA, TpropA, TBA, and TPA all interact with OCT2, as shown by the ability of each of these compounds to inhibit OCT2-mediated transport of radiolabeled TEA (and other organic cations) (18Suhre W.M. Ekins S. Chang C. Swaan P.W. Wright S.H. Mol. Pharmacol. 2005; 67: 1067-1077Crossref PubMed Scopus (91) Google Scholar). To determine whether the presence of a substrate/inhibitor within the binding surface/region influences the ability of maleimide-PEO2-biotin to access one or more of the reactive thiols within the hOCT2 sequence, we tested the effect of exposing cells to these quaternary ammonium compounds on precipitation of transport protein. The addition of TMA, TEA, TpropA, TBA, or TPA to the biotinylation reaction almost completely prevented the precipitation of hOCT2 (Fig. 2A). Furthermore, exposure to TPA ameliorated the reduced level of TEA transport elicited by exposure of hOCT2-expressing cells to maleimide-PEO2-biotin (Fig. 2B). These data suggest that maleimide-PEO2-biotin interacts with one or more cysteine residues that are near to or part of the binding surface for the quaternary ammonium compounds. Fig. 3 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 (15Zhang X. Shirahatti N.V. Mahadevan D. Wright S.H. J. Biol. Chem. 2005; 280: 34813-34822Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Between the N- and C-terminal halves of the protein sits a large hydrophilic cleft that is proposed to contain the binding surface for organic cations (15Zhang X. Shirahatti N.V. Mahadevan D. Wright S.H. J. Biol. Chem. 2005; 280: 34813-34822Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). TMHs 1, 2, 4, 5, 7, 8, 10, and 11 of OCT2 (Fig. 3A, blue) form the hydrophilic cleft, whereas the other TMHs (i.e. 3, 6, 9, and 12 (green)) are peripheral, are not exposed to solvent, and likely assist in anchoring the protein in the membrane. In TMHs 10 and 11, Glu-488 and Asp-475 (Glu-447 and Asp-474 in the rabbit ortholog) (Fig. 3, B and C, red) are oriented toward the hydrophilic cleft; site-directed mutagenesis studies have shown that these residues exert a profound influence on substrate affinity and selectivity (14Popp C. Gorboulev V. Muller T. Gorbunov D. Shatskaya N. Koepsell H. Mol. Pharmacol. 2005; 67: 1600-1611Crossref PubMed Scopus (121) Google Scholar, 15Zhang X. Shirahatti N.V. Mahadevan D. Wright S.H. J. Biol. Chem. 2005; 280: 34813-34822Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 16Gorboulev V. Shatskaya N. Volk C. Koepsell H. Mol. Pharmacol. 2005; 67: 1612-1619Crossref PubMed Scopus (79) Google Scholar, 17Gorboulev V. Volk C. Arndt P. Akhoundova A. Koepsell H. Mol. Pharmacol. 1999; 56: 1254-1261Crossref PubMed Scopus (101) Google Scholar). Using the homology model of rbOCT2, Zhang et al. (15Zhang X. Shirahatti N.V. Mahadevan D. Wright S.H. J. Biol. Chem. 2005; 280: 34813-34822Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) incorporated several residues known to influence substrate binding (e.g. Glu-447 and Asp-474) to develop a 5-Å docking surface within the cleft (Fig. 3D). The model of the docking surface did surprisingly well in predicting the interaction of the transport protein with 14 different compounds, including high affinity organic cations and low affinity organic anions. As noted earlier, of the 13 cysteine residues found in hOCT2, six are found in the long extracellular loop (“loop” cysteines (yellow triangles)). Of the remaining seven cysteines, three are present in the peripheral TMHs (“peripheral” cysteines (orange circles)), whereas four are distributed in the cleft (“cleft” cysteines (yellow circles)), i.e. within TMHs 10 and 11, at amino acid positions 437, 451, 470, and 474 (Fig. 3C). We hypothesized that maleimide-PEO2-biotin interacts with one or more of the four residues in TMHs 10 and 11 (Fig. 3D). Cysteine Accessibility in the Hydrophilic Cleft of hOCT2— 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 was restored at each individual position (Cys-437, Cys-451, Cys-470, and Cys-474). All of the mutant transporters were expressed in the plasma membrane and displayed considerable transport activity, albeit at a reduced level compared with the wild-type transport protein (TEA uptake 3-6-fold lower than wild type) (Fig. 4). Of the mutant transporters, only the mutant containing Cys-474 exhibited TEA transport activity that was sensitive to maleimide-PEO2-biotin (Fig. 5A). Indeed, biotinylation experiments showed that only wild-type hOCT2 and the Cys-474 add-back mutant could be precipitated with maleimide-PEO2-biotin. As noted previously for wild-type hOCT2, the reduction caused by maleimide-PEO2-biotin of TEA transport by the Cys-474 add-back mutant was prevented by co-treatment with TPA (Fig. 5B). Precipitation of the quadruple mutant and the Cys-437, Cys-451, and Cys-470 add-back mutants was rescued following permeabilization of the plasma membrane with 0.1% saponin (supplemental Fig. 3). This observation confirms that maleimide-PEO2-biotin does not readily permeate the plasma membrane under the experimental procedures used. The precipitation of the transport protein following permeabilization probably reflects the interaction of maleimide-PEO2-biotin with one or more of the cl" @default.
• W2021001573 created "2016-06-24" @default.
• W2021001573 creator A5008199212 @default.
• W2021001573 creator A5010971240 @default.
• W2021001573 creator A5017318940 @default.
• W2021001573 creator A5033940581 @default.
• W2021001573 date "2006-11-01" @default.
• W2021001573 modified "2023-10-15" @default.
• W2021001573 title "Cysteine Accessibility in the Hydrophilic Cleft of Human Organic Cation Transporter 2" @default.
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