QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.107.125716v1 jpet.107.125716v2 jpet.107.125716v3 323/2/720    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mason, J. N. Articles by Blakely, R. D. Search for Related Content PubMed PubMed Citation Articles by Mason, J. N. Articles by Blakely, R. D.

NEUROPHARMACOLOGY

Desvenlafaxine Succinate Identifies Novel Antagonist Binding Determinants in the Human Norepinephrine Transporter

Departments of Pharmacology (J.N.M., R.J.R., R.D.B.) and Psychiatry (R.D.B.) and Center for Molecular Neuroscience (R.D.B.), Vanderbilt University School of Medicine, Nashville, Tennessee; and Women's Health Research (D.C.D., R.C.W.) and Chemical and Screening Sciences (G.S., P.E.M., E.T.), Wyeth Pharmaceuticals, Collegeville, Pennsylvania

Received May 12, 2007; accepted August 1, 2007.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Desvenlafaxine succinate (DVS) is a recently introduced antagonist of the human norepinephrine and serotonin transporters (hNET and hSERT, respectively), currently in clinical development for use in the treatment of major depressive disorder and vasomotor symptoms associated with menopause. Initial evaluation of the pharmacological properties of DVS (J Pharmacol Exp Ther 318:657–665, 2006) revealed significantly reduced potency for the hNET expressed in membranes compared with whole cells when competing for [³H]nisoxetine (NIS) binding. Using hNET in transfected human embryonic kidney-293 cells, this difference in potency for DVS at sites labeled by [³H]NIS was found to distinguish DVS, the DVS analog rac-(1-[1-(3-chloro-phenyl)-2-(4-methylpiperazin-1-yl)-ethyl]cyclohexanol (WY-46824), methylphenidate, and the cocaine analog 3-(4-iodophenyl)tropane-2-carboxylic acid methyl ester (RTI-55) from other hNET antagonists, such as NIS, mazindol, tricyclic antidepressants, and cocaine. These differences seem not to arise from preparation-specific perturbations of ligand intrinsic affinity or antagonist-specific surface trafficking but rather from protein conformational alterations that perturb the relationships between distinct hNET binding sites. In an initial search for molecular features that differentially define antagonist binding determinants, we document that Val148 in hNET transmembrane domain 3 selectively disrupts NIS binding but not that of DVS.

Although more than a decade has elapsed since the initial cloning of the biogenic amine transporters, only recently have specific determinants of antagonist recognition been elucidated. Progress on the interaction of selective 5-HT reuptake inhibitors (SSRIs) with hSERT proteins has localized high-affinity binding determinants of these agents to TMs 1 and 3 (Barker et al., 1998; Henry et al., 2006b), regions that also support substrate interactions as identified in structure-function studies (Barker et al., 1999; Adkins et al., 2001) and crystallographic resolution of leucine binding in LeuT_Aa (Yamashita et al., 2005; Henry et al., 2006a). Less is understood concerning the interactions of hNET with its antagonists or with the class of 5-HT/NE reuptake inhibitors, typified by agents such as venlafaxine (Stahl et al., 2005; Vis et al., 2005; Dell'Osso et al., 2006) and the recently described analog desvenlafaxine succinate (DVS) (Alfinito et al., 2006; Deecher et al., 2006). Previously, we showed that DVS binds to and inhibits both hNET and hSERT using in vitro bioassays. Furthermore, using in vivo microdialysis studies, we demonstrated that DVS elevates extracellular levels of both NE and 5-HT in rat hypothalamus and frontal cortex. In our initial characterization of DVS, we noted differences in DVS potency for hNET in membrane preparations versus whole cells for displacement of [³H]nisoxetine (NIS) binding. In this report, we further explored this finding to understand whether loss of potency is a general feature of NET antagonists, whether it is supported by ligand-induced hNET trafficking, and/or whether it reflects unique properties of hNET antagonist binding sites that can be further defined using site-directed mutagenesis.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Compounds. DVS and the DVS analog WY-46824 (Fig. 1, A–D) (Husbands and Stack, 1989a,b) were synthesized by the Chemical and Screening Sciences group of Wyeth Research (Princeton, NJ). Desipramine (catalog number D-3900), mazindol (catalog number M-2017), imipramine (catalog number I7379), methylphenidate (catalog number M2892), and cocaine hydrochloride (catalog number C5776) were purchased from Sigma-Aldrich (St. Louis, MO). RTI-55 was a gift from F. Ivy Carrol (RTI International, Research Triangle Park, NC).

View larger version (11K): [in this window] [in a new window]   Fig. 1. Structural illustrations of norepinephrine reuptake inhibitors DVS (A) DVS analog WY-46824 (B), cocaine analog RTI-55 (C), and NIS (D). Inset box notes the position of tritium (T) labeling sites of [3H]WY-46824.

Radioligands. [³H]NE (catalog number NET-048; 5–15 Ci/mmol), [³H]nisoxetine (catalog number NET-1084; 85.5 Ci/mmol), [¹²⁵I]RTI-55 (catalog number NEX-272; 2200 Ci/mmol) and scintillation cocktail (Ultima Gold; catalog number 6013329) were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). The [³H]WY-46824 (Fig. 1, inset) was radiolabeled by the Chemical and Screening Science group (Wyeth Research). 1-[1-(3-Chlorophenyl)-2-(4-methylpiperazin-1-yl)-2-oxoethyl]cyclohexanol was reduced with B[³H₃] (Than et al., 1995) (3 Eq) in tetrahydrofuran at 70°C to produce [³H]WY-46824 (specific activity, 22 Ci/mmol, as determined by UV at 249 nm).

Cell Culture, Transfection, and Reagents. The hNET-HEK-293 cells, stably transfected with hNET (Pacholczyk et al., 1991; Galli et al., 1995) or hNET mutants were cultured in growth medium containing high-glucose Dulbecco's modified Eagle's medium (catalog number 11995; Invitrogen, Carlsbad, CA), 10% fetal bovine serum (dialyzed, heat-inactivated, lot FBD1129HI; U.S. Bio-Technologies, Parkerford, PA) and 500 µg/ml Geneticin (G-418, catalog number 10131; Invitrogen). Cells were routinely plated at 300,000 cells/T75 flask, and they were split twice weekly. For evaluation of the impact of mutation on antagonist binding, HEK-293 cells were plated at a density of 10,000 cells per well in a 96-well culture plate. Cells were transfected with hNET or hNET mutant constructs with TransIT transfection reagent (4 ml/mg DNA; Mirus Inc., Madison, WI). Mutation of hNET in pcDNA3.1 to generate mutants V148M and F72Y has been described previously (Henry et al., 2006b). Following transfection (24–48 h), cells were washed with Krebs-Ringer-HEPES buffer (120 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 2.2 mM CaCl₂, and 10 mM HEPES, pH 7.4), and they were assayed either as whole cells or used to make membrane preparations in the radioligand binding assays described below.

Membrane Preparation and Radioligand Binding Assays. The preparation of membranes for binding assays were done by dispensing 3 x 10⁶ hNET-HEK-293 cells into 80-cm² Nunc tissue culture flasks (Fisher Scientific, Pittsburgh, PA) containing growth medium, and they were maintained for 2 days before harvest. Cells were harvested using cell scrapers, and the cell debris was collected from one flask using phosphate-buffered saline (PBS) solution devoid of calcium and magnesium. The cells were centrifuged at 3000g for 10 min at 4°C to remove residual cell media and to pellet the cells. The supernatant was discarded, and the cell pellet was resuspended in 5 ml of binding buffer (50 mM Tris-HCl, 300 mM NaCl, and 5 mM KCl, pH 7.4), and the it was homogenized with a tissue tearer (BioSpec Products, Inc., Bartlesville, OK) at a setting of 2. Cell membranes were centrifuged at 4°C for 10 min at 19,000g in a Biofuge Stratus centrifuge (Sorvall, Newton, CT). The resulting pellet was resuspended in 5 ml of binding buffer, and the above-mentioned steps were repeated twice more. Fresh membranes were suspended in binding buffer (4°C) and prepared to approximately 3 ± 1 µg of protein per 200-µl aliquot. Protein assays were performed (Bio-Rad, Hercules, CA) using bovine serum albumin as standard. Binding reactions were performed in 12- x 75-mm borosilicate glass tubes (Fisherbrand catalog number 14-961-26; Fisher Scientific). Cell membranes (200 µl) were added to each reaction tube, followed by 50 µl of compound solution. Desipramine (200 nM final concentration) was added to duplicate tubes, to assess nonspecific binding. Total radioligand bound was defined using [³H]NIS (GE Healthcare, Little Chalfont, Buckinghamshire, UK) at 5 nM, [³H]WY-46824 (Wyeth Research) at 200 nM, or [¹²⁵I]RTI-55 (Amersham Corp.) at 8 nM. Stock solutions of WY-46824 and mazindol were prepared in dimethyl sulfoxide/H₂O (1:1) at concentrations from 100 nM to 10 mM. RTI-55, nisoxetine, imipramine, desipramine, cocaine, and methylphenidate were prepared in binding buffer. On day of assay, compounds were diluted in assay buffer according to test range (10^–9–10^–2 nM). Compounds and membranes were preincubated at 4°C for 15 min before addition of radioligand, incubating for 2 h at 4°C. Assays were terminated by rapid filtration on a cell harvester (Brandel Inc., Gaithersburg, MD) through glass fiber filters preequilibrated in 0.3% polyethylenimine. Filters containing ³H transporter were counted in a Tri-Carb 2900TR liquid scintillation analyzer (PerkinElmer Life and Analytical Sciences, Boston, MA) whereas the ¹²⁵I-labeled transporters were analyzed on a Gamma 4000 gamma counter (Beckman Coulter, Fullerton, CA).

Whole-Cell Radioligand Binding Assays. Twenty-four hours before assay, cells were plated in 96-well plates at 3000 to 5000 cells/well in growth medium, and they were maintained in a cell incubator at 37°C, 5% CO₂. On day 2, growth medium was replaced with 80 µl of assay buffer (25 mM HEPES, 120 mM NaCl, 5 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgSO₄, and 2 mg/ml glucose, pH 7.4, at 37°C) containing 0.2 mg/ml ascorbic acid and 1 µM pargyline. A 10-µl aliquot of compound was added to each assay well. Competition radioligand binding assays were conducted with [³H]NIS or [¹²⁵I]RTI-55 (5 and 8 nM final concentration, respectively). Saturation binding studies with [³H]NIS and [¹²⁵I]RTI-55 used increasing concentrations of radioligand with nonspecific binding defined at each point with 200 nM desipramine. The DVS analog [³H]WY-46824 displayed too low an affinity in membranes to be used in saturation analyses; thus, use of this label was restricted to competition studies performed at 200 nM final radioligand concentration. Compounds and membranes were preincubated at 37°C for 15 min before initiating the binding reaction by addition of radioligand. The cells in the assay buffer with test compound and radioligand were incubated for 2 h at 37°C. The cells were washed twice with 200 µl of assay buffer at room temperature to remove unbound radioligand. After binding, cells were incubated for 1 h in MicroScint 20 scintillation fluid (PerkinElmer Life and Analytical Sciences), and radiolabeled ligand binding was quantified using a TopCount plate scintillation counter (PerkinElmer Life and Analytical Sciences).

[³H]NE Uptake Assays. Cells were plated at 3000 cells/well in a 96-well plate in growth medium and maintained at 37°C, 5% CO₂. On day 2, growth medium was replaced with 80 µl of assay buffer (25 mM HEPES, 120 mM NaCl, 5 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgSO₄, and 2 mg/ml glucose, pH 7.4, at 37°C) containing 0.2 mg/ml ascorbic acid and 10 µM pargyline. Cells were equilibrated in 90 ml of assay buffer for 10 min at 37°C before addition of compounds. Desipramine was delivered to triplicate wells (final concentration 200 nM) to define nonspecific NE uptake. A 10-µl aliquot of vehicle or various concentrations of antagonist were added directly to triplicate wells containing cells in 80 µl of assay buffer. The cells were preincubated with test compound for 15 min at 37°C. To initiate transport, [³H]NE (1-[7,8³H]norepinephrine, 30 Ci/mmol; Amersham Biosciences) was diluted in assay buffer (50 nM final assay concentration), and it was delivered in 10-µl aliquots to each well. Then, the plates were incubated for 10 min at 37°C. Medium was then removed from wells, and cells were washed twice with 200 µl of assay buffer to remove unincorporated [³H]NE label. The wells containing cells were then incubated for 1 h in 80 µl of MicroScint 20 scintillation fluid (PerkinElmer Life and Analytical Sciences), and the [³H]NE accumulation quantified using a TopCount scintillation counter (PerkinElmer Life and Analytical Sciences).

Cell Surface Biotinylation Assays. To examine possible antagonist-induced alterations in transporter surface expression, surface proteins were labeled with the lysine-directed, membrane-impermeant biotinylating reagent sulfo-NHS-SS-biotin (Pierce Chemical, Rockford, IL) as described previously (Apparsundaram et al., 1998). The hNET-HEK-293 cells were plated into six-well dishes (500,000 cells/well), and the following day cells were washed four times with PBS/CM (137 mM NaCl, 2.7 mM KCl, 1.5 mM KH₂PO₄, 8.1 mM Na₂HPO₄, 0.1 mM CaCl₂, and 1.0 mM MgCl₂) at 4°C and incubated with 1.5 mg/ml sulfo-NHS-SS-biotin in PBS/CM for 30 min at 4°C. To terminate biotinylation reactions and quench unreacted biotinylating reagent, cells were washed twice with 100 µM glycine in PBS/CM and incubated for 20 min at 4°C in glycine/PBS/CM containing buffer. The buffer was removed and solubilized in RIPA buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, and 1% sodium deoxycholate) containing protease inhibitors (1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µM pepstatin, and 250 µM phenylmethylsulfonyl fluoride), by shaking for 30 min at 4°C. The supernatants were removed and cleared of insoluble material by pelleting at 20,000xg for 20 min at 4°C. ImmunoPure immobilized streptavidin beads (Pierce) were washed three times with 100 mM glycine /PBS/CM and then four times with RIPA buffer, finally resuspending the beads in RIPA buffer. A portion of the supernatant that constituted total solubilized protein was removed for subsequent analysis. A 210-µl aliquot of bead/RIPA slurry was added to 750 µl of supernatant, and the contents were gently mixed 1 h at room temperature. The streptavidin beads were then washed four times with RIPA buffer, and biotinylated proteins were eluted from the beads with 50 µl of Laemmli sample buffer (Laemmli, 1970) and analyzed in parallel with total samples on a 10% SDS-polyacrylamide gel electrophoresis gel. Proteins were transferred electrophoretically to Immobilon-P membrane (Millipore Corporation, Billerica, MA), and the membranes were incubated with a monoclonal antibody directed against hNET (NET17-1; Mab Technologies, Inc., Stone Mountain, GA) at a dilution of 1:400 followed by incubation with a goat anti-mouse peroxidase-conjugated secondary antibody (Jackson ImmunoResearch laboratories Inc., West Grove, PA) at a dilution of 1:5000. Visualization of immunoreactivity was achieved using Western Lightning Chemiluminescent Reagent (PerkinElmer Life and Analytical Sciences). Films were scanned at multiple exposures to ensure linearity of exposure. The values were averaged from three experiments to obtain mean values with statistical tests comparing vehicle- and antagonist-treated preparations.

Statistical Analysis and Data Presentation. Competition and saturation isotherms were generated using Prism software (GraphPad Software Inc., San Diego, CA). Calculation of the dissociation constant (K_i) values was based on the dissociation constant (K_D) values of the radioligand for each bioassay (Cheng and Prusoff, 1973). Data analyses of the equilibrium radioligand binding assays were done using nonlinear regression curve fitting based on a multisite equation for saturation binding. Because Hill terms proved nonsignificantly different from 1, the Hill term was set to 1, and the analysis was repeated to estimate the K_D and maximal binding capacity (B_max) values. Likewise, fits to competition data used a single-site model to determine IC₅₀ values before K_i conversion. Values reported are the mean of at least three independent experiments ± S.E.M. Potency comparisons and levels of total/surface hNET proteins were made using an unpaired Student's t test. A P 0.05 was used as a threshold for statistical significance.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Affinity Differences of NET Antagonists in Membranes Compared with Whole Cells Containing the hNET
Radioligand Binding Competition Assays Using [³H]NIS. Our previous report demonstrated a reduction in DVS potency when competing for [³H]NIS binding at hNET expressed in Madin-Darby canine kidney cells in membrane preparations versus whole cells. To extend these studies, we examined the hNET expressed in stably transfected HEK-293 cells. The affinities of DVS and other NET antagonists were compared in [³H]NIS radioligand binding competition assays using either whole cells or membrane preparations. The structures of the primary NET antagonists studied in these experiments were selected due to their structural diversity (Fig. 1). As initially reported with hNET-Madin-Darby canine kidney cells, we observed that DVS exhibited lower potency (6-fold) when competing for [³H]NIS labeling of hNET in membrane preparations compared with whole cells expressing the hNET (Table 1). In contrast, NIS, mazindol, and the tricyclic antidepressants imipramine and desipramine exhibited no change in potency (Table 1). The potency of the ADHD medication methylphenidate (Ritalin; Sigma-Aldrich) was also negatively affected by membrane preparation (8-fold shift). Although cocaine displayed no shift in potency, the cocaine analog RTI-55 demonstrated a 23-fold loss in potency when competing [³H]NIS for hNET in whole cells versus membrane preparations (Table 1). The potency shift for the DVS analog WY-46824 was the greatest among compounds tested (275-fold), and the shift bracketed potency shifts of other structurally related analogs of DVS and WY-46824 (data not shown).

Affinity Comparisons from Competition Assays Using Either [³H]NIS Binding or [³H]NE Uptake Assays. Competition curves depicting DVS, WY-46824, and desipramine for [³H]NIS labeling in either whole cells or membrane preparations are compared with [³H]NE uptake inhibition performed in whole cells containing hNET (Fig. 2, A–C). Antagonist potencies determined using the [³H]NE uptake bioassay closely correlate to those affinities reported when [³H]NIS is used to label hNET in whole cells (Table 1). These data suggest that the membrane preparation introduces a specific change in the hNET that can be revealed in [³H]NIS radioligand competition assays when a subset of hNET antagonists are used as competitors.

View larger version (11K): [in this window] [in a new window]   Fig. 2. Competition assays DVS (A), WY-46824 (DVS analog; B), and desipramine (C) for [3H]NIS binding reveal differences in affinities dependent on the source of hNET. Intact HEK-293 cells (open circles) or membranes (closed circles) containing the hNET were incubated with 5 nM [3H]NIS and increasing concentrations of desvenlafaxine succinate, WY-46824, or desipramine for 2.5 h at 37°C (whole cells) or 4°C (membranes) as described under Materials and Methods. Nonspecific binding was defined by 200 nM desipramine. Norepinephrine uptake (open squares) was performed with HEK-293 cells expressing hNET incubated with 50 nM [3H]NE for 5 min as described under Materials and Methods. Nonspecific uptake was defined with 1 µM desipramine. Results are the average of three separate experiments, with individual points assayed in triplicate. The Ki values for these experiments are reported in Table 1.

View larger version (9K): [in this window] [in a new window]   Fig. 3. Lack of affinity differences when using [125I]RTI-55 in hNET competition assays with either isolated membranes or intact cells. DVS (A), WY-46824 (B), and desipramine (C) competition for [125I]RTI-55 binding at hNET expressed in HEK-293 intact cells and membranes. The HEK-293 intact cells (open symbols) or membranes (closed circles) stably expressing hNET were incubated with 5 nM [125I]RTI-55 and increasing concentrations of desvenlafaxine succinate, WY-46824, or desipramine for 2.5 h at 37°C (intact cells) or 4°C (membranes) as described under Materials and Methods. Nonspecific binding was defined using 200 nM desipramine. Results are the average of three separate experiments, with individual points assayed in triplicate. The Ki values for these depicted competition curves are reported in Table 2.

Equilibrium Radioligand Binding Assays Using [¹²⁵I]RTI-55 and [³H]Nisoxetine Reveal Different Binding Densities in Membrane Preparations
Determination of the K_D for [¹²⁵I]RTI-55 and [³H]NIS. The observations of differences in antagonist potencies in membranes depending on the radioligand used led us to assess the intrinsic affinities of hNET sites labeled by [³H]NIS or [¹²⁵I]RTI-55. Thus, we generated saturation binding isotherms for [³H]NIS (Fig. 4, A and C) and [¹²⁵I]RTI-55 (Fig. 4, B and D) at hNET in both whole cell and membrane preparations. Our studies revealed no significant differences in the K_D values of [³H]NIS or [¹²⁵I]RTI-55 at hNET comparing the affinities obtained using whole cells versus membrane preparations (Table 3).

View larger version (7K): [in this window] [in a new window]   Fig. 4. The number of hNET binding sites differ in membranes compared with whole cells, dependent on radioligand used, with no apparent affinity differences using equilibrium binding assays. A to D, saturation analyses of [3H]NIS (open circles) and [125I]RTI-55 binding (closed circles) to hNET in transfected HEK-293 whole cells and membranes. Whole cell and membranes from HEK-293 cells stably expressing the hNET were incubated with increasing concentrations of either [3H]NIS or [125I]RTI-55 for 2.5 h as described under Materials and Methods. Nonspecific binding was defined 200 nM desipramine. Results are the average of three separate experiments, with individual points assayed in triplicate. Graphs are representative plots of single experiments with average values for KD and Bmax values derived from single-site hyperbolic fits (Prism). The KD and Bmax values for these depicted competition curves are reported in Table 3.

Determination of the B_max for [¹²⁵I]RTI-55 and [³H]Nisoxetine. Interestingly, the B_max values for [³H]NIS and [¹²⁵I]RTI-55 in whole cells were equivalent, whereas the number of hNET binding sites labeled by [¹²⁵I]RTI-55 in membrane preparations is approximately 3.5 times that established for [³H]NIS, suggesting a preparation-specific exposure of sites for the cocaine analog (Table 3).

A DVS Analog Identifies Affinity Differences of NET Antagonists in Membranes Compared with Whole Cells Containing hNET
Radioligand Binding Competition Assays Using [³H]WY-46824. Because membrane preparation does not seem to alter the intrinsic affinity of sites labeled by [³H]NIS or [¹²⁵I]RTI-55, we sought evidence as to whether membrane preparation alters the intrinsic affinity of DVS-like compounds. To address this objective, the DVS analog WY-46824 was radiolabeled (Fig. 1, inset). Preliminary evaluation was completed to determine the specific binding of [³H]WY-46824 for the hNET in whole cells and membrane preparations. Although we could establish the presence of hNET-specific sites using comparisons to nontransfected cells (data not shown), the specific binding in hNET-transfected membranes across a concentration range was too low to generate adequate saturation plots due to increasing nonspecific binding. However, we were able to obtain competition binding data with [³H]WY-46824 in both preparations (Fig. 5). A loss of potency for both WY-46824 (50-fold) and NIS (256-fold) was observed when competing for [³H]WY-46824 (200 nM), in membrane preparations compared with whole cells (Fig. 5, B and C). Interesting, both NIS and WY-46824 showed similar affinities when competed for [³H]WY-46824 using whole cells (Table 4). We performed these assays in parallel with [³H]NIS competition assays and found, as noted above in both competition and saturation, that NIS loses no potency comparing these two preparations (Fig. 5A). These striking data underscore a specific sensitivity of the site labeled by [³H]WY-46824 to membrane preparations that 1) reduces intrinsic WY-46824 affinity and 2) occludes interactions of NIS with the [³H]WY-46824 site.

View larger version (9K): [in this window] [in a new window]   Fig. 5. A decrease in affinity was noted for nisoxetine when competing for [3H]WY-46824 binding to hNET in membranes. NIS (A and B) and WY-46824 (C) were competed for [3H]NIS (A) and [3H]WY-46824 binding using either whole cells or membranes containing the human norepinephrine transporter. The HEK-293 whole cells (open circles) or membranes (closed circles) stably expressing hNET were incubated with 5 nM [3H]NIS or 200 nM [3H]WY-46824 and increasing concentrations of unlabeled NIS or WY-46824 2.5 h at 37°C (whole cells) or 4°C (membranes) as described under Materials and Methods. Nonspecific binding was defined by 200 nM desipramine. Results are the average of three separate experiments, with individual points assayed in triplicate. The Ki values for these depicted competition curves are reported in Table 4.

Lack of a Role of Antagonist-Modulated NET Trafficking in Loss of Antagonist Potency in Membranes
Western Blot Analyses of hNET. We considered the possibility that DVS-like compounds trigger a specific loss of hNET surface expression in the whole cell context, leading to assays that reflect only a subset of NET binding sites (with possibly distinct properties) compared with those monitored in membranes. However, treatments of intact cells with 5 µM WY-46824 or desipramine for 30 min and 2 h, followed by surface protein biotinylation (Fig. 6A), purification, and Western blot analysis revealed no changes in either NET total (Fig. 6B) or surface density (Fig. 6C). These studies reinforce the idea that conformational changes in hNET that target distinct antagonist binding sites account for the potency changes observed between whole cell and membrane preparations.

View larger version (17K): [in this window] [in a new window]   Fig. 6. The total and cell surface protein levels of hNET do not change after antagonist incubations. HEK-293 cells stably expressing hNET were cultured in the presence of desipramine (DMI) or WY-46824 (WY) or vehicle (control) and biotinylated as described under Materials and Methods. Aliquots containing equal amounts of protein were taken from each sample before streptavidin extraction of surface protein for total hNET protein. Blots were probed with monoclonal antibody to hNET(NET-17-1) followed by a goat anti-mouse and horseradish peroxidase-conjugated secondary antibody and chemiluminescent detection. Results from a representative blot are shown (A). Molecular mass (kilodaltons) indicated is from prestained standards run in parallel. Quantitative analyses from three replicate experiments are shown for total (A) and biotinylated (B) samples.

View larger version (8K): [in this window] [in a new window]   Fig. 7. The mutation V148M affects the affinity of nisoxetine binding without altering the binding affinity of WY-46824 in competition studies. Evaluation of mutations in the human norepinephrine transporter were completed in radioligand competition assays using [3H]nisoxetine and [3H]WY-46824. A to D, hNET(wild type) (closed symbols) and hNET(V148M) (open symbols) were evaluated using [3H]NIS or [3H]WY-46824 in HEK-293-transfected cells as described under Materials and Methods. Cells were incubated with increasing concentrations of [3H]WY-46824 and [3H]NIS and unlabeled compounds for 2.5 h as described under Materials and Methods. Nonspecific binding was defined using 200 nM desipramine. Results are the average of three separate experiments, with individual points assayed in triplicate. The Ki values for these depicted competition curves are reported in Table 5.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Modifying extracellular levels of NE by the blockade of NET is an important clinical target for the treatment of mood disorders (Blier, 2001), ADHD (Corman et al., 2004), and menopausal symptoms (Deecher et al., 2006). It is important to understand whether all NET antagonists attenuate NE transport via common or distinct mechanisms. Although the crystal structure of the bacterial Slc6 transporter LeuT_Aa, cocrystallized with the substrate leucine in the binding pocket, is known (Yamashita et al., 2005), little information can be used to define the binding sites for many transporter antagonists, in particular those for hNET. Past studies have indicated that NET antagonists may interact allosterically, observable as a change in antagonist dissociation rates in the presence of a distinct antagonist (Plenge and Mellerup, 1997), findings also observed with certain SERT antagonists (Plenge et al., 1991; Chen et al., 2005). These findings may be explained through the interdependent interactions of individual antagonists at distinct binding sites on a single protein, or they may reflect the simultaneous and cooperative occupancy of a common antagonist binding site on a transporter multimer. Although physical evidence for multimers exists among members of the SLC6 family (Kilic and Rudnick, 2000; Hastrup et al., 2001), including the hNET (Hahn et al., 2003), no evidence as yet relates multimer assembly to the formation or interactions of antagonist binding sites.

We have followed up on observations that a novel class of NET antagonists displays altered potency when cells are disrupted to produce membranes for binding assays (Deecher et al., 2006), supporting the existence of distinct binding sites for typical NE reuptake inhibitors such as NIS and the tricyclic desipramine compared with DVS series compounds. Our studies here used DVS and a DVS analog, WY-46824, but we have observed similar behavior with a large number of other DVS-related analogs (D. Deecher, unpublished data). The use of multiple radiolabeled hNET antagonists was instrumental in our identification of physical distinctions between binding sites. Thus, whereas DVS and the DVS analog WY-46824, as well as RTI-55 and methylphenidate, shift potency in membranes when [³H]NIS is used to label hNET, the same is not true when [¹²⁵I]RTI-55 is used to label hNET. Because the K_D value of NIS is are not altered when assessed directly in whole cell versus membrane environments, these findings suggest that membrane preparation is benign with respect to the integrity of the latter antagonist binding site and either specifically disrupts the other sites or their spatial relationship with the NIS site. The most striking evidence for this idea arose when we examined the ability of NIS to inhibit [³H]WY-46824 binding. Whereas NIS potency against [³H]NIS is unaltered by membrane preparation, NIS loses potency against [³H]WY-46824 much in the same way that unlabeled WY-46824 loses potency against [³H]NIS labeling. Although we could not obtain reliable saturation isotherms for [³H]WY-46824, unlabeled WY-46824 exhibits much lower potency against [³H]WY-46824 in competition studies, suggesting that a critical determinant for binding potency of WY-46824 is altered by membrane preparation. One caveat to our interpretations is that the low affinity of [³H]WY-46824 results in relatively low signal-to-noise ratios for whole cell binding assays (50%), which is even lower in membrane preparations (20%). Because our whole cell potency values for unlabeled NIS and WY46824 match those obtained with [³H]NIS, we think that [³H]WY-46824 labels a partially overlapping site to that interacting with classical NET antagonists. Interestingly, a potency shift is not evident for DVS or WY-46824 if [¹²⁵I]RTI-55 is used to label hNET. However, we note that in these assays the potency for DVS and WY-46824 is much lower that when these antagonists compete for [³H]NIS or whole cell [³H]WY-46824 binding. Because the potency of desipramine is the same when using either ligand, these data suggest that [³H]NIS and [¹²⁵I]RTI-55 labeling define distinct but overlapping sites, both of which can be accessed by desipramine but nonequivalently by DVS and WY-46824. These data seem most explainable in terms of three mutually interacting and physically distinct sites, each of which has the capacity to immobilize the transporter or physically occlude the substrate binding pocket.

With respect to the presence of distinct hNET antagonist binding sites, we provide evidence that at least one residue, Val148, can differentiate the binding of NIS and WY-46824. This is a particularly important finding, because our saturation studies gave little evidence for multiple sites except when we compare between ligands or binding preparations. The Val148 residue is the homolog of Ile172 in hSERT that supports the high-affinity binding of the SSRIs citalopram and fluoxetine (Henry et al., 2006b). Interestingly, although the I172M mutation shifts the potency of SERT for citalopram by 2 orders of magnitude, it does not affect interactions with 5-HT nor the SSRI paroxetine (Henry et al., 2006b). In the crystal structure of LeuT_Aa (Yamashita et al., 2005), the residue homologous to this site lies in TM3 adjacent to the substrate binding pocket. Whereas the I172M substitution in hSERT does not affect 5-HT transport rates, in hNET, the V148M mutation eliminates transport of NE, although the transporter is expressed and it reaches the cell surface at wild-type levels (Henry et al., 2006b). This finding suggests a somewhat distinct environment or contribution of this site to transport compared with hSERT. What seems most relevant is the fact that this site in both hNET and hSERT supports antagonist-selective interactions [citalopram versus paroxetine in hSERT (Henry et al., 2006b), NIS versus WY-46824 in hNET (this study)]. Although we provide evidence of a NIS-selective contact at Val148, we do not think that this residue is itself responsible for the differential effects of membrane preparation on the potency of NIS- and DVS-type compounds, but rather that the differential impact of membrane preparation compelled us to seek a physical basis for distinct antagonist binding sites, the basis of which seems to include Val148. A specific site sensitive to membrane preparation awaits further investigations. Regardless, we suggest that the tricyclic antidepressants and NIS may bind at or near the substrate binding pocket, whereas the DVS class compounds bind to more distal sites, although more complex scenarios that take into account multimer-based allosterism can be envisaged.

One important issue not addressed by these studies relates to whether the differences in the modes of binding of NIS and DVS in whole cell versus membrane environments or as distinguished by V148M have important lessons for the development of NET antagonists with differential clinical utility. One possibility that we are excited to consider is that the binding differences observed in the membrane and whole cell preparations could mirror conformational states achieved through normal modes of transporter regulation or through interactions with transporter accessory proteins. NETs have been found to exhibit changes in surface distribution with chronic stress (Miner et al., 2006), and possibly conformations of internal and surface pools could differentially detect or be stabilized by the binding of NIS and DVS analogs. Studies are now underway to examine this possibility. The hNET is also known to associate with a number of scaffolding or regulatory proteins, including PICK1 (Torres et al., 2001), syntaxin 1A (Sung et al., 2003), PP2A (Bauman et al., 2000), and 14-3-3 proteins (Sung et al., 2005). Possibly, these transporter-protein associations, thought to support the trafficking and/or intrinsic activity of hNET, may be sensitive to conformational states stabilized by some transporter antagonists but not others. Support for this concept has recently arisen with evidence that different dopamine transporter antagonists influence the transporter's capacity for phosphorylation (Gorentla and Vaughan, 2005). This concept is all the more plausible given that the initial observations of differences in reduced potency for our antagonists were noted in the membrane preparations. Loss of key protein associations during the process of membrane harvest or normalization of membrane potential could bias conformations selectively for one class of antagonists over another. Having access to reagents that bind to distinct sites and exhibit context-specific reuptake blockade could greatly extend the therapeutic range of biogenic transporter-based therapeutics.

	Acknowledgements

 
We thank Tammy Jessen, Qiao Han, Jane Wright, and Angela Steele for general laboratory maintenance and expert technical assistance. We are grateful to L. Keith Henry for assistance and insight with properties and binding assays related to hNET F72Y and hNET V148M.

	Footnotes

 
J.N.M. and D.C.D. contributed equally to this work.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.125716.

ABBREVIATIONS: NE, norepinephrine; NET, norepinephrine transporter; hNET, human norepinephrine transporter; ADHD, attention-deficit hyperactivity disorder; TM, transmembrane domain; 5-HT, 5-hydroxytryptamine (serotonin); SERT, serotonin transporter; hSERT, human serotonin transporter; SSRI, 5-hydroxytryptamine reuptake inhibitor; DVS, desvenlafaxine succinate; WY-46824, rac-(1-[1-(3-chloro-phenyl)-2-(4-methylpiperazin-1-yl)-ethyl]cyclohexanol; RTI-55, 3-(4-iodophenyl)tropane-2-carboxylic acid methyl ester; HEK, human embryonic kidney; PBS, phosphate-buffered saline; RIPA, radioimmunoprecipitation assay.

Address correspondence to: Dr. Randy D. Blakely, Center for Molecular Neuroscience, Suite 7140 MRBIII, Nashville, TN 37232-8548. E-mail: randy.blakely{at}vanderbilt.edu

	References

Top Abstract Materials and Methods Results Discussion References

 

Adkins EM, Barker EL, and Blakely RD (2001) Interactions of tryptamine derivatives with serotonin transporter species variants implicate transmembrane domain I in substrate recognition. Mol Pharmacol 59: 514–523.[Abstract/Free Full Text]

Alfinito PD, Huselton C, Chen X, and Deecher DC (2006) Pharmacokinetic and pharmacodynamic profiles of the novel serotonin and norepinephrine reuptake inhibitor desvenlafaxine succinate in ovariectomized Sprague-Dawley rats. Brain Res 1098: 71–78.[CrossRef][Medline]

Apparsundaram S, Schroeter S, Giovanetti E, and Blakely RD (1998) Acute regulation of norepinephrine transport: II. PKC-modulated surface expression of human norepinephrine transporter proteins. J Pharmacol Exp Ther 287: 744–751.[Abstract/Free Full Text]

Barker EL, Moore KR, Rakhshan F, and Blakely RD (1999) Transmembrane domain I contributes to the permeation pathway for serotonin and ions in the serotonin transporter. J Neurosci 19: 4705–4717.[Abstract/Free Full Text]

Barker EL, Perlman MA, Adkins EM, Houlihan WJ, Pristupa ZB, Niznik HB, and Blakely RD (1998) High affinity recognition of serotonin transporter antagonists defined by species-scanning mutagenesis. An aromatic residue in transmembrane domain I dictates species-selective recognition of citalopram and mazindol. J Biol Chem 273: 19459–19468.[Abstract/Free Full Text]

Bauman AL, Apparsundaram S, Ramamoorthy S, Wadzinski BE, Vaughan RA, and Blakely RD (2000) Cocaine and antidepressant-sensitive biogenic amine transporters exist in regulated complexes with protein phosphatase 2A. J Neurosci 20: 7571–7578.[Abstract/Free Full Text]

Blier P (2001) Norepinephrine and selective norepinephrine reuptake inhibitors in depression and mood disorders: their pivotal roles. J Psychiatry Neurosci 26 (Suppl): S1–S2.[Medline]

Bönisch H and Brüss M (2006) The norepinephrine transporter in physiology and disease. Handb Exp Pharmacol 175: 485–524.[Medline]

Chen F, Larsen MB, Sanchez C, and Wiborg O (2005) The S-enantiomer of R,S-citalopram, increases inhibitor binding to the human serotonin transporter by an allosteric mechanism. Comparison with other serotonin transporter inhibitors. Eur Neuropsychopharmacol 15: 193–198.[CrossRef][Medline]

Cheng Y and Prusoff WH (1973) Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol 22: 3099–3108.[CrossRef][Medline]

Corman SL, Fedutes BA, and Culley CM (2004) Atomoxetine: the first nonstimulant for the management of attention-deficit/hyperactivity disorder. Am J Health Syst Pharm 61: 2391–2399.[Abstract/Free Full Text]

Deecher DC, Beyer CE, Johnston G, Bray J, Shah S, Abou-Gharbia M, and Andree TH (2006) Desvenlafaxine succinate: a new serotonin and norepinephrine reuptake inhibitor. J Pharmacol Exp Ther 318: 657–665.[Abstract/Free Full Text]

Dell'Osso B, Nestadt G, Allen A, and Hollander E (2006) Serotonin-norepinephrine reuptake inhibitors in the treatment of obsessive-compulsive disorder: a critical review. J Clin Psychiatry 67: 600–610.[Medline]

Galli A, DeFelice LJ, Duke BJ, Moore KR, and Blakely RD (1995) Sodium-dependent norepinephrine-induced currents in norepinephrine-transporter-transfected HEK-293 cells blocked by cocaine and antidepressants. J Exp Biol 198: 2197–2212.[Medline]

Gether U, Andersen PH, Larsson OM, and Schousboe A (2006) Neurotransmitter transporters: molecular function of important drug targets. Trends Pharmacol Sci 27: 375–383.[CrossRef][Medline]

Gorentla BK and Vaughan RA (2005) Differential effects of dopamine and psychoactive drugs on dopamine transporter phosphorylation and regulation. Neuropharmacology 49: 759–768.[CrossRef][Medline]

Hahn MK, Robertson D, and Blakely RD (2003) A mutation in the human norepinephrine transporter gene (SLC6A2) associated with orthostatic intolerance disrupts surface expression of mutant and wild-type transporters. J Neurosci 23: 4470–4478.[Abstract/Free Full Text]

Hastrup H, Karlin A, and Javitch JA (2001) Symmetrical dimer of the human dopamine transporter revealed by cross-linking Cys-306 at the extracellular end of the sixth transmembrane segment. Proc Natl Acad Sci U S A 98: 10055–10060.[Abstract/Free Full Text]

Henry LK, Defelice LJ, and Blakely RD (2006a) Getting the message across: a recent transporter structure shows the way. Neuron 49: 791–796.[CrossRef][Medline]

Henry LK, Field JR, Adkins EM, Parnas ML, Vaughan RA, Zou MF, Newman AH, and Blakely RD (2006b) Tyr-95 and Ile-172 in transmembrane segments 1 and 3 of human serotonin transporters interact to establish high affinity recognition of antidepressants. J Biol Chem 281: 2012–2023.[Abstract/Free Full Text]

Husbands GEM and Stack GP (1989a) inventors; American Home Products, assignee. Substituted 1-(aralkyl-piperazinoalkyl) cycloalkanols. U.S. Patent 4826844. 1989 May 2.

Husbands GEM and Stack GP (1989b) inventors; American Home Products, assignee. Substituted cycloalkanol. U.S. Patent EP310268. 1989 May 2.

Keller NR, Diedrich A, Appalsamy M, Miller LC, Caron MG, McDonald MP, Shelton RC, Blakely RD, and Robertson D (2006) Norepinephrine transporter-deficient mice respond to anxiety producing and fearful environments with bradycardia and hypotension. Neuroscience 139: 931–946.[CrossRef][Medline]

Keller NR, Diedrich A, Appalsamy M, Tuntrakool S, Lonce S, Finney C, Caron MG, and Robertson D (2004) Norepinephrine transporter-deficient mice exhibit excessive tachycardia and elevated blood pressure with wakefulness and activity. Circulation 110: 1191–1196.[Abstract/Free Full Text]

Keller NR and Robertson D (2006) Familial orthostatic tachycardia. Curr Opin Cardiol 21: 173–179.[Medline]

Kilic F and Rudnick G (2000) Oligomerization of serotonin transporter and its functional consequences. Proc Natl Acad Sci U S A 97: 3106–3111.[Abstract/Free Full Text]

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.[CrossRef][Medline]

Lorang D, Amara SG, and Simerly RB (1994) Cell-type-specific expression of catecholamine transporters in the rat brain. J Neurosci 14: 4903–4914.[Abstract]

Miner LH, Jedema HP, Moore FW, Blakely RD, Grace AA, and Sesack SR (2006) Chronic stress increases the plasmalemmal distribution of the norepinephrine transporter and the coexpression of tyrosine hydroxylase in norepinephrine axons in the prefrontal cortex. J Neurosci 26: 1571–1578.[Abstract/Free Full Text]

Pacholczyk T, Blakely RD, and Amara SG (1991) Expression cloning of a cocaine- and antidepressant-sensitive human noradrenaline transporter. Nature 350: 350–354.[CrossRef][Medline]

Plenge P and Mellerup ET (1997) An affinity-modulating site on neuronal monamine transport proteins. Pharmacol Toxicol 80: 197–201.[Medline]

Plenge P, Mellerup ET, and Laursen H (1991) Affinity modulation of [³H]imipramine, [³H]paroxetine and [³H]citalopram binding to the 5-HT transporter from brain and platelets. Eur J Pharmacol 206: 243–250.[CrossRef][Medline]

Schroeder C, Adams F, Boschmann M, Tank J, Haertter S, Diedrich A, Biaggioni I, Luft FC, and Jordan J (2004) Phenotypical evidence for a gender difference in cardiac norepinephrine transporter function. Am J Physiol Regul Integr Comp Physiol 286: R851–R856.[Abstract/Free Full Text]

Stahl SM, Grady MM, Moret C, and Briley M (2005) SNRIs: their pharmacology, clinical efficacy, and tolerability in comparison with other classes of antidepressants. CNS Spectr 10: 732–747.[Medline]

Sung U, Apparsundaram S, Galli A, Kahlig KM, Savchenko V, Schroeter S, Quick MW, and Blakely RD (2003) A regulated interaction of syntaxin 1A with the antidepressant-sensitive norepinephrine transporter establishes catecholamine clearance capacity. J Neurosci 23: 1697–1709.[Abstract/Free Full Text]

Sung U, Jennings JL, Link AJ, and Blakely RD (2005) Proteomic analysis of human norepinephrine transporter complexes reveals associations with protein phosphatase 2A anchoring subunit and 14-3-3 proteins. Biochem Biophys Res Commun 333: 671–678.[CrossRef][Medline]

Than C, Morimoto H, Andreas H, and Williams PG (1995) Preparation, NMR characterization, and labeling reactions of tritiated borane-THF complex at high specific radioactivity. J Org Chem 60: 7503–7507.[CrossRef]

Torres GE, Yao WD, Mohn RR, Quan H, Kim K, Levey AI, Staudinger J, and Caron MG (2001) Functional interaction between monoamine plasma membrane transporters and the synaptic PDZ domain-containing protein PICK1. Neuron 30: 121–134.[CrossRef][Medline]

Usera PC, Vincent S, and Robertson D (2004) Human phenotypes and animal knockout models of genetic autonomic disorders. J Biomed Sci 11: 4–10.[CrossRef][Medline]

Vis PM, van Baardewijk M, and Einarson TR (2005) Duloxetine and venlafaxine-XR in the treatment of major depressive disorder: a meta-analysis of randomized clinical trials. Ann Pharmacother 39: 1798–1807.[Abstract/Free Full Text]

Xu F, Gainetdinov RR, Wetsel WC, Jones SR, Bohn LM, Miller GW, Wang YM, and Caron MG (2000) Mice lacking the norepinephrine transporter are supersensitive to psychostimulants. Nat Neurosci 3: 465–471.[CrossRef][Medline]

Yamashita A, Singh SK, Kawate T, Jin Y, and Gouaux E (2005) Crystal structure of a bacterial homologue of Na⁺/Cl-dependent neurotransmitter transporters. Nature 437: 215–223.[CrossRef][Medline]

This article has been cited by other articles:

				L. K. Henry and R. D. Blakely Distinctions between Dopamine Transporter Antagonists Could be Just around the Bend Mol. Pharmacol., March 1, 2008; 73(3): 616 - 618. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.107.125716v1 jpet.107.125716v2 jpet.107.125716v3 323/2/720    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mason, J. N. Articles by Blakely, R. D. Search for Related Content PubMed PubMed Citation Articles by Mason, J. N. Articles by Blakely, R. D.