Binding and Transport in Norepinephrine Transporters

REAL-TIME, SPATIALLY RESOLVED ANALYSIS IN SINGLE CELLS USING A FLUORESCENT SUBSTRATE*

Joel W. Schwartz, Randy D. Blakely, and Louis J. DeFeliceDagger

From the Department of Pharmacology, Center for Molecular Neuroscience, Vanderbilt University Medical Center, Nashville, Tennessee 37232-8548

Received for publication, September 24, 2002, and in revised form, December 17, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Monoamine transporters, the molecular targets for drugs of abuse and antidepressants, clear norepinephrine, dopamine, or serotonin from the synaptic cleft. Neurotransmitters, amphetamines, and neurotoxins bind before being transported, whereas cocaine and antidepressants bind to block transport. Although binding is crucial to transport, few assays separate binding from transport, nor do they provide adequate temporal or spatial resolution to describe real-time kinetics or localize sites of active uptake. Here, we report a new method that distinguishes substrate binding from substrate transport using single-cell, space-resolved, real-time fluorescence microscopy. For these studies we use a fluorescent analogue of 1-methyl-4-phenylpyridinium, a neurotoxic metabolite and known substrate of monoamine transporters, to assess binding and transport with 50-ms, sub-micron resolution. We show that ASP+ (4-(4-(dimethylamino)styrl)-N-methylpyridinium) has micromolar potency for the human norepinephrine transporter, that ASP+ accumulation is Na+-, Cl--, cocaine-, and desipramine-sensitive and temperature-dependent, and that ASP+ competes with norepinephrine uptake. Using this method we demonstrate that norepinephrine transporters are efficient buffers for substrate, with binding rates exceeding transport rates by 100-fold. Furthermore, substrates bind deep within the transporter, isolated from both the bath and the lipid bilayer. Although transport per se depends on Na+ and Cl-, binding is independent of Na+ and actually increases in low Cl-. We further demonstrate that ASP+ interacts with transporters not only in transfected cells but in cultured neurons. ASP+ is also a substrate for dopamine and serotonin transporters and therefore represents a powerful new technique for studying the biophysical properties of monoamine transporters, an approach also amenable to high throughput assays for drug discovery.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Noradrenergic signaling in the central nervous system modulates attention, mood, arousal, learning, and memory (1-4). Norepinephrine transporters (NETs)1 attenuate neuronal signaling via rapid neurotransmitter clearance, thus NETs are implicated in the pathology of major depression, post-traumatic stress disorder, and attention deficit disorder (5-10). Therapeutic agents that inhibit NET elevate extracellular NE in the brain and periphery (5, 11-13). Noradrenergic signaling in the peripheral nervous system influences blood pressure and heart rate (14-16), and NET inhibitors such as cocaine and antidepressants induce cardiac complications (17-19). Consequently, new methods for the analysis of NET function are needed to understand fundamental mechanisms supporting substrate translocation. In addition, protocols that are amenable to high throughput drug screens may prove valuable in the development of therapeutic agents for depression, hypertension, and drug abuse.

NET is a member of a large family of Na+ and Cl--dependent transporters (20) with sub-micromolar potency for substrates. NET concentrates NE by coupling its transport to co-transported ions in a proposed stoichiometry of 1 NE:1 Na+:1 Cl- (21-23) with a turnover rate of one transport cycle per second (24). Synaptic transmission, however, occurs at significantly faster time scales; e.g. dopaminergic midbrain neurons fire one action potential every 10 ms (25). Thus transport rates are incongruous with synaptic transmission, and it has been proposed that transporters may serve the additional function of buffering neurotransmitter in the synaptic cleft prior to transport. Indeed, glutamate binding occurs on a much faster time scale than a complete transport cycle (26, 27). Although the buffering hypothesis is current in the literature, there has been no direct test for the monoamine transporters, and until now there is no method that compares binding and transport in the same assay. Radiolabeled high affinity antagonist displacement and rotating disk voltammetry have been used to measure serotonin (5HT) and dopamine (DA) binding to their respective transporters, SERT and DAT (28-30). Antagonist displacement methods suggest that 5HT binding to SERT is Na+-independent (28). DAT high affinity antagonist displacement via DA is dependent on Na+ from 0 to 20 mM but is Na+-independent from 20 to 120 mM (31, 32). The requirement of Na+ for substrate binding to NET has not previously been studied. Finally, although the Cl- dependence of monoamine transport is well known, the Cl- dependence of binding has received limited evaluation (21, 28, 31).

Here we describe a novel method based on fluorescence microscopy to assess the ionic dependence of substrate binding. The fluorescence approach relies on the availability of fluorescent substrates, which have not been readily available. Hardrich and colleagues (33) generated fluorescent NE and nisoxetine analogues; however, these compounds do not separate substrate binding from actual transport. We conjectured that NET might transport 4-(4-dimethylaminostyrl)-N-methylpyridinium (ASP+), a fluorescent analogue of the neurotoxin MPP+. MPP+ is a substrate for the organic cation ion transporter, OCT (34, 35), and, importantly, DAT, where it induces neurotoxic degeneration of the substantial nigra (36). MPP+ is also a substrate for NET (37-41) and SERT (42), making ASP+ an attractive candidate as a fluorescent substrate for monoamine transporters. This report investigates ASP+ as a substrate, particularly for NET, and evaluates fluorescence microscopy as an investigative tool for studying neurotransmitter transporters. We show that binding and transport are distinguishable in this assay with a high degree of spatial and temporal resolution. Here we report the effects of co-transported ions, cocaine, antidepressants, and temperature on binding and transport. We find that transporters have binding rates 100-fold greater than transport rates, supporting the hypothesis that transporters serve as buffers for transmitters. The accessibility of quenchers to ASP+ demonstrates that substrates bind deep within the transporter and are isolated from the bath and lipid membrane. We further show that in hNET, substrate binding is independent of Na+, and, surprisingly, binding is stronger in Cl--free conditions. Finally, this assay reveals that ASP+ accumulation occurs non-uniformly along the membrane of neurites in cultured neurons, supporting the idea that NET proteins may localize to specific plasma membrane sub-domains. ASP+ is also a substrate for DATs and SERTs, which, taken together with the above data, argues that ASP+ fluorescence microscopy may benefit the study of monoamine transporters and provide a useful platform for automated, non-isotopic drug discovery assays.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- HEK-293 cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum (v/v), 2 mM glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin (Invitrogen). The stable cells lines that we used for the human norepinephrine transporter (HEK-hNET) and the human serotonin transporter (HEK-hSERT) were previously described (43, 44). The stable cell lines that we used for the human dopamine transporter (HEK-hDAT) cells are a gift from Michelle Mazei.

Radiolabeled Transport Assay-- All experiments were performed at room temperature (22 °C) unless otherwise indicated. HEK-hNET cells were plated on poly-L-lysine-coated 24-well tissue culture plates at 105 cells per well 3 days prior to performing transport assays (90% confluence on the third day). The medium was removed by aspiration. Cells were then preincubated for 10 min in Krebs-Ringer-Hepes (KRH (in mM): 130 NaCl, 1.3 KCl, 2.2 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 10 Hepes, and 1.8 g/liter glucose, pH 7.4) medium with or without 10 µM desipramine. Desipramine, a specific NET blocker, was used to establish nonspecific activity in hNET cells. Pargyline (10 µM) and ascorbic acid (10 µM) were added to prevent metabolism and oxidation of NE, respectively. The assay mixture was aspirated after 10 min, and cells were washed three times with 4 °C KRH buffer. Accumulated [3H]NE was determined by liquid scintillation of 1% (w/v) SDS-solubilized cells.

Primary Tissue Culture-- SCG neurons were dissociated by trituration followed by digestion with 0.25% trypsin and 0.3% collagenase. Non-neuronal cells were removed by pre-plating on uncoated, Falcon 60-mm plates. Neurons were cultured on poly-L-ornithine/laminin/poly-D-lysine-coated MatTek dishes at a density of 3000-4000 cells/well in F-14+ media containing 5% fetal calf serum, 2 mM L-glutamine, 60 ng/ml progesterone, 16 mg/ml putrescine, 400 ng/ml L-thyroxine, 38 ng/ml sodium selenite, 340 ng/ml tri-iodothyroxine, 5 mg/ml insulin/penicillin/streptomycin, 10 µM fluorodeoxyuridine, and 20 ng/ml nerve growth factor. The neurons were maintained for 3-5 days in the presence of nerve growth factor before use.

Microscopy-- HEK-hNET cells were plated on 35-mm glass bottom Petri dishes (MatTek, Ashland, MA) coated with poly-L-lysine 3 days prior to experimentation. The culture medium was aspirated, cells were immediately mounted on a Zeiss 410 confocal microscope, and the microscope was focused on the center of the monolayer of cells. During the confocal measurement, cells remain without buffer for approximately 30 s. Background autofluorescence was established by collecting images for 10 s prior to the addition of KRH (see "Radiolabeled Transport"), 1.8 mg/liter glucose, 10 µM ascorbic acid, 10 µM pargyline, 10 µM tropolone (Sigma), and ASP+. The argon laser was tuned to 488 nm; the emitted light was filtered with a 580- to 630-nm band pass filter (lambda max = 610 nm). The gain (contrast) and offset (brightness) for the photomultiplier tube was set to avoid detector saturation at the highest ASP+ concentration used in these experiments (10 µM). The effects of photobleaching on ASP+ accumulation were determined by examining the rate of ASP+ accumulation and decay at various acquisition rates. Acquisition rates greater than 0.3 Hz degraded sequestered ASP+ (3.33 s/image).

Image Analysis-- The fluorescent images were processed using MetaMorph imaging software (Universal Imaging Corp., Downington, PA). Fluorescent accumulation was established by measuring the average pixel intensity of time-resolved fluorescent images within a specified region identified by the differential interference contrast image. Average pixel intensity is used to normalize among cells. HEK-293 cells and SCG neurons possess endogenous mechanisms for ASP+ accumulation (35). NET-mediated ASP+ accumulation is defined as the fluorescence of HEK-hNET cells or neurons minus background fluorescence.

Fluorescence Anisotropy-- To evaluate ASP+ binding to the surface membranes, HEK-hNET cells were exposed to 2 µM ASP+ with horizontal polarizer (Fig. 5C), with the polarizer rapidly switching to the vertical position. Cells were imaged with alternating polarizations for 3 min to measure light intensity in the horizontal (Ih) and vertical (Iv) positions to calculate the anisotropy ratio, r = (Iv - gIh)/(Iv + 2gIh). The factor g was determined by using a half-wave plate as described by Blackman et al. (45). In this formulation, r = 0.4 implies an immobile light source (45). Surface anisotropy was measured at the cell circumference over 1 pixel width (0.625 µm). Cytosolic anisotropy was measured near the center of the cell, ~5 pixel widths from the membrane.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

MPP+ and ASP+ Inhibit NE Accumulation-- To test whether ASP+ interacts with hNET, we initially exposed HEK-hNET cells to increasing concentrations of ASP+ in the presence of a fixed concentration of radiolabeled NE. MPP+ was used as a control, because MPP+ is a known substrate for NET (37, 46) and has similar structure to ASP+ (Fig. 1). Fig. 1 shows that increasing MPP+ or ASP+ inhibits the DS-defined accumulation of radiolabeled NE. The inhibition constants (Ki) for MPP+ and ASP+ are 600 ± 67 and 780 ± 77 nM (n = 5), respectively; thus MPP+ and ASP+ interact with HEK-hNET cells in the sub-micromolar range.


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Fig. 1.   MPP+ and ASP+ inhibit NE accumulation. [3H]NE accumulation was measured in hNET-transfected HEK-293 cells in the presence of increasing ASP+ or MPP+ and normalized to similar data in the absence of inhibitor. Nonspecific activity was determined by application of 10 µM desipramine. The data were fit to NE transport remaining by the equation, y = 100/(1 + ([I]/IC50)n), where [I] is the concentration of ASP+ or MPP+ and Ki values were determined using Cheng-Prusoff correction for substrate concentration. The fits yield Ki (ASP+) = 780 ± 77 nM and Ki (MPP+) = 600 ± 67 nM. Values are represented as means ± S.E., n = 5.

Cells That Express hNET Accumulate ASP+-- Confocal slices through the monolayer (lower panels in Fig. 2) indicate that ASP+ accumulation in HEK-hNET divides into two phases: a rapid phase I that ensues immediately after ASP+ addition and appears localized to the cell surface (S), followed by a slower phase II localized to the cell interior (I). Line scans of similar images (see below) will amplify this segregation. Concomitant differential interference contrast (DIC) images (upper panels in Fig. 2) help to register the confocal images. In the first 3 s after adding ASP+ (Fig. 2B), the surface of some cells becomes noticeably bright, whereas the internal compartment shows less accumulation. Subsequently the cell interior becomes brighter and begins to fill in, but surface brightness remains constant. This pattern is the same whether we employ brief or long exposures of ASP+. These qualitative features are also observed in hDAT- and hSERT-transfected cells, as illustrated in Fig. 3. The weakest responder to ASP+ is hSERT (panels G-I in Fig. 3); however, the accumulation pattern in HEK-hSERT cells is significantly above parental cells (J-K). The remainder of this report focuses on hNET-transfected cells.


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Fig. 2.   Cells that express hNET accumulate ASP+. Single cells are visualized by DIC microscopy (top row), and ASP+ accumulation is measured by an increase in ASP+ fluorescence under confocal microscopy (bottom row). Images were taken at 0, 3, and 180 s after exposure to 800 nM ASP+ (panels A-C). In the upper panels, fluorescence images were projected onto the DIC image of the corresponding cells below to identify cell surface (S) and interior (I). The color gradient represented in panel A denotes the ASP+ intensity values (red being the most intense).


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Fig. 3.   Monoamine transporters interact with ASP+. Like HEK-hNET cells hDAT- and hSERT-transfected cells accumulate ASP+. hNET (A-C), hDAT (D-F), hSERT (G-I), and parental HEK-293 (J-L) were exposed to 2 µM ASP+ for the times indicated in panels J-L. The color gradient represented in panel J denotes the corresponding ASP+ intensity values.

In Fig. 4 we quantify the initial rapid rise (phase I) and the slower phase II by plotting the average pixel intensity integrated over the entire cell against time after adding ASP+. Line scans (see below) will identify phases I and II with the cell surface and cell interior, respectively. Fig. 4A shows that, if we acquire fluorescence data at 0.3 Hz (or lower), the rate of rise of phase II accumulation is arrested immediately after removing ASP+. Because ASP+ binds mitochondria (35); thus we expect flux to be largely unidirectional and cells to retain ASP+ after it enters the cell. Acquiring data at 12 Hz (or higher) results in a decline in brightness after removing ASP+. The decay time constant is linearly proportional to the acquisition rate and represents ASP+ photobleaching, which we can also observe in the absence of HEK-hNET cells. Photobleaching thus sets a limit on the frequency of image acquisition. In the presence of a constant pool of ASP+, however, the rate of accumulation in phase I is independent of sampling rate at 12 Hz. By this method we are able to acquire data up to 20 Hz, permitting analysis of phase I at 50-ms resolution. HEK-293 cells also demonstrate phase I and II ASP+ accumulation; however, both the rapid and slow phases are negligible in parental cells compared with hNET-transfected cells. Fig. 4B data are collected at 100-ms time resolution. From similar data on 400 cells (4 independent dishes with 100 cells per dish), the average slope of phase I was 18 ± 3.46 arbitrary fluorescent units (AFUs)/s, and the average slope of phase II was 0.28 ± 0.0057 AFUs/s.


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Fig. 4.   Photobleaching and phase I kinetics. Transfected HEK-hNET and parental HEK-293 cells were exposed to 2 µM ASP+ for 180 s, followed by a wash. A, images were taken from at least 40 individual cells per dish (cells defined by the corresponding DIC image) from four separate dishes. The average pixel intensity ± S.E. over all cells is plotted on the y axis in arbitrary fluorescence units (AFUs). After ASP+ was removed, photobleaching was assessed at 0.3 and 12 Hz for hNET-293 cells and 12 Hz for HEK-293 cells. Similar data were obtained up to 20 Hz and normalized to the fluorescence intensity maximums before and after ASP+ removal. B, phase I binding measured at high acquisition rates. HEK-hNET and HEK-293 cells were exposed to 2 µM ASP+ and images were collected every 100 ms. Photobleaching does not significantly effect accumulation in the presence of constant ASP+. Data represent average pixel intensity and standard error of the mean.

Phase I Represents Binding; Phase II Represents Transport-- Fig. 5 shows a series of line scans taken across individual cells at different times after adding ASP+. Notice that initial rapid accumulation peaks at the cell edges, as identified in the DIC images (arrows), remains constant between 3 and 180 s, whereas the brightness at the cell center (between arrows) gradually increases. Furthermore, accumulation at the center occurs at the same rate as phase II (compare phase II slope in Fig. 4A with center elevation in Fig. 5A). HEK-293 cells subjected to similar line scans do not display sharply localized fluorescence pattern (Fig. 5B). These data support the interpretation that phase I represents a surface interaction with hNET, and phase II represents ASP+ transport. To further test this interpretation we compared the anisotropy of polarized light from the edges and centers of cells, which assesses the mobility of ASP+ molecules in these regions. Light from the edge demonstrates significant divergence between horizontal (0°) and vertical (90°) polarization, whereas light from the center is less divergent (Fig. 5C). From these data we calculate the fluorescence anisotropy (see "Experimental Procedures") across the line scan. The anisotropy measurements summarized in Fig. 5D demonstrate that light from regions near the membrane arises from an immobile source (0.30 r < 0.32) compared with light from the cytosol (0.21 < r < 0.24). Solution ASP+ shows even lower anisotropy (0.14 < r < 0.15). Thus ASP+ at the cell surface is effectively bound. Finally, preincubation with 1 mM citalopram, a weak blocker of hNET transport, significantly reduces the slope of phase I, as expected if ASP+ has fewer available binding sites. These data indicate two distinct populations of the fluorescent compound in HEK-hNET cells exposed to ASP+: a pool of rapidly immobilized ASP+ molecules located at or near the cell surface, which we interpret as bound substrate, and a second more slowly accumulating, mobile pool of ASP+ located in the cell interior, which we propose are transported molecules.


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Fig. 5.   Phase I represents ASP+ binding; phase II represents ASP+ transport. Line scans across the center of HEK-hNET cells (A) and HEK-293 cells (B) were taken at 0, 3, and 180 s after exposure to 2 µM ASP+. By this method we observe that in hNET cells the rapid increase in ASP+ fluorescence, named phase I, is localized to the cell surface (arrows), as identified in DIC images, and the slower phase II registers with the cytosol (between arrows). In panel C, HEK-hNET cells were exposed to 2 µM ASP+ under blue polarized light and red images were collected at 0° and 90° with respect to the incident polarization. Polarized images from ASP+ in solution, in the cytosol, and at the cell surface were collected from at least 40 cells in four dishes in panel D.

To further investigate the possibility that our assay indeed detects bound and transported populations of ASP+ molecules in HEK-hNET cells, we administered a potent blocker of transport, desipramine (DS), at various times during phase II. Because mitochondria sequester ASP+, the substrate does not efflux from the cell. By quantifying the sample data shown in Fig. 6 (A, without DS, and B, with DS), we observe that the DS reduces the total light intensity by exactly the amplitude of phase I (arrows in Fig. 6C). Thus the rate of phase II transport (top slope) equals the rate of ASP+ accumulation inside cells (bottom slope), because DS rapidly replaces ASP+ on the cell surface and halts further uptake. As a final test, we note that substrate binding is less sensitive to temperature than transport (47); thus, if our interpretation is correct, reduced temperature should affect phase I less than it affects phase II. This phenomenon is observed in Fig. 6D. Together these data provide strong evidence that phase I represents ASP+ binding and phase II represents transport.


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Fig. 6.   Desipramine displaces phase I ASP+ and arrests phase II transport. The top panels show confocal images of confluent HEK-hNET cells exposed to 2 µM ASP+ for 60 s (A) followed by application of 10 µM desipramine (B). From similar data, panel C shows three separate HEK-hNET cells exposed to 2 µM ASP+ for 60, 120, or 180 s followed by rapid application of 10 µM desipramine. Data integrated over the entire cell were collected at 0.3 Hz to avoid photobleaching. The top dashed line represents the slope of phase II, and the bottom dashed line represents the corresponding increase in sequestered ASP+ at each time (only three of six time measurements are shown). Panel D shows the temperature dependence of phase I and II slopes in 2 µm ASP+. The slopes of phase I and II are normalized to room temperature. The color gradient in panel B represents ASP+ intensity.

ASP+ Pharmacology-- To test whether ASP+ has pharmacological properties similar to hNET HEK-hNET, cells were preincubated for 10 min with 10 µM desipramine, 10 µM cocaine, or 30 µM NE. After preincubation with inhibitor alone, we added 2 µM ASP+ to the inhibitor solution. In Fig. 7A, data are separated into phase I and II slopes as previously described. Compared with HEK-hNET cells, phase I is small in HEK-293 cells and insignificant in the presence of antagonists cocaine or DS, or in the presence of competing NE. Phase II is small in HEK-293 cells and in the presence of competing NE and is insignificant in the presence of cocaine. Finally, phase I and phase II saturate at similar ASP+ bath concentrations, and they have similar Michaelis-Menten constants, although ASP+ is slightly more potent for phase I (Fig. 7, B and C). Phase I: Vmax = 10.5 ± 0.94 AFUs/s, Km = 850 ± 186 nM, and n = 1.15 ± 0.30; phase II: Vmax = 0.32 ± 0.014 AFUs/s, Km = 480 ± 60 nM, and n = 1.51 ± 0.35). The above parameters from ASP+ accumulation are comparable to those obtained with NE, the endogenous substrate (43), underscoring the utility of ASP+ as a measure of NET activity.


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Fig. 7.   ASP+ pharmacology. The bars in panel A correspond to normalized slopes of phase I and II under various treatments (DS 10 µM, cocaine 10 µM, NE 30 µM). Panel B shows the average pixel intensity from whole-cell confocal images as described in Fig. 3. External ASP+ is varied as indicated. Panel C plots the kinetics of phase I and II as a function of ASP+ concentration. Values are represented at normalized slopes ± S.D. of four experiments with 100 cells per experiment.

Binding Depends on Cl- but Not Na+-- To determine the ionic dependence of binding, we exposed HEK-hNET cells to bath solutions in which Na+ or Cl- were replaced with NMDG (N-methyl-D-glucamine) or acetate, respectively. Fig. 8 shows, whereas Na+ replacement does not significantly alter binding, Cl- replacement enhances binding. Na+ replacement dramatically reduces transport to background levels, defined by DS, although Cl- replacement is less effective in blocking transport, in agreement with previous results from radiometric assays (12).


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Fig. 8.   Na+ and Cl- dependence of ASP+ binding. Na+ and Cl- are replaced with NMDG+ and acetate, respectively. Values are represented at normalized slopes ± S.D. of four experiments with 100 cells per experiment. *, significantly different compared with control values (Student's t test, p = 0.05).

Substrates Are Inaccessible to Hydrophobic and Hydrophilic Fluorescence Quenchers-- To determine the accessibility of bound substrate to the bath or the lipid bilayer, HEK-hNET cells were exposed to ASP+ (2 µM) premixed with various concentrations of trypan blue (hydrophilic) or 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL, hydrophobic). Trypan blue and TEMPOL are collision (dynamic) quenchers, which transiently interact with excited states in fluorescent molecules and directly transfer their energy without radiation (48-50). Energy normally released as a photon from ASP+ is transferred to the quenching agent, thus reducing ASP+ fluorescence. To be effective, the distance between the quenching agent and the fluorescent molecule must be less than <RAD><RCD> <OVL><IT>&Dgr;x<SUP>2</SUP></IT></OVL></RCD></RAD><IT>=</IT><RAD><RCD><IT>2D&tgr;</IT></RCD></RAD><IT>,  D=kT/6&pgr;&ngr;R</IT> , where D is the diffusion coefficient, tau  is the lifetime of ASP+, k is the Boltzmann constant, T is temperature (Kelvin), eta  is viscosity of solution, and R is the atomic radius of the molecule (51). Accordingly, trypan blue and TEMPOL must be separated from ASP+ by ~30-50 Å (determined from the above equation). Fig. 9 shows a Stern-Volmer analysis (51) in which the y axis is the degree of quenching of ASP+ by trypan blue or TEMPOL. The presence of trypan blue does not affect phase I, implying that the bound substrate is inaccessible to the bath. Trypan blue does not affect phase II, because it is membrane-impermeable (data not shown). The presence of TEMPOL does not affect phase I, implying that the bound substrates are also inaccessible to the lipid bilayer. Although membrane permeable TEMPOL reduces phase II, the intracellular concentration was not determined (data not shown).


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Fig. 9.   Bound ASP+ is spatially isolated from bath and membrane. A, ASP+ fluorescence measured in solution in increasing concentration of trypan blue. Phase I is measured using the same ASP+/trypan blue solutions. B, ASP+ solution fluorescence measured in increasing concentrations of 4-hydroxy-tempo (TEMPOL). Phase I is measured with the same ASP+/TEMPOL solutions. Data fit to the Stern-Volmer equation, Fo/F = 1 + KD[Q], where Fo is fluorescence in the absence of quencher, F is the fluorescence in the presence of quencher, KD is the quenching constant, and [Q] is the concentration.

ASP+ Accumulation in SCG Neurons-- Lastly we investigated ASP+ interactions with NET in primary tissue culture neurons. Superior cervical ganglia (SCG) neurons endogenously express NET (52). To test for ASP+ accumulation in SCG neurons, we exposed dissociated cells to 2 µM ASP+ after preincubation for 10 min in the absence and presence of 10 µM desipramine. SCG neurons also contain OCTs, which could contribute to total ASP+ accumulation and reduce the signal-to-noise ratio in SCGs compared with HEK-hNET cells. However, 10 µM DS inhibits NET without affecting OCT activity (53), thus a difference between ASP+ accumulation in the absence and presence of DS would support the identification of NET-mediated ASP+ accumulation in neurons. Fig. 10D quantifies primary data illustrated in Fig. 10 (A and C) and reveals a separation of the integrated light signal after adding DS to the preparation. ASP+ accumulation in the cell body of neurons is qualitatively similar to hNET-HEK cells, and accumulation in neurites appears markedly punctate (Fig. 10C). The interrupted ASP+ pattern in the neurites is similar to the pattern of NET protein expression on noradrenergic fibers previously defined by immunohistochemical techniques (52), indicating that ASP+ accumulation likely functionally marks regions of NET surface expression. Thus ASP+ appears amenable to the investigation of NET not only in heterologous expression cultures where monoamine transporters are overexpressed but also at naturally occurring expression levels, thus affording the opportunity to study the location and activity of transporters in authentic neurons.


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Fig. 10.   ASP+ accumulates in SCG neurons. Panel A quantifies DS-sensitive accumulation by monitoring the increase in intracellular fluorescence within individual neurons. Comparing panels A (without DS) and B (with DS) shows that ASP+ accumulation in superior cervical ganglia (SCG) cells is desipramine-sensitive. Panel C shows a neurite at higher magnification. The color gradient represented in panel A denotes the color range corresponding to the intensity values. Panel D quantifies DS-sensitive accumulation in SRG neurons by monitoring ASP+ fluorescence in the absence and presence of DS.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the present study we demonstrate that the fluorescent molecule, ASP+, is a substrate for monoamine transporters. Visualization of ASP+ accumulation can report temporal and spatial information about transporters in both heterologous and native expression systems. Although ASP+ is structurally dissimilar to the endogenous substrate, it is similar to MPP+, a neurotoxic metabolite of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and well-studied substrate for monoamine transporters (38, 39, 41, 54, 55). Monoamine oxidase makes MPP+ from MPTP, the agent responsible for producing Parkinson-like paralysis in drug addicts who had used impure batches of synthetic heroin. MPP+ enters neurons via monoamine transporters and inhibits respiratory mitochondria. Similar to MPP+ in structure and function, the fluorescent analogue ASP+ competes effectively for specific NET-mediated NE transport, and ASP+ accumulation kinetics are similar to radiolabeled NE uptake kinetics.

Fluorescence anisotropy indicates the presence of two populations of ASP+ with distinct rotational movements: rapid phase I, which is localized to the cell surface and relatively immobile, and slow phase II, which represents mobile ASP+. These two populations are readily distinguished by sequential administration of desipramine, which shows a parallel increase of phase II accumulation with sequestered ASP+, strongly supporting the interpretation that phase I measures binding and phase II measures transport. Spatial patterning and temperature dependence strengthen this view, as does the pharmacological dissection of phase I and II. The biphasic phase I and phase II patterns do not represent transporter endocytosis, because NET-mediated ASP+ accumulation is unaffected by a 30-min preincubation with concanavalin A or 0.45 mM sucrose (data not shown).

We have shown that agents that inhibit substrate transport, such as desipramine or cocaine, also inhibit substrate binding. Although, cocaine is reported to inhibit transporters through a uncompetitive mechanism (56, 57). From the literature, Na+ removal has no effect on 5HT and DA displacement of high affinity SERT and DAT antagonists (28, 31, 32). Our assay reveals that ASP+ binding is independent of Na+ and actually increases in zero Cl-. Thus, although Na+ and Cl- are both significant to co-transport, only Cl- is significant to substrate binding in NETs. We recognize that native amine substrates may display characteristics distinct to ASP+, and our assay provides a means to explore these differences. Moreover, our data show that bound substrate is segregated from the bath and membrane, i.e. unavailable to quenchers localized to these compartments. Therefore, the transporter protein isolates bound substrate from bath and membrane, revealing for the first time that a monoamine transporter substrate binds deep within the transporter.

In summary, binding and transport in single mammalian cells and neurons, impossible to study with radiometric techniques, is readily measured with ASP+ fluorescence microscopy. The method also permits analysis of many cells while retaining spatial and kinetic information derived from single cells and is thus amenable to automated, multiwell protocols. Cells expressing hSERT and hDAT also accumulate ASP+ significantly above background, suggesting ASP+ as a general tool for the investigation of monoamine transporters. Single cell assays are important for mechanistic studies. For example, Prasad and Amara (25) conclude that dopamine accumulation in midbrain dopaminergic neurons does not depend on voltage. However, the uptake data and the voltage clamp data leading to their conclusion are from different cell populations, and the authors describe their inability to measure uptake in single mammalian cells under voltage clamp as a major hindrance. The opportunity to assess the spatial segregation of monoamine transport activity along the processes of living neurons should also permit the examination of regulatory mechanisms affecting transporter trafficking and catalytic function.

    ACKNOWLEDGEMENTS

We thank Hongping Yuan for technical assistance, Dr. Bruce D. Carter for assistance with the superior cervical ganglia neurons, Dr. Tapasree Goswami for editing and discussing this manuscript at various stages of its development, Michelle Mazei and Dr. Urlik Gether for providing HEK-hDAT cells, and the Cell Imaging Core at Vanderbilt University.

    FOOTNOTES

* This work was supported by National Institutes of Health (NIH) Grants NS-34075 (to L. J. D.) and MH 58921 (to R. D. B.). Analyses were performed in part in the Vanderbilt University Medical Center Cell Imaging Core Resource under the supervision of Dr. Sam Wells (supported by NIH Grant CA68485).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232-8548. Tel.: 615-343-6278; Fax: 615-343-1679; E-mail: lou.defelice@vanderbilt.edu.

Published, JBC Papers in Press, December 23, 2002, DOI 10.1074/jbc.M209824200

    ABBREVIATIONS

The abbreviations used are: NET, norepinephrine transporter; hNET, human NET; NE, norepinephrine; 5HT, serotonin; DA, dopamine; SERT, serotonin transporter; hSERT, human SERT; DAT, dopamine transporter; hDAT, human DAT; ASP+, 4-(4-(dimethylamino)styrl)-N-methylpyridinium; OCT, organic cation ion transporter; DS, desipramine; DIC, differential interference contrast; TEMPOL, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl; SCG, superior cervical ganglia; AFU, arbitrary fluorescence unit; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MPP+, 1-methyl-4-phenylpyridinium.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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