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.
DeFelice
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 |
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 |
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 |
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
(
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 |
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.

View larger version (16K):
[in this window]
[in a new window]
|
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.

View larger version (42K):
[in this window]
[in a new window]
|
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).
|
|

View larger version (85K):
[in this window]
[in a new window]
|
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.

View larger version (14K):
[in this window]
[in a new window]
|
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.

View larger version (24K):
[in this window]
[in a new window]
|
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.

View larger version (84K):
[in this window]
[in a new window]
|
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.

View larger version (20K):
[in this window]
[in a new window]
|
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).

View larger version (15K):
[in this window]
[in a new window]
|
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
, where D is the diffusion coefficient,
is the lifetime
of ASP+, k is the Boltzmann constant,
T is temperature (Kelvin),
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).

View larger version (14K):
[in this window]
[in a new window]
|
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.

View larger version (26K):
[in this window]
[in a new window]
|
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 |
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.
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 |
1.
|
Aston-Jones, G.,
Rajkowski, J.,
and Cohen, J.
(1999)
Biol. Psychiatry
46,
1309-1320[CrossRef][Medline]
[Order article via Infotrieve]
|
2.
|
Coull, J. T.,
Buchel, C.,
Friston, K. J.,
and Frith, C. D.
(1999)
Neuroimage
10,
705-715[CrossRef][Medline]
[Order article via Infotrieve]
|
3.
|
Skrebitsky, V. G.,
and Chepkova, A. N.
(1998)
Rev. Neurosci
9,
243-264[Medline]
[Order article via Infotrieve]
|
4.
|
Hatfield, T.,
and McGaugh, J. L.
(1999)
Neurobiol. Learn. Mem.
71,
232-239[CrossRef][Medline]
[Order article via Infotrieve]
|
5.
|
Axelrod, J.,
and Kopin, I. J.
(1969)
Prog. Brain Res.
31,
21-32[Medline]
[Order article via Infotrieve]
|
6.
|
Pacholczyk, T.,
Blakely, R. D.,
and Amara, S. G.
(1991)
Nature
350,
350-354[CrossRef][Medline]
[Order article via Infotrieve]
|
7.
|
Ressler, K. J.,
and Nemeroff, C. B.
(1999)
Biol. Psychiatry
46,
1219-1233[CrossRef][Medline]
[Order article via Infotrieve]
|
8.
|
Southwick, S. M.,
Bremner, J. D.,
Rasmusson, A.,
Morgan, C. A., III,
Arnsten, A.,
and Charney, D. S.
(1999)
Biol. Psychiatry
46,
1192-1204[CrossRef][Medline]
[Order article via Infotrieve]
|
9.
|
Dow, B.,
and Kline, N.
(1997)
Ann. Clin. Psychiatry
9,
1-5[Medline]
[Order article via Infotrieve]
|
10.
|
Biederman, J.,
and Spencer, T.
(1999)
Biol. Psychiatry
46,
1234-1242[CrossRef][Medline]
[Order article via Infotrieve]
|
11.
|
Bonisch, H.
(1984)
Naunyn-Schmiedebergs Arch. Pharmacol.
327,
267-272[Medline]
[Order article via Infotrieve]
|
12.
|
Corey, J. L.,
Quick, M. W.,
Davidson, N.,
Lester, H. A.,
and Guastella, J.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
1188-1192[Abstract]
|
13.
|
Fleckenstein, A. E.,
Haughey, H. M.,
Metzger, R. R.,
Kokoshka, J. M.,
Riddle, E. L.,
Hanson, J. E.,
Gibb, J. W.,
and Hanson, G. R.
(1999)
Eur. J. Pharmacol.
382,
45-49[CrossRef][Medline]
[Order article via Infotrieve]
|
14.
|
Jones, S. L.
(1991)
Prog. Brain Res.
88,
381-394[Medline]
[Order article via Infotrieve]
|
15.
|
Jacob, G.,
Shannon, J. R.,
Costa, F.,
Furlan, R.,
Biaggioni, I.,
Mosqueda-Garcia, R.,
Robertson, R. M.,
and Robertson, D.
(1999)
Circulation
99,
1706-1712[Abstract/Free Full Text]
|
16.
|
Hartzell, H. C.
(1980)
J. Cell Biol.
86,
6-20[Abstract]
|
17.
|
Watanabe, H.,
Furukawa, Y.,
and Chiba, S.
(1981)
Jpn. Heart J.
22,
977-985[Medline]
[Order article via Infotrieve]
|
18.
|
Clarkson, C. W.,
Chang, C.,
Stolfi, A.,
George, W. J.,
Yamasaki, S.,
and Pickoff, A. S.
(1993)
Circulation
87,
950-962[Abstract]
|
19.
|
Glassman, J. N.,
Dugas, J. E.,
Tsuang, M. T.,
and Loyd, D. W.
(1985)
J. Nerv. Ment. Dis.
173,
573-576[Medline]
[Order article via Infotrieve]
|
20.
|
Masson, J.,
Sagne, C.,
Hamon, M.,
and El Mestikawy, S.
(1999)
Pharmacol. Rev.
51,
439-464[Abstract/Free Full Text]
|
21.
|
Gu, H. H.,
Wall, S.,
and Rudnick, G.
(1996)
J. Biol. Chem.
271,
6911-6916[Abstract/Free Full Text]
|
22.
|
Ramamoorthy, S.,
Prasad, P. D.,
Kulanthaivel, P.,
Leibach, F. H.,
Blakely, R. D.,
and Ganapathy, V.
(1993)
Biochemistry
32,
1346-1353[Medline]
[Order article via Infotrieve]
|
23.
|
Bonisch, H.,
and Harder, R.
(1986)
Naunyn-Schmiedebergs Arch. Pharmacol.
334,
403-411[Medline]
[Order article via Infotrieve]
|
24.
|
Gu, H.,
Wall, S. C.,
and Rudnick, G.
(1994)
J. Biol. Chem.
269,
7124-7130[Abstract/Free Full Text]
|
25.
|
Prasad, B. M.,
and Amara, S. G.
(2001)
J. Neurosci.
21,
7561-7567[Abstract/Free Full Text]
|
26.
|
Diamond, J. S.,
and Jahr, C. E.
(1997)
J. Neurosci.
17,
4672-4687[Abstract/Free Full Text]
|
27.
|
Otis, T. S.,
and Kavanaugh, M. P.
(2000)
J Neurosci
20,
2749-2757[Abstract/Free Full Text]
|
28.
|
Humphreys, C. J.,
Wall, S. C.,
and Rudnick, G.
(1994)
Biochemistry
33,
9118-9125[Medline]
[Order article via Infotrieve]
|
29.
|
Earles, C.,
and Schenk, J. O.
(1998)
Anal. Biochem.
264,
191-198[CrossRef][Medline]
[Order article via Infotrieve]
|
30.
|
Earles, C.,
Wayment, H.,
Green, M.,
and Schenk, J. O.
(1998)
Methods Enzymol.
296,
660-675[Medline]
[Order article via Infotrieve]
|
31.
|
Li, L. B.,
and Reith, M. E.
(2000)
J. Neurochem.
74,
1538-1552[CrossRef][Medline]
[Order article via Infotrieve]
|
32.
|
Li, L. B.,
Cui, X. N.,
and Reith, M. A.
(2002)
Naunyn-Schmiedebergs Arch. Pharmacol.
365,
303-311[CrossRef][Medline]
[Order article via Infotrieve]
|
33.
|
Hadrich, D.,
Berthold, F.,
Steckhan, E.,
and Bonisch, H.
(1999)
J. Med. Chem.
42,
3101-3108[CrossRef][Medline]
[Order article via Infotrieve]
|
34.
|
Hohage, H.,
Stachon, A.,
Feidt, C.,
Hirsch, J. R.,
and Schlatter, E.
(1998)
J. Pharmacol. Exp. Ther.
286,
305-310[Abstract/Free Full Text]
|
35.
|
Stachon, A.,
Schlatter, E.,
and Hohage, H.
(1996)
Cell. Physiol. Biochem.
6,
72-91
|
36.
|
Gainetdinov, R. R.,
Fumagalli, F.,
Jones, S. R.,
and Caron, M. G.
(1997)
J. Neurochem.
69,
1322-1325[Medline]
[Order article via Infotrieve]
|
37.
|
Buck, K. J.,
and Amara, S. G.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
12584-12588[Abstract/Free Full Text]
|
38.
|
Sitte, H. H.,
Hiptmair, B.,
Zwach, J.,
Pifl, C.,
Singer, E. A.,
and Scholze, P.
(2001)
Mol. Pharmacol.
59,
1129-1137[Abstract/Free Full Text]
|
39.
|
Sitte, H. H.,
Scholze, P.,
Schloss, P.,
Pifl, C.,
and Singer, E. A.
(2000)
J. Neurochem.
74,
1317-1324[Medline]
[Order article via Infotrieve]
|
40.
|
Pifl, C.,
Agneter, E.,
Drobny, H.,
Sitte, H. H.,
and Singer, E. A.
(1999)
Neuropharmacology
38,
157-165[CrossRef][Medline]
[Order article via Infotrieve]
|
41.
|
Scholze, P.,
Zwach, J.,
Kattinger, A.,
Pifl, C.,
Singer, E. A.,
and Sitte, H. H.
(2000)
J. Pharmacol. Exp. Ther.
293,
870-878[Abstract/Free Full Text]
|
42.
|
Wall, S. C.,
Gu, H.,
and Rudnick, G.
(1995)
Mol. Pharmacol.
47,
544-550[Abstract]
|
43.
|
Galli, A.,
DeFelice, L. J.,
Duke, B. J.,
Moore, K. R.,
and Blakely, R. D.
(1995)
J. Exp. Biol.
198,
2197-2212[Medline]
[Order article via Infotrieve]
|
44.
|
Ramamoorthy, S.,
Giovanetti, E.,
Qian, Y.,
and Blakely, R. D.
(1998)
J. Biol. Chem.
273,
2458-2466[Abstract/Free Full Text]
|
45.
|
Blackman, S. M.,
Cobb, C. E.,
Beth, A. H.,
and Piston, D. W.
(1996)
Biophys. J.
71,
194-208[Abstract]
|
46.
|
Smith, N. C.,
and Levi, R.
(1999)
J. Pharmacol. Exp. Ther.
291,
456-463[Abstract/Free Full Text]
|
47.
|
De Oliveira, A. M.,
Schoemaker, H.,
Segonzac, A.,
and Langer, S. Z.
(1989)
Neuropharmacology
28,
823-828[CrossRef][Medline]
[Order article via Infotrieve]
|
48.
|
Rasmussen, S. G.,
Carroll, F. I.,
Maresch, M. J.,
Jensen, A. D.,
Tate, C. G.,
and Gether, U.
(2001)
J. Biol. Chem.
276,
4717-4723[Abstract/Free Full Text]
|
49.
|
Eftink, M. R.,
and Ghiron, C. A.
(1981)
Anal. Biochem.
114,
199-227[Medline]
[Order article via Infotrieve]
|
50.
|
Eftink, M. R.,
and Ghiron, C. A.
(1981)
Arch. Biochem. Biophys.
209,
706-709[Medline]
[Order article via Infotrieve]
|
51.
|
Lakowicz, J.
(1999)
Principles of Fluorescence Spectroscopy
, 2nd Ed.
, Kluwer Academic/Plenum Publishers, New York
|
52.
|
Schroeter, S.,
Apparsundaram, S.,
Wiley, R. G.,
Miner, L. H.,
Sesack, S. R.,
and Blakely, R. D.
(2000)
J. Comp. Neurol.
420,
211-232[CrossRef][Medline]
[Order article via Infotrieve]
|
53.
|
Wu, X.,
George, R. L.,
Huang, W.,
Wang, H.,
Conway, S. J.,
Leibach, F. H.,
and Ganapathy, V.
(2000)
Biochim. Biophys. Acta
1466,
315-327[Medline]
[Order article via Infotrieve]
|
54.
|
Javitch, J. A.,
D'Amato, R. J.,
Strittmatter, S. M.,
and Snyder, S. H.
(1985)
Proc. Natl. Acad. Sci. U. S. A.
82,
2173-2177[Abstract]
|
55.
|
Javitch, J. A.,
and Snyder, S. H.
(1984)
Eur. J. Pharmacol.
106,
455-456[CrossRef][Medline]
[Order article via Infotrieve]
|
56.
|
Earles, C.,
and Schenk, J. O.
(1999)
Synapse
33,
230-238[CrossRef][Medline]
[Order article via Infotrieve]
|
57.
|
McElvain, J. S.,
and Schenk, J. O.
(1992)
Biochem. Pharmacol.
43,
2189-2199[CrossRef][Medline]
[Order article via Infotrieve]
|
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.