From the Division of Cellular and Molecular
Physiology, Department of Medical Physiology, The Panum Institute,
University of Copenhagen, DK-2200 Copenhagen N, Denmark,
§ Chemistry and Life Sciences, Research Triangle
Institute, Research Triangle Park, North Carolina, 27709, and
the ¶ Medical Research Council Laboratory of Molecular Biology,
Cambridge, CB2 2QH United Kingdom
Received for publication, September 4, 2000, and in revised form, November 1, 2000
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ABSTRACT |
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To explore the biophysical properties
of the binding site for cocaine and related compounds in the serotonin
transporter SERT, a high affinity cocaine analogue
(3 Cocaine is one of the most widely abused psychostimulants, causing
major medical and socioeconomic problems (1). Currently, there is no
effective treatment against cocaine addiction available; therefore,
clarifying the molecular mechanisms underlying the psychostimulatory
effects and addictive properties of cocaine should prove critical for
potential development of future therapeutic strategies. Cocaine and
related drugs act by inhibiting clearance of released monoamine
neurotransmitters from the synaptic cleft (2-4). This clearance of
monoamines occurs via three distinct but highly homologous monoamine
transporters, the serotonin transporter (SERT),1 the dopamine
transporter (DAT), and the norepinephrine transporter (NET) (2-4).
Cocaine binds with high affinity to all three transporters and is
generally believed to act as a competitive blocker of substrate translocation (4, 5). Several studies have provided evidence that
inhibition of the DAT is the predominant mechanism behind the
stimulatory effects and addictive properties of cocaine (6-8). However, this hypothesis has been challenged by recent studies on mice
in which the DAT gene has been deleted (1). Despite the absence of the
DAT gene, it was surprisingly observed that these mice
self-administered cocaine, indicating a possible important role of also
the SERT and NET (9, 10).
The SERT belongs together with DAT and NET to a family of
Na+/Cl In this study we have investigated the biophysical nature of the
cocaine binding site in the rat SERT (rSERT). For this purpose, a
cocaine analogue, which contained the environmentally sensitive fluorescent moiety, nitrobenzoxadiazol, were synthesized (see Fig. 1).
Moreover, an epitope-tagged version of the rSERT was expressed in Sf-9
insect cells and purified to obtain a transporter preparation that
provided a sufficiently high signal-to-noise ratio for characterizing
the fluorescent properties of the ligand bound to the rSERT. Our
subsequent spectroscopic analysis of the bound fluorescent ligand
provided evidence for a highly hydrophobic binding pocket. However,
collisional quenching experiments showed that, despite the hydrophobic
character of the binding crevice, the bound cocaine analogues were
still accessible for aqueous quenching consistent with a partially
exposed binding site. These findings contrast the observations for
small-molecule ligand binding sites in other membrane proteins, such
as, for example, G protein-coupled receptors, where the binding sites
for small-molecule ligands are known to be deeply embedded in the
transmembrane core of the molecule and entirely inaccessible to aqueous
quenching (21, 22).
Synthesis of 3 Expression of the SERT in Sf-9 Insect cells--
The rSERT,
tagged at the N terminus with the c-myc epitope and at the C
terminus with 10 histidines, was inserted into baculovirus expression
vector pVL1392 (Invitrogen, San Diego, CA) as described (24). The
resulting construct was expressed in Sf-9 insect cells, and baculovirus
encoding the tagged rSERT was isolated by plaque purification followed
by several rounds of amplification to obtain a high titer virus stock
(~109 plaque-forming units/ml) (24). Baculovirus encoding
canine calnexin was generated as described previously (24). For
purification, insect cells were grown in 1000-ml cultures in SF 900 II
medium supplemented with 5% (v/v) heat-inactivated fetal calf serum
and 0.1 mg/ml gentamicin (all products purchased from Life
Technologies, Inc. The cell cultures were infected at a density of
2 × 106 cells/ml by inoculating a 1:25 dilution of a
rSERT high titer virus stock plus 1:15 dilution of a high titer
calnexin virus stock. The cells were harvested 48 h later by
centrifugation (10 min at 5000 × g) and kept at
Membrane Preparation from Sf-9 Insect Cells--
25 ml of Sf-9
cell cultures at a density of 3 × 106 cells/ml in
125-ml disposable Erlenmeyer flasks (Costar, Acton, MA) were infected
for 48 h with 1.0 ml of a high titer rSERT virus stock encoding
rSERT. The cells were harvested by centrifugation (2000 × g for 5 min), washed once in phosphate-buffered saline, and homogenized using 25 strokes with a Dounce homogenizer in 50 mM Na2HPO4/NaH2PO4,
pH 7.4, containing 1 mM EDTA, 10 µg/ml benzamidine (Sigma), 10 µg/ml leupeptin (Sigma), and 0.5 mM
phenylmethylsulfonyl fluoride(Sigma). The lysate was centrifuged for 5 min at 500 × g, and the resulting supernatant was
centrifuged at 40,000 × g for 30 min at 4 °C. The
membrane pellet was resuspended in ice-cold sodium phosphate buffer (50 mM
Na2HPO4/NaH2PO4,
pH 7.4, 1 mM EDTA) containing the above-mentioned protease
inhibitors followed by an additional round of centrifugation and
resuspension. Protein was determined using the Bio-Rad DC protein assay
kit (Bio-Rad). The membranes were diluted to 1 mg of protein/ml in
buffer before storage at Purification of the SERT--
The transporter was purified using
a two-step purification procedure, which will be described in further
detail elsewhere.2 Briefly,
one pellet of Sf-9 cells from a 1000-ml infected culture was
resuspended in ice-cold lysis buffer (25 mM Hepes, pH 7.5, with 10 µM desipramine (Research Biochemicals
International, Natick, MA), 10 µg/ml leupeptin (Sigma), 10 µg/ml benzamidine (Sigma), and 1 mM phenylmethylsulfonyl
fluoride (Sigma)) followed by centrifugation at 30,000 × g for 30 min at 4 °C. The lysed cells were resuspended in
solubilization buffer (25 mM Hepes, pH 7.5, with 1%
digitonin (Calbiochem), 30% glycerol, 100 mM NaCl, 10 µM desipramine, 10 µg/ml leupeptin, 10 µg/ml
benzamidine, and 1 mM phenylmethylsulfonyl fluoride),
homogenized in a Dounce homogenizer, and stirred for 2 h at
4 °C. Upon centrifugation (30,000 × g for 30 min at
4 °C), the supernatant containing the solubilized transporter was
purified by nickel chromatography using chelating Sepharose (Amersham
Pharmacia Biotech). Binding to the resin was carried out in batch for
2 h at 4 °C under constant rotation. The transporter was eluted from the nickel resin in 200 mM imidazole. The eluted
transporter was further purified by concanavalin A chromatography.
Binding to the concanavalin A resin (Amersham Pharmacia Biotech) was
carried out in batch, and elution was done in 250 mM
Ligand Binding Assays--
Binding experiments on rSERT
expressed in Sf-9 insect cell membranes and of the purified transporter
were performed using 125I-RTI-55 (PE Biosystems) as
radioligand. In competition binding assays on membranes, 5 µg of
membrane protein was assayed in a total volume of 250 µl using a
sodium phosphate buffer (50 mM Na2HPO4/NaH2PO4,
pH 7.4) containing 0.25 nM 125I-RTI-55 and
increasing concentrations of competing ligands, 5-HT, citalopram,
cocaine, RTI-233, or RTI-55. Citalopram was kindly provided by Klaus
Gundertofte, Lundbeck A/S, Denmark. Cocaine and 5-HT were obtained from
Research Biochemicals International. The membranes were
incubated for 2 h at room temperature before separation of bound
from unbound by rapid filtration over glass fiber filters (FilterMat B,
Wallac, Turku, Finland) using a Tomtec 96-well cell harvester. MeltiLex
Melt-on scintillator sheets (Wallac) were used for counting of the
filter in a Wallac Tri-Lux Fluorescence Spectroscopy--
Fluorescence spectroscopy was
performed on a SPEX Fluoromax-2 spectrofluorometer connected to a PC
equipped with the Datamax 2.2 software package (Jobin Yvon Inc.,
Edison, NJ). In all experiments, the excitation and emission bandpass
were set at 5 nm. For the emission scan, quenching, and anisotropy
experiments, 20 pmol of purified rSERT was incubated in 100 µl of
digitonin buffer (25 mM Hepes, pH 7.5, with 0.1%
digitonin, 100 mM NaCl) in the presence of 1 µM RTI-233 and, when indicated, 1 mM 5-HT, 10 µM RTI 55, or 10 µM citalopram for 30 min
at 4 °C before separation of bound from unbound on 2 ml of Sephadex
G-50 columns. The fraction of bound was obtained by eluting with 1000 µl of ice-cold digitonin buffer. A 400-µl sample of the eluate was
transferred to a 5 × 5-mm quartz cuvette (Helma, Mulheim,
Germany) for the subsequent spectroscopic measurements. The absorption
by the nonfluorescent ligands, 5-HT, citalopram, and RTI-55 is less
than 0.01 at the used concentrations excluding any "inner filter" effect.
Emission Scan Experiments--
The emission scans were performed
either on 400 µl of Sephadex G-50-separated samples obtained as
described above or directly on buffer/dioxane samples containing
RTI-233. Excitation was 480 nm, and emission was measured from 495 to
625 nm with an integration time of 0.3 s/nm. All emission spectra are
averages of three consecutive scans. Photobleaching was negligible
under the experimental conditions used. The emission spectra were
corrected for any background fluorescence by routinely subtracting
control spectra on buffer alone.
Collisional Quenching Experiments--
Stock solutions (1.0 M) of the hydrophilic quencher potassium iodide containing
10 mM Na2S2O3 were
prepared freshly for each round of experiments. The hydrophobic
quencher 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) was
dissolved in 10% Me2SO at a concentration of 100 mM and immediately used. The experiments were performed on
either 400 µl of the Sephadex G-50-separated samples and prepared as described above or directly on buffer samples containing RTI-233. To
correct for dilution/ionic strength effects on fluorescence, measurements were performed in parallel using a 1.0 M stock
of potassium chloride (KCl) and a 10% Me2SO stock for the
potassium iodide-and TEMPO-quenching experiments, respectively. Ten
µl of quencher (potassium iodide or TEMPO) or control solution
(potassium chloride or 10% Me2SO) was added sequentially
followed by thorough mixing after each addition and subsequent
recording of fluorescence using the Constant Wavelength Analysis
program in the Datamax software package. The excitation wavelength was
480 nm, and the emission wavelength was either 532 nm for the
recordings on RTI-233 bound to rSERT or 536 nm for the recordings on
RTI-233 alone in digitonin buffer. A complete experiment was performed
in 8 min, during which dissociation of ligand was negligible. In the
experiments with TEMPO, the fluorescence intensities were corrected as
described (26) for inner filter effects caused by the absorption by
TEMPO at the used excitation and emission wavelengths. The corrected data were plotted according to the Stern-Volmer equation,
Fo/F = 1 + Ksv[Q], where
Fo/F is the ratio of fluorescence intensity in the absence and presence of quencher (Q), and
Ksv is the Stern-Volmer quenching constant (27).
Fluorescence Anisotropy--
The SPEX Fluoromax-2 fluorometer
was equipped with an automated L-format polarization accessory
including two Glan-Thomson UV polarizers placed in the sample chamber
to enable polarized excitation and emission detection. The anisotropy
measurements were carried out using the Constant Wavelength Analysis
program with the excitation set at 480 nm and emission measured at 532 nm for RTI-233 bound to rSERT and 536 nm for free RTI-233 in digitonin buffer (integration time 10 s). Concurrent measurements of the emission intensity with the excitation-side polarizer in the vertical position (V) and the emission-side polarizer in either the V or horizontal position (H) were carried out. The measurements were collected at 4, 20, and 37 °C by perfusion of water with adequate temperature through the thermostatted cuvette holder. The data were
converted to anisotropy according to the equation, A = (IVV Synthesis of a Fluorescent Cocaine Analogue--
To obtain a
fluorescent analogue of cocaine with preserved high affinity for the
rSERT, the environmentally sensitive fluorescent moiety
7-nitrobenzo-2-oxa-1,3-diazole (NBD) was connected to the 2 Binding of RTI-233 to the Purified rSERT--
The rSERT, expressed
in Sf-9 insect cells, was purified by nickel chromatography followed by
concanavalin A chromatography. The purified transporter bound RTI-233
with an affinity that was almost identical to that measured in the Sf-9
cell membranes (Fig. 2 and Table I). The KI for RTI-233 was 14 nM for the purified versus 6 nM for
the transporter in membranes, as determined from competition binding
assays with 125I-RTI-55 (Fig. 2 and Table I). Similarly,
the KI values for serotonin and the two blockers RTI-55 and
citalopram for inhibition of 125I-RTI-55 binding to the
purified transporter were similar to those observed in the membranes
(Fig. 2 and Table I). These data suggest that the overall conformation
of the transporter is conserved upon purification and support the
contention that the purified rSERT in detergent micelles can be used as
a model system for exploring the biophysical characteristics of the
binding site for cocaine-like blockers.
Spectral Properties of the Fluorescent Cocaine Analogue--
The
emission from RTI-233 is highly sensitive to the polarity of the
environment. In water, the Evidence for a Highly Hydrophobic Microenvironment in the Binding
Site for Cocaine-like Blockers--
To characterize the spectral
properties of RTI-233 bound to the transporter, 1 µM
RTI-233 was preincubated with purified rSERT (200 nM) for
30 min at 4 °C in the absence or presence of an excess of serotonin,
citalopram, or RTI-55. Bound ligand was separated from unbound by rapid
gel filtration using 2-ml G-50 gel filtration columns as described
under "Experimental Procedures." Subsequent fluorescence
spectroscopy analysis revealed a strong fluorescence signal if no
competing nonfluorescent ligand was present during precincubation (Fig.
3B). However, if the transporter was preincubated with
RTI-233 together with an excess of either serotonin, citalopram, or
RTI-55, the measured fluorescence was negligible (illustrated by a
representative curve in Fig. 3B). These data are consistent with the ability of these three structurally distinct compounds to
compete with RTI-233 for the rSERT and supports that the spectrum obtained in the absence of these compounds is derived from RTI-233 specifically bound to the rSERT. As shown in Fig. 3B, this
emission spectrum of bound RTI-233 displayed a Evidence for Decreased Rotational Freedom of RTI-233 upon Binding
to the rSERT--
Anisotropy represents a measure of molecular motions
at a nanosecond time scale (27). Accordingly, steady-state anisotropy measurements were carried out to investigate the rotational freedom of
unbound RTI-233 as compared with RTI-233 bound to the rSERT (Fig.
4). The anisotropy of unbound RTI-233 in
water was 0.057 ± 0.002 (means ± S.E., n = 3) at 20 °C, consistent with a substantial rotational freedom. In
digitonin buffer, the anisotropy of unbound RTI-233 increased to
0.173 ± 0.002 (means ± S.E., n = 3) at
20 °C (Fig. 4). This increase reflects most likely some degree of interaction of the ligand with the detergent molecules, causing inhibition of the rotational freedom. Importantly, however, the anisotropy was further increased upon binding of RTI-233 to the rSERT
(0.329 ± 0.004 at 20 °C; means ± S.E., n = 3; Fig. 4), indicating a significantly constrained rotational freedom
of RTI-233 when bound to the transporter as compared with free in
solution. As expected, the anisotropy was
temperature-dependent, consistent with increasing
rotational mobility at increasing temperatures (Fig. 4). The anisotropy
was stable for more than 15 min at 20 °C, indicating negligible
dissociation of the ligand under the experimental conditions used.
Probing Ligand Accessibility by Collisional Quenching--
The
accessibility of bound RTI-233, as compared with free RTI-233, was
evaluated in collisional quenching experiments. Collisional quenching
requires a bimolecular interaction between the quencher and the
fluorophore, and therefore, such experiments can determine the
"availability" of the fluorophore to the surrounding solvent (27).
The aqueous quencher iodide (I Although cocaine is one the most widely abused psychostimulants,
only little is known about the molecular mechanisms underlying the
inhibitory effect of cocaine at the monoamine transporters (SERT, DAT,
and NET). Most remarkable, the binding site for cocaine and related
analogues in the transporters is still unknown. In this study, we have
obtained new insight into the biophysical nature of this binding site
for cocaine-like blockers using fluorescence spectroscopy techniques.
To carry out the studies, we developed a fluorescent 3-phenyltropane
(cocaine-like) analogue (RTI-233) that retained high affinity for the
SERT despite the incorporation of the fluorescent NBD moiety. The NBD
moiety is characterized by a high sensitivity of the emission to the
polarity of the surrounding solvent, allowing the possibility of using
the ligand as reporter of the biophysical environment in the blocker
binding pocket (27, 28). Initially, we tried to perform the
spectroscopic measurements on Sf-9 cell membranes expressing the SERT;
however, the nonspecific background fluorescence was too high to detect
specific binding of RTI-233 (data not shown). Therefore, a purification
scheme for SERT expressed in Sf-9 cells was developed. The purified
SERT displayed the same pharmacological profile as that observed in Sf-9 cell membranes (Fig. 2 and Table I). Furthermore, this
pharmacological profile is similar to that observed for rSERT both in
brain tissue (29, 30) and in transfected mammalian cell lines (13, 14, 31). This suggests that the overall structure of the SERT is conserved
upon purification and, thus, that the purified preparation can be used
as an appropriate model system for exploring the binding site for
cocaine-like blockers as well as for studying other structure-function relationships in SERT. We should note that it cannot be excluded that
the absence of a transmembrane ion gradient and the use of temperatures
lower than 37 °C in our experiments to some extent could affect the
conformation of the binding site. However, we have no indication that
such effects are of any significance. Hence, the Several observations supported a highly hydrophobic microenvironment in
the binding pocket for cocaine-like blockers. First, it was observed
that the emission spectrum of RTI-233 bound to the SERT was similar to
that for the free ligand in 80% dioxane. Second, the bound ligand was
quenched substantially stronger by the lipid-soluble nitroxide
compound, TEMPO, than the free ligand. Third, the accessibility of
bound RTI-233 to the aqueous quencher iodide was markedly decreased in
comparison to free RTI-233. A noteworthy observation was, nevertheless,
that RTI-233 bound to rSERT could still be quenched to some degree by
the aqueous quencher iodide. It is interesting to compare this
observation for a small-molecule inhibitor binding site in a
transporter protein with data obtained for small-molecule ligands in
other membrane proteins, such as G protein-coupled receptors. Notably,
similar studies of inhibitor binding sites have not been carried before
in Na+-coupled transporter proteins. In the G
protein-coupled Many previous studies have aimed at defining the binding site for
cocaine and other blockers in the monoamine transporters. Generation of
chimeric transporters have been used to identify the domain important
for the selective recognition of, for example, tricyclic
anti-depressants, but due to lack of cocaine selectivity among the
monoamine transporters, chimeric studies have provided little insight
into determinants of cocaine binding (33, 34). Surprisingly,
substitution of multiple single residues has provided only limited
additional information about residues important for recognition of
cocaine-like substances. Nonconservative substitutions of a series of
prolines and phenylalanines caused only minor changes in the affinities
for the cocaine analogue WIN 35,528, making it highly difficult to
assess whether these changes are due to direct effects or indirect
structural perturbations (17, 18). Similarly, mutation of an aspartic
acid in the cytoplasmic half of TM 1 (Asp-98), which is conserved among
the monoamine transporters and believed to be critical for substrate
recognition, only marginally affected cocaine affinity (14). It did,
however, cause a more than 100-fold decrease in the apparent affinity
of citalopram, suggesting a pivotal role of this residue in binding
this inhibitor (14). Additional residues have been implicated in
binding of other inhibitors but not cocaine-like substances. These
include Tyr-95 in TM 1 and Phe-596 in TM 12 that were found to dictate the species selectivity of mazindol and imipramine between the human
and rat transporter. However, substitution of these residues hardly
affected cocaine binding affinity (13, 35). Rudnick and co-workers (20)
recently carried out a systematic cysteine mutagenesis scan of TM3 and
identified two residues that could be in proximity to the cocaine
binding pocket. This conclusion was based on the observation that
derivatization of cysteines inserted in these two positions (Ile-172
and Tyr-176) with the positively charged methanethisulfonate
reagent, MTSET ([2-(Trimethylammonium)ethyl]-methanethiosulfonate), severely inhibited binding of the cocaine analogue,
2 In summary, we have carried out a biophysical characterization of the
binding site for cocaine-like blockers in the rSERT. For this purpose
we have synthesized a fluorescent cocaine analogue that, to the best of
our knowledge, is the first fluorescent cocaine analogue displaying
high affinity for the rSERT. Furthermore we have developed a
purification scheme for the rSERT expressed in Sf-9 insect cells. In
addition, to provide the first direct information about the
microenvironment of the binding pocket for cocaine-like blockers, the
fluorescent ligand together with the purification procedure should
prove important tools in future studies. Such studies could include,
for example, further mapping of the binding site for cocaine-like
blockers based on measurements of fluorescent resonance energy transfer
(FRET) between the bound ligand and cysteine-reactive fluorescent
probes site-selectively incorporated into the purified transporter molecule.
-(4-methylphenyl)tropane-2
-carboxylic acid
N-(N-methyl-N-(4-nitrobenzo-2-oxa-1,3-diazol-7-yl)ethanolamine ester hydrochloride (RTI-233); KI = 14 nM) that contained the environmentally sensitive
fluorescent moiety 7-nitrobenzo-2-oxa-1,3-diazole (NBD) was
synthesized. Specific binding of RTI-233 to the rat serotonin
transporter, purified from Sf-9 insect cells, was demonstrated by the
competitive inhibition of fluorescence using excess serotonin, citalopram, or RTI-55
(2
-carbomethoxy-3
-(4-iodophenyl)tropane). Moreover, specific
binding was evidenced by measurement of steady-state fluorescence
anisotropy, showing constrained mobility of bound RTI-233 relative to
RTI-233 free in solution. The fluorescence of bound RTI-233 displayed
an emission maximum (
max) of 532 nm, corresponding to a
4-nm blue shift as compared with the
max of RTI-233 in
aqueous solution and corresponding to the
max of RTI-233 in 80% dioxane. Collisional quenching experiments revealed that the
aqueous quencher potassium iodide was able to quench the fluorescence of RTI-233 in the binding pocket (KSV = 1.7 M
1), although not to the same
extent as free RTI-233 (KSV = 7.2 M
1). Conversely, the hydrophobic
quencher 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO)
quenched the fluorescence of bound RTI-233 more efficiently than free
RTI-233. These data are consistent with a highly hydrophobic microenvironment in the binding pocket for cocaine-like uptake inhibitors. However, in contrast to what has been observed for small-molecule binding sites in, for example, G protein-coupled receptors, the bound cocaine analogue was still accessible for aqueous
quenching and, thus, partially exposed to solvent.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-dependent solute carriers
that are characterized functionally by their dependence on the presence
of Na+ and Cl
in the extracellular fluid (3,
11). All Na+/Cl
-dependent
carriers are believed to share a common topology characterized by the
presence of 12 transmembrane segments connected by alternating extracellular and intracellular loops with an intracellular location of
the N and C terminus (3, 4, 11). Despite intense efforts, including
many mutagenesis studies (12-18) and studies using photoaffinity labeling (19), surprisingly little is known about the binding site for
cocaine-like substances in the monoamine transporters. Although
cysteine-scanning mutagenesis of transmembrane segment 3 in the SERT
has suggested that two residues (Ile-172 and Tyr-176) in the middle of
the transmembrane segment could be in close proximity to the cocaine
binding site (20), no direct contact sites have been established
between cocaine and specific transporter residues. Hence, it is not yet
clear whether the cocaine binding site is deeply buried inside the
transmembrane core of the transporter molecule or if the binding site
is partially or fully exposed on the transporter surface.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-(4-Methylphenyl)tropane-2
-carboxylic Acid
N-(N-methyl-N-(4-nitrobenzo-2-oxa-1,3-diazol-7-yl)ethanolamine Ester
Hydrochloride (RTI-233)--
Oxalyl chloride (2.0 M in
CH2Cl2; 0.80 ml, 1.60 mmol) was added dropwise
to a stirred solution of
3
-(4-methylphenyl)tropane-2
-carboxylic acid (RTI-374) (23)
(200 mg, 0.77 mol) in CH2Cl2 (20 ml) under an
argon atmosphere at 25 °C. After stirring for 1 h, the
CH2Cl2 was removed by reduced pressure. A
solution of the acid chloride and
N-methyl-N-(4-nitrobenzo-2-oxa-1,3-diazol-7-yl)aminoethanol (458 mg, 1.93 mmol) in CH2Cl2 (10 ml) under
argon was treated slowly with triethylamine (0.5 ml, 3.5 mmol) and
stirred at 25 °C for 38 h. Removal of
CH2Cl2 by rotary evaporation afforded an
orange-red residue, which was purified by column chromatography on
silica gel (4.5 × 18 cm) with an ethyl acetate/methyl alcohol gradient elution (100:0-50:50; 100 ml). Removal of the eluant by
reduced pressure gave a red solid (270 mg, 73%), melting point 184-186 °C. The free base (270 mg) in
CH2Cl2 was treated with ethereal HCl (1.0 M, 3 ml) to yield the hydrochloride salt (282 mg), melting
point >250 °C (decomposition);
[
]Dd20
35° (C = 0.1g/100
ml methanol). The analysis of
C25H30ClN5O5·0.5 H2O was C, 57.23; H, 5.95; N, 12.35 (calculated) and C,
57.22; H, 6.32; N, 12.18 (experimental).
80 °C until purification. A significant fraction of the rSERT
expressed in Sf-9 cells was previously found to be misfolded (24, 25).
However, coinfection with the molecular chaperone, calnexin, was
demonstrated to increase the fraction of correctly folded protein
through assisted folding in the endoplasmatic reticulum of properly
glycosylated SERT, which stabilizes the transporter at the cell surface
(24, 25).
80 °C.
-methyl-mannopyranoside (Sigma). The purified transporter was
concentrated using Centricon-30 spin concentrators (Amicon, Beverly,
MA). The specific activity of the purified transporter was ~3 nmol/mg
of protein, corresponding to around 20% purity (active protein).
Protein was determined using the detergent-insensitive Bio-Rad DC
protein assay kit (Bio-Rad). The purified transporter was routinely
analyzed by 10% SDS-polyacrylamide gel electrophoresis and visualized
by standard silver staining from which functional protein
(125I-RTI-55 binding activity) were assessed to be around
two-thirds of total SERT on the stain. In general, ~2 nmol of
purified SERT could be obtained from a 1000-ml culture.
scintillation counter. Competition
binding experiments on purified transporter (15 fmol of transporter)
were performed in digitonin buffer (25 mM Hepes, pH 7.5, containing 0.1% digitonin, and 100 mM NaCl) in a total
volume of 100 µl using 0.25 nM of 125I-RTI-55
and increasing concentrations of unlabeled ligands. The binding assays
were incubated at room temperature for 30 min before separation of
bound from unbound on 2 ml of Sephadex G-50 columns (Amersham Pharmacia
Biotech). The eluate was collected directly in 4-ml counting vials
(Wallac) using 1000 µl of ice-cold digitonin buffer. Scintillation
fluid (HiSafe, Wallac) was added, and the vials were counted in a
Wallac Tri-Lux
scintillation counter. All determinations in the
binding assays were done in triplicate. Binding data were analyzed by
nonlinear regression analysis using Prism 2.0 from GraphPad Software,
San Diego, CA.
GIVH)/(IVV + 2GIVH), where IVV is the
intensity measured with both the excitation-side and emission-side
polarizer in the vertical position (V), IVH is
the intensity measured with the excitation-side polarizer in the
vertical position (V) and the emission-side polarizer in the horizontal
position (H), and G is the ratio of the sensitivities of the
detection system for vertically and horizontally polarized light
(SV/SH) (27). The
anisotropy was stable for at least 15 min at the indicated
temperatures, indicating negligible dissociation of ligand under the
experimental conditions used.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
position
of the tropane backbone in RTI-374
(3
-(4-methylphenyl)tropane-2
-carboxylic acid) using
N-methylethanolamine as the linker as described under "Experimental Procedures" (Fig. 1)
(5). Importantly, very large 2
-carboalkoxy groups have been shown to
be rather well tolerated in both cocaine and in the 3-phenyltropane
analogue, RTI-55 (5). Notably, the fluorescent compound, RTI-233,
differed, like RTI-55 and the parent 2
-carbomethoxy compound RTI-374
(3
-(4-methylphenyl)tropane-2
-carboxylic acid), from cocaine by
having the aromatic ring directly connected to the 3 position of the
tropane ring (Fig. 1). The 3-phenyltropane-type of structure was chosen
since it is known to increase binding affinity as compared with that of
cocaine itself (5). As shown in Fig. 2,
RTI-233 bound with high affinity to the rSERT expressed in Sf-9 cell
membranes displaying a KI value of 6 nM for
inhibition of 125I-RTI-55 binding (Fig. 2 and Table
I). In comparison, the
structurally related compound, RTI-55, and cocaine itself
displayed KI values of 0.22 and 109 nM,
respectively, for inhibition of 125I-RTI-55 binding.
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Fig. 1.
Synthesis of RTI-233. The cocaine
analogue RTI-233 was synthesized as described under "Experimental
Procedures" by connecting the environmentally sensitive fluorescent
moiety NBD to the 2 position of the 3-phenyltropane backbone using
an N-methylethanolamine linker. RTI-233 differs, like
RTI-55, from cocaine by having the phenyl ring directly connected to
the 3-position of the tropane ring.
View larger version (33K):
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Fig. 2.
Pharmacological profile of purified rSERT
compared with rSERT in Sf-9 cell membranes. Competition binding
experiments were performed on Sf-9 cell membranes (A) and on
the purified transporter (B) as described under
"Experimental Procedures." Binding of 125I-RTI-55 (0.25 nM) to the transporter were competed with unlabeled RTI-55
(closed squares), RTI-233 (open squares), cocaine
(closed circles), 5-HT (open circles), and
citalopram (open triangles). Data are percentage of maximum
125I-RTI-55 binding (means ± S.E., n = 3).
Binding properties of purified rSERT in comparison to rSERT in Sf-9
membranes
[radioligand]. The IC50 values used for calculation of
Ki values were obtained from means of pIC50
values determined by nonlinear regression analysis using Prism from
GraphPad software (San Diego, CA) and the S.E. interval from
pIC50 ± S.E. Ki values were calculated
according to the equation Ki = IC50/(1 + L/Kd) where L is concentration of
radioligand.
max (wavelength at which maximum emission occurs) of RTI-233 was 543 nm when the optimal excitation wavelength of 480 nm was used (Fig.
3A and Table
II). Decreasing the polarity of the
solvent by adding increasing concentrations of dioxane caused a
significant blue shift in
max and a concomitantly dramatic increase in the fluorescence quantum yield from RTI-233. In
90% dioxane,
max was 528 nm, and the fluorescence
quantum yield was more than 5-fold higher than in water (Fig.
3A and Table II). Notably, in buffer containing 0.1%
digitonin, the
max was 536 nm, consistent with a lowered
polarity due to the presence of the detergent molecules (Table II). The
strong dependence of the fluorescence from RTI-233 on the polarity of
the surrounding solvent corroborated the possibility of using RTI-233
as molecular reporter of the microenvironment in the cocaine binding
crevice of the rSERT.
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[in a new window]
Fig. 3.
Fluorescent properties of RTI-233 free in
solution and bound to the purified rSERT. A, emission
spectra of free RTI-233 in dioxane/H20. The fraction of
dioxane ranges from 0 to 90% (v/v) with 10% intervals. B,
emission spectrum of RTI-233 bound to rSERT ( max = 532 nm) with control emission spectra obtained by preincubation with an
excess of 5-HT, citalopram, or RTI-55. The spectra shown are
representative of nine experiments. In all experiments the excitation
wavelength was 480 nm, with a 5-nm bandpass for both excitation and
emission.
Fluorescent properties of RTI-233 in solvents of different polarity and
bound to rSERT
max) of RTI-233 in water, in water/dioxane
mixtures containing increasing amounts of dioxane (% v/v), in
digitonin buffer, and bound to rSERT are shown. The fluorescence
intensities relative to water at
max are indicated for the
water/dioxane mixtures. The emission maxima for RTI-233 in digitonin
buffer and bound to rSERT are means ± S.E. of the indicated
number of experiments.
max of
532 nm, representing a 4-nm blue shift as compared with that observed
for RTI-233 free in the buffer (Table II). According to the emission
spectra of RTI-233, in different concentrations of the hydrophobic
solvent dioxane, a corresponding blue shift was observed for free
RTI-233 in 80% dioxane (Fig. 3A and Table II).
View larger version (17K):
[in a new window]
Fig. 4.
Fluorescence anisotropy of free and bound
RTI-233. Fluorescence anisotropy of RTI-233 in water or digitonin
buffer and of RTI-233 bound to the transporter at 4, 20, and 37 °C.
The anisotropy was determined as described under "Experimental
Procedures." Data are the means ± S.E. of three independent
experiments. In all experiments the excitation was set at 480 nm, and
emission was recorded at 532 nm for the SERT-bound RTI-233 and at 543 or 536 nm for RTI-233 in water or digitonin buffer, respectively.
) was found to be a strong
quencher of the fluorescence from RTI-233 free in buffer. This is
illustrated by the linear Stern-Volmer plot in Fig.
5A, in which
Fo/F is plotted against the potassium
iodide concentration. The slope of the line represents the Stern-Volmer
constant (KSV), which was 7.2 M
1 for the free ligand. The
iodide quenching of bound RTI-233 was substantially smaller but still
apparent with a linear Stern-Volmer plot and a
KSV value of 1.7 M
1 (Fig. 5A). The
accessibility of RTI-233 was further examined by comparing quenching
produced by the lipid-soluble, nitroxide radical compound, TEMPO. The
addition of increasing concentrations of TEMPO efficiently quenched
both unbound and bound RTI-233. However, as shown in Fig.
5B, TEMPO was a stronger quencher of bound RTI-233 than of
free RTI-233. Whereas the KSV value was 37 M
1 for the bound ligand, it was reduced to 16 M
1 for the unbound ligand (Fig.
5B). It is important to note that the addition of potassium
iodide up to a concentration of 200 mM and TEMPO up to a
concentration of 20 mM did not affect binding of
125I-RTI-55 to the purified transporter (data not
shown).
View larger version (18K):
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Fig. 5.
Stern-Volmer plots of collisional quenching
of free RTI-233 and RTI-233 bound to rSERT. A,
quenching of RTI-233 fluorescence by the aqueous quencher potassium
iodide (KI). B, quenching of RTI-233 fluorescence by the
lipid-soluble quencher TEMPO. Open circles, RTI-233 bound to
rSERT; closed circles, free RTI-233. The quenching
experiments were carried out, and data were plotted as described under
"Experimental Procedures." The Stern-Volmer quenching constants
(Ksv) were 1.7 ± 0.13 M 1 and 7.2 ± 0.2 M
1 (means ± S.E.,
n = 3) for bound and unbound RTI-233 in the potassium
iodide quenching experiments, respectively, and 37 ± 3 M
1 and 16 ± 1.6 M
1 in the TEMPO quenching
experiments, respectively. In all experiments the excitation was 480 nm, and emission was recorded at 532 nm for the bound RTI-233 and at
536 nm for RTI-233 in digitonin buffer.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
max for
RTI-233 bound to the transporter is the same at 20° and at 37°,
suggesting that the microenvironment is not changing upon changes in
temperature (data not shown).
2-adrenergic receptor, the fluorescent
antagonist carazolol was used to probe the biophysical properties of
the ligand binding pocket (21). The fluorescence emission spectrum of
the bound carazolol was consistent with an extremely hydrophobic
environment in the binding site of the receptor, and exposure to
collisional quenchers demonstrated that carazolol bound to the purified
2-adrenergic receptor was not accessible to the solvent
at all (21). Furthermore, the fluorescence of bound carazolol was not
quenched by exposure to sodium nitrite, a Forster energy acceptor
having an R0 value of 11.7 Å with carazolol
(21). It was concluded, therefore, that the antagonist binds to the
2-adrenergic receptor in a rigid hydrophobic environment
buried deep within the transmembrane core of the protein (21). Similar
results were obtained for a fluorescently derivatized small-molecule
nonpeptide antagonist of the NK-1 (substance P) receptor (22). The
fluorescence emission of the bound nonpeptide antagonist was not
sensitive to aqueous quenchers, consistent with a deeply buried binding
site similar to that of carazolol in the
2-adrenergic
receptor (22). The spectral properties of the fluorescent nonpeptide
antagonist were compared with those of a fluorescently derivatized
substance P analogue. In contrast to what was observed for the
nonpeptide compound, the fluorescence from this fluorescent peptide
bound to the receptor was readily quenched by aqueous quenchers,
consistent with the predicted binding of the larger peptide ligands on
the extracellular face of the receptors (22). Taken together, our
current data differ from the observations for small ligands in G
protein-coupled receptors in that the binding site for cocaine-like
blockers is only partially buried and, thus, cannot be deeply embedded
in the protein interior. This conclusion is interesting in light of the
recent finding that cocaine can protect Cys-135 and Cys-342 in the
first and third intracellular loop from reaction with the
sulfhydryl-reactive methanethiosulfonate reagent, MTSEA
(2-Aminoethyl methanethiosulfonate) (32). It was concluded that the
protection by cocaine was due to an indirect conformational
effect; however, it is intriguing to consider the possibility
that the two cysteines in the intracellular loops are in close
proximity to the cocaine binding site of the monoamine transporters.
-carbomethoxy-3
-4-[125I]iodophenyl)tropane
(20). Moreover, this inhibition was blocked by cocaine itself
(20). It was nonetheless difficult to assess whether the observed
effects were direct or indirect; hence, it is clear that specific
interactions between cocaine and individual residues in the
transporters remains to be identified. Although we have to take into
consideration that the RTI-233 and cocaine binding pockets may not be
entirely identical, the current data could be an indication that the
further search for direct cocaine interactions should not be restricted
to residues that are deeply embedded in the predicted core of the
transporter molecule but may also include residues closer to the
transporter surface.
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ACKNOWLEDGEMENT |
---|
Dr. Brian Kobilka is thanked for helpful comments on the manuscript.
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FOOTNOTES |
---|
* The study was supported by the Danish Natural Science Research Council, National Institutes of Health Grants P01 DA 12408 and R37 DA05477, the Lundbeck Foundation, and the NOVO Nordisk Foundation.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.
Recipient of an Ole Rømer Associate Research Professorship
from the Danish Natural Science Research Council. To whom
correspondence should be addressed: Div. of Cellular and Molecular
Physiology, Dept. of Medical Physiology 12-5-22, The Panum Institute,
University of Copenhagen, DK-2200 Copenhagen N, Denmark. Tel.: 45 3532 7548; Fax: 45 3532 7555; E-mail: gether@mfi.ku.dk.
Published, JBC Papers in Press, November 2, 2000, DOI 10.1074/jbc.M008067200
2 S. G. F. Rasmussen and U. Gether, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are:
SERT, serotonin transporter;
rSERT, rat SERT;
NET, norepinephrine
transporter;
DAT, dopamine transporter;
NBD, 7-nitrobenzo-2-oxa-1,3-diazole;
5-HT, 5-hydroxytryptamine;
TEMPO, 2,2,6,6-tetramethylpiperidine-N-oxyl;
RTI55, 2-carbomethoxy-3
(4-iodophenyl)tropane;
RTI-233, 3
-(4-methylphenyl)tropane-2
-carboxylic acid
N-(N-methyl-N-(4-nitrobenzo2-oxa-1,3-diazol-7-yl)ethanolamine
ester.
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REFERENCES |
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