From the § Institute of Pharmacology, University of
Vienna Medical School, Währingerstrasse 13a, A-1090 Vienna,
Austria and the Department of Vascular Biology and
Thrombosis Research, University of Vienna Medical School,
Brunnerstrasse 59, A-1235 Vienna, Austria
Received for publication, August 14, 2000, and in revised form, October 13, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recent biochemical studies indicate that the
serotonin transporter can form oligomers. We investigated whether the
human serotonin transporter (hSERT) can be visualized as an oligomer in
the plasma membrane of intact cells. For this purpose, we generated
fusion proteins of hSERT and spectral variants of the green fluorescent protein (cyan and yellow fluorescent proteins, CFP and YFP,
respectively). When expressed in human embryonic kidney 293 cells, the resulting fusion proteins (CFP-hSERT and YFP-hSERT) were
efficiently inserted into the plasma membrane and were functionally
indistinguishable from wild-type hSERT. Oligomers were visualized by
fluorescence resonance energy transfer microscopy in living cells using
two complementary methods, i.e. ratio imaging and donor
photobleaching. Interestingly, oligomerization was not confined to
hSERT; fluorescence resonance energy transfer was also observed between
CFP- and YFP-labeled rat It is widely accepted that tyrosine kinases and related receptors
signal as dimers (1). Similarly, the oligomeric nature of
voltage-dependent and ligand-gated ion channels is firmly
established. In addition, over recent years it has been
determined that other integral membrane proteins, which were
originally thought to exist in monomeric form, actually form homo- and
hetero-oligomers. For instance, this is true for several G
protein-coupled receptors (2-5) and for a number of transporters,
e.g. the erythrocyte glucose transporter-1 and the brain
glutamate transporter (6, 7). The structural organization of
transporters is likely to determine their function. This consideration
is particularly relevant in understanding the transporters that mediate
the re-uptake of neurotransmitters from the synaptic cleft (8). These
proteins depend on the presence of Na+ and Cl However, it is at present unknown whether SERT exists as an oligomer in
the membrane in situ and whether this is the preferred conformation. To address these issues, we applied a nondestructive method to visualize hSERT in living cells. This has become possible by
the recent development of green fluorescent protein (GFP) variants that
are suited to monitor close associations of fusion proteins by means of
fluorescence resonance energy transfer (FRET), a quantum physical
phenomenon that was first described by Förster (18).
The human SERT was tagged on its amino terminus with cyan fluorescent
proteins (CFP) and yellow fluorescent proteins (YFP), respectively;
these spectral variants of GFP have an appropriate spectral overlap of
the donor (CFP) emission and the acceptor (YFP) excitation (19). Our
observations show that hSERT exists as an oligomer in the membrane of
living cells and that this is the preferred conformation. Moreover,
this finding was not confined to the hSERT alone. A similar
homoassociation exists with the rat GABA transporter 1 (rGAT1).
Materials--
Tissue culture reagents were from Life
Technologies, Inc. [3H]5-Hydroxytryptamine (HT) was from
PerkinElmer Life Sciences. The following drugs were kindly
donated: citalopram (Lundbeck A/S, Kobenhavn, Denmark), paroxetine
(SmithKline Beecham, Worthing, United Kingdom), and cocaine (Dolda AG,
Basel, Switzerland). Imipramine, para-chloroamphetamine, and
serotonin were from Sigma. Methylenedioximethamphetamine ("ecstasy") was from Research Biochemical International (Natick, MA). All other chemicals were from commercial sources.
Plasmid Construction--
hSERT cDNA was a generous gift of
Dr. R. D. Blakely (Vanderbilt University, Nashville, TN). A
HindIII/XbaI fragment (encompassing the coding
region) was inserted into the plasmid pRC/CMV (Invitrogen), ligated into HindIII/XbaI-digested pEGFP-C1
(CLONTECH, Palo Alto, CA) to produce
hSERT-pEGFP-C1, and transferred from this vector using XhoI
to the plasmids pECFP-C1 and pEYFP-C1 (CLONTECH) to produce the plasmids hSERT-pECFP-C1 and hSERT-pEYFP-C1, respectively. GFP, CFP, or YFP is fused to the NH2 terminus of hSERT and
resides in the cytoplasm.
A HindIII/StuI fragment of the human dopamine
D2-receptor (hD2R), encompassing the coding
region and lacking eight COOH-terminal amino acids, was ligated into
HindIII/SmaI-digested pYFP-N1 to produce the
plasmid hD2R-YFP; YFP is fused to the COOH terminus of
hD2R and situated in the cytoplasm of the cell.
As a positive control for FRET imaging, we constructed a fusion protein
of CFP and YFP. The yellow variant, GFP10C, was a generous gift of Dr.
Roger Tsien (University of California, San Diego, CA) and was subcloned
into pEGFP-C1 COOH-terminal to the enhanced GFP, which was subsequently
replaced by CFP (derived from pECFP-C1), thus resulting in a plasmid
coding for a CFP-YFP tandem.
The cDNA encoding the rGAT1 was a generous gift of Dr. Patrick
Schloss (ZI für Seelische Gesundheit, Mannheim, Germany). The
coding region was excised using BbrPI and
KpnI; blunt ends were generated at the SalI
restriction site of the vector pEYFP-C1 (CLONTECH),
which was digested afterward with KpnI. The rGAT1-cDNA was then inserted to result in the construct YFP-rGAT1. Subsequently, the rGAT1-cDNA was subcloned into the vector pECFP-C1
(CLONTECH) using the enzymes SacI and
KpnI to generate the construct CFP-rGAT1.
Cell Culture and Transfection--
Human embryonic kidney 293 (HEK-293) and HeLa cells were grown in minimal essential medium with
Earle's salts and L-alanyl-L-glutamine (L-GlutaMAX ITM, Life Technologies, Inc.), 10%
fetal bovine serum, and 50 mg/liter gentamicin on 10-cm diameter cell
culture dishes at 37 °C in an atmosphere of 5% CO2,
95% air. One day before transfection, cells were replated to obtain
subconfluent cultures either on glass coverslips (22 mm in diameter and
placed into 6-well plates) or on 12-well plates (2 × 105 cells/well plate) for uptake experiments. Transient
transfections were performed with equal amounts of CFP and YFP plasmids
using the CaPO4 precipitation method or LipofectAMINE
PlusTM as described (20).
Transport of [3H]5-Hydroxytryptamine--
Uptake
experiments were performed as described earlier (21). Cells transfected
with either hSERT-wild type, CFP-hSERT, YFP-hSERT, or pECFP-C1,
pEYFP-C1 vectors were incubated for 5 min at 22 °C in 0.5 ml
of Krebs-Ringer-Hepes buffer (10 mM Hepes, 120 mM NaCl, 3 mM KCl, 2 mM
CaCl2, 2 mM MgCl2, 20 mM glucose, final pH 7.3) containing 2 µCi of
[3H]5-HT (specific activity adjusted with unlabeled
5-HT). The amount of accumulated radioactivity was determined by liquid
scintillation counting.
FRET Microscopy (Ratio Imaging and Donor Photobleaching FRET
Microscopy)--
Transfected HEK-293 or HeLa cells were investigated 1 day after transfection on a Nikon Diaphot TMD microscope using filter sets, which discriminate between CFP and YFP fluorescence (Omega Optical Inc., Brattleboro, VT) (CFP filter set: excitation, 440 nm;
dichroic mirror, 455 nm; emission 480 nm; YFP filter set: excitation,
500 nm; dichroic mirror, 525 nm; emission, 535 nm), and a cooled
CCD-camera (Kappa GmbH, Gleichen, Germany). For ratio imaging FRET
microscopy, images were taken with the donor filter set (for CFP) and a
FRET filter set (XF88, Omega Optical) with excitation of the donor (440 nm), a 455 nm dichroic mirror, and an emission filter for the acceptor
(535 nm). Images were captured with both filter sets under identical
conditions. This choice of settings was based on initial experiments
with cells expressing CFP and YFP. These experiments were employed to
verify that the imaging parameters did not result in spurious ratio
images. Ratio images that are suited to detect a decrease in donor and
an increase in acceptor fluorescence were calculated by dividing the
acceptor filter image by the donor image (20, 22) using the NIH
image software version 1.62. In principle, black (zero) ratio images are obtained even if an equal intensity of fluorescence is recorded with the donor and FRET filters. Nevertheless, to prevent the detection
of false positive FRET images, the imaging conditions were adjusted to
favor donor emission over acceptor emission. These settings introduce a
bias against acceptor emission and, furthermore, underestimate FRET. We
chose to sacrifice the sensitivity for detection of FRET for the sake
of increasing the specificity of the signal.
Quantification of fluorescence ratios was achieved by imaging the
transfected cells at lower magnification (× 10 objective) with donor
and acceptor filter sets and by subsequently determining the integrated
density values (ID = number of pixels × (mean intensity background)) for the calculation of the YFP:CFP fluorescence ratio at
donor excitation.
Photobleaching FRET microscopy was done by continuous illumination with
a 100-watt-mercury lamp and the CFP filter set with time series imaging
for 1 min (with the acquisition of one image every 2 s), which was
sufficient to bleach the donor to an extent of less than 20%. Regions
of interest were selected over the membrane, and fluorescence emission
intensities were quantified using the NIH image software. The resulting
decay curves were fitted to the equation for a single exponential decay
approaching a constant value: fluorescence intensity = A0*e Fluorometry--
Extracts were prepared from cells expressing
CFP-hSERT and YFP-hSERT or CFP-hSERT and YFP by solubilizing the
particulate fraction in phosphate-buffered saline containing 0.5%
Nonidet P-40 and protease inhibitors (10 µg/ml of aprotinin, 20 µg/ml of phosphoramidon, 40 µg/ml Pefabloc, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 mM EDTA from 1000× stock solutions).
The insoluble material containing cell nuclei was removed by
centrifugation at 14,000 × g for 15 min, and the
supernatant was measured on a Jasco FP-920 spectrofluorometer. Emission
wavelength scans were performed with excitation at 436 nm (for CFP and
FRET measurements) or at 500 nm (for YFP). The bandwidth of excitation
was 18 nm, and that of the emission was set to 10 nm. Emission scans
with an excitation at 500 nm were performed to determine the amount of
YFP without the influence of FRET. The minor contribution of YFP
fluorescence to the emission curve at the CFP excitation wavelength (436 nm), which results from coexcitation of YFP at that wavelength (instead of sensitized fluorescence due to FRET), was subtracted from
the emission scans, and the curves were normalized to equal CFP peak
fluorescence at 476 nm. By that means, FRET can be detected by a shift
of the emission scan at the acceptor wavelength showing the increase in
acceptor fluorescence.
FPLC Analysis--
Extracts were prepared from CFP-hSERT and
YFP-hSERT expressing cells as described above and subjected to gel
filtration on a Pharmacia FPLC system using a Superdex 200 matrix
equilibrated with extraction buffer at a flow rate of 2 ml/min. The
column (diameter = 16 mm, length = 2 m, volume = 402 ml) was calibrated with appropriate standards (Bio-Rad catalog no.
151-1901; 670, 158, 44, 17, and 1.35 kDa). Fluorescent proteins were
detected during FPLC elution with a Jasco FP-920 spectrofluorometer in the kinetics mode using a 16-µl flow cell, excitation at 436 nm, and
emission at 510 nm with an 18-nm bandwidth, respectively. Recording of
the fluorescence signal was started briefly before the flow through of
the void volume with read outs every 30 s for a total of 2 h.
Statistics--
All results are expressed as means ± S.E.
values. The significance of differences among the means of various
groups was determined by Student's t test for independent samples.
The major aim of this study was to investigate the serotonin
transporter in living cells by using GFP chimeras of this
pharmacologically important transporter molecule. For that purpose, it
is essential to verify the functional integrity of the fusion protein.
This was achieved on several levels. Initially, we confirmed that the chimeric proteins comprising hSERT and different variants of GFP were
inserted into the plasma membrane. Upon expression of CFP-hSERT and
YFP-hSERT in HEK-293 cells (or in HeLa cells, data not shown), the bulk
of the fluorescence was recorded over the plasma membrane (Fig.
1 and see Fig. 3G). The
imaging of CFP-hSERT expressing cells with the YFP filter set showed no
fluorescence signal. The same was found with YFP-hSERT expressing cells
and image acquisition with the CFP filter set, proving the specificity
of the filters and the imaging conditions (Fig. 1). Uptake measurements
showed that the fusion proteins of hSERT with GFP variants transported the natural substrate 5-HT with Vmax and
Km values similar to those of wild-type hSERT (Fig.
2A;
Vmax = 17 ± 1 versus 22 ± 2 pmol/min/well and Km = 0.99 ± 0.23 versus 0.94 ± 0.37 µM for cells
transfected with YFP-hSERT/CFP-hSERT and hSERT, respectively;
n = 3). Cells transfected with CFP and YFP alone showed
no specific uptake of 5-HT (Fig. 2A). Similarly, the
inhibition of 5-HT influx by imipramine exhibited no significant
difference in both cases (IC50 = 16 ± 2 and 19 ± 3 nM, n = 3 for cells expressing YFP-hSERT/CFP-hSERT and hSERT, respectively, Fig. 2B). Thus,
CFP- and YFP-tagged fusion proteins of hSERT are functional with
characteristics that are very similar to the wild-type protein.
-aminobutyric acid transporter. The bulk of
serotonin transporters was recovered as high molecular weight complexes
upon gel filtration in detergent solution. In contrast, the monomers of
CFP-hSERT and YFP-hSERT were essentially undetectable. This indicates
that the homo-oligomeric form is the favored state of hSERT in living cells, which is not significantly affected by coincubation with transporter substrates or blockers. Based on our observations, we
conclude that constitutive oligomer formation might be a general property of Na+/Cl
-dependent
neurotransmitter transporters.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
and generate a current during transport, i.e. they may share properties similar to ion channels (9-12) that are known to be organized as oligomeric complexes. The human serotonin transporter (hSERT)1 is a prototypic
member of this family; its properties are of considerable clinical
interest because the inhibitors are useful as antidepressants, and
substrates that induce the reversal of transport (e.g.
"ecstasy") are abused (13). The complexity of the transport
reaction is suggestive of a higher level of organization, and recent
biochemical experiments on the SERT of different species indicate that
the transporter can, in principle, form oligomeric structures
(14-17).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Kt + offset, where A0 denotes the starting value,
offset denotes the final fluorescence signal, and K is the
decay constant. The time constant
(fluorescence lifetime) is
defined as 1/K.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (34K):
[in a new window]
Fig. 1.
Fluorescence microscopy. HEK-293 cells
were transfected with plasmids encoding CFP-hSERT (top row)
or YFP-hSERT (bottom row). The next day images (× 40 magnification) were taken using the CFP filter set (left
column), the YFP filter set (middle column) and the
FRET filter set (right column) as outlined under
"Experimental Procedures." The images are representative of two
different transfections with aquisition of five images with each
different filter set.
View larger version (16K):
[in a new window]
Fig. 2.
Functional properties of YFP-hSERT and
CFP-hSERT constructs. A, saturation analysis of 5-HT
uptake. HEK-293 cells transfected with plasmids encoding CFP and YFP
( ), wild-type hSERT (
), or equal amounts of cDNA encoding
CFP-hSERT and YFP-hSERT (gray square) were incubated with
the indicated concentrations of [3H]5-HT for 5 min.
B, inhibition of uptake by imipramine. HEK-293 cells
transfected with plasmids encoding wild-type hSERT (
) or CFP-hSERT
and YFP-hSERT (gray square) were incubated with increasing
concentrations of imipramine to inhibit [3]5-HT
uptake.
To visualize the oligomeric state of hSERT in living cells, we applied
FRET microscopy using ratio imaging. The negative control consisted of
cells that expressed CFP and YFP. In these cells, the donor image (Fig.
3A) showed a higher
fluorescence signal than the FRET image (Fig. 3B) by using
the imaging conditions as described under "Experimental
Procedures." This observation results in a black ratio image (Fig.
3C). As a positive control, we used cells expressing the
YFP-CFP tandem. Given the known Förster distance (~50 Å for
CFP and YFP), the short tether in the CFP-YFP tandem, and its soluble
nature, a strong intramolecular FRET was to be expected in the cytosol.
The fluorescence intensity recorded with the FRET filter set (Fig.
3E) exceeded the intensity measured with the donor filter
set (Fig. 3D), resulting in an intense cytosolic FRET signal
in the ratio image (Fig. 3F).
|
If hSERT oligomerized in the plasma membrane, the intermolecular FRET
ought to be seen in cells coexpressing YFP-hSERT and CFP-hSERT. These
experiments were performed by the transient transfection of two
different cell lines (HEK-293 and HeLa cells) with clear-cut evidence
for FRET in each case. At the wavelength for donor excitation, fluorescence emission recorded with the CFP filter set (Fig.
3G) was obviously lower than the signal obtained with the
FRET filter (Fig. 3H). This loss in donor emission is
indicative of FRET. Accordingly, the ratio image revealed a signal
confined to the cellular membrane (Fig. 3I). Cells
expressing CFP-hSERT or YFP-hSERT alone did not exhibit any positive
ratio image, thereby further supporting the specificity of this FRET
imaging technique (Fig. 1). The clear positive ratio image of CFP-hSERT
and YFP-hSERT coexpressing cells can only be accounted for by
intermolecular FRET due to oligomerization of the fluorescent-tagged
hSERT proteins. This oligomerization was detected not only in cells
expressing CFP- and YFP-hSERT but also in cells expressing the CFP- and
YFP-tagged rGAT1, which is a member of the same subfamily of
Na+/Cl-dependent neurotransmitter
transporter proteins (sharing about 50% sequence homology) (Fig. 3,
bottom line, K-M).
We also quantified FRET at a lower magnification in several independent experiments to obtain an average for a high number of transfected cells. The corresponding fluorescence ratio was significantly higher for images taken from cells expressing the YFP-CFP tandem (1.94 ± 0.37, n = 7) and from cells coexpressing YFP-hSERT and CFP-hSERT (1.56 ± 0.13, n = 22) or YFP-rGAT1 and CFP-rGAT1 (1.61 ± 0.18, n = 9) than the ratio obtained for the negative control cells (i.e. coexpressing CFP and YFP, 0.54 ± 0.09, n = 13).
The interaction between CFP- and YFP-tagged hSERT was further verified
in vitro by means of emission scanning fluorometry. If CFP
and YFP were in close proximity due to oligomerization of tagged hSERT
molecules, this would result in FRET between CFP and YFP. Hence, an
increase of the YFP fluorescence (sensitized acceptor fluorescence)
ought to be observed at the excitation wavelength of CFP
(i.e. the FRET donor), and this effect ought to be clearly
detectable in emission wavelength scans. To test for sensitized
acceptor fluorescence, we prepared extracts from cells expressing
either CFP- and YFP-hSERT or CFP-hSERT with untagged YFP as control and
recorded emission wavelength scans at the donor and acceptor
excitation, respectively. Normalized emission scans of these extracts
revealed a distinct sensitized acceptor fluorescence for the
CFP-hSERT/YFP-hSERT sample as compared with control extracts (Fig.
4), which is clearly indicative of FRET,
and this can arise only due to oligomerization of CFP- and YFP-hSERT.
|
The detection of intermolecular FRET unequivocally demonstrated the
propensity of hSERT to form an oligomer in the cellular membrane of
intact cells; however, it was not possible to estimate the relative
proportion of transporters in the oligomeric or monomeric form by FRET.
To verify that oligomers represented a sizable fraction of the total,
CFP-hSERT and YFP-hSERT were solubilized from the plasma membranes in
the presence of 0.5% Nonidet P-40 and subjected to gel filtration. The
bulk of the transporter protein eluted in the high molecular
mass range with a peak at about 600-800 kDa (Fig.
5); two shoulders were also evident (at
about 550 kDa and 320 kDa, respectively), which may represent distinct
multimers (e.g. dimers and tetramers). A detailed
spectroscopic analysis of the fluorescent peak using wavelength scans
was not feasible because of the high dilution of tagged hSERT in the
peak, which required detector settings that are not appropriate to
determine FRET unequivocally. However, it seems unlikely that these
high molecular mass complexes comprising CFP- or YFP-tagged hSERT are monomeric forms of the transporter molecule surrounded by detergent micelles because the size of Nonidet P-40 micelles is expected to be
only 60-90 kDa based on the average number of detergent molecules in
micelles () and the molecular mass of 603 for the Nonidet P-40
monomer. Nevertheless, we cannot rule out that hSERT may associate with
additional membrane proteins, and these may contribute to the formation
of large molecular weight complexes. In fact, it is very likely that
hSERT forms a complex with synaptic proteins because it is specifically
targeted to synaptic specializations in neurons (and in differentiated
PC12 cells).2 Furthermore, we
cannot formally exclude the possibility that nonfunctional hSERT
aggregates are incorporated in the high molecular weight peak. However,
we note that we did not detect a significant portion of hSERT monomers
by employing native gel electrophoresis. In contrast, in the denatured
form hSERT migrated with the relative molecular mass predicted for the
monomer (data not shown). Thus, taken together, these data are
consistent with the notion that hSERT is a constitutive oligomer.
|
It is worth pointing out that the existence of higher order complexes has also been postulated based on molecular sieve chromatography, cross-linking, and coimmunoprecipitation experiments (14, 15, 17). Our observations confirm and extend these findings by directly visualizing hSERT oligomers in the membranes of living cells.
To rule out the possibility that the FRET signal we detected between CFP- and YFP-tagged hSERT resulted from transient collision events within the membrane (and presumably nonspecific interaction), we aimed to investigate CFP-hSERT in combination with other transmembrane proteins fused to YFP. However, a different method has to be used for the detection of FRET in this case because the distinct nature of different transmembrane proteins might result in differences in expression levels or subcellular distribution. The prerequisite for the calculation of FRET ratio images is that both fluorophores exhibit the same intracellular distribution and that they are present in equal amounts. These requirements are met by employing essentially identical plasmids encoding homotypic proteins, such as CFP and YFP or CFP-hSERT and YFP-hSERT, which differ only by nine amino acids within the core region of the fluorophore but are otherwise identical. They may not be fulfilled if the proteins that are coexpressed differ substantially in amino acid composition. Thus, we employed the method of donor photobleaching FRET microscopy (2), which relies on the fact that the bleaching of a donor fluorophore is slower in the presence of a FRET acceptor because energy is transferred to the acceptor (and thus unavailable for bleaching the donor). The time constant of the fluorescence decay is independent of fluorophore concentration (as long as the acceptor is in excess) and is not affected by the differences in intracellular distribution (2).
If high levels of illumination were applied through the donor filter
set, donor fluorescence emission rapidly declined (Fig. 6A), and this process was
monoexponential (Fig. 6B). As expected, the time constant of
fluorescence decay was significantly lower in cells coexpressing CFP
and YFP than in cells expressing the CFP-YFP tandem, the time constant
being 22.0 ± 1.9 s (n = 10) and
31.7 ± 2.1 s (n = 14), respectively
(p = 0.015, see Fig. 6C). Similarly, the
fluorescence decay of CFP-hSERT alone significantly was faster than
CFP-hSERT in the presence of YFP-tagged hSERT (24.4 ± 0.5 s,
n = 7, and 29.9 ± 0.6 s, n = 31, respectively; p = 0.02). The fact that YFP-hSERT
slowed the decay in the fluorescence of CFP-hSERT was indicative of
intermolecular FRET and thus represented an independent confirmation of
the ratio imaging results. As a prototypical structurally unrelated
transmembrane protein, we chose the human dopamine D2
receptor fused to YFP (hD2R-YFP). In cells expressing
CFP-hSERT and hD2R-YFP (in excess), a time constant
of
24.3 ± 1.0 s (n = 6) was calculated,
i.e. similar to
of CFP-hSERT alone. This means that
there is no significant interaction between hSERT and hD2R
in the cellular membrane. Hence, statistical transient collision events
between structurally unrelated transmembrane proteins cannot account
for the FRET signal observed between CFP- and YFP-hSERT. However, it
was reported that the hD2R is able to hetero-oligomerize
with another membrane protein, the human somatostatin receptor (23), as
detected by FRET microscopy as well.
|
An intriguing question was whether different agonistic or antagonistic
drugs acting at hSERT would interfere with the oligomeric organization
of the transporter as detected by intermolecular FRET. To answer this
question, we applied donor-bleaching FRET microscopy of CFP- and
YFP-hSERT expressing cells before and 3, 6, and 9 min after the
addition of drugs (at a concentration of about 10× IC50).
As antagonistic substances, we used citalopram, cocaine, imipramine,
and paroxetine as transporter substrates 5-HT,
methylenedioximethamphetamine ("ecstasy"), and
para-chloroamphetamine were applied. We could not detect any
significant alteration of the values and thus no change of the
intermolecular FRET, indicating that these compounds do not
considerably modify the homo-oligomerization of CFP- and YFP-hSERT
(data not shown).
In conclusion, we unambiguously found that the human serotonin
transporter forms homo-oligomers within the cellular membrane of living
cells. Moreover, a similar homo-oligomerization was found for the
structurally related rat GABA transporter 1. It is therefore attractive
to speculate that homo-oligomer formation is a property that is common
to all members of the subfamily of the
Na+/Cl-dependent neurotransmitter transporters.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Julia Zwach and Sonja Novak for excellent technical assistance, Herwig Just, Markus Klinger, and Ulrik Gether for helpful discussions, and Franz Hammerschmid for indispensable help with the FPLC analysis.
![]() |
FOOTNOTES |
---|
* This work was supported by Austrian Science Foundation Grants P-13183 (to E. A. S.), P-14509 (to H. H. S), P-13097 (to M. F.), and SFB5-12 (to J. A. S.).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: Inst. of Pharmacology, University of Vienna Medical School, Währinger Str. 13a, A-1090 Vienna, Austria. Tel.: 43-1-4277-64188; Fax: 43-1-4277-64122; E-mail: harald.sitte@univie.ac.at.
Published, JBC Papers in Press, November 8, 2000, DOI 10.1074/jbc.M007357200
2 H. Just, M. Freissmuth, and H. H. Sitte, unpublished observation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
hSERT, human
serotonin transporter;
GFP, green fluorescent protein;
FRET, fluorescence resonance energy transfer;
CFP, cyan fluorescent protein;
YFP, yellow fluorescent protein;
GABA, -aminobutyric acid;
rGAT1, rat GABA transporter 1;
5-HT, 5-hydroxytryptamine (= serotonin);
hD2R, human dopamine D2-receptor;
HEK-293, human embryonic kidney cells 293;
FPLC, fast protein liquid
chromatography.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Heldin, C. H. (1995) Cell 80, 213-223[Medline] [Order article via Infotrieve] |
2. | Gadella, T. W., Jr., and Jovin, T. M. (1995) J. Cell Biol. 129, 1543-1558[Abstract] |
3. | Jordan, B. A., and Devi, L. A. (1999) Nature 399, 697-700[CrossRef][Medline] [Order article via Infotrieve] |
4. | Overton, M. C., and Blumer, K. J. (2000) Curr. Biol. 10, 341-344[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Rocheville, M.,
Lange, D. C.,
Kumar, U.,
Sasi, R.,
Patel, R. C.,
and Patel, Y. C.
(2000)
J. Biol. Chem.
275,
7862-7869 |
6. |
Pessino, A.,
Hebert, D. N.,
Woon, C. W.,
Harrison, S. A.,
Clancy, B. M.,
Buxton, J. M.,
Carruthers, A.,
and Czech, M. P.
(1991)
J. Biol. Chem.
266,
20213-20217 |
7. |
Haugeto, O.,
Ullensvang, K.,
Levy, L. M.,
Chaudhry, F. A.,
Honore, T.,
Nielsen, M.,
Lehre, K. P.,
and Danbolt, N. C.
(1996)
J. Biol. Chem.
271,
27715-27722 |
8. | Rudnick, G. (1997) in Neurotransmitter Transporters: Structure, Function, and Regulation (Reith, M. E. A., ed) , pp. 73-100, Humana Press, Inc., Totowa, NJ |
9. | Bruns, D., Engert, F., and Lux, H. D. (1993) Neuron 10, 559-572[Medline] [Order article via Infotrieve] |
10. | Mager, S., Min, C., Henry, D. J., Chavkin, C., Hoffman, B. J., Davidson, N., and Lester, H. A. (1994) Neuron 12, 845-859[Medline] [Order article via Infotrieve] |
11. | Sitte, H. H., Huck, S., Reither, H., Boehm, S., Singer, E. A., and Pifl, C. (1998) J. Neurochem. 71, 1289-1297[Medline] [Order article via Infotrieve] |
12. | Petersen, C. I., and DeFelice, L. J. (1999) Nat. Neurosci. 2, 605-610[CrossRef][Medline] [Order article via Infotrieve] |
13. | Schloss, P., and Williams, D. C. (1998) J. Psychopharmacol. 12, 115-121[Medline] [Order article via Infotrieve] |
14. | Ramamoorthy, S., Leibach, F. H., Mahesh, V. B., and Ganapathy, V. (1993) Placenta 14, 449-461[Medline] [Order article via Infotrieve] |
15. | Jess, U., Betz, H., and Schloss, P. (1996) FEBS Lett. 394, 44-46[CrossRef][Medline] [Order article via Infotrieve] |
16. | Chang, A. S., Starnes, D. M., and Chang, S. M. (1998) Biochem. Biophys. Res. Commun. 249, 416-421[CrossRef][Medline] [Order article via Infotrieve] |
17. |
Kilic, F.,
and Rudnick, G.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
3106-3111 |
18. | Förster, T. (1948) Ann. d. Physik (Leipzig) 2, 55-75 |
19. | Pollok, B. A., and Heim, R. (1999) Trends Cell Biol. 9, 57-60[CrossRef][Medline] [Order article via Infotrieve] |
20. |
Schmid, J. A.,
Birbach, A.,
Hofer-Warbinek, R.,
Pengg, M.,
Burner, U.,
Furtmüller, P. G.,
Binder, B. R.,
and de Martin, R.
(2000)
J. Biol. Chem.
275,
17035-17042 |
21. | Sitte, H. H., Scholze, P., Schloss, P., Pifl, C., and Singer, E. A. (2000) J. Neurochem. 74, 1317-1324[Medline] [Order article via Infotrieve] |
22. | Periasamy, A., and Day, R. N. (1999) Methods Cell Biol. 58, 293-314[Medline] [Order article via Infotrieve] |
23. |
Rocheville, M.,
Lange, D. C.,
Kumar, U.,
Patel, S. C.,
Patel, R. C.,
and Patel, Y. C.
(2000)
Science
288,
154-157 |