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J. Biol. Chem., Vol. 276, Issue 31, 29012-29018, August 3, 2001
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From the
Received for publication, December 29, 2000, and in revised form, June 1, 2001
The binding affinity of the cocaine analog
[3H]2 The dopamine (DA)1
transporter (DAT) in neuronal cells clears DA from the extracellular
space (1, 2), thereby playing a pivotal role in dopaminergic
neurotransmission (3). DAT is also thought to mediate the uptake of
potentially neurotoxic agents into DA cells (4) and to be a principal
target for psychostimulant drugs such as cocaine (5-10).
A number of studies have demonstrated that sulfhydryl reagents
inhibit DA uptake by and ligand binding to DAT (11-17). Protection against modification of DAT by sulfhydryl reagents has been
predominantly interpreted as an indication of whether the reactive
cysteine residues lie inside or outside the binding domain for the
protecting ligand (13-17). Differences in the protective abilities of
various compounds have therefore been interpreted as evidence that the binding sites are not completely overlapping.
Other experimental evidence, however, also implicates conformational
changes in drug action at DAT, thereby complicating the interpretation
of these protection assays. Bonnet et al. (18), based on a
thorough thermodynamic analysis, suggested that conformational changes
occur upon binding to DAT of blockers but not substrates. Héron
et al. (19) reported that the interaction between DAT and
blockers requires more time than that for substrates, implying that the
binding of blockers involves a significant conformational change.
Similarly, Do-Rego et al. (20) observed a two-step reaction of 1-(2-(diphenylmethoxy)-ethyl)-4-(3-phenyl-2-propenyl) piperazine (GBR 12783) with DAT.
More recently, Ferrer and Javitch (21) provided evidence for
conformational changes of DAT upon exposure to cocaine. Specifically, cocaine increased the reaction of Cys-90 with charged
methanethiosulfonate (MTS) reagents, thereby augmenting the stimulatory
effect of this reaction on the binding of the cocaine analog,
[3H]2 The present work focuses on the ability of a number of drugs to alter
the reaction of endogenous cysteines in DAT with MTS ethyltrimethylammonium (MTSET), a positively charged sulfhydryl reagent
(22). To focus on the role of the various reactive cysteine residues,
we used DAT constructs in which Cys-90, Cys-135, or Cys-342 were
replaced, one at a time, into an MTSET-insensitive mutant DAT construct
(X) in which five endogenous cysteine residues had been substituted
(C90A, C135A, C306A, C319F, and C342A) (see Fig.
1) (21). The results showed intriguing
differences between the potencies with which particular drugs inhibited
ligand binding to DAT and the potencies with which they modulated the
effects of MTSET on ligand binding. The DAT inhibitors benztropine and cocaine appear to affect differently the reaction of Cys-90 and of
Cys-135, suggesting that inhibitors do not all stabilize the same
conformational state.
Materials--
[3H]WIN 35,428 (84.5-86.0 Ci/mmol)
and [3H]mazindol (23.5 Ci/mmol) were from PerkinElmer
Life Sciences. Unlabeled WIN 35,428 was from the NIDA (Research
Triangle Institute, Research Triangle Park, NC), and
N-[1-(2-benzo[b]thiophenyl)cyclohexyl]piperidine was obtained from Research Biochemicals Inc. (Natick, MA). Mazindol was
a gift from Sandoz (Basel, Switzerland), and D-amphetamine was from Smith Kline & French Laboratories. MTSET was from Toronto Research Chemicals (Toronto, Ontario, Canada). Bovine serum was from HyClone (Logan, UT). All other chemicals were from Sigma or
Fisher. Glass fiber filter mats and Betaplate Scint scintillation mixture for the binding assays were from Wallac Inc. (Gaithersburg, MD). The experiments were conducted with human wild-type (WT) and
mutant DAT stably expressed in HEK-293 cells as described previously
(21).
Treatment with Protector and MTSET and Wash-out--
Cell
membranes were prepared as described previously (14). The general
procedures for treatment with sulfhydryl reagent and wash-out were also
as described in that study, with the following modifications. Membranes
were pretreated in assay buffer (see below) with varying concentrations
of the protecting compound at 21 °C for 5 min. MTSET or vehicle was
added, and after 15 min all reagents were removed in three
centrifugation steps. For compounds that were more difficult to wash
out (benztropine and mazindol) five centrifugation steps were applied.
The wash buffer used in these steps was the assay buffer (see below)
minus tropolone. This wash in itself did not alter the pharmacological
profile of [3H]WIN 35,428 binding to DAT, with 0 and 3 washes resulting in the same affinities for cocaine, WIN 35,428, benztropine, and DA in WT DAT (23) (data not shown).The membrane
protein concentration in the MTSET treatment phase was set at ~0.6
mg/ml for the experiments with 0.3 and 1.0 mM MTSET and at
2 mg/ml for those with 10 mM MTSET.
[3H]WIN 35,428 and [3H]Mazindol
Binding--
The washed pellets obtained above were homogenized with a
Brinkmann Instruments Polytron (setting 6, 15 s) in 0.4 ml of
assay buffer: 30 mM sodium phosphate buffer resulting from
mixing primary and half-strength secondary sodium phosphate buffer to
pH 7.4 at room temperature, containing also (in mM): 122 NaCl, 5 KCl, 1.2 MgSO4, 10 glucose, 1 CaCl2,
and 0.1 tropolone. The tropolone was added to inhibit
catechol-O-methyltransferase. Aliquots of 20 µl of the
membrane suspension (25-50 µg of protein) were assayed in triplicate
for [3H]WIN 35,428 binding in a final volume of 200 µl
of assay buffer in 96-well plates using a cell harvester and liquid
scintillation counter as described previously (24). For
[3H]mazindol binding, 30 µl of suspension (40-75 µg
of protein) was assayed in a final volume of 300 µl.
In addition to the binding assays in the protection design described
above, inhibitory potencies of compounds were assessed in separate
experiments under conditions identical to those used for protection.
Inhibition curves consisted of six concentrations evenly spaced around
the IC50 value of the test compound.
For routine protection and inhibition experiments, binding assays were
conducted at 21 °C with 4 nM [3H]WIN
35,428 for 15 min or with 0.5 nM [3H]mazindol
for 10 min. Nonspecific binding was defined with 100 µM
cocaine. For saturation analysis, [3H]WIN 35,428 was
present at 2 nM, along with varying concentrations of
nonradioactive WIN 35,428 (0, 3, 10, 30, 100, 300, 1,000, 3,000, and
10,000 nM) ("varying cold" design). In saturation
experiments with [3H]mazindol, the radioligand was added
at 0.03, 0.09, 0.22, 0.75, 2.2, 7, 22, and 75 nM with or
without 100 µM cocaine for nonspecific binding
("varying hot" design).
Analysis of the Effects of MTSET on Binding--
Because
reaction of Cys-90 and of Cys-306 stimulates binding, whereas reaction
of Cys-135 and of Cys-342 inhibits binding (21), the overall ability of
a drug to reduce MTSET-induced inhibition of binding will also depend
on the effect of the drug on the MTSET-induced stimulation of binding
(see below). These effects will obviously differ in the different
mutants. Thus, we defined EC50 empirically as the
concentration of a compound that reduces the MTSET-induced inhibition
of [3H]WIN 35,428 binding by 50%. The effect of a
compound on MTSET-induced inhibition of [3H]WIN 35,428 binding was calculated as ((sample
In DAT constructs in which Cys-90 and Cys-306 were mutated and
stimulation of binding by MTSET does not occur, we quantified the
potency of a compound to protect against MTSET-induced inhibition of
binding relative to its potency to inhibit [3H]WIN 35,428 binding by EC50/IC50, which is inversely
related to the protective activity (see Refs. 14, 17, and 19). The ALLFIT equation (25) was used for calculation of IC50 and
EC50 values.
KD and Bmax were determined
by the nonlinear regression program LIGAND (27) as described previously
(28); for the "varying hot only" experiments, the specific binding
at each radioligand concentration was entered into the program. In WT upon MTSET treatment, and in C90A regardless of treatment, a low affinity binding component was observed with highly variable
KD values between 1 µM and 1 mM. The present study focuses on the high affinity
component obtained by LIGAND in two-site fits, or in one-site fits with
floating nonspecific binding, with similar results observed previously
(29).
All results are expressed as mean ± S.E. Comparisons were made by
one-way analysis of variance followed by Tukey-Kramer Multiple Comparisons Test and by one-sample Student's t test for
testing significance of protection. For comparison of
EC50/IC50 ratios, the EC50 value
for each experiment was divided by the average IC50 value
observed for the compound, grouped, and tested. Data were
log-transformed for homogeneity of variance when required. The accepted
level of significance was 0.05.
Effect of MTSET in the Presence or Absence of Various Compounds in
WT DAT--
In WT DAT, 10 mM MTSET caused approximately a
50% reduction in [3H]WIN 35,428 binding (Fig.
2). A number of DAT inhibitors and substrates were tested for their ability to reduce this effect of 10 mM MTSET. The concentrations needed for 50% reduction of MTSET-induced inhibition of binding (EC50) ranged from
nanomolar (WIN 35,428) to sub-millimolar (DA) (Table
I). For each compound, the affinity with
which it inhibited [3H]WIN 35,428 binding to DAT
(IC50) was measured under the same conditions, and in
comparison with the IC50, the EC50 was somewhat higher affinity (cocaine and WIN 35,428) or lower affinity (mazindol, benztropine, and DA).
Incubation with various DAT inhibitors (WIN 35,428, cocaine, and
mazindol) or a substrate (DA) prior to and during MTSET treatment potentiated the binding of [3H]WIN 35,428 (4 nM) above the control level observed without drug or MTSET
(Fig. 3A). In contrast,
although the substrate D-amphetamine and the inhibitor
benztropine restored binding to control values, even higher
concentrations of these compounds did not potentiate binding beyond
that of control (Fig. 3A). Potentiation of binding above
control values requires that part of the effect of reaction with MTSET
be a stimulation of binding and that exposure to DAT compounds uncovers
this stimulatory effect by protecting against the inhibitory effects of
reaction elsewhere and/or enhances the stimulatory effect. Indeed,
saturation analysis with WIN 35,428 showed that treatment of WT with
MTSET greatly reduced the Bmax (~6-fold),
which is reflected as a decrease in overall binding, but raised
the affinity of the residual binding (~5-fold increase in affinity)
(Table II). A similar 6-fold
effect on Bmax and 5-fold effect on
KD was observed when binding of
[3H]mazindol to DAT was assessed after reaction with
MTSET (data not shown).
Thus, 1 µM cocaine or 3 mM DA, in conjunction
with MTSET, increased [3H]WIN 35,428 binding ~2-fold
(Fig. 3A), through a dual mechanism. Both the binding
affinity and the number of binding sites were increased
(KD values were reduced by 29%, and
Bmax values were enhanced by 80% in the case of
cocaine and MTSET), compared with MTSET treatment alone (Table II). The
analogous effects on [3H]mazindol binding for cocaine
were an 18% reduction in KD and 62% enhancement in
Bmax values (data not shown).
Effect of MTSET in the Presence or Absence of Various Compounds in
C90A DAT--
Because Cys-90 has been implicated in the stimulatory
effects of MTSET on binding (21), the C90A mutant was examined for a
loss of stimulation of binding upon MTSET treatment. MTSET at 10 mM caused approximately a 70% reduction in
[3H]WIN 35,428 binding, greater than the inhibition seen
in WT (Fig. 2). The potentiation seen in WT with various compounds upon
co-incubation with 10 mM MTSET was not observed in C90A,
even at concentrations at least 10 times the C90A EC50
values (Fig. 3B and Table I). The various compounds still
reversed the MTSET-induced inhibition of binding (Fig. 3B)
but less potently than in WT, due to the absence of the Cys-90-induced
stimulation, which affects the entire curve in WT. Even so, the rank
order of potency in reducing the MTSET-induced binding inhibition among
compounds was similar to that observed in WT: WIN 35,428 > cocaine > benztropine, DA (Table I). Consistent with these
observations with [3H]WIN 35,428 as the radioligand,
cocaine also increased the binding of [3H]mazindol above
control in WT but not in C90A, and cocaine displayed a greatly
increased EC50 in C90A (data not shown).
C90A still contains Cys-306, which also reacts with MTSET to stimulate
binding. Indeed, MTSET reduced the KD value of
[3H]WIN 35,428 binding in C90A, but the magnitude of the
effect was smaller (~3-fold) than in WT (~5-fold) (Table II).
Similar to WT, the Bmax value of C90A was
reduced ~5-fold by MTSET. The analogous effects on
[3H]mazindol binding in C90A were a ~2-fold reduction
in KD and a 7-fold reduction in
Bmax values (data not shown). Cocaine (200 µM) and dopamine (3 mM) before and during
reaction with MTSET did not further decrease the KD
value of [3H]WIN 35,428 binding, compared with MTSET
alone, consonant with the lack of enhancement of reaction of Cys-306,
but did increase the Bmax value by ~100%
(Table II), reflecting protection against inhibition. A comparable lack
of effect on KD and a 262% increase in
Bmax values, respectively, were observed for
cocaine on [3H]mazindol binding (data not shown).
Effect of MTSET in the Presence or Absence of Various Compounds in
X-A342C DAT--
In the following experiments, we used a background
DAT construct with five cysteines removed (X), which in itself is not
reactive with the concentrations of MTSET used here (21) (see
Introduction). X-A342C, in which Cys-342 has been substituted back into
its original position, reacted with 0.3 mM MTSET to inhibit
[3H]WIN 35,428 binding by 60% (Fig. 3A). In
contrast to WT, no potentiation of binding was observed in the presence
of any of the inhibitors or substrates (Fig.
4A), consistent with the lack
of Cys-90 and Cys-306 in this DAT construct (21). Thus, the effect of
the compounds was simply to protect Cys-342 from reaction. The
protective ratios (EC50/IC50) ranged from 7.4 for cocaine to 70 for DA (Table III).
Most significantly, benztropine had a protective ratio of 32, only
about 4-fold less than that for cocaine (Table III).
In X-342C KD values for WIN 35,428 with or without
MTSET were not significantly different (Table
IV). Cocaine (200 µM) or DA
(3 mM) also had no effect on the KD but
restored the Bmax toward control values (Table
IV).
Effect of MTSET in the Presence or Absence of Various Compounds in
X-A135C DAT--
X-A135C, in which Cys-135 has been substituted into
its original position, reacted with 1 mM MTSET to inhibit
[3H]WIN 35,428 binding by 50% (Fig. 3A). As
in X-A342C, no potentiation of binding was observed by any of the
compounds (Fig. 4B). The protective ratios
(EC50/IC50) ranged from 6.3 for WIN 35,428 to >1000 for benztropine (Table III). Remarkably, benztropine did not
show protection at concentrations as high as 300 µM
(Table III and Fig. 4B). In comparison with X-A342C, the
protective ratios in X-A135C were similar for WIN 35,428 and cocaine,
somewhat lower for mazindol and DA, and much higher for benztropine.
Effect of MTSET in the Presence or Absence of Various Compounds in
X-A90C DAT--
In contrast to the other mutant constructs, X-A90C
displayed increases in [3H]WIN 35,428 binding upon
exposure to MTSET (Fig. 2). Cocaine (1 µM), WIN 35,428 (0.6 µM), mazindol (10 µM), and DA (3 mM) significantly augmented the stimulation of binding seen
in the presence of 10 mM MTSET (Fig.
5) In contrast, benztropine (100 µM) was completely ineffective (Fig. 5).
The present results emphasize the complexity encountered in
experiments in which compounds are used to protect against the reaction
of a sulfhydryl reagent, particularly when reaction is assessed through
an effect on function. In WT DAT, the effects of MTSET (10 mM) on [3H]WIN 35,428 binding were clearly a
mixture of inhibition and stimulation. With increasing concentrations
of protecting ligand, the stimulation by MTSET became more evident,
thereby resulting in binding as much as 400% of control values in the
absence of MTSET. Thus, after MTSET treatment of WT there was a large
loss of binding sites (for either [3H]WIN 35,428 or
[3H]mazindol), but the residual sites had a higher
affinity than the control preparation. Thus we propose that reaction of
MTSET with Cys-135 and/or Cys-342 blocks high affinity binding, whereas in the fraction of transporters in which Cys-135 and Cys-342 have not
reacted, binding can be potentiated by the reaction of MTSET with
Cys-90 and/or Cys-306. Cocaine or DA increases binding by protecting
Cys-135 and Cys-342 from reaction and also by increasing the reaction
of Cys-90, thereby resulting in "protection." Consistent with this
interpretation, cocaine further lowered the KD in
WT, but not in C90A, for both [3H]WIN 35,428 (Table II)
and [3H]mazindol binding (data not shown). Evidence for
such an enhanced reaction of Cys-90 in X-A90C was seen with all
compounds tested except benztropine (Fig. 5, see below).
C90A displayed 3-4-fold lower affinity than WT for benztropine. This
effect must be indirect, however, because benztropine did not protect
Cys-90 from reaction with MTSET. Moreover, because the tested compounds
(except benztropine) enhance the accessibility of Cys-90 to MTSET,
these effects must result from conformational changes that expose
and/or increase the reactivity of Cys-90 and not from a direct effect.
Benztropine was able to protect Cys-342 but not Cys-135 even at
concentrations 1000-fold greater than its IC50. The results taken together suggest that benztropine is weak in "protecting" binding in WT from MTSET because (i) it does not enhance reaction of
MTSET with Cys-90 to further stimulate binding, and (ii) it cannot
protect Cys-135 from inhibition of binding. This also explains why
benztropine was very weak and/or unable to protect C90A, which contains
Cys-135. Under the conditions of the present experiments with WT and
C90A, full protection of inhibitory Cys-342 and Cys-135 was not
achieved, as evidenced by the only partial restoration of the
Bmax values by cocaine or DA pretreatment
(Tables III and IV). This also explains why C90A, despite the presence
of a stimulatory cysteine (Cys-306), did not show net potentiation of
binding after pretreatment with cocaine, DA, or other compounds (Fig.
4B).
Several observations argue against the possibility of affinity or
off-rate, playing a role in the protection of Cys-342 and Cys-135 by
compounds. Cocaine and benztropine, despite their similar affinities in
competition with ligand binding to DAT, displayed dramatically
different protective indices in X-A342C and X-A135C. The same applies
to WIN 35,428 and mazindol in X-A342C, although the difference was
smaller. In addition, DA had a much lower apparent affinity than
mazindol, but these compounds showed relatively similar protective ratios.
There have been previous reports on drug interactions with DAT that
suggest complexities beyond simple competition for a common or shared
binding domain. Thus, although mazindol and the cocaine analog WIN
35,428 inhibited each other's binding in cells expressing the human
DAT in a competitive fashion quantitatively in agreement with their
affinity (28), Schenk and co-workers (30) found evidence for linked
sites where mazindol and cocaine interact to inhibit DA uptake into rat
striatal tissue, with the relationship between these sites conforming
to a negative allosteric or hyperbolic competitive model (see Ref. 28).
In addition, Bonnet et al. (18) found that binding of
inhibitors, but not substrates, was associated with a substantial
reduction in entropy and inferred that binding of blockers must result
in conformational changes in DAT (18, 19). Curiously, in the study of
Bonnet et al. (18) the apparent affinity of benztropine
exhibited the lowest dependence on temperature of all the inhibitors
tested, which included amineptine, mazindol, nomifensine, pyrovalerone,
methylphenidate, GBR 12783, GBR 12909, cocaine, and benztropine. Thus
the ratio between apparent KI values at 37 and
0 °C was 1.8 for substrates such as DA and amphetamine, 2.3 for
benztropine, and 5.3-33 for the other inhibitors. Kinetic (20) and
protection (19) approaches by the same group also supported
conformational changes in the binding of inhibitors. Along similar
lines, a recent study inferred conformational motions of transmembrane
domains of DAT from synergistic or antagonistic interactions between
the effect of mutations in different transmembrane domains on Gibbs free energy changes for the binding of DA or WIN 35,428 (31).
Cys-342 and Cys-135 are in the 3rd and 1st intracellular loops,
respectively (Fig. 1). Intracellular loops are not generally thought to
participate in forming the binding site of ligands in neurotransmitter
transporters, but this impression is based more on analogy to
G-protein-coupled receptors that bind ligand in the transmembrane
domain than on actual data. Thus, although the protective effect of
compounds against reaction of Cys-342 and Cys-135 with MTSET most
likely results from conformational changes that reduce exposure and/or
reactivity of the cysteines to MTSET, it is also possible that one or
both of these loops physically participate in forming the binding site.
Since both cocaine and benztropine protect Cys-342 from reaction with
MTSET, these compounds either produce the same conformational change at
this position or both bind near this region and protect sterically. In
contrast, benztropine does not protect Cys-135, suggesting either that
it does not produce a conformational change at this position or that it
binds in a different orientation than cocaine and is unable to protect directly.
Transporter proteins can be conceptualized as containing a channel-like
lumen flanked by extracellular and intracellular gates that open and
close sequentially in the translocation process (32). Residues that are
part of binding domains for substrates or blockers will be occluded
upon binding of substrate or blocker and therefore be protected from
reaction with MTSET. In contrast, gating residues could be extremely
sensitive to conformational changes during substrate transport with
gates moving in and out of regions of the plasma membrane sequestered
from the medium (33). In the case of DAT, Cys-90 could be considered a
candidate for being associated with an extracellular gate, and Cys-342
and Cys-135 with an intracellular gate, but the membrane preparation used in the current experiments does not allow observations of phenomena linked to substrate translocation as described by Chen et al. (34) for Cys-342.
These present results underscore the usefulness of MTS reagents for
detecting cysteine residues in conformationally sensitive regions of
biogenic amine transporter proteins (33, 34). We observed a diversity
in the ability of compounds to induce conformational changes in DAT
that could not have been predicted merely on the basis of their
apparent affinity for DAT. Thus, the rank order for protection was
quite different for different ligands in different DAT constructs,
owing to the different effects of the ligands on the reaction of the
various endogenous cysteines with MTSET. The most striking difference
was the inability of benztropine to protect Cys-135 from reaction or to
enhance the reaction of Cys-90. It is noteworthy that previous studies
have suggested the existence of different modes of binding for
benztropine and cocaine. These compounds are likely to be positioned in
a binding site in a different way, based on structure-activity studies
showing that the difference between R- and
S-enantiomers observed in analogs of cocaine was reversed in
fluorinated analogs of 2-carbomethoxy-benztropine (35). Vaughan
et al. (36) observed incorporation of a benztropine-based photoaffinity ligand into a DAT domain encompassing transmembrane regions 1 and 2, as opposed to incorporation of a cocaine-based ligand
into a domain containing transmembrane helices 4-7, again suggesting
that the orientation of the compounds in the binding site may differ
(35). Moreover, there are differences in the psychomotor stimulant-like
effects of cocaine and benztropine in rodents (37) and
self-administration of these compounds in rhesus monkeys (38). The
disparity in the latter two tests were shown to be unrelated to the
anticholinergic activity of benztropine (37, 38), and it is conceivable
that the different behavioral effects stem from a different molecular
action on DAT.
*
This work was supported by National Institute on Drug Abuse
Grants DA 08379 (to M. E. A. R.), MH 57324, and DA 11495 (to
J. A. J.).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 Biomedical and
Therapeutic Sciences, College of Medicine University of Illinois, Box
1649, Peoria, IL 61656. Tel.: 309-671-8545; Fax: 309-671-8403; E-mail:
MaartenR@uic.edu.
Published, JBC Papers in Press, June 6, 2001, DOI 10.1074/jbc.M011785200
The abbreviations used are:
DA, dopamine;
DAT, DA transporter;
GBR 12783, 1-(2-(diphenylmethoxy)-ethyl)-4-(3-phenyl-2-propenyl) piperazine;
MTSET, methanethiosulfonate ethyltrimethylammonium;
WIN 35, 428,
2
The Uptake Inhibitors Cocaine and Benztropine Differentially
Alter the Conformation of the Human Dopamine Transporter*
§,,, Department of Biomedical and Therapeutic
Sciences, University of Illinois College of Medicine,
Peoria, Illinois 61656 and ¶ Center for Molecular Recognition
and Departments of Pharmacology and Psychiatry, College of Physicians
and Surgeons, Columbia University, New York, New York 10032
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-carbomethoxy-3-(4-fluorophenyl) tropane
(WIN) for the dopamine transporter (DAT) is increased by the reaction
of Cys-90, at the extracellular end of the first transmembrane segment,
with methanethiosulfonate (MTS) reagents. Cocaine enhances the reaction
of Cys-90 with the sulfhydryl reagents, thereby augmenting the increase
in binding. In contrast, cocaine decreases the reaction of Cys-135 and
Cys-342, endogenous cysteines in cytoplasmic loops, with MTS reagents. Because this reaction inhibits [3H]WIN binding, cocaine
protects against the loss of binding caused by reaction of these
cysteines. In the present work, we compare the abilities of DAT
inhibitors and substrates to affect the reaction of Cys-90, Cys-135,
and Cys-342 with MTS ethyltrimethylammonium (MTSET). The results
indicate that the different abilities of compounds to protect against
the MTSET-induced inhibition of binding are attributable to
differences in their abilities to attenuate the inhibitory effects of
modification of Cys-135 and Cys-342 as well as to enhance the reaction
with Cys-90 and the resulting potentiation of binding. The inhibitor
benztropine was unique in its inability to protect Cys-135. Moreover,
whereas cocaine, WIN, mazindol, and dopamine enhanced the reaction of
Cys-90 with MTSET, benztropine had no effect on this reaction. These
two features combine to give benztropine its weak potency in protecting
ligand binding to wild-type DAT from MTSET. These results indicate that different inhibitors of DAT, such as cocaine and benztropine, produce
different conformational changes in the transporter. There are
differences in the psychomotor stimulant-like effects of these compounds, and it is possible that the different behavioral effects of
these DAT inhibitors stem from their different molecular actions on
DAT.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-carbomethoxy-3-(4-fluorophenyl) tropane (WIN
35,428). In contrast, cocaine decreased the reaction of Cys-135 and
Cys-342 with MTS reagents, a reaction that inhibited
[3H]WIN 35,428 binding. This divergence in the effects of
cocaine suggested that differences in the protective abilities of
various compounds against the modification of DAT by sulfhydryl
reagents relate to differences in the ability of these compounds to
alter the conformation of DAT and hence the reactivities of the various endogenous cysteine residues.
View larger version (22K):
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Fig. 1.
Hydrophobicity-based model of DAT
protein structure with 12 transmembrane domains. Five of the 13 cysteines present in human DAT (indicated as circles with
position numbers) have been removed to give the
MTSET-insensitive DAT construct X (Ferrer and Javitch (21)). X is the
background for placing back cysteine residues, one by one, to yield
X-A90C, X-A135C, and X-A342C.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mt)/(total mt)) × 100, where sample is the binding after MTSET
treatment in the presence of compound, total is the binding in the
absence of MTSET/compound, and mt is the binding after MTSET
treatment in the absence of compound. In a small number of cases,
correction for residual, incompletely washed out compound was
applied at the highest concentration point as described previously
(26). Compounds that together with MTSET increased binding (sample) over that observed in the control without MTSET or compound (total) give values greater than 100%.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (17K):
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Fig. 2.
Concentration-dependent effect of
MTSET on [3H]WIN 35,428 binding. As described under
"Experimental Procedures," DAT membranes were prepared and exposed
to MTSET at ~2 mg of membrane protein/ml. After wash-out, residual
[3H]WIN 35,428 binding was measured. DAT clones were as
described by Ferrer and Javitch (21). Points shown are from a typical
experiment, assayed in triplicate, that was repeated twice with similar
results.
Potency of compounds in reducing MTSET-induced inhibition of
[3H]WIN 35,428-binding sites (EC50) and in inhibiting
[3H]WIN 35,428 (4 nM) binding (IC50) in
WT and C90A DAT
View larger version (32K):
[in a new window]
Fig. 3.
Concentration-dependent effects
of drugs in reducing MTSET-induced inhibition of [3H]WIN
35,428 binding in WT and C90A DAT. As described under
"Experimental Procedures" DAT was exposed to drug and 10 mM MTSET at ~2 mg of membrane protein/ml. After
wash-out, residual [3H]WIN 35,428 (4 nM)
binding was measured. Experimental data are the means ± S.E.
(vertical bar shown where greater than the symbol itself) of
3-5 independent experiments assayed in sextuplicate. The
dotted-dashed line indicates a 100% effect, i.e.
restoration of binding to the control value observed without MTSET. *
indicates p < 0.05 for the value being greater than
100% (one-sample Student's t test), representing
potentiation of binding.
Effect of MTSET with or without pretreatment with cocaine (Coc) or DA
on high affinity [3H]WIN 35,428 binding in WT and C90A DAT
View larger version (28K):
[in a new window]
Fig. 4.
Concentration-dependent effects
of drugs in reducing MTSET-induced inhibition of [3H]WIN
35,428 binding in X-A342C and X-A135C DAT. As described under
"Experimental Procedures" DAT was exposed to drug and 0.3 (X-A342C)
or 1.0 (X-A135C) mM MTSET at ~0.6 mg of membrane
protein/ml. After wash-out, residual [3H]WIN 35,428 (4 nM) binding was measured. Experimental data are the
means ± S.E. (vertical bar shown where greater than
the symbol itself) of 3-5 independent experiments assayed in
sextuplicate. The dotted-dashed line indicates a 100%
effect, i.e. restoration of binding to the control value
observed without MTSET.
Potency of compounds in reducing MTSET-induced inhibition of
[3H]WIN 35,428 binding sites (EC50) and in inhibiting
[3H]WIN 35,428 (4 nM) binding (IC50) in
X-A342C and X-A135C DAT
Effect of MTSET with or without pretreatment with cocaine (Coc) or DA
on high affinity [3H]WIN 35,428 binding in X-A342C DAT
View larger version (18K):
[in a new window]
Fig. 5.
Effect of MTSET on [3H]WIN
35,428 binding to X-A90C DAT after pre-exposure to compounds. As
described under "Experimental Procedures," DAT was exposed to MTSET
(10 mM) with or without drug at ~2 mg of membrane
protein/ml. After wash-out, residual [3H]WIN 35,428 (4 nM) binding was measured. Experimental data are the
means ± S.E. (vertical bar) of n (between
parentheses following drug notation) independent experiments
assayed in sextuplicate. The results are expressed as % over the
effect of MTSET alone. * indicates p < 0.05 for the
value being greater than 0% (one-sample Student's t
test).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
FOOTNOTES
ABBREVIATIONS
-carbomethoxy-3-(4-fluorophenyl) tropane;
WT, wild-type;
MTS, methanethiosulfonate.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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