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The Journal of Neuroscience, June 1, 2002, 22(11):4399-4405
Peroxynitrite Inactivates the Human Dopamine Transporter by
Modification of Cysteine 342: Potential Mechanism of Neurotoxicity in
Dopamine Neurons
Samuel U.
Park1,
Jasmine V.
Ferrer4, 5,
Jonathan A.
Javitch4, 5, and
Donald M.
Kuhn1, 2, 3
1 Department of Psychiatry and Behavioral
Neurosciences, 2 Center for Molecular Medicine and
Genetics, Wayne State University School of Medicine, and
3 The John D. Dingell Veterans Affairs Medical
Center, Detroit, Michigan 48201, and 4 Departments of
Pharmacology and Psychiatry and 5 Center for Molecular
Recognition, Columbia University College of Physicians and Surgeons,
New York, New York 10032
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ABSTRACT |
Peroxynitrite (ONOO ) has been
implicated as a causative factor in dopamine neuronal damage resulting
from exposure to methamphetamine and
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and it may be involved in the etiology of Parkinson's Disease.
ONOO causes a concentration-dependent and
irreversible reduction in dopamine uptake by EM4 cells
stably expressing the human dopamine transporter (hDAT). The effect of
ONOO is manifested as a reduction in
Vmax. Cysteine, dithiothreitol, glutathione,
and N-acetyl-cysteine, reagents that interact directly with ONOO, prevent this inhibition, whereas a
scavenger of hydroxyl radical (dimethylsulfoxide), hydrogen peroxide
(catalase), and superoxide (superoxide dismutase) did not. Dopamine in
the extracellular medium protects the hDAT from
ONOO, whereas intracellular dopamine does not.
Parachloromercuribenzoic acid and 2-aminoethyl
methanethiosulfonate (MTSEA), which share with
ONOO the ability to modify cysteine sulfhydryls,
also inhibit hDAT function. ONOO treatment lowers
cysteine-specific labeling of the hDAT by MTSEA-biotin, suggesting that
ONOO reacts with one or more cysteines in hDAT. A
mutant of hDAT (X7C) in which all intracellular and extracellular loop
cysteines were mutated was resistant to inhibition by
ONOO. Sensitivity to ONOO was
restored in mutants of hDAT in which reduced cysteines were present
only in the first (C135) and third (C342) intracellular loops (CD-DAT),
or in which C342 alone had been reintroduced into X7C (X7C-M342C).
These results indicate that the hDAT is inhibited by
ONOO through oxidation of cysteine 342. Our
studies also substantiate the possibility that drugs known to decrease
DAT function in vivo (e.g., methamphetamine and MPTP)
may exert their effects through ONOO-mediated
oxidative stress.
Key words:
dopamine transporter; peroxynitrite; cysteine
residues; dopamine; Parkinson's disease; neurotoxic amphetamines
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INTRODUCTION |
The dopamine transporter (DAT) is an
integral membrane protein in dopamine (DA) nerve endings where it
subserves the critical function of terminating the synaptic activity of
DA through transport into the presynaptic process (Amara et al., 1998 ;
Chen and Reith, 2000). Drugs or pathological conditions that disrupt
the function of the DAT could have profound effects on behaviors and
physiological processes that are mediated by DA (Jaber et al., 1997).
Methamphetamine (Cubells et al., 1994; Sulzer et al., 1995) and MPP+
(Javitch and Snyder, 1984; Javitch et al., 1985; Gainetdinov et al.,
1997) are substrates for inward transport by the DAT. Once inside the DA nerve terminal, these drugs displace DA from storage vesicles into
the cytoplasm and eventually into the synapse through reverse outward
transport (Cubells et al., 1994; Sulzer et al., 1995; Lotharius and
O'Malley, 2000). In vitro studies have shown that reactive
oxygen species (Fleckenstein et al., 1997b; Hanson et al., 1998;
Haughey et al., 1999) have reversible inhibitory effects on the DAT
that simulate closely the in vivo effects of methamphetamine on it (Haughey et al., 2000; Sandoval et al., 2001), leading to the
conclusion that DAT function is acutely responsive to oxidative insult.
Methamphetamine (Davidson et al., 2001) and
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Blum
et al., 2001; Schmidt and Ferger, 2001) are known to exert toxic
effects on DA neurons, including persistent reductions in DAT function.
Nitric oxide and superoxide have been implicated in the damaging
effects of these drugs on DA neurons (Cadet et al., 1994; Schulz et
al., 1995; Di Monte et al., 1996; Przedborski et al., 1996; Przedborski and Jackson-Lewis, 1998; Itzhak et al., 1999). However, the coincident production of nitric oxide and superoxide almost certainly results in
the production of peroxynitrite (ONOO)
(Koppenol et al., 1992). ONOO is a
powerful oxidant that can modify proteins, nucleotides, lipids, and
cell organelles, properties that are thought to underlie its cytotoxic
potential (Beckman and Koppenol, 1996). Perhaps the best known
posttranslational modification of proteins caused by
ONOO is the nitration of tyrosine
residues (Ischiropoulos and al-Mehdi, 1995; Crow and Ischiropoulos,
1996). The appearance in brain of nitrotyrosine immunoreactivity after
administration of methamphetamine (Imam et al., 1999; Imam and Ali,
2001) or MPTP (Ferrante et al., 1999; Pennathur et al., 1999) suggests
that these drugs cause the production of
ONOO.
In light of evidence correlating ONOO
generation with drug-induced reductions in DAT function and
neurotoxicity, we hypothesized that ONOO
might inactivate DAT. The DAT contains a total of 13 cysteine residues
(Giros et al., 1991, 1992) that are arrayed throughout its
transmembrane domains, and extracellular and intracellular loops, and
these cysteines are potential targets for
ONOO reaction. The present studies use
EM4 cells stably expressing the wild-type hDAT and selected cysteine
mutants to establish that DA transport is inhibited by
ONOO via its action on cysteine 342 in
the third cytoplasmic loop of the hDAT.
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MATERIALS AND METHODS |
Materials. The following materials were obtained from
Sigma (St. Louis, MO): DA, N-acetyl-DA, dimethylsulfoxide
(DMSO), cocaine, dithiothreitol (DTT), glutathione (GSH), cysteine,
H2O2, sodium periodate,
p-chloromercuribenzoic acid (pCMB),
N-acetylcysteine, superoxide dismutase (SOD), anti-Flag
monoclonal antibodies (M2), and Triton X-100. Catalase was a product of
Boehringer Mannheim (Indianapolis, IN), and a monoclonal antibody
against nitrotyrosine was from Cayman Chemical (Ann Arbor, MI).
3,4-[7-3H]dihydroxy-phenylethylamine
([3H]DA) (31.6 Ci/mmol) was obtained
from NEN Life Science Products (Boston, MA). 2-aminoethyl
methanethiosulfonate hydrobromide (MTSEA), [2-(trimethylammonium)ethyl] methanethiosulfonate bromide
(MTSET), and MTSEA-biotin were obtained from Toronto Research Chemicals Inc. (Toronto, Canada). Nomifensine and
2- -carbomethoxy-3--(4-fluorophenyl) tropane (-CFT) were
obtained from Research Biochemicals International (Natick, MA). Fetal
bovine serum was purchased from HyClone (Logan, UT), and all cell
culture media was from Invitrogen (Carlsbad, CA). Lactate
dehydrogenase (LDH) assay kits were purchased from Promega (Madison,
WI). Ultralink immobilized neutravidin plus agarose was from Pierce
Endogen (Rockford, IL), and PVDF membranes were from Bio-Rad
Laboratories (Hercules, CA).
Site-directed mutagenesis and stable transfection of hDAT.
EM4 cells, which are HEK 293 cells stably transfected with the macrophage embryonic receptor to increase their adherence to tissue culture plastic (Robbins and Horlick, 1998), were used as host cells
for stable expression of hDAT (Ferrer and Javitch, 1998; Chen et al.,
2000; Hastrup et al., 2001). All forms of hDAT used presently were
tagged at the N terminus with the tandem Flag-HA epitope (18 amino
acids) as previously described (Hastrup et al., 2001). Introduction of
this epitope tag removed the first 22 amino acids of the hDAT from the
amino terminus of all constructs, including C6, and did not
significantly affect expression or transport (Saunders et al., 2000;
Hastrup et al., 2001). In addition to wild-type hDAT, the following
constructs, with mutations specified, were used: X7C (C90A, C135A,
C306A, C319M, C342M, C581L); cysteine-depleted (CD)-DAT (C90A, C243A,
C306A, C319M, C463S, C523S, C530A, C581L); and X7C-M342C (C342
reintroduced into X7C). In descriptive terms, X7C lacks cysteines in
all of its putative intracellular and extracellular loops and in the
sixth transmembrane domain (C319); CD-DAT lacks all putative
extracellular and transmembrane free sulfhydryls, and contains
disulfide-bridged C180-C189, C135 in the first intracellular loop and
C342 in the third intracellular loop; and X7C-M342C is X7C (above) into
which C342 has been re-introduced. Stably transfected cells were grown
in DMEM supplemented with 10% fetal bovine serum (HyClone) at 37°C
and 5% CO2.
Treatment of DAT with ONOO and
cysteine sulfhydryl reagents. Cells were grown to 90% confluence
(~0.8 × 106 cells) on 24 well
plates. Media was removed, and cells were washed three times with
Krebs'-Ringer's-HEPES (KRH) containing (in mM): 150 NaCl, 4.7 KCl, 2.2 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 10 HEPES, and 10 glucose, pH 7.4. ONOO was synthesized by
the quenched-flow method of Beckman et al. (1994), and its
concentration was determined by the extinction coefficient
 302 = 1670 M/cm-1. The hydrogen
peroxide contamination of ONOO was
removed by manganese dioxide chromatography and filtration (Beckman et
al., 1994). ONOO was added to
hDAT-expressing cells with rapid mixing and cells were incubated at
25°C for 10 min. Cells were washed three times with KRH, and uptake
was assessed as described below. Concentrated solutions of
ONOO were allowed to decompose at room
temperature in phosphate buffer at pH 7.4 (confirmed
spectrophotometrically) before addition to cells and this reverse-order
addition served as a control for the
ONOO solvents. LDH activity was measured
in cells and culture media in some experiments to ensure that
ONOO was not causing cell lysis. For
experiments aimed at assessing the specificity of
ONOO actions on the hDAT, antioxidants
and/or reducing agents (cysteine, DTT, GSH, and
N-acetyl-cysteine) or scavengers of reactive oxygen species
(DMSO, catalase, and SOD) were added 5 min before
ONOO.
The ability of DA to modulate the effects of
ONOO on hDAT was assessed by two
different methods. First, hDAT-expressing cells were incubated with
various concentrations of DA (10-100 µM) for 10 min at
37°C after which they were washed and exposed to
ONOO. Second, cells were incubated with
DA as described above, but were exposed to
ONOO without washing. These experiments
were designed to determine the ability of intracellular or
extracellular DA, respectively, to modulate the effects of
ONOO on hDAT function. Treatments with
cysteine sulfhydryl reagents (pCMB, MTSEA, MTSET) or dopamine quinone
were performed as described for ONOO.
Stable o-quinones were formed by oxidizing DA or
N-acetyl-DA with one equivalent of
NaIO4 immediately before addition to cells (Graham, 1978; Graham et al., 1978).
[3H] DA uptake. Cells were
incubated with 3 µM DA containing 25 nM [3H] DA for 2 min at room temperature. Nonspecific uptake was defined in the presence
of 10 µM nomifensine and was typically ~5%
of total uptake. Cells were washed and solubilized in 1% Triton X-100, and tritium was counted by liquid scintillation spectrometry. Kinetic
analysis of hDAT (control and
ONOO-treated) was performed by
incubating cells with increasing concentrations of unlabeled DA and a
constant concentration of [3H] DA.
Apparent Vmax and
Km values were estimated using
nonlinear regression curve fitting with a one-site binding equation
(GraphPad Prism).
MTSEA-biotin labeling of hDAT. The wild-type hDAT used in
the present studies has a Flag-HA epitope-tag (18 amino acids)
replacing the first 22 amino acids of the N terminus (Saunders et al.,
2000; Hastrup et al., 2001), and this tag was used for immunoblotting. MSTEA-biotin was used to label cysteine residues in hDAT as previously described (Daniels and Amara, 1998; Javitch, 1998), after treatment of
intact cells with ONOO or other
sulfhydryl reagents. Reductions in MTSEA-biotin labeling of hDAT caused
by ONOO serves as a nonquantitative
index of cysteine modification. Intact cells were treated
with ONOO or other
sulfhydryl reagents and washed three times with KRH. Cells were
homogenized in PBS, and washed membranes were solubilized in 1%
Triton X-100. Biotinylated proteins were isolated from solubilized membranes by adsorption to neutravidin agarose for 60 min at room temperature, and after three washes, agarose beads were eluted with an
SDS-stop solution. Proteins were resolved by SDS-PAGE (Laemmli, 1970),
blotted to PVDF membranes, and probed with anti-Flag M2 antibodies (to
measure hDAT). In some experiments, membranes from
ONOO-treated cells were
immunoprecipitated with an antibody against nitrotyrosine
(MacMillan-Crow and Thompson, 1999) and subsequently probed with
anti-Flag antibodies to determine if the hDAT had been
tyrosine-nitrated.
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RESULTS |
Effects of ONOO on hDAT function
Exposure of intact EM4 cells stably expressing the hDAT to
ONOO for 10 min caused a
concentration-dependent decrease in DA uptake, as shown in Figure
1. DAT function was reduced by ~10% at
a concentration of 0.5 mM, and concentrations of 1.0-1.5
mM reduced DA uptake by 50-90%, respectively. At 2.0 mM ONOO, hDAT function was
completely inhibited. If ONOO was
allowed to decompose before addition to cells, this reverse order
addition at a reagent concentration of 2 mM did not inhibit DA uptake. The overall effect of ONOO
concentration on DAT function was significant (p < 0.01; ANOVA). The inhibitory effects of 1-2
mM ONOO were also
significant (p < 0.05; Bonferroni's test).
ONOO did not cause lysis of cells as
measured by LDH release into the cell culture media (data not shown).
If cells were preloaded with 3H-DA and
then exposed to ONOO, we did not observe
a release of DA (data not shown). The kinetic parameters of DA uptake
were determined after exposure to 1 mM ONOO and revealed a reduction in
Vmax from 22.9 pmol/min per well to
10.5 pmol/min per well (46% control; p < 0.05, Bonferroni's test). The apparent Km
for DA uptake was not significantly altered by 1 mM ONOO (1.35 µM for control compared with 1.25 µM after
ONOO).
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Figure 1.
Effects of ONOO on hDAT
activity. Intact EM4 (~0.8 × 106) cells
stably expressing wild-type hDAT were exposed to the indicated
concentrations of ONOO for 10 min at 25°C in 24 well tissue culture plates. Cells were washed 3× with KRH, and the
uptake of 3H-DA was determined immediately as described in
Materials and Methods. The open circle above the 2 mM ONOO concentration marker
represents the effects of decomposed ONOO on DA
uptake. Data represent means ± SEM for four experiments run in
triplicate. The effect of ONOO on hDAT activity is
reported as percentage of control (control levels of DA uptake were
13.5 pmol/min per well). The overall effect of
ONOO on DA uptake was significant
(p < 0.01; ANOVA). Concentrations of
ONOO of 1 mM and higher were also
significantly different from control levels of DA uptake
(*p < 0.05; Bonferroni's test).
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Specificity of the effect of ONOO on hDAT
Reagents known to react directly with
ONOO, including cysteine, DTT, GSH, and
N-acetyl-cysteine (Radi et al., 1991; Halliwell et al.,
1999), provided almost complete protection against the inhibitory
effects of ONOO on hDAT. Figure
2A shows that 1 mM concentrations of each reactant, with the
exception of GSH, reduced the effect of
ONOO from a 50% inhibition to ~20%
inhibition. GSH provided complete protection against
ONOO. These reagents did not alter hDAT
activity in the absence of ONOO.
Attempts to reverse the effect of ONOO
on hDAT with these same reagents were not successful (data not shown).
Figure 2B shows that a scavenger of hydroxyl radical
(DMSO), hydrogen peroxide (catalase), or superoxide (SOD), was
ineffective in preventing ONOO-induced
inhibition of hDAT. DMSO, catalase, and SOD were devoid of effects on
hDAT activity in the absence of ONOO
(data not shown).
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Figure 2.
Effects of antioxidants and radical scavengers on
ONOO-induced inhibition of the hDAT. Intact EM4
cells expressing wild-type hDAT were exposed to
ONOO (1 mM) for 10 min at 25°C. The
indicated reagents were added to cells 5 min before
ONOO and remained present during treatment.
A shows the effects of antioxidants and reducing agents
(1 mM concentrations for all reagents) on the
ONOO-induced inhibition of hDAT activity.
B shows the effects of a scavenger of hydroxyl radical
(DMSO, 50 mM), hydrogen peroxide (catalase [Cat], 10 U/ml), and superoxide (SOD, 10 U/ml) on the
ONOO-induced (1 mM) inhibition of the
hDAT. The results represent mean ± SEM of four or five
experiments run in triplicate for each condition. *Indicates that the
effects of that treatment were significantly different from
ONOO alone (p < 0.05;
Bonferroni's test).
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Effects of DA on ONOO-induced inhibition
of hDAT
The hDAT is known to undergo a substrate-induced conformational
change that increases the accessibility of cysteine 342 to sulfhydryl
reagents (Chen et al., 2000). Therefore, we tested the effects of
extracellular and intracellular DA on
ONOO-induced inactivation of hDAT.
Figure 3 shows that extracellular DA
provided almost complete protection of hDAT from inactivation by
ONOO. Concentrations of DA from 50-100
µM reduced the inhibitory effect of 1 mM
ONOO from 52% to 10-15%. By contrast,
when cytoplasmic DA levels were increased via hDAT-mediated uptake, and
extracellular DA was removed by rapid washing just before the addition
of ONOO, the effect of
ONOO was only slightly altered. Thus,
preincubation of intact cells with 50-100 µM DA followed
by washing reduced the inhibitory effects of
ONOO by only ~10%. The effect of
extracellular DA on ONOO inactivation of
the hDAT was significant (p < 0.05; ANOVA),
whereas the effect of intracellular DA was not. Nomifensine, -CFT,
and cocaine, antagonists of the hDAT, did not prevent
ONOO-induced inactivation of the hDAT
(data not shown).
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Figure 3.
Effects of DA on the
ONOO-induced inhibition of hDAT. Intact EM4 cells
stably expressing the hDAT were treated with ONOO
(1 mM) in the presence of extracellular or intracellular
DA. Cells were incubated for 10 min at 25°C with the indicated
concentrations of DA. ONOO was added to cells with
(intracellular DA) or without (extracellular DA) removal of exogenous
DA from the incubation medium and treatment was continued for 10 min.
Cells were then washed three times with KRH, and uptake of
3H-DA was determined. The data are means ± SEM of
four or five experiments run in triplicate. Controls for intracellular
and extracellular DA on hDAT activity omitted ONOO
treatment. The overall effect of extracellular DA on the
ONOO-induced inhibition of hDAT was significant
(p < 0.05; ANOVA), whereas the effect of
intracellular DA was not.
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Effects of cysteine-sulfhydryl reagents on hDAT
Various reagents that react specificity with cysteine residues in
membranes (van Iwaarden et al., 1992) were tested for their effects on
hDAT. Figure 4 shows that pCMB caused
significant reductions in DA uptake. At a concentration of 100 µM, pCMB was more inhibitory (95% inhibition of hDAT)
than ONOO (55% inhibition). In
agreement with previous results (Ferrer and Javitch, 1998; Chen et al.,
2000), the cell membrane-permeable cysteine reactant MTSEA (1 mM) lowered DA uptake to 30% of control, whereas equimolar
concentrations of the impermeable MTSET had no effect on DA uptake.
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Figure 4.
Effects of cysteine sulfhydryl reagents on hDAT
activity. Intact EM4 cells stably expressing wild-type hDAT were
exposed to ONOO (1 mM), pCMB (100 µM), MTSEA (1 mM), or MTSET (1 mM) for 10 min at 25°C for 10 min. Cells were washed
three times with KRH, and the uptake of 3H-DA was
determined. Results represent the mean ± SEM of four or five
experiments run in triplicate for each treatment. *Indicates that the
treatment was significantly different from untreated controls
(p < 0.05; Bonferroni's test).
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Effect of ONOO on cysteine labeling
with MTSEA-biotin
MTSEA-biotin labels reduced cysteines in proteins and we used it
presently to determine if the reduction in hDAT activity caused by
ONOO was related to oxidation of
cysteine residues. Intact cells were exposed to 1 mM
ONOO and washed membranes were isolated
and labeled with MTSEA-biotin. The results are presented in Figure
5 and show that untreated hDAT was
readily detected by MTSEA-biotin labeling.
ONOO treatment caused a substantial
reduction in labeling of the hDAT by MTSEA-biotin, indicative of
cysteine modification. Treatment of cells with pCMB or MTSEA reduced
MTSEA-biotin labeling of hDAT much like
ONOO (Fig. 5). Digital scans of the data
in Figure 5 establish that ONOO reduced
MTSEA-biotinylation of the hDAT by ~80%. The reductions in labeling
caused by pCMB (70%) and MTSEA (45%) were somewhat smaller in
magnitude when compared with ONOO. We
did not observe evidence of ONOO-induced
tyrosine nitration of the hDAT under the same conditions where it
reduced MTSEA-biotin labeling (data not shown).
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Figure 5.
The effect of ONOO on
MTSEA-biotin labeling of cysteine residues in hDAT. Intact EM4 cells
stably expressing wild-type hDAT were exposed to
ONOO (1 mM), pCMB (100 µM), or MTSEA (1 mM) for 10 min at 25°C.
Controls were treated with decomposed ONOO. Cells
were washed three times with KRH and then homogenized in PBS. Membranes
pelleted by a 15 min centrifugation step at 40,000 × g were washed three times with PBS and exposed to 1 mM MTSEA-biotin for 30 min at 4°C. Unreacted MTSEA-biotin
was removed by washing, and membranes were solubilized in 1% Triton
X-100. Biotinylated proteins were separated from unlabeled proteins by
adsorption to neutravidin agarose beads for 60 min at room temperature.
Beads were washed, and adsorbed proteins were eluted with SDS-stop
solution. Proteins were resolved by SDS-PAGE, blotted to PVDF
membranes, and probed for hDAT with the use of antibodies against the
Flag-epitope tag (8 µg/ml). The hDAT was visualized with enhanced
chemiluminescence. This experiment was repeated on four other occasions
with the same result.
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Identification of the cysteine residues in hDAT that are targeted
by ONOO
The data in Figure 5 suggested a relative loss of reduced
cysteines in hDAT after ONOO treatment,
but stopped short of identifying the most critical cysteine target or
targets. Therefore, we used mutant forms of the hDAT to identify the
cysteine targets for ONOO attack. The
results in Figure 6 show that X7C (a
mutant of hDAT in which all intracellular and extracellular cysteines
and one transmembrane cysteine were mutated), was relatively resistant to inhibition by ONOO. Wild-type hDAT
was inhibited by 50% after treatment with 1 mM ONOO, but the X7C mutant was inhibited
by only 11% after the same treatment. The effect of
ONOO on X7C was significantly different
from its effect on wild-type hDAT (p < 0.001;
Bonferroni's test). The CD-DAT mutant, which only contains reduced
cysteines in its first (C135) and third (C342) intracellular loops, was
inhibited by ONOO as was a mutant in
which C342 alone had been reintroduced into X7C (X7C-M342C).
ONOO reduced DA uptake mediated by
CD-DAT and X7C-M342C to ~50% of control, the same extent as seen in
wild-type hDAT.
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Figure 6.
The effect of ONOO on
cysteine mutants of hDAT. Intact EM4 cells stably expressing wild-type
hDAT or the indicated cysteine mutants were exposed to
ONOO (1 mM) for 10 min at 25°C.
Cells were washed three times with KRH, and the uptake of
3H-DA was determined. X7C is a mutant of the wild-type
(WT) hDAT in which all intracellular and
extracellular loop cysteines were mutated (see Materials and Methods).
CD-DAT is a cysteine-deficient mutant that contains reduced cysteines
only in its first (C135) and third (C342) intracellular loops.
X7C-M342C is a mutant into which only cysteine 342 has been
reintroduced into X7C. Data are expressed as a percentage of control,
where each mutant (untreated) served as its own control for
ONOO treatment. The results represent mean ± SEM of five to seven experiments run in triplicate. *Indicates that the
effect of ONOO was significantly different from
its effect on WT hDAT (p < 0.05;
Bonferroni's test).
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DISCUSSION |
ONOO is a powerful oxidant and
cytotoxin whose production has been associated with conditions that
result in damage to DA neurons. For example, the neurotoxic
amphetamines (Imam et al., 1999, 2001a,b; Imam and Ali, 2001) and MPTP
(Schulz et al., 1995; Ferrante et al., 1999; Pennathur et al., 1999)
are thought to exert toxicity to DA neurons, at least in part, through
the production of ONOO. The appearance
of nitrotyrosine immunoreactivity in postmortem brain from individuals
with Parkinson's disease and other neurodegenerative conditions
(Beckman and Koppenol, 1996; Good et al., 1996, 1998; Heales et al.,
1999) also serves as indirect evidence of
ONOO production. Methamphetamine
(Fleckenstein et al., 1997a,c; Bennett et al., 1998; Hanson et al.,
1998; Kokoshka et al., 1998; Metzger et al., 1998; Sandoval et al.,
2000) and MPTP (Kilbourn et al., 2000; Poyot et al., 2001) cause
reductions in DAT function, and the levels of hDAT are reduced in
Parkinson's disease as well (Kaufman and Madras, 1991; Guttman et al.,
1997; Marek et al., 2001; Varrone et al., 2001). Although the DAT is
known to be sensitive to inhibition by certain reactive oxygen species
(Fleckenstein et al., 1997b; Hanson et al., 1998; Davidson et al.,
2001; Sandoval et al., 2001), the effects of
ONOO on DAT have not been investigated directly.
Our results establish that ONOO causes a
selective and irreversible reduction in hDAT function. DA uptake into
intact EM4 cells stably expressing the hDAT was diminished by
ONOO through a mechanism that reflected
a reduction in Vmax.
ONOO did not cause cell lysis, and it
did not provoke release of DA from preloaded cells, suggesting that its
effects were mediated by a direct effect on the inward transport
properties of the hDAT. The hDAT has a total of 13 cysteine residues
arrayed throughout its 12 transmembrane domains and intracellular and
extracellular loops (Amara et al., 1998; Chen and Reith, 2000). Recent
molecular studies using a number of mutants of the hDAT have revealed
the importance of its cysteines in DA transport and ligand binding (Ferrer and Javitch, 1998; Chen et al., 2000; Hastrup et al., 2001;
Whitehead et al., 2001). In particular, cysteine 342, located in the
third intracellular loop, is critical to hDAT function. These results
provided ample precedence to focus attention on cysteine residues as
targets for the inhibitory effects of
ONOO.
Several lines of evidence establish that
ONOO inactivates the hDAT through
modification of cysteine residues. First,
ONOO is a powerful cysteine oxidant
(Radi et al., 1991), and cysteine residues in proteins are among the
most reactive amino acids with ONOO
(Alvarez et al., 1999). Second, cysteine residues in the hDAT are known
to be critical determinants of its function (Ferrer and Javitch, 1998;
Javitch, 1998; Chen et al., 2000; Hastrup et al., 2001; Whitehead et
al., 2001). We found that the membrane-permeable sulfhydryl reagents
pCMB and MTSEA inhibited hDAT function like ONOO. MTSET, a membrane-impermeable
sulfhydryl reagent, had no effect on hDAT activity, in agreement with
previous results (Chen et al., 2000). Third, MTSEA-biotin labeling of
hDAT was substantially reduced by a concentration of
ONOO that significantly inhibits hDAT
function. Taken together, these findings support the conclusion that
ONOO modifies hDAT function through its
effects on cysteine residues.
Intact cell membranes are not thought to present barriers to
ONOO (Beckman et al., 1994; Beckman and
Koppenol, 1996), so we tested a series of cysteine mutants of hDAT to
determine the potential inhibitory site of action of
ONOO. The X7C mutant, in which all
intracellular and extracellular loop cysteines were mutated, was
relatively resistant to inhibition by
ONOO. The CD-DAT mutant, which only
contains reduced cysteines 135 and 342, was sensitive to inhibition to
ONOO. The X7C-M342C, a mutant into which
C342 alone had been reintroduced into X7C, was also inhibited by
ONOO to the same extent as wild-type
hDAT. These results point to cysteine 342 as the site in hDAT at which
ONOO acts to lower its activity.
Cysteine 342 is not the only cysteine residue modified by
ONOO (Fig. 5) or other oxidants, but it
appears that modification of cysteine 342 has the most severe
consequences on hDAT function.
DA and other substrates that are transported by the DAT are known to
cause a conformational alteration in hDAT that makes cysteine 342 more
accessible to sulfhydryl reagents (Chen et al., 2000). Therefore, we
predicted initially that intracellular DA would make hDAT more
susceptible to inhibition by ONOO. It
was found, however, that when cells were loaded with DA before ONOO treatment (and extracellular DA was
removed by washing), the effect of ONOO
on hDAT was neither increased nor decreased. On the other hand, when
cells remained exposed to external DA during
ONOO treatment, inhibition of hDAT was
completely prevented. The ability of extracellular DA to prevent
ONOO-induced inactivation of hDAT
appears to be the result of a detoxification of
ONOO through its reaction with DA. It
has recently been observed that DA can react with
ONOO to produce the o-quinone
of DA (Pannala et al., 1997; Kerry and Rice-Evans, 1999).We did not
find evidence that the DA quinone is a substrate for uptake by the hDAT
(our unpublished observations), so we conclude that the reaction
of DA-quinone with external hDAT cysteines, like that shown by MTSET,
would not lead to inhibition of uptake. It may seem paradoxical that 10 µM DA could block the effects of 1 mM ONOO. However,
ONOO is extremely unstable at neutral
pH, where it has an apparent half-life of 1.9 sec (Beckman et al.,
1990). Taking this into account, the bolus addition of 250 µM ONOO is
approximately equivalent to a steady-state level of 1 µM maintained for 7 min (Beckman et al., 1994).
Therefore, it is likely that cells were exposed to an effective
concentration of ONOO that is in the
same range as that of DA (i.e., micromolar). The situation is different
for intracellular cysteine residues in hDAT. DA-quinone, synthesized by
electrochemical oxidation of DA, is known to prevent ligand binding to
hDAT as a result of DA-quinone-mediated modification of cysteine 342 (Whitehead et al., 2001). Thus, our results are entirely consistent
with those of Whitehead et al. (2001) and suggest that
ONOO reacts with intracellular DA to
create the DA-quinone, which in turn modifies cysteine 342 and causes
partial loss of transport function.
The reduced transport Vmax caused by
ONOO treatment is most likely mediated
by a direct effect on uptake of modification of Cys342. DAT activity
can also be altered by changes in its membrane trafficking. For
example, amphetamine leads to a reduction in surface expression of the
hDAT and diminished DA uptake (Saunders et al., 2000). Nonetheless, the
time of exposure to ONOO was relatively
brief (10 min), and cell surface labeling of the hDAT remained strong
after treatment with ONOO (our
unpublished observations). These results represent the first characterization of the effects of ONOO
on the hDAT, and they establish the possibility that drug- and disease-induced oxidative stress can alter hDAT function.
ONOO has been implicated as a causative
factor under conditions in which DA neurons are damaged, including the
neurotoxic amphetamines and in Parkinson's disease.
| |
FOOTNOTES |
Received Jan. 31, 2002; revised March 14, 2002; accepted March 19, 2002.
This work was supported by National Institutes of Health Grants
DA06067 (S.U.P.), DA10756 (D.M.K.), DA11495 (J.A.J.), DA14942 (J.A.J.),
and MH57324 (J.A.J.), and by a Veterans Affairs Merit Award
(D.M.K.).
Correspondence should be addressed to Dr. Donald M. Kuhn,
Department of Psychiatry and Behavioral Neurosciences, Wayne State University School of Medicine, 2125 Scott Hall, 540 East Canfield, Detroit, MI 48201. E-mail: donald.kuhn{at}wayne.edu.
| |
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