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Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York (W.W., H.S., C.K.S.); Vollum Institute, Oregon Health and Science University, Portland, Oregon (M.S.S.); Medications Discovery Research Branch, Intramural Research Program, National Institute on Drug Abuse, Baltimore, Maryland (M.K.K.); Department of Pharmacology and Toxicology, Duquesne University, Pittsburgh, Pennsylvania (O.T.U., C.K.S.)
Received December 3, 2002; accepted May 2, 2003
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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3-fold less potent in uptake than in
binding assays. Apparent affinities for substrates were unaffected by the D79E
mutation unless the catechol moiety was modified. Strikingly, potencies for
nonsubstrate inhibitors in uptake and binding assays matched for D79E DAT,
because of a 3-fold lowering of binding affinities relative to WT DAT. The
present findings reveal a complex role for D79 in determining substrate
specificity and high-affinity binding of DAT inhibitors. We propose that at
least two discrete inhibitor-binding DAT conformations or populations exist
and that the DAT conformation/population responsible for inhibitor
high-affinity binding is less responsible for dopamine uptake. The findings
may be extensible to other psychostimulants and antidepressants that display
discrepancies between binding affinity and monoamine uptake inhibition potency
and may be relevant to development of a long-sought "cocaine
antagonist".
;
Hoffman et al., 1991;
Kilty et al., 1991;
Pacholczyk et al., 1991;
Shimada et al., 1991) has
suggested candidate amino acid residues for direct contact with the monoamine
substrates. Scanning the Na+/Cl- dependent transporter
family, it is quickly noted that an aspartic acid residue in the first
putative transmembrane domain (TM) of the plasma membrane monoamine
transporter is not shared by transporters for GABA, glycine, proline, betaine,
or the other members of this group (Fig.
1).
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In a previous study, replacement of the rat DAT TM1 aspartic acid residue
(D79) with alanine, glycine, or glutamic acid decreased the dopamine uptake
Vmax value, increased the Km value,
and decreased binding of the cocaine analog [3H]WIN 35,428
(Kitayama et al., 1992). The
authors proposed that the D79 residue directly contacts the positively charged
amino group of dopamine, an idea borrowed from a model involving agonist
docking to a G protein-coupled receptor (GPCR), the 
-adrenergic receptor
(Strader et al., 1988).
Indeed, ionic competition for D79 has been viewed as the mechanism of cocaine
inhibition of dopamine uptake; this postulate has served as the central tenet
for DAT-ligand interactions (Carroll et
al., 1992). More recently, neutral 8-oxa analogs of WIN 35,428 and
cocaine have proven to be quite potent DAT inhibitors relative to the charged,
nitrogen-based parent compounds (Madras et
al., 1996; Kozikowski et al.,
1999), which places in doubt the necessity of a D79-cocaine ion
pair.
The postulate that the dopamine amino group forms an ionic bond with D79 is
inconsistent, however, with amino acid sequence homology and substrate
commonalities within the Na+/Cl- dependent transporter
family. Transporters for GABA, glycine, proline, creatine, betaine, and
taurine possess a glycine residue at the analogous position
(Fig. 1) (Guastella et al.,
1990,
1992;
Fremeau et al., 1992;
Yamauchi et al., 1992;
Uchida et al., 1992;
Guimbal and Kilimann, 1993),
yet all of these substrates possess the very same positively charged amino
group as dopamine. Given that dopamine, norepinephrine, epinephrine and
serotonin share a phenolic moiety not shared by the other six substrates
above, a more logical role for the aspartate side chain would be to form an
intramolecular contact that supported a phenolic (or aromatic) binding pocket.
The fact that this charged, hydrophilic residue is one of only two
such residues located in the hydrophobic lipid bilayer further
implies its importance to DAT function; thus, a better understanding of the
contribution of D79 to substrate and inhibitor binding is essential.
No direct contact points have been unequivocally established between a
transporter in this family and either its cognate neurotransmitter or cocaine,
although specific serotonin transporter residues have been identified as
contributors to cocaine or mazindol binding sites
(Chen et al., 1997;
Barker et al., 1998). Toward
the goal of establishing discrete dopamine-DAT points of contact, we have
tested the premise of a dopamine-DAT D79 ionic bond serving as the governing
point of contact by generating mutants that alter the charge, hydrogen bonding
potential, or length of the D79 side chain. Use of a dopamine analog that is
one carbon shorter at D79E DAT did not re-establish wild-type (WT) levels of
apparent substrate uptake, and WT and D79E DAT displayed a very similar
affinity for dopamine, findings that did not support the postulated
D79-dopamine interaction. To directly and accurately compare ligand binding
affinities and dopamine uptake inhibition potencies, binding and uptake
inhibition assays were carried out under the same conditions. High-affinity
binding of WIN 35,428 was lost at the D79E DAT mutant; paradoxically,
potencies for this and other DAT blockers in inhibiting dopamine transport
were considerably less affected by the mutation. This result led to the
inference that two DAT populations or conformations recognize DAT inhibitors
and to the unexpected conclusion that the DAT population/conformation
responsible for high-affinity cocaine analog binding is not primarily
responsible for dopamine uptake.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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85 Ci/mmol) and
[3H]dopamine (29 Ci/mmol) were obtained from PerkinElmer Life
Sciences (Boston, MA). Nonradioactive WIN 35,428, cocaine, methylphenidate,
and amphetamine were obtained from Research Triangle Institute (Research
Triangle Park, NC) via the National Institute on Drug Abuse Division of Basic
Research. Nonradioactive dopamine and ascorbic acid were obtained from Sigma
Chemical Co. (St. Louis, MO); mazindol, norepinephrine, and the tyramines were
obtained from RBI/Sigma (Natick, MA); and dihydroxybenzylamine was obtained
from Aldrich (Milwaukee, WI). Scintillation counting materials were from
Fisher Scientific (Pittsburgh, PA), and GF/B paper was from Brandel
(Gaithersburg, MD). Anti-DAT antibody (rat) was obtained from Chemicon
(Temecula, CA), biotinylation reagents were obtained from Pierce Chemical Co.
(Rockford, IL), and all other Western blotting reagents were from Bio-Rad
(Hercules, CA). COS-7 and CHO-K1 cell lines were obtained from American Type
Culture Collection (Manassas, VA).
Mutagenesis and Cell Transfections. All site-directed mutagenesis
was conducted as described previously
(Spivak et al., 1997). COS-7
cells were cultured in Dulbecco's minimal essential medium plus 10% fetal
bovine serum, 10% penicillin/streptomycin, and 20 mM L-glutamine at
37°C in 5% CO2 in 750-cm2 flasks. Cells were
distributed in 24-well plates such that the monolayer would be 95% confluent
when the transfection commenced. For an individual well of the 24-well plate,
1 µg of plasmid and 1.5 ml of "LIPO 2000" LipofectAMINE
suspension were preincubated 20 min before adding to the well in Opti-MEM
medium. After 5 h, an equal volume of Dulbecco's minimal essential medium plus
10% fetal bovine serum, 10% penicillin/streptomycin, and 20 mM
L-glutamine was added to the well, and cells were used for
experiments approximately 24 h after initiating the transfection.
Stably-transfected DAT-CHO cell lines were prepared by LipofectAMINE-mediated
transfection and selection of stable transfectants in the presence of 500
µg/ml G-418 in Ham's F-12 medium; cell lines were maintained in the same
medium containing 100 µg/ml G-418.
Cell Surface Labeling of Expressed DAT Proteins. Stably transfected
CHO cells or transiently transfected COS-7 cells were grown to confluence on
six-well (35 mm/well) polylysine-free plates. After washing the cell monolayer
3 x 10 min with ice-cold phosphate-buffered saline supplemented with 0.1
mM CaCl2 and 1 mM MgCl2 (PBS/Ca/Mg buffer)
(Ramamoorthy et al., 1998), 1
ml of a 1.5 mg/ml solution of freshly-prepared
sulfosuccinimidyl-2-(biotinamido-)ethyl-1,3-dithiopropionate in PBS/Ca/Mg
buffer was added for 25 min at 4°C. Free biotinylation reagent was
quenched by washing thrice with cold PBS/Ca/Mg containing 100 mM glycine.
After two more washes with PBS/Ca/Mg alone, cells were lysed with 0.2 ml of
0.1% SDS, 1% Triton X-100, 150 mM NaCl, and 1 mM EDTA in 10 mM Tris-HCl, pH
7.4, for 30 min on ice. Lysates were cleared by centrifugation at
14,000g for 10 min at 4°C. The upper 150 µl of supernatant was
transferred to a new tube and combined with 50 µl of NeutrAvidin resin (50%
slurry), incubating overnight at 4°C and mixing with end-over-end
rotation. The resin was concentrated to a pellet with 5000g
centrifugation for 4 min at 4°C. The pellet was washed first three times
by resuspending the resin in the lysis buffer, and then two times more in 0.1%
Triton X-100, 500 mM NaCl and 5 mM EDTA in 50 mM Tris-HCl, pH 7.5; each wash
was centrifuged as before with all steps at 4°C. After a final wash with
50 mM Tris-HCl, pH 7.5, the supernatant was completely removed, and
biotinylated proteins were released from the NeutrAvidin resin by addition of
standard SDS gel loading buffer containing 2-mercapto-ethanol. Biotinylated
DAT was separated from other biotinylated proteins by SDS-polyacrylamide gel
electrophoresis, and identified with Western blotting (standard conditions,
using a rat anti-DAT primary antibody diluted 1:4000). To test that
biotinylation was specific for cell surface proteins, the same blot was
stripped of antibodies with 100 mM 2-mercaptoethanol and 2% SDS in 62.5 mM
Tris-HCl, pH 6.7, for 1 min at 40°C and subsequently reprobed with a
monoclonal antibody against actin, an intracellular marker protein.
[3H]Dopamine Uptake. Assays were conducted with cell monolayers in six-well plates. The monolayer was washed 2 x 2 ml with KRH buffer (25 mM HEPES, pH 7.3, 125 mM NaCl, 4.8 mM KCl, 1.3 mM CaCl2, 1.2 mM Mg2SO4, 1.2 mM KH2PO4, and 5.6 mM glucose), and uptake was typically initiated by addition of 1 ml of 10 nM [3H]dopamine and 50 µM ascorbic acid in KRH to duplicate or triplicate cell monolayers. A time course was conducted for each DAT mutant to determine when uptake velocity was in the linear phase, typically occurring at 5 to 10 min at 22°C. Uptake was quenched by washing the monolayer with 2 x 2 ml of KRH + ascorbic acid. Cell monolayers were solubilized in 0.5 ml of 1% SDS, and transferred to scintillation vials for determination of incorporated tritium. DAT mutants demonstrating the ability to transport dopamine in this assay were studied in uptake "saturation" experiments in which final concentrations of 0.1 to 16 µM dopamine were employed, and [3H]dopamine was diluted with nonradioactive dopamine to obtain a specific activity of 0.1 Ci/mmol. Nonspecific uptake was assessed by inclusion of 30 µM cocaine. Uptake inhibition experiments used 2 to 20 µM [3H]dopamine plus nonradioactive DAT uptake blockers at the following concentration ranges: cocaine, 0.01 to 100 µM; WIN 35,428, 3 to 10,000 nM; mazindol, 3 to 10,000 nM; methylphenidate, 3 to 10,000 nM; and dihydroxybenzylamine (DHBA), 0.3 to 100 mM. All nonsubstrate inhibitors were preincubated 10 min with the cell monolayer before adding [3H]dopamine. Km and Vmax values for transport and Ki values for uptake inhibition were determined with Prism 2.0 (GraphPad Software, San Diego, CA).
Ligand Binding Assays. [3H]WIN 35,428, structurally similar to cocaine but much more stable in vitro, was the radioligand employed for all experiments. With the exception of the membrane binding experiment referred to under Results, all binding assays were conducted exactly as described above for the dopamine uptake assay except that [3H]dopamine was replaced with 1 nM [3H]WIN 35,428, and radioligand and nonradioactive competitor were incubated with cells for 15 min (the same incubation period allowed for an uptake blocker in the uptake assay). Nonradioactive competitor concentrations were as indicated above for uptake inhibition. Nonspecific binding was assessed by addition of 10 µM mazindol except when mazindol was the drug tested, in which case 50 µM cocaine was used to assess nonspecific binding. For the membrane binding experiment, membranes of CHO cells stably transfected with WT or D79E DAT cDNAs were prepared by suspending 2.5 x 108 cells in 40 ml of 10 mM HEPES, pH 8.0, 50 mM MgCl2 buffer. The suspension was processed in a Polytron homogenizer (Kinematica AG, Basel, Switzerland), and the homogenate was centrifugated for 20 min at 48,000g (all steps at 4°C). After protein quantitation, membranes were resuspended in KRH buffer and combined with 2 to 20 nM [3H]WIN 35,428 and increasing concentrations of nonradioactive WIN 35,428 for 60 min at 22°C. Membranes were aspirated with a Brandel harvester onto GF/B glass fiber filter paper, washed three times with 5 ml of ice-cold KRH, and the filters were measured for radioactivity via liquid scintillation counting. For all binding assays, data were analyzed with the Prism 2.0 software to obtain Kd, Ki, and Bmax values.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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-actin protein was not detected with an
anti-actin monoclonal antibody at intact cells, and was observed only for cell
lysates (data not shown), meaning that the immunoreactivity of DAT proteins
could not be explained by intracellular biotinylation due to cell permeation
with biotin.
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To more thoroughly characterize D79E DAT relative to WT DAT, stably
transfected CHO-K1 cells were prepared. Biotinylation experiments again
established that WT and D79E DAT proteins were present at the cell surface,
but total, intracellular, and cell surface expression of D79E DAT were
approximately half that of WT DAT (Fig.
3b). The kinetics of [3H]dopamine uptake were
determined for each stable cell line (Fig.
4). The WT DAT turnover rate in CHO cells was in agreement with
that reported for hDAT in oocytes (Sonders
et al., 1997) and rDAT in LLC-PK cells
(Gu et al., 1994), and 7-fold
higher than that for D79E DAT CHO cells
(Table 1). Half of the
Vmax decrease at D79E DAT cells could be attributed to
fewer transporter proteins at the cell surface, the latter evidenced by the
biotinylation data (Fig. 3b)
and supported by the Bmax values for [3H]WIN
35,428 binding (Table 1). Thus,
the D79E mutation rendered a decrease of approximately 4-fold in DAT dopamine
turnover rate (Table 1). A
decrease in transport capacity was also observed for the analogous TM1 SERT
mutant D98E (Barker et al.,
1999).
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On the other hand, this mutation had little effect on the Km value for dopamine uptake (Fig. 4 and Table 2). Because this value cannot be equated with an affinity constant, dopamine affinity was assessed by its ability to displace [3H]WIN 35,428 binding at WT and D79E DAT. (Binding affinity Ki values for all other substrates and inhibitors in the study were also assessed in this way, and this Ki value will hereafter be referred to as the "binding affinity" of the ligand for WT or D79E DAT.) By this measure, dopamine displayed an almost identical binding affinity for WT and D79E DAT proteins (Fig. 5 and Table 2). This result may be inconsistent with the idea that the D79 residue is the governing element of the DAT dopamine-binding pocket because the added steric bulk of the glutamate side chain would leave less room for dopamine (assuming that dopamine does not adopt an energetically unfavorable conformation to accommodate the mutation). To further address this steric issue, DHBA was employed as a potential DAT substrate. This compound is identical to dopamine except for the absence of one of two methylene groups between the amino and dihydroxyphenyl groups. If the governing DAT-dopamine point of contact were at D79 and the role of the D79 residue were simply to form an interaction with the amino group of dopamine, the extra methylene group in the glutamic acid side chain would exactly compensate for the shorter DHBA ligand. Binding and uptake of DHBA by D79E DAT should thus be as efficient as dopamine binding and uptake at WT DAT and superior to that of dopamine at D79E DAT. This was not the case; DHBA binding affinities were identical at WT and D79E DAT, and DHBA remained a quite inferior inhibitor of [3H]dopamine uptake (Fig. 5 and Table 2). The potency of DHBA in inhibiting [3H]dopamine uptake was 7-fold higher at D79E DAT compared with WT DAT. Still, this Ki value for DHBA at D79E DAT was 460-fold higher than the dopamine Km value at WT DAT and 377-fold higher than the Ki value for dopamine binding at WT DAT. The DHBA Ki value for [3H]dopamine uptake inhibition at D79E DAT agrees well with that for inhibition of [3H]WIN 35,428 binding; curiously, the [3H]dopamine uptake inhibition potency of DHBA at WT DAT was 7-fold less than its apparent affinity for inhibiting [3H]WIN 35,428 binding. Ligand binding assays were conducted under conditions that matched those for assaying uptake inhibition.
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Other dopamine analogs assessed the effect of adding substituents to the
methylene groups or of altering the catechol hydroxyl groups
(Table 2). Norepinephrine
(addition of a -OH group to dopamine) was equally effective at WT and
D79E DAT in inhibiting dopamine uptake but was 2-fold less effective at
displacing [3H]WIN 35,428 at D79E DAT than at WT DAT. The dopamine
uptake inhibition potencies of m- or p-tyramine (each
lacking one of the catechol OH groups) were unchanged or 2-fold lower as a
result of the D79E mutation, respectively, yet the mutation decreased binding
affinities for both tyramines by more than 5-fold. Of the DAT substrates
tested, the D79E mutation most dramatically affected the dopamine uptake
inhibition potency (5-fold loss) and binding affinity (9-fold loss) of the
more psychoactive isomer of amphetamine (lacking both catechol OH groups and
adding an 
-methyl group), S(+)-amphetamine. For
R(-)-amphetamine, however, dopamine uptake inhibition potency was not
significantly affected by the mutation.
The dopamine uptake inhibition potencies of the classic uptake inhibitors cocaine, WIN 35,428, mazindol, and methylphenidate were compared at WT and D79E DAT cells. Of the four inhibitors, only the dopamine uptake inhibition potency of cocaine was altered (2-fold decrease) in a statistically significant fashion by the mutation (Fig. 6 and Table 3). Binding affinities for each inhibitor were also determined, using intact cell monolayers and under conditions essentially identical to those for dopamine uptake assays. All inhibitors were shown to reach equilibrium with WT or D79E DAT within 15 min; Ki values did not differ from those obtained after 60- or 120-min binding assays (data not shown). For all four inhibitors, the potency of inhibition of [3H]WIN 35,428 binding matched the potency for inhibition of [3H]dopamine uptake at D79E DAT, as would be expected if occupancy of the [3H]WIN 35,428 binding site prevented substrate translocation. In contrast to the D79E mutant, inhibitory potencies derived from uptake assays and binding assays did not match for intact cells stably expressing WT DAT: WIN 35,428, mazindol, methylphenidate, and cocaine were 3- to 4-fold more potent as inhibitors of [3H]WIN 35,428 binding than as inhibitors of [3H]dopamine uptake (Fig. 6 and Table 3). These results suggest the existence of a conformational state or binding site in WT DAT with high affinity for WIN 35,428 that is not detectable in D79E DAT.
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Interestingly, specific binding of [3H]WIN 35,428 at D79E DAT was undetectable using either suspended cells (the monolayer was scraped from the plate) or a cell membrane preparation in the KRH uptake buffer, whereas [3H]WIN 35,428 binding affinity at WT DAT was unaffected by these conditions (Kd = 20 ± 1, 19 ± 1, and 18 ± 4 nM for attached, suspended, and membrane-prepared WT DAT CHO cells, respectively). No appreciable specific uptake of [3H]dopamine was observed for suspended cells possessing either transporter protein (data not shown).
The results with WT DAT cells suggested that a high-affinity binding site for uptake inhibitors may have been masked in the dopamine uptake assay. To search for two inhibitor sites at WT DAT, 24-point concentration curves were employed for cocaine inhibition of [3H]dopamine uptake. A reproducible second WT DAT site was indeed suggested from visual inspection of the curve (but was not sufficiently defined for GraphPad analysis), and this site seemed to be in the vicinity of the 128 nM Ki value previously measured for high-affinity binding. To a lesser extent, D79E DAT also occasionally displayed this high-affinity cocaine site (Fig. 7). Similarly, DHBA typically displayed a mild deviation from the monophasic uptake inhibition curve for WT DAT that was consistent with its measured higher affinity (800 µM) binding site (data not shown).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Based on models for dopamine interaction with its G protein-coupled
receptor, related GPCR findings, and DAT mutagenesis, the negatively charged
carboxylate moiety of the D79 DAT residue has been postulated to contact the
positively charged amino group of dopamine
(Kitayama et al., 1992). In
agreement with Kitayama et al.
(1992), we observed that of
several substitutions at the D79 position, the glutamic acid mutation was
least deleterious to dopamine transport via DAT. However, we did not detect an
appreciable difference in the dopamine Km values at WT and
D79E DAT (Table 2), contrasting
with the previously observed 4-fold increase in Km value
at the mutant. Dopamine affinity was assessed by inhibition of
[3H]WIN 35,428 binding and was again found not to differ between WT
and D79E DAT in a statistically-significant fashion
(Table 2).
Experiments with DHBA, identical to dopamine except shorter by a methylene
group, more directly addressed the role of the D79 residue in substrate
recognition. The 7-fold increase in dopamine uptake inhibition potency
observed for DHBA resulting from the D79E mutation was hardly an indication
that the putative D79-substrate ion pair had been re-established, because it
did not significantly approach the increase of more than 3300-fold needed to
meet the level of dopamine transport by WT DAT. Moreover, DHBA affinities at
WT and D79E DAT were identical (Table
2). This 7-fold increase in dopamine uptake inhibition potency for
the shorter DHBA molecule may reflect some alleviation of substrate steric
hindrance created by the D79E substitution, without necessarily involving an
interaction between substrate amino group and D79 carboxylate. It is possible
that the residue contributes to the tertiary structure of the transporter or
even formation of the dopamine binding pocket without actually contacting
dopamine. During the initial phase of our experiments, a study involving the
analogous TM1 aspartic acid residue of SERT was reported
(Barker et al., 1999). In
concert with the present DAT results, only the SERT glutamic acid substitution
at this position (D98E) displayed detectable substrate uptake, and
Km values for serotonin uptake were essentially the same
for wild-type and D98E SERT. The mutation effected a compensatory 12-fold
affinity increase for a "shorter" serotonin analog employed to
accommodate the longer glutamic acid side chain. By itself, the result might
suggest that an ionic bond forms between D98 and serotonin; however, serotonin
was equipotent at wild-type and D98E SERT. It is conceivable that serotonin
contacts the D98E side chain, but it should be noted that a less dramatic
mutation-induced gain-of-function was required to approximate the potency of a
"full-length" serotonin analog at wild-type SERT compared with the
potency difference of more than 3000-fold between dopamine and DHBA at WT DAT.
Still, it must be acknowledged that of all the DAT ligands tested in our
study, only DHBA displayed an increase in dopamine uptake inhibition potency
as a result of the D79E mutation.
The possibility that D79 is involved in recognition of some feature of the substrate catechol ring moiety was also tested. The dopamine uptake inhibition potencies for the tyramines and (-)-amphetamine at WT versus D79E DAT indicated, however, that loss of one or both catechol hydroxyl groups was essentially tolerated by D79E DAT. Binding affinities for these three substrates, on the other hand, were reduced severalfold by the mutation. The result suggests that one DAT conformation or population is more sensitive to alterations in the substrate catechol moiety than is a second DAT conformation/population. As explained in the Introduction, the aromatic moiety of dopamine would be the most logical substrate pharmacophore to show dependence on the D79 DAT residue. It should be noted that after correcting for differences in surface protein levels, D79E DAT displayed a 4-fold lower turnover number (Vmax/Bmax) than WT DAT (Table 1). Thus, the role of the D79 side chain in dopamine transport would seem to be more complex than merely contributing to the dopamine binding site.
The D79E mutation had no effect on the dopamine uptake inhibition potency
of WIN 35,428, mazindol, or methylphenidate, and cocaine potency was decreased
by only half. The binding affinity for each inhibitor was assessed under
conditions identical to those employed in the dopamine uptake inhibition
assay, and binding affinity constants matched those for uptake inhibition for
D79E DAT. Curiously, this correlation was not seen for WT DAT; binding
affinities were higher than dopamine uptake inhibition potencies for all
inhibitors tested (Fig. 6 and
Table 3). Moreover, D79E DAT
did not display significant [3H]WIN 35,428 affinity unless cells
bearing this protein were attached to the culture plate as a monolayer. The
requirement for an attached cell monolayer for [3H]WIN 35,428
binding at D79E DAT is currently not understood. It is possible that the
"off rate" of WIN 35,428 binding at this mutant protein is too
rapid for detection with glass-fiber vacuum filtration. Another possibility is
that scraping the CHO cells disrupts a DAT association with either another
protein, e.g., phosphatase 2A, syntaxin-1A, PICK1, or -synuclein
(Bauman et al., 2000;
Deken et al., 2000;
Lee et al., 2001;
Torres et al., 2001), or
itself, as an oligomer (Kilic and Rudnick,
2000; Hastrup et al.,
2001), and that such an association is required for 20 nM
[3H]WIN 35,428 binding.
Our results suggest that for each uptake inhibitor tested there exists a discrete site, conformation, or population for WT DAT that exhibits "higher" or "lower" binding affinity for the drug. The D79E mutation largely eliminates the high-affinity component (Fig. 8). Considering that the uptake inhibition potencies for three of four inhibitors 1) were insensitive to the mutation and 2) matched the binding affinities at D79E DAT, it appears that the lower affinity component for each drug at WT DAT was principally responsible for blocking translocation of dopamine into the cell. It is understandable why the [3H]WIN 35,428 binding curve for WT DAT did not display 2 obvious phases. In binding displacement assays, use of a low concentration of radioligand favors detection of the site with highest affinity, at the expense of lower affinity sites. Thus, the apparently monophasic WT DAT curve corresponding to the 20 nM Kd value may be a weighted average between the lower affinity component and a component with an affinity greater than 20 nM.
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On the other hand, such explanations do not account for the disparity
between uptake inhibition potency and binding affinity observed for all
inhibitors tested at WT DAT. Because the gap between these two values was
greatest for cocaine, this drug was chosen for 24-point uptake inhibition
experiments that sought to detect a high-affinity component. A second phase of
the WT DAT curve corresponding to the high-affinity value was frequently
present using this assay and occasionally present, to a lesser extent, in the
D79E DAT curve (Fig. 7).
Although visually apparent, this phase was not sufficiently prominent to be
detected with the regression analysis. The WT DAT population that recognizes
inhibitors with higher affinity may be a poorer transporter of dopamine than
the population containing the lower affinity inhibitor binding site
(Fig. 8). This explanation
accounts for the under-representation of the "high-affinity" DAT
population in the [3H]dopamine uptake inhibition assay but not in
the [3H]WIN 35,428 binding assay. Interestingly, syntaxin-1A
modulates GABA uptake via direct contact with the GABA transporter
(Deken et al., 2000); it is
possible that association of DAT with a cytoskeletal protein (or another DAT
molecule) modulates uptake and at the same time creates the higher affinity
binding site for classic inhibitors. Alternatively, it cannot be ruled out
that the "high-affinity" DAT population is a minority for WT DAT
and nearly undetectable in the case of D79E DAT.
The lack of correlation between cocaine potencies for binding and uptake
inhibition at WT DAT has been noted previously. Using COS cells
transiently-transfected with DAT, Pristupa et al.
(1994) reported
Ki values of 240 and 743 nM for cocaine binding and uptake
inhibition, respectively, numbers very close to our own. Using DAT-transfected
human embryonic kidney 293 cells, Eshleman et al.
(1999) observed a 2- to 3-fold
greater cocaine potency for uptake inhibition than for binding. Literature
values for dopamine uptake inhibition potency of cocaine range from 149 nM
(Eshleman et al., 1999) to
137,000 nM (Reith and Coffey,
1994); this variance seems to be dependent on cell/tissue type and
experimental conditions. Again, DAT interactions with DAT binding proteins or
DAT post-translational modifications are among possibilities that may be
relevant to the differences in cocaine potency. We have observed cocaine
dopamine uptake inhibition potency to fluctuate within a 3-fold range as a
function of cell state when employing CHO cells stably transfected with WT DAT
(C. K. Surratt, in preparation).
Our findings may be of great relevance to ongoing efforts in the
development of anti-cocaine medications. Despite decades of investigation, the
search for a therapy against dependence on cocaine has been largely
unsuccessful. The principal focus in the development of potential anti-cocaine
medications has been to inhibit high-affinity cocaine or WIN 35,428 binding
while attempting to spare dopamine uptake. Our finding that the potency of
high-affinity inhibitor binding to WT DAT diverges from that for inhibition of
dopamine uptake suggests that development of a "cocaine
antagonist" therapeutic should not be focused on blocking high-affinity
cocaine binding. The D79E DAT mutant may thus serve as an important diagnostic
tool in the development of a clinically useful cocaine antagonist. Future
experiments will attempt to further define the dopamine uptake inhibition DAT
site for various classic inhibitors, and will include mapping the cocaine and
WIN 35,428 binding sites of D79E DAT using the substituted cysteine
accessibility method (Ferrer and Javitch,
1998). Localizing drug binding sites relevant to dopamine uptake
inhibition should markedly enhance the prospects for computer-aided rational
design of anti-cocaine medications.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: DAT, dopamine transporter; SERT, serotonin
transporter; TM, transmembrane domain; WIN 35,428,
(-)-3-(4-fluorophenyl)-tropan-2-carboxylic acid methyl ester
tartrate; WT, wild type; CHO, Chinese hamster ovary; PBS, phosphate-buffered
saline; KRH, Krebs-Ringers-HEPES; DHBA, dihydroxybenzylamine.
1 The N-terminally epitope-tagged DAT was pharmacologically indistinguishable
from wild-type DAT (data not shown). The introduced epitope was not employed
in the experiments reported within, because antisera directed against an
authentic DAT N-terminal epitope proved superior for the biotinylation
studies. 
Address correspondence to: Dr. Christopher K. Surratt, Division of Pharmaceutical Sciences, Duquesne University, 453 Mellon Hall, 600 Forbes Avenue, Pittsburgh, PA 15282. E-mail: surratt{at}duq.edu.
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) or D79E (
) DAT were incubated with
increasing concentrations of a fixed ratio of [3H]dopamine:
nonradioactive dopamine. A representative result of three separate experiments
is shown.
