From the Department of Pharmacology and Center for Molecular
Neuroscience, Vanderbilt University School of Medicine,
Nashville, Tennessee 37232-6600, the Department of
Medicinal Chemistry and Molecular Pharmacology, Purdue University
School of Pharmacy, West Lafayette, Indiana 47907, the
§ Charles A. Dana Research Institute, Drew University,
Madison, New Jersey 07940, and the ¶ Department of Psychiatry
and Laboratory of Molecular Neurobiology, Clarke Institute of
Psychiatry, Toronto, Ontario M5T 1RS, Canada
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ABSTRACT |
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Human and Drosophila melanogaster serotonin (5-HT) transporters (SERTs) exhibit similar 5-HT transport kinetics and can be distinguished pharmacologically by many, but not all, biogenic amine transporter antagonists. By using human and Drosophila SERT chimeras, major determinants of potencies of two transporter antagonists, mazindol and citalopram, were tracked to the amino-terminal domains encompassing transmembrane domains I and II. Species-scanning mutagenesis, whereby amino acid substitutions are made switching residues from one species to another, was employed on the eight amino acids that differ between human and Drosophila SERTs in this region, and antagonist potencies were reassessed in 5-HT uptake assays. A single mutation in transmembrane domain I of human SERT, Y95F, shifted both citalopram and mazindol to Drosophila SERT-like potencies. Strikingly, these potency changes were in opposite directions suggesting Tyr95 contributes both positive and negative determinants of antagonist potency. To gain insight into how the Y95F mutant might influence mazindol potency, we determined how structural variants of mazindol responded to the mutation. Our studies demonstrate the importance of the hydroxyl group on the heterocyclic nucleus of mazindol for maintaining species-selective recognition of mazindol and suggest that transmembrane domain I participates in the formation of antagonist-binding sites for amine transporters.
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INTRODUCTION |
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5-HT1 is a biogenic
amine neurotransmitter that mediates many biological processes in the
periphery and central nervous system, playing an important role in a
variety of behaviors including mood, appetite, sleep, libido, and
memory (1-3). The neurotransmitter transporter that removes 5-HT from
the extracellular space is of much interest as a molecular target for
many antidepressants, including the tricyclics such as imipramine and
amitriptyline, as well as the more recently introduced
serotonin-selective reuptake inhibitors such as citalopram, fluoxetine,
and paroxetine. SERTs also are targets for substances of abuse
including cocaine and addictive amphetamines such as
3,4-methylenedioxy-methamphetamine (MDMA, "ecstasy") (3-5). Rodent
(4-6), guinea pig (7), ovine (8), human (9), and Drosophila
melanogaster (10, 11) SERTs have been cloned with characterization
in heterologous expression systems confirming pharmacological
properties similar to native SERTs. All SERTs are members of the larger
Na+/Cl cotransporter gene family first
established with the cloning of the
-aminobutyric acid (12) and
norepinephrine (13) transporters. Like other members of this gene
family (14), the SERT amino acid sequence predicts 12 TMDs, a large
extracellular loop between TMD III and TMD IV with multiple sites for
N-linked glycosylation, and multiple consensus sequences in
the intracellular domains for possible phosphorylation by protein
kinases. Although inconsistencies have been reported for the 12 TMD
model (15, 16), the predicted 12 TMD topology for SERT has been
supported by biochemical and mutagenesis studies. For example, the
location of a cysteine residue in the proposed extracellular loop
between predicted TMDs I and II of SERT has been confirmed (17), and
the extracellular locations of other putative domains including the
large N-glycosylated loop between TMDs III and IV have been
verified for both SERT (18, 19) and NET (20-22) using
transporter-specific antibodies. Further studies are underway to
validate other aspects of the predicted SERT model.
Knowledge of SERT primary structure provides opportunities for probing structural determinants of SERT function and may contribute to a better understanding of ligand recognition and structure for all of the biogenic amine transporters. Thus, we have focused on identifying which components of the proposed 12 TMD structure of SERT participate in forming the binding pocket for substrates and antagonists. In this regard, conserved amino acid sequences are thought to be responsible for common properties among homologous structures, whereas sequence differences can reveal areas responsible for the unique properties of a protein. Mutagenesis and chimera studies with the catecholamine transporters, NET and DAT, have suggested that the major determinants of the various functions of these transport proteins can be attributed to discrete domains of the proteins structure (23-27). A useful strategy for identifying ligand binding domains of G protein-coupled receptors and ion channels has been the exploitation of species differences in antagonist potencies, in which cross-species chimeras and site-directed mutagenesis localize domains and residues critical for ligand interaction (28-30). Previously, such cross-species comparisons of SERT demonstrated differences in tricyclic antidepressant and amphetamine potency between rat and human SERTs localized by chimera studies to carboxyl-terminal regions of the protein (31). Follow-up studies using site-directed mutagenesis identified a single amino acid in TMD XII responsible for the species selectivity of the tricyclic antidepressants (32).
We reasoned that analyses of pharmacologic differences evident between more distantly related SERTs, such as Drosophila and human SERTs (9-11), might allow for additional insights into key structural domains for ligand recognition. dSERT is a protein of 622 amino acids with the same proposed 12 TMD topology modeled for mammalian SERTs. The sequences of the dSERT and hSERT share an overall amino acid identity of 49% which increases to 58% when considering only the TMDs (10). In order to compare the properties of hSERT and dSERT expressed in the same host cells, we have performed 5-HT uptake assays in HeLa cells transiently transfected with either SERT species homolog. These studies confirmed that hSERT and dSERT have markedly different sensitivities to some, but not all, biogenic amine transporter antagonists, although 5-HT transport kinetics are essentially equivalent. Specifically, most antagonists such as fluoxetine and citalopram were much less potent at dSERT with the one exception being the NET/DAT-selective antagonist mazindol which exhibited greater potency for dSERT as compared with hSERT. Using hSERT/dSERT chimeras and species-scanning mutagenesis, we have tracked the determinant of species-specific recognition of the antagonists mazindol and citalopram to an aromatic residue located in TMD I. Selective effects on the potency of cocaine induced by substitutions at this position support a contribution of TMD I to the binding pocket for phenyltropane antagonists as well. SERT species homologs and mutants also were analyzed using multiple mazindol derivatives, thereby identifying critical functional groups of mazindol likely to participate in TMD I interactions with SERTs. Our findings reveal molecular information regarding potential ligand-protein interactions between mazindol and monoamine transporters and support the role of TMD I in the formation of antagonist-binding sites.
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EXPERIMENTAL PROCEDURES |
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Construction of HA-hSERT
Antibodies targeted to the carboxyl tail of hSERT have been described (19) and could be used for detection of chimeric proteins when a chimera contained the hSERT carboxyl tail (19). Unfortunately, antibodies against the amino-tail of hSERT or any portion of dSERT suitable for immunoblotting do not exist, hindering our ability to perform protein analysis on expression of other chimeras. Therefore, an epitope (YPYDVPDYA) derived from a viral hemagglutinin (HA) protein (33) was added to the amino terminus of the hSERT cDNA for immunochemical detection of the protein using commercial anti-HA antibodies (Babco). The HA epitope was introduced into the parental hSERT using a polymerase chain reaction (PCR) overlap extension method (34, 35) using Vent DNA polymerase for 30 cycles of 94 °C for 1 min, 45 °C for 2 min, and 72 °C for 3 min, with a 10-min extension time at cycles 1 and 30. The oligonucleotide primers (sense, 5'-TATCCATATGATGTTCCAGATTATGCTGAGACGACGCCCTTGAATTC-3'; antisense, 5'-AGCATAATCTGGAACATCATATGGATACATCCTGCTGGTTAGTAAATG-3') that encoded the HA tag also introduced a unique NdeI restriction endonuclease site that allowed for easy identification of HA tag-containing plasmids. The 650-base pair PCR product and the parental hSERT cDNA were digested with XcmI restriction enzyme, and the two fragments were ligated using T4 DNA ligase after gel purification. Dideoxy sequencing (36) of this ligated region confirmed the presence of the HA tag in the proper orientation and confirmed that no other sequence changes were introduced during construction.
Chimera Formation
Human and Drosophila SERT chimeras were generated
using the previously described restriction site-independent method of
chimera formation (37, 38). Briefly, the parental cDNAs, HA/hSERT and dSERT, were subcloned into a tandem tail-to-head construct in the
vector pBluescriptII KS. The tandem insert construct T7 HA/hSERT-dSERT was generated by transferring the HA/hSERT cDNA DraIII-KpnI fragment into the dSERT plasmid
upstream of the dSERT cDNA. The T7 HA/hSERT-dSERT
construct was then digested between the two SERT cDNAs using
StuI and KpnI, and the linearized DNA containing
the two SERT cDNAs was gel-purified. 100-500 ng of purified DNA
was transformed into competent DH5
bacteria and plated on selective
media to obtain colonies containing recircularized plasmids. In a
similar manner, the complimentary tandem plasmid, T7
dSERT-HA/hSERT, was constructed using the
DraIII-XbaI fragment of dSERT inserted upstream
to HA/hSERT cDNA. The tandem construct was then linearized with
NdeI and XbaI and transformed as described previously. Colonies were screened for chimeric DNA by subjecting DNA
to agarose gel electrophoresis immediately following alkaline lysis
(cracking preparation). Colonies with plasmid sizes indicating retention of only a single transporter sized insert were isolated by
miniprep DNA preparations and plasmid DNA diagnostically digested with
restriction enzymes to localize the region of the internal chimeric
switch point. Dideoxy sequencing confirmed the location of the chimera
switch point and established that the junction was formed in-frame with
respect to the coding sequences of the parental transporters. Two
in-frame chimeras were recovered and characterized as follows:
D1-136H137-625 (amino acids 1-136 from dSERT
with remainder from hSERT) and H1-118 D119-627 (amino acids 1-118 from
hSERT with remainder from the Drosophila homolog) (Fig. 1).
An additional chimera H1-449D450-631 was
generated by using complementary restriction enzyme sites found in the
two SERT species variants. Initially, a BclI restriction site was introduced at position 1730 of dSERT using the Chameleon site-directed mutagenesis kit (Stratagene). The new BclI
site does not alter the coding sequence of the dSERT cDNA but
corresponds to the BclI site at position 1409 of the
HA/hSERT cDNA. After digesting the two SERT cDNA constructs
with XmnI and BclI, the 2346-base pair dSERT
fragment and the 2465 HA/hSERT fragment were ligated together to create
the chimera H1-449D450-631 (amino acids
1-449 from hSERT and the remaining amino acids from dSERT) (Fig.
1).
Site-directed Mutagenesis
Point mutations were introduced into the wild-type hSERT or dSERT cDNAs by using of the QuikChange mutagenesis kit (Stratagene). The wild-type hSERT cDNA was used instead of the HA/hSERT cDNA for mutation constructs because the HA epitope was not required for identification of the point mutants, and the properties of the two SERTs were identical. A "species-scanning" mutagenesis strategy was used which consists of changing residues in one species of SERT to the corresponding identity in the other species variant. Oligonucleotides used for the mutagenesis introduced both an amino acid mutation and a unique restriction site to provide for facile screening of mutants. The following mutations were made to introduce a dSERT identity in the hSERT cDNA: in TMD I, S91A, and Y95F, and in TMD II, L119V, T122C, I123L, M124F, A125L, and I130L. In addition, we converted dSERT Phe90 to Tyr and hNET Phe72 to Tyr, the reciprocal mutation of hSERT Y95F. Complementary sense and antisense primers were annealed to the cDNA of interest and extended with Pfu polymerase. Following the QuikChange kit protocol, the nicked, circular strands were digested with DpnI to eliminate the methylated, nonmutant parental strands. The DNA was transformed into XL1-Blue supercompetent Escherichia coli cells (Stratagene), and colonies were screened using PCR primers on either side of the mutation and then digested with the diagnostic restriction enzyme. Once mutant colonies were identified, the plasmid DNA was isolated and sequenced through the mutation-containing region. The fragment containing the mutations in hSERT was isolated by digestion with XbaI and NsiI, gel-purified, and ligated into the Xba/Nsi-digested wild-type hSERT vector. A similar digestion and ligation procedure was performed in the formation of the dSERT F90Y mutant using BsmI and NarI. Ligated DNA was transformed into supercompetent XL1-Blue cells, PCR-screened again for the presence of diagnostic restriction sites, and sequenced before use in 5-HT transport assays.
Transient Expression of SERTs in HeLa Cells
In order to compare directly the pharmacological profiles of
hSERT (9), dSERT (10), as well as chimeric and mutant SERTs, transient
expression of the transporters was achieved using the recombinant
vaccinia virus T7 expression system in HeLa cells (39, 40).
The parental cDNAs, hSERT and dSERT, were previously cloned into
the plasmids pBluescript II KS and pBluescript II
SK+, respectively. In each case, sense RNA is transcribed
by the plasmid-encoded T7 RNA polymerase promoter. HeLa
cells were cultured in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum, 2 mM
L-glutamine, and 1% penicillin/streptomycin at 37 °C in
a humidified 5% CO2 incubator. Cells plated in 24-well culture plates (100,000 cells per well) were infected with recombinant VVT7-3 vaccinia virus encoding T7 RNA
polymerase at 10 plaque-forming units per cell as described (39, 40).
Virus infection of the cells proceeded for 30 min in serum-free
Opti-MEM I containing 55 µM 2-mercaptoethanol at
37 °C. Following virus infection, SERT cDNA constructs were
transfected into virus-infected HeLa cells using liposome-mediated
transfection (Lipofectin reagent) at 100 ng of DNA per well at a ratio
of 1 µg of DNA to 3 µg of Lipofectin (mixed in Opti-MEM
I/
-mercaptoethanol).
[3H]5-HT Transport Assay
At 6 h postinfection, HeLa cells were washed with a
Krebs-Ringer-Hepes (KRH) buffer (120 mM NaCl, 4.7 mM KCl, 2.2 mM CaCl2, 10 mM Hepes, 1.2 mM
KH2PO4, 1.2 mM MgSO4,
pH 7.4). Cells were preincubated for 10 min at 37 °C in KRH
containing 0.18 g/liter D-glucose and then transport assays
with 10 nM [3H]-5-HT, 100 µM
pargyline, and 100 µM L-ascorbic acid were
performed for 10 min at 37 °C. Inhibitors were added in the
preincubation step. Saturation kinetics were determined using
increasing concentrations of [3H]5-HT with the specific
activity diluted to ~0.1 Ci/mmol with unlabeled 5-HT. Uptake was
terminated by three washes with ice-cold KRH buffer. The level of
accumulated [3H]5-HT was determined either by
solubilizing cells in 1% SDS and analysis by liquid scintillation
spectrometry or by solubilizing directly in scintillant (Optiphase
SuperMix) with direct counting of culture plate in a Wallac MicroBeta
plate reader. Nonspecific [3H]5-HT transport was assessed
by parallel transfections with the host plasmid pBluescript SK II()
alone and subtracted from the total counts. Substrate
Km and antagonist Ki values were
derived by nonlinear least-square fits (Kaleidagraph; Synergy Software)
using either the Hill equation for a rectangular hyperbola or the
four-parameter logistic equation with necessary adjustments of
IC50 values for substrate concentration to determine apparent Ki values (41). Experiments were performed in duplicate or triplicate and repeated in 2-3 separate assays. Means
were compared using two-sided Student's t tests (GraphPad InStat for MacIntosh, version 2.03) or one-way analysis of variance (GraphPad PRISM for MacIntosh, version 2.0).
Synthesis of Mazindol Derivatives
Synthesis of mazindane (42), MAZ-10 (43), MAZ-60 (44), MAZ-130 (45), and MAZ-110 (46) has been described previously. General methods: melting points were uncorrected. NMR data for 1H NMR were taken at 300 MHz and 13C NMR at 75.5 MHz. IR spectra were determined using KBr pellets. Mass spectra MS were obtained by a desorption chemical ionization method using ammonia or isobutane as the reagent gas. Elemental analyses for carbon, hydrogen, and nitrogen were within ±0.4% of theory unless noted otherwise. If not otherwise specified chemicals and reagents were obtained from Aldrich. Solvents were of the reagent grade and dried prior to use. Reaction progress and purity of final products were determined on Merck silica gel 60 chromatography plates.
Synthesis of MAZ-85
(6-(p-Chlorophenyl)-2,3,4,6-tetrahydropyrimido[2,1-a]isoindol-3,6-diol)--
A
mixture of 2-(p-chlorobenzoyl)-benzaldehyde (3.68 g, 0.015 mol) and 1,3-diamino-2-propyl alcohol (2.70 g, 0.03 mol) in xylene (100 ml) was stirred and refluxed in a flask equipped with a Dean-Stark tube. After the "water layer" in the side arm remained constant (~6 h), the solvent was removed in vacuo. The resultant
viscous yellow oil was dissolved in i-propyl alcohol (50 mL) and
stirred at room temperature in the presence of air for ~26 h. A white solid was filtered off and crystallized from 90% EtOH to give 2.10 g (44%) of MAZ-85, m.p. 217-218 °C; MS 315 (100, MH+); 1H NMR
(Me2SO-d6) 2.72 (m, 1H, H-4),
3.03 (m, 1H, H-3), 3.34 (m, 2H, H-2), 3.55 (d, 1H, OH), 3.97 (m, 1H,
H-4), 7.16 (m, 1H, H-7), 7.26-7.55 (m, 7H, 6 ArH, OH), 7.65 (m, 1H,
H-10); IR (KBr) cm
1 3421 (OH), 1657 (CN).
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(Eq. 1) |
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Synthesis of MAZ-89
(5-(3-Methylphenyl)-2,3-dihydro-5H-imidazo[2,1-a]isoindol-5-ol)
(47)--
A stirred solution of 2-phenylimidazoline (1.46 g, 0.01 mol)
and TEMED (2.56 g, 0.022 mol) in dry tetrahydrofuran (20 ml) under a
N2 atmosphere was treated dropwise with 2.5 M
n-butyl lithium (8.85 ml, 0.022 mol) over a 0.25-h period at
room temperature. The resultant mixture was stirred for an additional
3 h and then treated dropwise with a solution of
methyl-4-methylbenzoate (2.25 g, 0.015 mol) in tetrahydrofura (15 ml).
After stirring overnight the mixture was cooled in an ice bath, treated
dropwise with saturated NH4Cl solution (3.5 ml), and a
white solid filtered off to give 1.02 g (38.5%) of MAZ-89, m.p.
212-213 °C decomposes (dimethyl formamide/i-propyl alcohol);
1H NMR (Me2SO-d6) 2.55 (s, 3H, CH3),2.95 (t, 1H, H-3), 3.34 (m, 1H, H-3),
4.18 (m, 2H, H-2), 7.06-7.62 (m, 8H, 7 ArH, OH), 7.75 (d, 1H, H-9); IR
(KBr) cm
1 3421 (OH), 1657 (CN); UV (95% EtOH) nm 203.20 (41, 390), 269.20 (6, 521).
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(Eq. 2) |
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Materials
Dulbecco's modified Eagle's medium was purchased from Fisher,
fetal bovine serum from HyClone, and HeLa cells from the American Type
Culture Collection. Trypsin, glutamine, penicillin, streptomycin, Opti-MEM I medium, DH5 bacteria, and Lipofectin were obtained from
Life Technologies, Inc., and cell culture plates were from Falcon/Becton-Dickinson Labware. The plasmids pBluescript II
KS
and SK+ and Chameleon Kit for
double-stranded site-directed mutagenesis were purchased from
Stratagene. Gel purification reagent QIAEX resin and Qiagen plasmid
prep kits were purchased from Qiagen. Magic Minipreps DNA purification
system were purchased from Promega. Dideoxy sequencing with Sequenase
version 2.0 was purchased from U. S. Biochemical Corp.
5-Hydroxy[3H]tryptamine trifluoroacetate
([3H]5-HT) at ~100 Ci/mmol, [35S]dATP at
>3000 Ci/mmol, and Hyperfilm ECL were purchased from Amersham
Pharmacia Biotech. Restriction enzymes and T4 DNA ligase were purchased from New England Biolabs; Ecoscint H and Optiphase SuperMix scintillation fluor was obtained from National Diagnostics and
Wallac, respectively. Vaccinia virus T7 RNA polymerase
(VTF7-3) was provided by Dr. Bernard Moss, NIAID, National
Institutes of Health. Cocaine hydrochloride and mazindol were gifts
from Dr. J. Justice, Emory University; RTI-55 was a gift from Dr. John Boja, National Institute on Drug Abuse; citalopram was provided by H. Lundbeck A/S, Denmark. All other drugs and materials were obtained from
either Sigma or Fisher and were of the highest grade possible.
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RESULTS |
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Evidence That Insertion of HA Epitope Does Not Alter hSERT
Function--
To allow for immunochemical analysis of chimeric SERT
protein, an HA epitope was introduced into the amino-terminal tail of hSERT as described under "Experimental Procedures." Insertion of
the HA epitope into the amino terminus of hSERT did not alter the
function of the protein. [3H]5-HT uptake in HeLa cells
transiently transfected with the HA/hSERT protein showed a
Km of 650 ± 50 nM and
Vmax of 1.1 ± 0.2 × 1017 mol/cell/min which was comparable to that observed
for wild-type hSERT (Table I). Immunoblot
analysis of HA/hSERT protein expressed in HeLa cells revealed a single
band that migrates slightly slower than wild-type hSERT, accountable by
the nine additional amino acids encoded for by the HA epitope (data not
shown). [3H]5-HT uptake by HA/hSERT was inhibited by SERT
antagonists with Ki values essentially identical to
those observed for hSERT (data not shown). Thus, HA/hSERT was used in
all subsequent comparisons with dSERT and for the construction of
cross-species transporter chimeras.
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Pharmacological Comparisons of Human and Drosophila
SERT--
Previous studies showed that the dSERT clone transiently
transfected in COS-7 cells mediates uptake of [3H]5-HT
but not [3H]DA or [3H]NE (10). By using
transiently transfected HeLa cells, we have demonstrated that
dSERT-mediated 5-HT uptake occurs in a
concentration-dependent and saturable manner with an
estimated Km of 1150 ± 350 nM and
a Vmax of 5.0 ± 2.6 × 1018 mol/cell/min, values that are comparable to hSERT
(Table I) (10). [3H]5-HT uptake by dSERT was sensitive to
various biogenic amine transporter antagonists (Table
II) with a rank order of potency distinct
from hSERT as previously noted (10). Although most antagonists,
including the tricyclic antidepressants fluoxetine and citalopram, were
less potent inhibitors of dSERT-mediated 5-HT uptake as compared with
hSERT-mediated uptake, the DAT/NET selective antagonist mazindol was
>10 times more potent at dSERT as compared with hSERT (Table II).
Cocaine showed little if any difference in potency across species
(hSERT Ki = 444 ± 51 nM
versus dSERT Ki = 392 ± 98 nM); however, the cocaine analog RTI-55 was >300-fold less
potent at dSERT (367 ± 125 nM) than hSERT (1.1 ± 0.2 nM) (Table II). The rank order of potency of
antagonists for dSERT was as follows: paroxetine = mazindol > fluoxetine > citalopram > RTI-55 = cocaine
5-HT > imipramine, whereas hSERT was paroxetine > RTI-55 > citalopram = fluoxetine
imipramine > mazindol > cocaine
5-HT.
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Human and Drosophila SERT Chimeras Identify TMDs I-II as Involved
with Mazindol and Citalopram Interactions--
Three chimeras between
HA/hSERT and dSERT were generated to localize the observed differences
in drug potencies to distinct regions of the SERT protein (Fig.
1). A restriction-site independent technique was used in which HA/hSERT and dSERT were combined to produce
two hybrid transporters as described under "Experimental Procedures." Saturation analysis with 5-HT showed that the chimeras transport the amine equivalently to parental HA/hSERT and dSERT (Table
I). Chimera H1-118D119-627 (Fig. 1) is hSERT
up to but not including TMD II (amino acids 1-118) with the remaining
portion contributed by dSERT. This chimera displayed a
Km for 5-HT transport of 1300 ± 210 nM and a Vmax comparable to that of
the parental SERTs (1.5 ± 0.6 × 1017
mol/cell/min). Chimera D1-136H137-625 is
virtually a mirror image of the former chimera being dSERT through TMD
II (amino acids 1-136) and hSERT through the remainder of the protein
(Fig. 1). This chimera displayed a Km for 5-HT
transport of 680 ± 210 nM and
Vmax of 3.7 ± 2.0 × 10
18 mol/cell/min (Table I). Formation of chimera
H1-449D450-631 appears to disrupt 5-HT
transport activity when expressed in either HeLa (Table I) or COS-7
(data not shown) cells. Immunoblots and cell-surface biotinylation
experiments (22, 48) on this nonfunctional chimera verified protein
synthesis at ~60% of levels observed for HA/hSERT with a
corresponding 39% loss in cell-surface expression (data not shown).
Clearly, the modest reductions in cell-surface protein fail to account
for the total lack of detectable transport activity in this
chimera.
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Site-directed Mutagenesis Implicating hSERT Tyr95 as
Responsible for Species Differences for Mazindol and Citalopram
Potency--
By using the species-scanning mutagenesis approach, we
substituted dSERT amino acid identities into each of the eight
divergent positions in hSERT in the TMDs I-II region implicated in the
cross-species chimera studies. Thus the following mutations were
introduced into hSERT: S91A, Y95F, L119V, T122C, I123L, M124F, A125L,
and I130L. Our strategy was to characterize the individual mutants to
determine if a single mutation could increase mazindol potency at hSERT
to that of dSERT and thus avoid a mutant screen based upon a loss of
antagonist potency. Only one hSERT mutation, Y95F, led to an increase
in mazindol potency essentially equivalent to that observed for dSERT
(Fig. 4A). All other hSERT
mutants demonstrated mazindol potencies similar to wild-type hSERT.
Remarkably, the hSERT Y95F mutant also selectively induced a reciprocal
loss of potency for citalopram (Fig. 4B). All other hSERT
mutants demonstrated high affinity interactions with citalopram like
wild-type hSERT. Thus, the single Tyr to Phe switch that occurs between
the human and Drosophila SERTs in TMD I can account for the
observed species differences in potency of both mazindol and
citalopram. Further analysis of the hSERT Y95F mutant revealed no
alterations in either 5-HT transport kinetics (Km = 490 ± 340 nM, Vmax = 1.6 ± 1.1 × 1017 mol/min/cell; mean ± S.D.,
n = 2) or in imipramine or fluoxetine potency (Table
III). If a phenylalanine at position 95 of hSERT alters mazindol and citalopram potencies to dSERT-like
sensitivities, then the complementary mutation in dSERT (F90Y) should
cause a decrease in mazindol potency and an increase in citalopram
potency. Surprisingly, the dSERT F90Y mutant demonstrated essentially
no specific 5-HT transport activity when transiently expressed in HeLa
cells making functional characterizations impractical (data not shown).
Expression of the dSERT F90Y mutant in Xenopus oocytes confirmed this disruption of transporter activity as assessed by both
5-HT uptake experiments as well as analysis of 5-HT-induced currents in
F90Y-expressing oocytes under two-electrode voltage clamp (data not
shown). However, oocyte experiments with dSERT F90Y did reveal a small
substrate-induced current as well as a transporter-associated leak
current suggesting that the mutant protein was synthesized and present
on the cell surface (data not shown). Further confirmation of protein
formation for the dSERT mutant was not possible due to the lack of
antibodies against dSERT.
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Analysis of Mazindol Derivatives to Identify Key Functional Groups
Involved with Species-selective Recognition of Mazindol--
To
develop evidence for a direct transporter-antagonist interaction, we
sought to identify functional groups of mazindol responsible for
interaction with the TMD I aromatic residue of human and
Drosophila SERT. Mazindol has been shown to exist in
solution in a pH-dependent tautomeric equilibrium between
carbinolamine and keto forms of the structure shown in Fig.
5A (50). The keto form is
favored in acidic pH and the carbinolamine in neutral or basic pH. This equilibrium prevents the isolation of the individual enantiomers of
mazindol or the analogs used in this work. Since the transport assays
were carried out at pH 7.4, the carbinolamine form should be
predominant for all compounds. Mazindol derivatives with slight structural modifications were selected to potentially identify important determinants of either absolute potency or rank order of
potency across species (Fig. 5). All derivatives essentially maintained
dSERT versus hSERT selectivity (Table III). All derivatives excluding MAZ-10, which differed from mazindol by substitution of
Cl on the phenyl ring with a Br
, and
mazindane were less potent than mazindol. Derivative MAZ-130, in which
the aromatic group was replaced with a methyl group, maintained species
selectivity, but lost 2-3 magnitudes of overall potency. MAZ-89
replaced the Cl
on the phenyl ring with a methyl group,
whereas MAZ-10 replaced the Cl
with a related halogen,
Br
. Both derivatives maintained species selectivity, but
absolute potency was only lost in derivative MAZ-89. This observation
may indicate the importance of the role of a halogen in this position for high affinity interactions with dSERT. Although derivative MAZ-60
only differs from mazindol by an additional hydroxyl group on the core
phenyl ring, an order of magnitude of absolute potency was lost, and
species selectivity was maintained. This additional hydroxyl group
could be interfering with the ability of mazindol to interact in the
proper orientation for high affinity binding with dSERT. Derivative
MAZ-110, which opened the molecule at the indole portion, lost 2-3
orders of magnitude of potency indicating the possible importance of
two regions interacting simultaneously with a dSERT-binding site that
is disrupted when the molecule is not intact. Derivative MAZ-85 made
the 5-member dinitrogen ring into a 6-member ring as well as adding a
hydroxyl group in the para position of that ring. This derivative lost
2-3 orders of magnitude of potency again indicating there to be
several areas of the molecule that are needed for this high affinity
binding to occur. In general, the hSERT Y95F mutant showed potencies
with all mazindol derivatives similar to the wild-type dSERT with the exception of MAZ-60. Although not statistically different than wild-type dSERT, the potency of MAZ-60 appears to fall between the two
parental SERTs suggesting that the addition of the hydroxyl group on
the phenyl ring of the ligand disrupts the high affinity interactions
associated with a phenylalanine at position 95 in the hSERT
background.
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DISCUSSION |
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By using species-scanning mutagenesis of hSERT and dSERT cDNAs in a common heterologous expression system, we have explored the molecular basis of species differences in the pharmacological profiles of two SERT species homologs. Whereas most SERT antagonists such as the tricyclic antidepressants, fluoxetine and citalopram, were much less potent at inhibiting 5-HT uptake at dSERT as compared with hSERT, the NET/DAT-selective antagonist mazindol showed the reverse species selectivity being more potent at the Drosophila transporter. Differences in antagonist potency between human and Drosophila SERTs are presumably attributable to divergence in their amino acid sequences either through the establishment of different binding conformations of the transporter or the presence or absence of residues involved directly with coordinating protein-ligand interactions. The use of cross-species chimeras formed between hSERT and dSERT suggested that the major determinants of species-specific potencies for most antagonists including paroxetine and fluoxetine lie distal to TMD II, whereas the TMDs I-II region contains primary sites of species discrimination for both mazindol and citalopram, two structurally distinct biogenic amine uptake inhibitors. Both chimeras D1-136H137-625 and H1-118D119-627 transported 5-HT with kinetic properties similar to their parental clones, and their pharmacological profiles generally reflected whichever parental clone contributed the majority of sequence for that chimera. However, this finding did not hold true for mazindol or citalopram. For mazindol, both chimeras exhibited Ki values (16.2 and 9.8 nM) similar to wild-type dSERT (6.6 nM). When inhibited by citalopram, chimera H1-118D119-627 rendered a Ki value for 5-HT uptake similar to the lower potency observed for wild-type dSERT; however, chimera D1-136H137-625 demonstrated an intermediate potency for citalopram as compared with the parental SERTs. Other antagonists as well as 5-HT exhibit unaltered potencies relative to the parental SERTs suggesting both an antagonist-specific decrease in potency for citalopram and a lack of chimera-induced global disruptions in transporter structure. Regardless, the increase in mazindol potency and the decrease in citalopram potency observed for both chimeras suggested a contact site critical for interactions with both antagonists is likely located in TMDs I-II.
A major advantage of cross-species comparisons is that once a region has been identified as involved in a particular function, mutagenesis studies are facilitated by the binary option of candidate residues. As such, only eight residues differ between hSERT and dSERT within TMDs I-II. Thus, we designed site-directed mutagenesis strategies to introduce dSERT identities into the corresponding positions in hSERT seeking to identify a single residue that could be responsible for the differences observed in mazindol and citalopram potency. Although no single hSERT mutation in the TMD II region altered mazindol or citalopram potency, introduction of dSERT identities into the two nonconserved residues identified in TMD I of hSERT revealed Tyr95 as responsible for the species-selective recognition of both mazindol and citalopram. Interestingly, introduction of the hSERT identity (Tyr95) into the corresponding position in dSERT (Phe90) yielded a transporter that lacked detectable 5-HT transport activity when expressed in mammalian cells. This was particularly surprising because the Tyr identity at this position was apparently tolerated in chimera H1-118D119-627. These findings suggest the aromatic hydroxyl of the tyrosine interacts with additional nonconserved residues in hSERT allowing retention of transport activity and suggest that this residue may indeed lie in a region critical to the translocation mechanism for 5-HT. The comparable position to Tyr95 in NET and DAT is occupied by a phenylalanine, like dSERT, and thus this residue may contribute to the high affinity recognition of mazindol by catecholamine transporters compared with the mammalian SERTs as suggested by the hNET F72Y mutant (13, 49, 51). This suggestion also is consistent with NET/DAT chimera studies that localized high affinity mazindol interactions with NET to a region encompassing TMDs I-III (25). Previously, species-scanning mutagenesis has revealed single amino acid residues as responsible for species-specific ligand recognition for transporters (32) and G protein-coupled receptors (29, 52). For example, we have previously shown that a valine to phenylalanine substitution in TMD XII of the rat and human SERTs, respectively, leads to high affinity interactions of tricyclic antidepressants characteristic of hSERT (31, 32). Our present studies further reinforce the power of coupling cross-species pharmacologic studies with molecular approaches to identify ligand-binding sites.
Despite the numerous strengths of cross-species chimera studies, a potential limitation to the method is that transporter chimeras can exhibit potencies for ligands that are intermediate to the parent molecules. As mentioned previously, the Ki value for citalopram at chimera D1-136H137-625 lies between hSERT and dSERT. Interestingly, imipramine also displayed a potency for chimera D1-136H137-625 that did not completely correspond to either parental SERT although the Ki value of chimera for imipramine (48 nM) was much closer to hSERT (8.1 nM) than dSERT (844 nM). Possible interpretations for the fact that citalopram and imipramine potency did not completely follow either parental clone include nonconserved contact sites that may exist on SERTs for citalopram and imipramine, additional site(s) needed for citalopram and imipramine recognition may be located in regions outside of TMDs I-II, or new junctions created by chimera formation may have disrupted the citalopram and imipramine-binding site. Also, it remains unclear as to why chimera H1-118D119-627 possessed dSERT-like potencies for both mazindol and citalopram despite possessing a tyrosine at position 95 like hSERT, but one possible explanation is that chimera formation alters the protein conformation stabilizing the transporter in a high affinity mazindol/low affinity citalopram state by modulating additional ligand contact sites to yield the observed changes in antagonist potencies. One additional problem with chimera studies is that chimeric junctions formed in regions critical for substrate translocation can disrupt transport activity (37) as was the case with chimera H1-449D450-631 whose junction point lies between TMDs VIII and IX, a region implicated in substrate translocation by NET/DAT chimeras (26, 27). Despite these potential caveats, cross-species analyses as demonstrated by the present studies can reveal important new structural information regarding transporter function and ligand recognition.
If the aromatic residue in TMD I interacts directly with mazindol, aspects of the molecular structure of mazindol that participate in these interactions are conceivably identifiable by using mazindol analogs. Mazindol itself resembles the indole structure of 5-HT with modifications that restrict high affinity interactions with the mammalian SERTs, thus making mazindol more selective for NET, DAT, and dSERT. Previous structure-activity studies have identified five main portions of the mazindol structure that contribute significant binding energy as follows: the two aromatic rings, the ring hydroxyl, the isoindole ring, and the imine nitrogen (43). Furthermore, modeling studies have highlighted the importance of the orientation of the two aromatic rings of mazindol for high affinity interactions with DAT and NET (53). Characterization of the mazindol analogs at human and Drosophila SERTs revealed that the common core region of mazindol appears to support selectivity of most mazindol derivatives for dSERT versus hSERT. Alterations to substituents on either phenyl ring or removal of the free phenyl side chain caused the greatest loss of potency confirming these aromatic groups as major contributors of binding energy for mazindol. Interestingly, the removal of the hydroxyl group from the heterocyclic ring core structure to yield mazindane led to an increase in potency at hSERT suggesting that a hydrogen bond involving this hydroxyl may disrupt interactions with the human transporter; however, since the modest (3-fold) species preference still noted for mazindane between hSERT and dSERT was eliminated in the hSERT Y95F mutation, an additional negative interaction exists which contributes to mazindol hSERT potency. Thus, one possible molecular mechanism for the loss of potency for hSERT for mazindol based upon the present hSERT Y95F mutant data is that the hydroxyl group of mazindol forms a hydrogen bond with the hydroxyl group of Tyr95 altering the orientation of the aromatic rings of mazindol needed for high affinity interactions. Disruption of this hydrogen bond by either removing the hydroxyl group on mazindol (i.e. mazindane) or on the transporter (hSERT Y95F, wild-type dSERT, NET, or DAT) allows the aromatic rings to interact in a high affinity conformation. Molecular modeling studies (MacSpartan, Wavefunction, Inc.) show that the ether oxygen of citalopram can be aligned with the heterocyclic hydroxyl group of mazindol (Fig. 5B) and hence also could form a hydrogen bond with Tyr95, but in this case stabilizing high affinity interactions with citalopram. Further tests of this hypothesis are limited by the lack of readily available citalopram analogs. The lack of a hydroxyl group on this residue in DAT or NET could contribute to the lower potency observed for citalopram at these transporters. Indeed, the hNET F72Y mutant, which possesses the phenolic hydroxyl in this position, demonstrated increased potency for citalopram as compared with wild-type hNET. Another possible explanation for the increased potency of mazindane is that removal of the hydroxyl group on the ring core allows for freer rotation of the nitrogen-containing indole ring that has been proposed to be protonated in the active "keto" form of mazindol (50, 53). This could promote interactions between the protonated amine group and the aspartate at position 98 that we have implicated in direct interactions with the amine group of 5-HT (see below).
The differing rank order of potency of antagonists for hSERT and dSERT, combined with close conservation of substrate affinity for the species homologs, suggests multiple ligand-specific contact sites exist on SERT. This hypothesis has been supported by our previous studies performed on rat/human SERT chimeras in which contact sites specific for tricyclic antidepressants and d-amphetamine were localized to the region in or near putative TMD XII of SERT (31, 32). Furthermore, identification of the hSERT Y95F mutant as being responsible for interactions with mazindol and citalopram does not imply that only one contact site exists for these ligands but simply that a contact site important for high affinity interactions of mazindol with dSERT and citalopram with hSERT exists in TMD I. With regard to contact sites distinct for other transporter ligands, two compounds demonstrated equivalent potency for both species implying that shared or convergent domains are responsible for their interactions with SERT. For example, the Km values for 5-HT transport are essentially identical for both cloned human (Ref. 9 and present study) and rat (4, 5) SERTs, thus suggesting no apparent differences in substrate recognition sites when 5-HT is used. Although hSERT and dSERT also exhibited equal potencies for cocaine, the hSERT Y95F mutant showed an approximately 5-fold increase in cocaine potency implicating this region as containing important determinants of cocaine recognition. A role for TMD I in the formation of the cocaine-binding site has previously been implicated by both mutation studies on DAT (24) as well as NET/DAT chimeras (25). Whereas cocaine displayed no species selectivity for any of the SERT homologs, the high affinity cocaine analog RTI-55 was much less potent at dSERT as compared with hSERT, suggesting that high affinity cocaine analogs recruit additional sites of interaction with hSERT that, based upon our chimera data, are distal to TMD II and are absent or negated in dSERT. This is consistent with the photoaffinity labeled cocaine analog RTI-82 labeling DAT in the region distal to TMD IV (54). Because the high affinity cocaine analogs like RTI-55 apparently possess contact sites on the transporter distinct from the parent compound cocaine, the sole use of these high affinity analogs in binding assays or drug screens designed to identify cocaine antagonists for therapeutic use in the treatment of cocaine addiction could identify compounds that target only analog-specific sites and that have no effect on cocaine itself.
Information obtained from our work with cross-species SERT chimeras
(31, 32) as well as NET/DAT chimeras (25-27) provide a basis for a
general model relating transporter structure to function. In general,
there are distinct regions of the transporters that have primary
influence over substrate recognition, substrate translocation, and
antagonist affinity. TMDs I-III appear to contribute primary
determinants for substrate recognition and modulatory sites for
recognition of some antagonists. Previous mutagenesis studies of the
dopamine transporter support this model showing a conserved aspartate
79 located in TMD I as being critical for dopamine uptake and cocaine
analog recognition (24). We have recently examined the corresponding
residue in rat SERT TMD I, aspartate 98 (Asp98), and we
obtained evidence that this residue may indeed serve as a direct
contact site for substrates and some antagonists, as well as line the
permeation pathway for ions through the
transporter.2 Interestingly,
an -helical model of TMD I would place Tyr95 one helical
turn away from Asp98, thus positioned on the same side of
the helix and lining a potential path for substrates. Introduction of a
tyrosine at position 90 of dSERT disrupts 5-HT transport further,
suggesting that this region may be important for translocation of
substrates, and even subtle perturbations within the context of dSERT
can disrupt transport activity. Thus, the residues predicted to line
this face of the helix may indeed compose a part of both the
antagonist-binding pocket and substrate permeation pathway. Currently,
we are using cysteine scanning mutagenesis to explore further the
participation of TMD I in recognition of substrates and antagonists. In
addition to implicating a role for TMDs I-III, previous transporter
chimera studies suggest that TMDs V-VIII are involved in translocation mechanisms and primary influences for antagonist affinity, and TMDs
IX-XII contain modulatory sites for both substrate and antagonist affinity. The disruption of transport activity observed for chimera H1-449D450-631, whose junction point lies
between TMDs VIII and IX, is consistent with the importance of these
domains for 5-HT translocation. Interestingly, tricyclic antidepressant
and cocaine potency can be increased by mutations in TMD XII (32),
consistent with a proximity of carboxyl-terminal domains to
antagonist-binding sites. In the present study, we present evidence
that cocaine can be influenced by the hSERT Y95F mutation in TMD I. If
TMDs I and XII can positively influence cocaine potency, then an
argument could be made that TMDs I and XII may lie exist in relatively
close proximity to one another within the membrane. In the absence of
crystal structure data, these chimera and mutant studies will continue
to guide modeling studies aimed at understanding the arrangement of
helix packing, mechanism of substrate translocation, as well as the molecular basis for inhibition of transporter activity.
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ACKNOWLEDGEMENTS |
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We thank Kimberly Moore and Fariborz Rakhshan for technical assistance and Christina Petersen for testing the dSERT F90Y mutant in Xenopus oocytes.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants R01-DA07390 (to R. D. B.), F32-DA05679 (to E. L. B.), and R03-DA08516-02 (to W. J. H.).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
Pharmacology and Center for Molecular Neuroscience, Vanderbilt
University School of Medicine, 412 MRB II, Nashville, TN 37232-6600. E-mail: randy.blakely{at}mcmail.vanderbilt.edu.
1 The abbreviations used are: 5-HT, 5-hydroxytryptamine or serotonin; SERT, serotonin transporter; TMDs, transmembrane domains; NET, norepinephrine transporter; DAT, dopamine transporter; dSERT, Drosophila SERT; hSERT, human SERT; HA, hemagglutinin; PCR, polymerase chain reaction; MS, mass spectra; TEMED, N,N,N',N'-tetramethylethylenediamine.
2 E. L. Barker and R. D. Blakely, manuscript in preparation.
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REFERENCES |
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