High Affinity Recognition of Serotonin Transporter Antagonists Defined by Species-scanning Mutagenesis
AN AROMATIC RESIDUE IN TRANSMEMBRANE DOMAIN I DICTATES SPECIES-SELECTIVE RECOGNITION OF CITALOPRAM AND MAZINDOL*

Eric L. BarkerDagger , Melody A. Perlman, Erika M. Adkins, William J. Houlihan§, Zdenek B. Pristupa, Hyman B. Niznik, and Randy D. Blakelyparallel

From the Department of Pharmacology and Center for Molecular Neuroscience, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6600, the Dagger  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

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 gamma -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.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

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 DH5alpha 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/beta -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) delta  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).
<UP>C<SUB>17</SUB>H<SUB>15</SUB>ClN<SUB>2</SUB>O<SUB>2</SUB></UP> (Eq. 1)
<AR><R><C><UP>Calculated:</UP></C><C><UP>C</UP> 64.87</C><C><UP>H</UP> 4.80</C><C><UP>N</UP> 8.90</C></R><R><C><UP>Found:</UP></C><C><UP>C</UP> 64.75</C><C><UP>H</UP> 4.95</C><C><UP>N</UP> 8.71</C></R></AR>

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) delta  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).
<UP>C<SUB>17</SUB>H<SUB>16</SUB>N<SUB>2</SUB>O</UP> (Eq. 2)
<AR><R><C><UP>Calculated:</UP></C><C><UP>C</UP> 77.25</C><C><UP>H</UP> 6.10</C><C><UP>N</UP> 10.60</C></R><R><C><UP>Found:</UP></C><C><UP>C</UP> 77.31</C><C><UP>H</UP> 6.22</C><C><UP>N</UP> 10.58</C></R></AR>

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, DH5alpha 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.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

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 × 10-17 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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Table I
5-HT uptake kinetics for wild-type and chimeric SERTs
Km and Vmax values for [3H]5-HT uptake in HeLa cells transiently transfected with the cloned HA/human (HA/hSERT), Drosophila (dSERT), or chimeric SERTs are shown. Saturation analysis for each clone was performed using increasing concentrations of [3H]5-HT with the specific activity diluted to ~0.1 Ci/mmol with unlabeled 5-HT as described under "Experimental Procedures." Data represent means ± S.E. from results of three experiments performed in duplicate. Means were compared to Drosophila SERT values using a two-sided Student's t test; no statistical differences in mean values were detected.

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 × 10-18 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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Table II
Pharmacologic characterization of chimeric human and Drosophila SERTs
Ki values for the inhibition by various compounds of [3H]5-HT uptake in HeLa cells transiently transfected with the cloned human (HA/hSERT), Drosophila (dSERT), or chimeric SERTs. Chimeric SERTs were generated as described under "Experimental Procedures" and illustrated in Fig. 1. Data represents means ± S.E. from results of three experiments performed in triplicate.

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 × 10-17 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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Fig. 1.   Models describing human and Drosophila SERT chimeras. Three chimeras were formed between hSERT and dSERTs as described under "Experimental Procedures." Chimera junction points are indicated with hSERT and dSERT domains represented by white and black, respectively. Chimera D1-136H137-625 contains dSERT sequence from amino acids 1-136 and hSERT amino acids 137-625. Chimera H1-118D119-627 contains hSERT sequence from amino acids 1-118 and dSERT amino acids 119-627. Chimera H1-449D450-631, which was non-functional, contains hSERT sequence from amino acids 1-449 and dSERT amino acids 450-631. Those chimeras containing hSERT sequence at the amino-terminal end have the HA epitope inserted as indicated.

Antagonist potencies for the two functional chimeras were determined by uptake assays performed in parallel in HeLa cells transiently expressing wild-type HA/hSERT, dSERT, or the chimeric proteins. In general, we found that antagonist potency was largely dictated by domains distal to TMD II (Table II). For example, the Ki value for RTI-55 at HA/hSERT was 1.1 ± 0.2 nM, and the chimera that contained mostly hSERT (D1-136H137-625) had a similar Ki of 0.5 ± 0.3 nM. Likewise, the Ki for RTI-55 at dSERT was 367 ± 125 nM, and the chimera that was mostly dSERT (H1-118D119-627) was comparable with a Ki of 376 ± 51 nM. This finding held true for most antagonists except mazindol and citalopram (Table II; Figs. 2 and 3). Mazindol exhibited a Ki for HA/hSERT of 84 ± 10 nM and 6.6 ± 1.3 nM for dSERT; interestingly both chimeras had mazindol Ki values similar to wild-type dSERT at 16.2 ± 6.8 and 9.8 ± 3.9 nM (Fig. 2). With regard to citalopram, both chimeras demonstrated potencies similar to wild-type dSERT (Fig. 3) although a complete shift to dSERT-like potency was not observed for chimera D1-136H137-625 (Table II). These data suggested that the region encompassing TMDs I-II of dSERT imparts both the more potent interactions of mazindol as well as the loss of potency for citalopram.


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Fig. 2.   Mazindol inhibition of 5-HT uptake at parental and chimeric SERTs. [3H]5-HT uptake assays were performed on transiently transfected HeLa cells, as described under "Experimental Procedures," with increasing concentrations of mazindol added 10 min before the addition of 10 nM [3H]5-HT. A, evaluation of mazindol potency for HA/hSERT (bullet , solid line), dSERT (black-square, solid line), and chimera H1-118D119-627 (black-triangle, dashed line). B, evaluation of mazindol potency for HA/hSERT (bullet , solid line), dSERT (black-square, solid line), and chimera D1-136H137-625 (black-triangle, dashed line). Mean nonspecific uptake was determined in HeLa cells transfected with the parent vector pBluescript SK II(-) alone and subtracted from total uptake to yield specific 5-HT uptake. Data were plotted as percentage of specific 5-HT uptake. All data plotted represent means ± S.E. of triplicate determinations and are representative of three separate experiments. Mean Ki values are represented in Table II.


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Fig. 3.   Citalopram inhibition of 5-HT uptake at parental and chimeric SERTs. [3H]5-HT uptake assays were performed on transiently transfected HeLa cells, as described under "Experimental Procedures," with increasing concentrations of citalopram added 10 min before the addition of 10 nM [3H]5-HT. A, evaluation of citalopram potency for HA/hSERT (bullet , solid line), dSERT (black-square, solid line), and chimera H1-118D119-627 (black-triangle, dashed line). B, evaluation of citalopram potency for HA/hSERT (bullet , solid line), dSERT (black-square, solid line), and chimera D1-136H137-625 (black-triangle, dashed line). Mean nonspecific uptake was determined in HeLa cells transfected with the parent vector pBluescript SK II(-) alone and subtracted from total uptake to yield specific 5-HT uptake. Data were plotted as percentage of specific 5-HT uptake. All data plotted represent means ± S.E. of triplicate determinations and are representative of three separate experiments. Mean Ki values are represented in Table II.

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 × 10-17 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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Fig. 4.   Pharmacological characterization of hSERT point mutants. All mutations were introduced into hSERT to substitute the identity of the equivalent position from dSERT, as described under "Experimental Procedures." A, mazindol inhibition of 5-HT uptake comparing hSERT mutants with wild-type hSERT and dSERT. B, citalopram inhibition of 5-HT uptake comparing hSERT mutants with wild-type hSERT and dSERT. In both graphs, the hSERT Y95F mutant is indicated by the dashed line. [3H]5-HT uptake assays were performed on transiently transfected HeLa cells, as described under "Experimental Procedures," with increasing concentrations of antagonist added 10 min before the addition of 10 nM [3H]5-HT. Mean nonspecific uptake was determined in HeLa cells transfected with the parent vector pBluescript SK II(-) alone and subtracted from total uptake to yield specific 5-HT uptake. Data were plotted as percentage of specific 5-HT uptake. All data plotted represent means of triplicate determinations and are representative of 2-3 separate experiments. Apparent Ki values (in nM) for mazindol at the hSERT mutants were as follows: S91A, 62 ± 23; Y95F, 12 ± 0.6; L119V, 101 ± 27; T122C, 156 ± 34; I123L, 103 ± 27; M124F, 98 ± 10; A125L, 90 ± 24; I130L, 133 ± 8. Apparent Ki values (in nM) for citalopram at the hSERT mutants were as follows: S91A, 6.0 ± 1.0; Y95F, 21 ± 2.5; L119V, 2.3 ± 0.1; T122C, 3.0 ± 0.7; I123L, 2.5 ± 0.3; M124F, 3.0 ± 0.8; A125L, 1.9 ± 0.3; I130L, 1.6 ± 0.2.

                              
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Table III
Potency comparisons of antagonists and mazindol derivatives for SERTs and human SERT Y95F mutant
Apparent Ki values for the inhibition of [3H]5-HT uptake by various antagonists or mazindol derivatives were obtained by parallel transfections with the cloned human (hSERT), Drosophila (dSERT), or human SERT Y95F mutant in HeLa cells as described under "Experimental Procedures," and thus values vary slightly from those in Table II. Mazindol derivative structures are illustrated in Fig. 5. Data represent means ± S.E. from results of three experiments performed in triplicate.

In the absence of the dSERT F90Y data, we attempted to further verify the role of the aromatic residue in TMD I in ligand recognition by extending our finding to another member of the monoamine transporter family. Both NET and DAT also possess a Phe at the corresponding position in TMD I similar to dSERT, thus the complementary mutation to hSERT Y95F was constructed in hNET (F72Y). Similar to the dSERT F90Y mutant, hNET F72Y should cause a decrease in mazindol potency and an increase in citalopram potency. hNET F72Y transported [3H]dopamine although the transport activity of the mutant was reduced approximately 50% as compared with wild-type hNET (data not shown). Consistent with the hSERT Y95F data, hNET F72Y demonstrated a loss of potency for mazindol (Ki values for [3H]dopamine uptake, wild-type hNET 0.32 ± 0.19 nM, hNET F72Y 7.0 ± 1.1 nM) and an increase in citalopram potency (Ki values for [3H]dopamine uptake, wild-type hNET 17.8 ± 7.1 µM, hNET F72Y 2.5 ± 0.8 µM) confirming the role of the aromatic residue in ligand recognition.

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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Fig. 5.   A, chemical structures of citalopram, mazindol, and mazindol derivatives used in cross-species structure-activity relationship studies. Mazindol derivatives synthesized as described under "Experimental Procedures." B, alignment of citalopram (left) and mazindol (right) structures with dashed line indicating spatial positioning of citalopram's ether oxygen and mazindol's hydroxyl group.

Perhaps the most revealing data were observed with mazindane, the derivative that lacks a hydroxyl group on the heterocyclic nucleus. Mazindane was the only derivative that demonstrated increased potency for the wild-type hSERT as compared with mazindol (Table III). In fact, removal of the hydroxyl group on the ligand to form mazindane led to an increase in potency equivalent to that observed with the hSERT Y95F mutant tested against mazindol. The Y95F mutant differs from wild-type hSERT only by the removal of the hydroxyl group on the aromatic side chain at position 95. Thus, removal of the hydroxyl group either on the ligand (mazindane) or the protein (Y95F) led to an increased potency for mazindol suggesting that these hydroxyl groups may interact negatively to dictate the lower affinity species-selective recognition of mazindol by hSERT. Further support for the negative role of the hydroxyl groups comes from the fact that mazindane and mazindol were equipotent at wild-type dSERT which normally lacks a hydroxyl group at this position (Phe90).

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha -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.

    ACKNOWLEDGEMENTS

We thank Kimberly Moore and Fariborz Rakhshan for technical assistance and Christina Petersen for testing the dSERT F90Y mutant in Xenopus oocytes.

    FOOTNOTES

* 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.

parallel 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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Top
Abstract
Introduction
Procedures
Results
Discussion
References

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