Báo cáo khoa học: A cocaine insensitive chimeric insect serotonin transporter reveals domains critical for cocaine interaction ppt

A cocaine insensitive chimeric insect serotonin transporter reveals domains critical for cocaine interaction Sumandeep K. Sandhu 1,2 , Linda S. Ross 2 and Sarjeet S. Gill 1,2 1 Environmental Toxicology Graduate Program and 2 Department of Cell Biology and Neuroscience, University of California, Riverside, USA Serotonin transporters are key target sites for clinical drugs and psychostimulants, such as fluoxetine and cocaine. Molecular cloning of a serotonin transporter from the cen- tral nervous system of the insect Manduca sexta enabled us to define domains that affect antagonist action, particularly cocaine. This insect serotonin transporter transiently expressed in CV-1 monkey kidney cells exhibits saturable, high affinity Na + and Cl – dependent serotonin uptake, with estimated K m and V max values of 436 ± 19 n M and 3.8 ± 0.6 · 10 )18 molÆcellÆmin )1 , respectively. The Manduca high affinity Na + /Cl – dependent transporter shares 53% and 74% amino acid identity with the human and fruit fly serotonin transporters, respectively. However, in contrast to serotonin transporters from these two latter species, the Manduca transporter is inhibited poorly by fluoxetine (IC 50 ¼ 1.23 l M ) and cocaine (IC 50 ¼ 12.89 l M ). To delineate domains and residues that could play a role in cocaine interaction, the human serotonin transporter was mutated to incorporate unique amino acid substitutions, detected in the Manduca homologue. We identified a domain in extracellular loop 2 (amino acids 148–152), which, when inserted into the human transporter, results in decreased cocaine sensitivity of the latter (IC 50 ¼ 1.54 l M ). We also constructed a number of chimeras between the human and Manduca serotonin transporters (hSERT and MasSERT, respectively). The chimera, hSERT1–146/MasSERT106– 587, which involved N-terminal swaps including trans- membrane domains (TMDs) 1 and 2, was remarkably insensitive to cocaine (IC 50 ¼ 180 l M ) compared to the human (IC 50 ¼ 0.431 l M )andManduca serotonin trans- porters. The chimera MasSERT1–67/hSERT109–630, which involved only the TMD1 swap, showed greater sen- sitivity to cocaine (IC 50 ¼ 0.225 l M ) than the human transporter. Both chimeras showed twofold higher serotonin transport affinity compared to human and Manduca sero- tonin transporters. Our results show TMD1 and TMD2 affect the apparent substrate transport and antagonist sen- sitivity by possibly providing unique conformations to the transporter. The availability of these chimeras facilitates elucidation of specific amino acids involved in interactions with cocaine. Keywords: serotonin; cocaine; antidepressants; transporter; Manduca. Among all the neurotransmitters, serotonin (also known as 5-hydroxytryptamine) remains historically the most inti- mately involved with neuropsychopharmacology. There is ample evidence that the serotonin system modulates a multitude of brain functions including sleep, mood, cogni- tion, sensory perception, motor activity, temperature regu- lation, nociception, appetite, sexual behavior and hormonal secretion. Disturbances in regulation of this system are associated with severe behavioral malfunctions such as depression, obsessive–compulsive disorder, and possibly panic disorder, eating disorders, obesity and alcoholism [1,2]. The major mechanism by which serotonin action in the synaptic cleft is terminated is by its removal back into presynaptic nerve terminal via an uptake mechanism involving specific membrane transporters. Widely pre- scribed antidepressant drugs like Prozac TM (fluoxetine) and Zoloft TM , which selectively inhibit this uptake, cause a profound increase in the concentration of serotonin at postsynaptic receptors and are used currently to treat various psychiatric disorders. Serotonin transporters have been cloned and functionally characterized from a number of organisms including human, rat, mouse and fruit fly [3–7]. These transporters belong to a high affinity Na + /Cl – -dependent plasma- membrane transporters super-family. The monoamine family of transporters that includes serotonin, dopamine and norepinephrine transporters (SERTs, DATs and NETs, respectively) share a high amino acid homology and display very distinct pharmacologies. These transporters are targets for the development of novel drugs. Consequently, the analyses of structural and functional features of these transporters have captivated the interest of many researchers [8–16]. Cloning of species variants and their comparative pharmacological studies Correspondence to S. S. Gill, Department of Cell Biology and Neuroscience, University of California, Riverside, CA 92521, USA. Fax: +1 909 787 3087, Tel.: +1 909 787 4621, E-mail: Sarjeet.gill@ucr.edu Abbreviations: TMD, transmembrane domain; EL, extracellular loop; IL, intracellular loop; GABA, c-amino butyric acid; SERT, serotonin transporter; NET, norepinephrine transporter; DAT, dopamine transporter Note: The sequence reported in this paper has been deposited in the GenBank database (accession no. AF384164). (Received 2 April 2002, revised 12 June 2002, accepted 20 June 2002) Eur. J. Biochem. 269, 3934–3944 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03084.x have made possible preliminary insights into the activity of these transporters [10,11,17,18], thereby providing further insights into designing more selective and hence safer pharmacotherapeutics. As with vertebrates, serotonin has a well-defined role as a neurotransmitter and neuromodulator in invertebrates [19]. The benefits of studying insect transporters are numerous, not only providing potential targets for new insecticide design, but also providing major insights into structure and function analyses of these proteins. In spite of being evolutionarily distant, vertebrate and invertebrate trans- porters show significant similarity at the primary protein sequence level, and yet are very distinct functionally [6,7,20]. For example, the GABA (c-amino butyric acid) transporter from Manduca sexta [20] possesses 58% identity to mam- malian GABA transporter GAT1 and yet displays very different sensitivities to classic GABA uptake inhibitors. We report here the molecular cloning and functional characterization of the first lepidopteran serotonin trans- porter (MasSERT) from the CNS of the tobacco hornworm Manduca sexta. In comparison to mammalian homologues, MasSERT has low sensitivity to cocaine and fluoxetine. Employing site-directed mutagenesis on the human SERT (hSERT), and chimeras between MasSERT and hSERT, this study also provides additional information regarding cocaine recognition, possibly by governing conformational changes in the transporter. We anticipate that cloning of MasSERT and the availability of hSERT/MasSERT chi- meras will contribute to ongoing efforts of many researchers in understanding the mechanism of action of psycho- stimulants and antidepressants at the molecular level. EXPERIMENTAL PROCEDURES Materials Vaccinia virus VTF-7 )3 was purified [21] using a crude stock obtained from Dr Bernard Moss (National Institute of Health, Bethesda, MD, USA). hSERT was kindly provided by H. Lester (California Institute of Technology, Pasadena, CA, USA). cDNA library screening The full length MasSERT clone was isolated from a size- selected (> 2 kb) cDNA library from the CNS of 5th instar, day 3 larvae of M. sexta. The cDNA library was constructed in pSPORT1 (Life Technologies, Gaithersburg, MD, USA) as described previously [20]. The cDNA library was screened by the ÔLimited growthÕ PCR method [22]. Screening was carried out by nested PCR using pSPORT1- specific and MasSERT-specific oligonucleotide primers, which were based on a partial MasSERT PCR product (654 bp) obtained from Manduca sexta CNS. The partial PCR product was isolated by using synthetic degenerate primers derived from short stretches of highly conserved amino acid residues from the first (NVWRFPY) and sixth (WIDAATQ) transmembrane domains of human norepi- nephrine transporter (hNET) and rat GABA transporters (rGAT1) as described previously [20]. A full length MasSERT cDNA was obtained subsequently, and sequenced in both directions by the dideoxy chain termin- ation method using an automated sequencer (Applied Biosystems Inc.). Analysis of the nucleotide and deduced amino acid sequence was performed using the Lasergene software and programs from the Genetics Computer Group. Sequence homology searches were done using BLAST . Expression construct To express MasSERT in CV-1 cells, a NcoI/FseIfragment containing the MasSERT ORF was cloned into an expres- sion vector pTM1 [23]. Similarly, hSERT was cloned into the pTM1 vector. Mutagenesis Comparison of MasSERT and hSERT sequences identified a number of unique amino acid differences. Specific point mutations were introduced into hSERT cDNA to convert these positions to give amino acids corresponding to the homologous sequences in MasSERT. Mutations performed were Y134F, YM134–135FL, 189 LA, 188A/189LA, FT191–192IN, F474Y, F515V, F551V (underlined residues indicate amino acid insertions). Mutagenesis was performed using the QuikChange TM site-directed mutagenesis kit as described by the manufacturer (Stratagene). For each mutation, two complementary primers which contained the desired mutation were used (Table 1). All mutant clones were sequenced completely to confirm the presence of the mutation and the absence of any errors introduced by the polymerase. Chimera construction Chimeras were made by substituting segments of MasSERT cDNA for homologous segments in hSERT cDNA. Six chimeras illustrated in Fig. 1 were constructed. hSERT cDNA was analysed for the presence of unique restriction enzyme cutting sites to be used as endpoints for the exchange of segments with MasSERT cDNA. Once iden- tified, these restriction sites were engineered into homolog- ous sites of MasSERT cDNA. This was accomplished by synthesizing PCR products of MasSERT cDNA using primers homologous to corresponding regions of MasSERT cDNA, with the addition of the respective restriction site at the 5¢ end of each primer. The KpnIsite was in the vector DNA and therefore common to both MasSERT and hSERT. The primers used for creating the restriction enzyme sites are shown in Table 2. The chimeras were sequenced completely to confirm chimera construction and the absence of any PCR errors. MasSERT antibody MasSERT immunoreactive serum was prepared by immunizing rabbits with an antigenic 14 amino acid peptide from the C-terminus of MasSERT and a N-terminal cysteine that was coupled to keyhole lymphet hemocyanin protein (Imject maleimide activated conjugation kit, Pierce). The MasSERT peptide corresponding to amino acids 571–84 (CQRPEVTSIPPADST) was synthesized by Research Genetics. The crude serum was purified using peptide-coupled columns (Sulfolink kit, Pierce) and specific IgGs were aliquoted and stored at )80 °C until needed. Ó FEBS 2002 Cocaine insensitive chimeric serotonin transporter (Eur. J. Biochem. 269) 3935 Detection of SERT expression For Western blot analysis, total cell membranes were prepared from the CNS of 5th instar M. sexta,and MasSERT cDNA and mock transfected CV-1 cells. Tissues from the CNS were homogenized in 10 vol. of ice-cold 50 m M Tris/HCl (pH 7.4), containing 0.32 M sucrose, 0.5 m M phenylmethanesulfonyl fluoride and protease inhibitor cocktail (Sigma), and centrifuged at 3000 g for 10 min at 4 °C. The supernatant was recentrifuged at 100 000 g for 1 h at 4 °C. The resulting pellet was suspended in 50 m M Tris/HCl (pH 7.4) containing protease inhibitors. MasSERT and mock transfected CV-1 cell membranes were similarly prepared following homogeniza- tion in buffer [50 m M Tris/HCl (pH 7.4), 150 m M NaCl, 1% NP40, 0.5% deoxycholate, 0.1% SDS, 1 m M phenyl- methylsulfonyl fluoride, 1 m M dithiothreitol and protease inhibitor cocktail]. Samples obtained were incubated at 42 °C for 30 min in sodium dodecyl sulfate (SDS) sample buffer (2% SDS; 62 m M Tris/HCl, pH 6.8; 10% glycerol; 0.77% dithiothreitol; 0.01% bromophenol blue) and separ- ated by SDS/PAGE using 3% stacking and 10% resolving gels. The gels were transferred to Immobilon-P membranes (Millipore corporation) by standard procedures [24]. The membranes were then treated with blocking buffer (1 · NaCl/P i ; 13.7 m M NaCl, 0.27 m M KCl, 0.43 m M Na 2 HPO 4 Æ7H 2 O, 0.14 m M KH 2 PO 4 ), 1% bovine serum albumin, 0.05% Tween (20) for 1 h at room temperature on a shaker. The membrane was then incubated overnight at 4 °C with MasSERT antibody, diluted to 1 : 1500 in Table 1. Primers used for generating mutations. Name of Mutant Mutation Primers Y134F Y to F 5¢-GGGGGAATCCCGCTCTTTTTCATGGAGCTCGCACTGG (forward) 5¢-CCAGTGCGAGCTCCATGAAAAAGAGCGGGATTCCCCC (reverse) YM134–135FL M to L 5¢-CCCGCTCTTTTTCCTGGAGCTCGCACTGGGAC (forward) 5¢-GTCCCAGTGCGAGCTCCAGGAAAAAGAGCGGG (reverse) 189 LA Insert L+A 5¢-CGCTATACTACCTCATCTCCTTAGCTTCCTTCACGGACCAGCTGC (forward) 5¢-GCAGCTGGTCCGTGAAGGAAGCTAAGGAGATGAGGTAGTATAGCG (reverse) 188 A/189LA Insert A 5¢-GCGCTATACTACCTCATCGCTTCCTTAGCTTCCTTCACG (forward) 5¢-CGTGAAGGAAGCTAAGGAAGCGATGAGGTAGTATAGCGC (reverse) YM134–135FL FT to IN 5¢-CTACCTCATCTCCTCCATCAACGACCAGCTGCCCTGGAC (forward) 5¢-GTCCAGGGCAGCTGGTCGTTGATGGAGGAGATGAGGTAG (reverse) F474Y F to Y 5¢-TGGTCATCACCTGCTACTTTGGATCCCTGGTCACCCTGAC (forward) 5¢-GTCAGGGTGACCAGGGATCCAAAGTAGCAGGTGATGACCA (reverse) F515V F to V 5¢-GTCGCTGTGTCTTGGGTCTATGGCATCACTCAGTTCTGCAGGG (forward) 5¢-CCCTGCAGAACTGAGTGATGCCATAGACCCAAGACACAGCGAC (reverse) F551V F to V 5¢-GCCCTCTGTTTCTCCTGGTCATCATTTGCAGTTTTCTGATGAGCC (forward) 5¢-GGCTCATCAGAAAACTGCAAATGATGACCAGGAGAAACAGAGGGC (reverse) Fig. 1. Hydrophobicity based models illustra- ting chimeras of MasSERT and hSERT. Six chimeras were constructed as described in Experimental procedures. All replacements represent exchanges with homologous regions of the respective cDNAs. Numbers refer to amino acids sequence of the respective transporters. (A) MasSERT(1-67)/ hSERT(109-630); (B) MasSERT(1-291)/ hSERT(333-630), (C) hSERT(1-108)/ MasSERT(68-291)/hSERT(333-630), (D) hSERT(1-146)/hSERT(106-291)/ hSERT(333-630), (E) hSERT(1-146)/ MasSERT(106-587), (F) hSERT(1-333)/ MasSERT(293-587). Chimeras A and E were functional. 3936 S. K. Sandhu et al.(Eur. J. Biochem. 269) Ó FEBS 2002 blocking buffer. As a control, another membrane contain- ing the same samples was incubated with MasSERT antibody preadsorbed with a 30-fold excess of the MasSERT peptide at 4 °C for 16 h. The immunoreactivity was detected using horseradish peroxidase-coupled donkey anti-(rabbit IgG) secondary Ig, in combination with the ECL detection system (Amersham). In vitro translation In vitro translation of MasSERT was performed using a TNT rabbit reticulocyte lysate kit (Promega) according to the manufacturer’s instructions using [ 35 S]methionine. The translated products were separated by SDS/PAGE as described above. The gel was then stained with Coomassie Brilliant Blue G-250, destained and treated with Entensify (NEN Research Products) as per manufacturer’s instruc- tions and exposed to Hyperfilm TM MP autoradiography film (Amersham Life Science) at )70 °C for 2 days. Expression in CV-1 cells CV-1 cells were maintained following standard procedures [21]. Transient expression of MasSERT and hSERT in CV-1 cells was carried out using recombinant vaccinia virus VTF-7 )3 expression system as described previously [25]. Transport assays Transfected cells were washed with KRTH buffer (10 m M Hepes, pH 7.4, 120 m M NaCl, 4.7 m M KCl, 5 m M TrisHCl, 5m M KH 2 PO 4 ,2m M CaCl 2 ,1.2m M MgSO 4 ,5.6m M glucose, 100 l ML -ascorbate, 100 l M pargyline) [25] and incubated with KRTH buffer for 10 min. For transport studies, cells were then incubated with either [ 3 H]serotonin (10.2 CiÆmmol )1 , Amersham) alone or as a mixture of unlabeled and [ 3 H]serotonin at the concentrations indicated. After a 15 min incubation at room temperature, the cells were washed twice with cold uptake buffer, then solubilized in 1% SDS and the radioactivity of cell extracts was measured by liquid scintillation counting. Inhibition studies were carried out similarly in the presence of varying concentrations of inhibitors and a constant amount of [ 3 H]serotonin (0.05 l M ) for 15 min at room temperature. For studies with cocaine, the esterase inhibitor, phenyl- methylsulfonyl fluoride, was used at a concentration of 100 l M in the uptake assay mixture to prevent degradation of cocaine. All incubations were carried out in duplicate, and experiments were replicated a minimum of three times. Because no difference was observed between nonspecific [ 3 H]serotonin uptake levels in cells transfected with the pTM1 vector containing the M. sexta GABA transporter andcellstransfectedwithnoDNA(mocktransfected),we chose to use mock-transfected cells as a negative control in all our experiments. Nonspecific uptake was defined in parallel wells in duplicates and was subtracted from the total uptake to yield the specific uptake. All data represent specific uptake. The reported K m and IC 50 values were obtained by analysing the data in ORIGIN (MicroCal Inc.) using the Levenberg–Marquardt algorithm and by fitting the curves using the simplex method for non-linear least squares. RESULTS The MasSERT cDNA clone isolated was 3717 bp in length. Based on the consensus start site sequence [26] and the longest ORF, the start methionine is predicted to be at position 151 bp. The ORF is 1764 bp with a deduced amino acid sequence of 587 amino acids, and a 1803 bp 3¢ UTR. The hydrophobicity profile of MasSERT indicates the presence of 12 putative transmembrane domains (TMD), characteristic of this superfamily of proteins. There are two putative N-glycosylation sites, one in the large extracellular loop between TMD 3–4 (amino acids 182–85) and another between TMD 7–8 (amino acids 350–53). The unglycosy- lated translated MasSERT is predicted to have a molecu- larmassof64.8kDa.However,thein vitro translated MasSERT migrates as a 45-kDa band in SDS/PAGE whereas native and heterologously expressed MasSERT migrated as 55 kDa and 90 kDa bands suggesting possible glycosylation of MasSERT and/or existence of dimers, respectively (Fig. 2). An additional 45 kDa band was detec- ted in CV-1 cells transfected with MasSERT cDNA that may account for the unglycosylated form of MasSERT (Fig. 2). Interestingly, no putative protein kinase C phosphoryla- tion sites were predicted in the MasSERT, whereas seven proline-directed protein kinase phosphorylation sites were predicted in the regions in cytoplasmic domains based on the proposed topology modeled for GAT1 [8,9]. Phospho- rylation of hSERT by protein kinase C results in a reduction in the number of transporters on the cell surface [27], implying MasSERT might be regulated differently. The N-terminus also contains three PXXP motifs that could bind SH3 domains [28], which play an important role in signal transduction. Sequence comparisons with other known members of the family indicate that it indeed is a member of Na + /Cl – - dependent neurotransmitter transporter family. Dendro- gram analyses based on sequence alignment with previously reported transporters show that it is most closely related to serotonin transporters (Fig. 3). MasSERT displays 53% and 74% amino acid identity to hSERT [5] and dSERT [6,7], respectively. Transient expression of MasSERT in CV-1 cells showed significant (15–40·) increase in cellular [ 3 H]serotonin levels Table 2. Primers used for generating chimeras. Restriction site Primers KpnI5¢-TTAGAAGGTACCCCATTGTATGGGATC (forward) NdeI5¢-ACGCATATGTTACCAGAATGGAGGCGGT (forward) 5¢-CGCCATATGTAGGGGAATCGCCACACGT (reverse) NsiI5¢-ATAATGCATCACTCTCTGGAAACGGATC (forward) BglII 5¢-TTAGATCTTCTTCTCGCTCGGTCCCGG (forward) 5¢-TTAGATCTGGGATGCCGCGTCAATCC (reverse) FseI5¢-CAGTACGGCCGGCCTCACAGGTT (reverse) Ó FEBS 2002 Cocaine insensitive chimeric serotonin transporter (Eur. J. Biochem. 269) 3937 as compared to the background levels of mock-transfected cells. MasSERT cDNA-transfected cells did not transport radiolabeled GABA, glycine, proline, glutamate or leucine above the background levels (data not shown). MasSERT showed strong substrate specificity for serotonin over other biogenic amine substrates including dopamine, norepineph- rine, octopamine, histamine and tyramine (Fig. 4A, Table 3). At 10 l M concentration, serotonin uptake in MasSERT-transfected CV-1 cells is linear until 20 minutes and reached a plateau at 45 minutes (Fig. 4B). The transport was saturable, which indicated the expression of a carrier- mediated uptake system (Fig. 4C). The Michaelis Menton constant (K m ) for serotonin uptake was 436 ± 19.2 n M (n ¼ 5) with a V max of 3.84 ± 0.61 · 10 )18 molÆcellÆmin )1 . Similar K m values for serotonin transport were determined for cloned and endogenous SERTs [4,5,29]. The high affinity transport of serotonin by MasSERT was dependent on extracellular Na + and Cl – ions (Fig. 4D). Substitution of Na + with choline and substitution of Cl – with acetate or gluconate in the transport buffer totally eliminated serotonin transport. Chloride ion requirement for MasSERT is thus different from dSERT, where Cl – facilitates serotonin transport but is not an absolute requirement [7]. In contrast to mammals, Manduca hemo- lymph is characterized by a high K + )Na + ratio [30]. Several species of phytophagous lepidopteran larvae have been reported to possess K + -coupled transport systems [31]. Based on these observations, we also tested the effect of extracellular K + concentration in driving the serotonin transport by MasSERT. Addition of 120 m M K + to the transport buffer as well as a complete depletion of K + from the transport media did not significantly affect serotonin transport into CV-1 cells (Fig. 4D), suggesting MasSERT is not a K + -coupled transporter. The pharmacological sensitivity of MasSERT to a variety of well characterized serotonin uptake blockers and sub- strates is shown in decreasing rank order of potencies in Table 3, along with the comparison of inhibition constants reported elsewhere for hSERT. Among the antagonists tested in this study, mazindol is the most potent inhibitor of MasSERT with an IC 50 of 153 n M that is similar to the IC 50 determined for hSERT but 40· less potent than that for dSERT and hDAT [6,7,32]. Mazindol is a potent inhibitor of norepinephrine and dopamine transporters with IC 50 values of 1 n M and 11 n M for hNET and hDAT, respect- ively [32,33]. Nomifensine, a selective norepinephrine uptake blocker, was an extremely weak inhibitor of MasSERT, with an IC 50 value of 7.9 l M . However, clomipramine and desipramine, two other tricyclic antidepressants, were better inhibitors with IC 50 values of 370 and 638 n M , respectively. Trypt- amine, which is a substrate for the endogenous platelet SERT, inhibited half maximal MasSERT-mediated sero- tonin uptake at 5.3 l M . Other potential substrates, octop- amine, dopamine, norepinephrine, tyramine, tryptophan and histamine did not inhibit MasSERT at concentrations up to 200 l M . Fluoxetine, an effective selective serotonin uptake inhi- bitor in clinical use, and imipramine, the tertiary amine tricyclic antidepressant drug, were very weak antagonists of MasSERT with IC 50 values of 1.23 l M and 1.76 l M , respectively. Similarly, cocaine, the most actively studied non-selective inhibitor of biogenic amine transporters and a psychostimulant, was unable to inhibit MasSERT-mediated serotonin transport at concentrations sufficient to inhibit half maximal transport of mammalian serotonin, dopamine and norepinephrine transporters. To confirm our findings, we performed inhibition assays with MasSERT and hSERT under identical experimental conditions. Our results showed similar inhibition profile of hSERT-mediated serotonin uptake by cocaine and fluoxetine as reported previously [5,7], with IC 50 values of 431 n M and 4.2 n M respectively, but a very weak inhibition of MasSERT-mediated serotonin uptake (Table 3, Fig. 5). PILEUP analysis of MasSERT with other members of the monoamine transporter family (Fig. 3) indicated that, despite having a high sequence identity to hSERT and dSERT, there are amino acid residues that are unique to MasSERT. Because MasSERT is 30· less sensitive to cocaine than hSERT, the role of these unique amino acid sequences in cocaine sensitivity was analysed. Our initial focus was on tyrosine and phenylalanine residues because aromatic rings of these amino acids form polar p–p stacking or cation–p interactions with aromatic ligands [34]. Studies with nicotinic acetylcholine [35] and tachykinin receptors [36] showed that substitution of these amino acids with one another at functionally important sites is not always tolerated. Moreover, cocaine analogues lacking phenyl Fig. 2. In vitro translation and Western blot analysis using affinity purified MasSERT antibody. In vitro translated MasSERT (lane 1) runs as a 45-kDa protein on SDS/PAGE. For western analysis, equal amounts of cell membranes (25 lgperlane)fromManduca sexta CNS, mock-transfected CV-1 cells and MasSERT-transfected CV-1 cells (lanes 2–4, respectively) were subjected to SDS/PAGE and im- munoblotted with affinity purified anti-MasSERT Ig as described in Experimental procedures. The anti-MasSERT Ig recognized 55 and 90 kDa bands in the CNS and MasSERT-transfected cells. An addi- tional immunoreactive 45 kDa band is detected in transfected cells. The 55 and 90 kDa proteins are likely to represent glycosylated MasSERT and/or dimers, respectively, with the 45 kDa being the unglycosylated form of MasSERT. This immunoreactivity can be com- peted when the MasSERT antibody is preadsorbed with MasSERT peptide (lanes 5–7 were loaded similar to lanes 2–4) showing that the interaction of MasSERT antibody is specific to MasSERT. 3938 S. K. Sandhu et al.(Eur. J. Biochem. 269) Ó FEBS 2002 Fig. 3. Amino acid sequence alignment of MasSERT with known monoamine transporters. Deduced amino acid sequences of hSERT [5], dSERT [6,7], hDAT [32], hNET [33], dDAT [40] were aligned using the GCG program. Identical amino acids are shown on a black background, while conserved residues are shaded. The putative TM membrane spanning domains are overlined. Fig. 4. Characterization of MasSERT-mediated [ 3 H]serotonin uptake in CV-1 cells. (A) Substrate specificity. CV-1 cells transfected with MasSERT cDNA were incubated with 0.1 l M 3 H-labelled substrates for 15 min as indicated. The data represents percentage of substrate uptake above control levels. (B) Time course of serotonin transport. CV-1 cells transfected with MasSERT cDNA were incubated with 10 lm serotonin for the indicated time. (C) Kinetics of serotonin uptake. MasSERT-transfected cells were incubated with [ 3 H]serotonin and increasing concentrations of unlabeled serotonin for 15 min as described in Experimental procedures. The Eadie–Hofstee analysis is depicted in the inset of the figure (K m ¼ 439 n M , V max ¼ 3.3 · 10 )18 mol per cell per min). The data represents specific serotonin transport, expressed as fmol per cell per hour, and is from a single experiment that was repeated 4 more times with similar results. (D) Ion dependence. MasSERT cDNA-transfected CV-1 cells were incubated for 10minwith50n M [ 3 H]serotonin. Non-specific uptake was determined in CV-1 cells transfected with no DNA and subtracted from each determination. The data are given as percentage of specific uptake above control levels using values from duplicate wells. The original assay buffer was changed according to the different ions tested. For the assay of cations, NaCl was replaced by equimolar concentration of choline chloride. For the assay of anions, chloride was replaced by equimolar salts of sodium and potassium gluconate and sodium and potassium acetate. For potassium dependence, the assay was done either in the buffer containing no KCl and KH 2 PO 4 , or buffer containing 120 m M KCl. This buffer also contained 120 m M NaCl. Ó FEBS 2002 Cocaine insensitive chimeric serotonin transporter (Eur. J. Biochem. 269) 3939 rings have extremely low affinity for the dopamine trans- porter [37,38]. Therefore a number of mutations were performed by focusing on unique aromatic substitutions in hSERT by substituting with the amino acid residues found in MasSERT at the corresponding positions. Thus the mutants Y134F, YM134–135FL, FT191–192IN, F474Y, F515V, F551V were made. In addition two more mutants in the EL2, 189 LA and 188A/189LA, were made that had three amino acid insertions, which extend that particular region of MasSERT EL2 when compared with the rest of the neurotransmitter transporter superfamily. All of these mutants were functional. All mutations involving aromatic residues showed no statistically signifi- cant difference in cocaine sensitivity compared to hSERT (Fig. 6A, Table 4). However, the mutant Y134F showed a lower level of sensitivity to cocaine than hSERT. The two EL2 mutants, 189 LA and 188 A/189LA, that had amino acid insertions, were inhibited at relatively higher concen- trations of cocaine with IC 50 values of 1163 ± 31 n M and 1542 ± 42 n M , respectively (Fig. 6B, Table 4). These val- ues are statistically significant at P <0.005. Because these initial mutations did not result in major changes in cocaine sensitivity of hSERT we then construc- ted chimeras of MasSERT and hSERT. Six chimeras were constructed making use of available restriction sites in hSERT (Fig. 1). Only two of the six chimeras, Mas- SERT(1-67)/hSERT(109-630) and hSERT(1-146)/Mas- SERT(106-587) were functional. The MasSERT(1-67)/ hSERT(109-630) chimera displayed a twofold increase in cocaine potency (IC 50 ¼ 225 ± 11 l M ) as well as higher substrate transport specificity (231 ± 11 n M , P <0.05 versus hSERT) for serotonin (Fig. 6C, Table 4) compared to wild type hSERT. The hSERT(1-146)/MasSERT(106- 587) chimera also had higher specificity for serotonin (197 ± 19.nM, P < 0.05 versus hSERT) but a dramatic decline in cocaine sensitivity (Fig. 6C, Table 3), compared not only to hSERT but also to MasSERT, exhibiting an IC 50 value of 180 ± 8.3 l M (n ¼ 3). These differences in cocaine sensitivity are statistically significant at P <0.005 compared to both hSERT and MasSERT. The substrate saturation experiments for MasSERT, hSERT and chi- meras were done at same time under identical kinetic Table 3. Pharmacological specificity of [ 3 H]serotonin uptake in CV-1 cells transfected with MasSERT cDNA. IC 50 (n M ) values for inhibition of [ 3 H]serotonin uptake for various antagonists and substrates are listed in accordance with their rank order of potency. CV-1 cells transfected with MasSERT cDNA were incubated for 15 minutes with [ 3 H]serotonin and various concentrations of the indicated compounds. Data represents the mean ± SEM of 3–5 independent experiments, each conducted in duplicate. For all experiments, [ 3 H]serotonin concentration was kept constant at 0.05 l M .IC 50 values for hSERT are also included from our study and/or previously published reports. ND, not determined. Compound MasSERT hSERT dSERT Mazindol 153 ± 8.68 98 ± 1.7 a 3.9 ± .02 a Clomipramine 370 ± 12.7 0.8 ± 0.05 c ND Desipramine 638 ± 11.7 174 ± 20 a,b 580 ± 147 a Serotonin 905 ± 47.07 463 ± 44 a 490 ± 35 a Fluoxetine 1 235 ± 122 4.2 ± 0.1, 3.0 ± 0.05 a , 5 b 73 ± 5.6 a Imipramine 1 765 ± 152 4.6 ± 0.9 a,b 1450 ± 280 a Tryptamine 5 340 ± 254 ND ND Nomifensine 7 900 ± 335 839 ± 20 a,b 1130 ± 183 a Cocaine 12 890 ± 740 431 ± 41, 611 ± 66 a 464 ± 31 a Octopamine > 200 000 > 10 000 a,b > 10 000 a Tyramine > 200 000 > 10 000 a,b > 10 000 a Dopamine > 200 000 > 10 000 a,b > 10 000 a Histamine > 200 000 > 10 000 a,b > 10 000 a Tryptophan > 200 000 ND ND Norepinephrine > 200 000 > 10 000 a,b ND a Demchyshyn et al., 1994, b Ramamoorthy et al., 1993, c Barker et al., 1994. Fig. 5. Fluoxetine inhibition of the [ 3 H]serotoninuptakeintoCV-1cells transfected with MasSERT and hSERT. Transfected cells were incu- bated with fluoxetine at the indicated concentrations for 15 min. The [ 3 H]serotonin concentration was kept constant at 50 n M . Non-specific uptake was subtracted from the total uptake to yield specific [ 3 H]serotonin uptake. The data is presented as the percentage mean values of [ 3 H]serotonin uptake in the absence or presence of the an- tagonists. The IC 50 values obtained from the inhibition curves are: fluoxetine, hSERT-3.7 n M ,MasSERT-1.08l M . The data represented is from a single experiment that was repeated at least five times with similar results. 3940 S. K. Sandhu et al.(Eur. J. Biochem. 269) Ó FEBS 2002 conditions. The K m values for hSERT and MasSERT are 460.5 ± 17.5 n M and 436 ± 19.2 n M respectively. DISCUSSION MasSERT described here is the only known Na + /Cl – - dependent serotonin transporter that displays a significant relative insensitivity to psychostimulants like cocaine and the antidepressant fluoxetine. However, in spite of the low sensitivity to cocaine, MasSERT has serotonin transport affinity similar to that observed with other SERTs. All mammalian and nonmammalian monoamine transporters identified so far are cocaine sensitive and have comparable inhibition constants [4–7,32,33,39] except for DATs from Drosophila melanogaster [40] and Caenorhabditis elegans [41], which have reported IC 50 values of 2.6 l M and 5 l M , respectively. The IC 50 values for human, mouse, rat and fruit fly SERTs reported by various groups fall in the range of 300–600 n M with minor differences attributable to differences in the experimental conditions. In contrast MasSERT shows no sensitivity to cocaine in this concen- tration range, but displayed 30 times less sensitivity than human SERT and DAT [4,5,32,42]. MasSERT was 300– 400 times less sensitive to fluoxetine than human SERT, for which fluoxetine is a potent inhibitor (IC 50 ¼ 3–5 n M ); it is also less sensitive than Drosophila SERT (IC 50  73 n M ). This difference in pharmacology is not entirely due to differences between insects and mammals, as the Drosophila SERT shows high sensitivity to both cocaine and fluoxetine. However, MasSERT is nearly equally sensitive as dSERT to the tertiary amine tricyclic antidepressants, imipramine and desipramine. These differences in pharmacology sug- gest MasSERT is likely to have unique structural domains, compared to other SERTs, making it insensitive to cocaine and fluoxetine. Cocaine abuse in the United States continues to remain a major socioeconomic and medical issue of modern society, with no effective treatment available for cocaine dependence [14,43]. DATs, SERTs and NETs are major targets for the reinforcing actions of cocaine [12,44–47]. It is known that Fig. 6. Cocaine inhibition of the [ 3 H]serotonin uptake into CV-1 cells transfected with hSERT, mutant hSERT and chimeras. Transfected cells were incubated with cocaine at the indicated concentrations for 15 min. The [ 3 H]serotonin concentration was kept constant at 50 n M . Unless otherwise specified, the data is presen- ted as the percentage mean values of [ 3 H]serotonin uptake in the absence or pres- ence of the antagonists. Non-specific uptake was subtracted from the total uptake to yield specific [ 3 H]serotonin uptake. The data rep- resented are from a single experiment that was repeated at least three times with similar results. The mean IC 50 values obtained from these inhibition curves are given in Table 4. (A) and (B) hSERT with specific amino acid mutants. (C) hSERT and MasSERT chimeras. Table 4. Cocaine inhibition of [ 3 H]serotonin in mutant hSERT and MasSERT/hSERT chimeras. CV-1 cells transfected with wild type or mutant cDNAs were incubated for 15 minutes with [ 3 H]serotonin and increasing concentrations of cocaine. Data represents the mean ± SEM of 3–4 independent experiments, each conducted in duplicate. For all experiments [ 3 H]serotonin concentration was kept constant at 0.05 lM. The Student’s t-test was performed for statistical analysis of IC 50 values compared to hSERT. P** < 0.005, P* < 0.02. The mutants F474Y, F515V, F551V also did not show substantial change in cocaine sensitivity. The K m and V max values for MasSERT, hSERT and chimeras are derived from Eadie–Hofstee analysis of kinetics of [ 3 H]serotonin transport. Transporter Protein Cocaine IC 50 (n M ) K m (n M ), V max (mol per cell per min) HSERT 431 ± 41 460 ± 17; 12.25 ± 1.1·10 )18 MasSERT 12 890 ± 740 436 ± 19; 3.84 ± 0.6·10 )18 MasSERT1-67/hSERT109-630 225 ± 11* 231 ± 11; 3.39 ± 0.8·10 )18 hSERT1-146/MasSERT106-587 180 000 ± 8300** 197 ± 19; 1.99 ± 0.5·10 )18 hSERT-Y134F, TM2 870 ± 68 ND hSERT-YM134-135FL, TM2 502 ± 122 ND hSERT-189 LA, EL2 1163 ± 31** ND hSERT-188 A/189LA, EL2 1542 ± 42** ND hSERT-FT191-192IN, EL2 441 ± 13 ND Ó FEBS 2002 Cocaine insensitive chimeric serotonin transporter (Eur. J. Biochem. 269) 3941 dopamine and serotonin transporters have distinct domains for substrate recognition and antagonist binding [11,18, 44,48,49]. Experiments involving protection of hDAT regions from alkylation with N-ethylmaleimide substrates show differential binding of dopamine and cocaine [49]. Cocaine and benztropine bring differential conformational changes in hDAT that makes amino acid C90 available to methanethiosulfonate reagents only in the presence of cocaine [50]. In spite of major advances made in this field using chimera construction and mutagenesis [8–10,13– 15,17,43,51,52], a cocaine-binding site on the serotonin or dopamine transporters has not been resolved. These studies have provided evidence for a possible role of TMD 1–2, 4–5, 8, 11 and 12 in cocaine recognition. However, due to a lack of cocaine selectivity among the monoamine transporters, the conclusions from these studies are based on small (2–8·) differences observed in cocaine potency. Our human SERT mutants, Y134F, YM134–135FL, FT191–192IN, F474Y, F515V, and F551V did not show any change in cocaine sensitivity compared to the wild type. Possibly these amino acid changes, which are unique to MasSERT, do not interact with cocaine but could contri- bute towards recognition and binding of other SERT antagonists. Phenylalanine and threonine in EL2 [FT(191- 192)] are absolutely conserved in all monoamine Na + /Cl – dependent transporters, except in MasSERT where they are substituted by isoleucine and asparagine, however, these residues are not important for cocaine sensitivity. Similarly YM134-135 in hSERT (FL in MasSERT) is absolutely conserved in amine transporter subfamily. Mutations at these amino acids also did not affect hSERT sensitivity to cocaine. Although the extracellular loops (EL) between trans- membrane domains do not appear to be responsible for substrate specificity and antagonist selectivity, these loops may provide the desired conformation required for proper transporter function [53,54]. It is interesting to note that EL2 in MasSERT carries two additional amino acid residuesA148andS149,whichextendthisregionofEL2 as compared to rest of the superfamily. The hSERT mutants, 189 LA and 188A/189LA, mutagenized to intro- duce the corresponding region, were less sensitive to cocaine (Fig. 6B, Table 4). Although the mutant 188 A/189LA did not show a dramatic shift in cocaine potency towards MasSERT, it might be sufficient to bring subtle conforma- tional change in the transporter or even make one of the many possible recognition sites for cocaine. As functional data from specific mutations in hSERT did not yield sufficient information to explore the binding sites for cocaine, we focused our attention on the classical approach of constructing chimeras between hSERT and MasSERT, using available restriction sites in hSERT. In chimera hSERT(1–146)/MasSERT(106–587) insertion of the N-terminus of hSERT in MasSERT, by replacing its first 105 amino acids, makes it 418· and 14· more resistant to cocaine than hSERT and MasSERT, respectively. It was expected that this chimera would either behave similarly to MasSERT in the presence of cocaine or its cocaine sensitivity curve would shift towards hSERT. Because this chimera was much more resistant to cocaine and yet had an improved transport affinity to serotonin, it makes an excellent tool to identify domains and amino acid residues which could be potentially involved in cocaine interaction. Interestingly, chimera MasSERT(1–67)/hSERT(109–630), which only contains the N-terminal 67 amino acids of MasSERT, was more sensitive to cocaine than hSERT, and it displays similar higher transport affinity to serotonin as observed for chimera hSERT(1–146)/MasSERT(106–587). These results suggest that the N-terminus, including TMD1–2, plays a substantial role in providing a unique conformation to the transporter thereby governing the substrate transport affinity, cocaine sensitivity and possibly sensitivities to other antagonists. Based on chimera design, it appears that TMD 1 of MasSERT and TMD1–2 of hSERT contain unique molecular determinants that interact differ- entially with the rest of the transmembrane domains of hSERT and MasSERT, respectively. Previous studies with cross-species chimeras have provided evidence that TMD1– 2 might play a critical role in antagonist recognition [10,52]. However, these chimeras displayed marginal or no differ- ences for cocaine potencies. For example, chimeras con- structed between hSERT and dSERT at similar positions, dSERT(1–136)/hSERT(137–625) and hSERT(1–118)/ dSERT(119–627), [10] exhibit comparable potencies for cocaine to those found in the parental transporters. Similarly, DAT and NET chimeras that intersect within or near TMD1 have been shown to have only slightly lower potency for cocaine than wild type DAT and NET [52]. The availability of transporters and chimeric transporters having a wide range of sensitivities to cocaine (225 n M to 180 l M ) facilitates a systematic probe of structural determinants. Efforts are underway to further investigate the pharmaco- logical properties of these two chimeras in order to precisely define the domains/amino acid residues important for bringing conformational changes to the transporter and antagonist binding. Taken together, it is evident that MasSERT is compar- atively less sensitive to cocaine and other pharmacological agents than most members of the monoamine transporter subfamily. 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M., Itokawa, M., Li, X.F., Wei, H.B., Wichems, C., Lesch, K.P., Murphy, D.L & Uhl, G.R (2001) Molecular mechanisms . 5¢-CGCTATACTACCTCATCTCCTTAGCTTCCTTCACGGACCAGCTGC (forward) 5¢-GCAGCTGGTCCGTGAAGGAAGCTAAGGAGATGAGGTAGTATAGCG (reverse) 188 A/ 189LA Insert A 5¢-GCGCTATACTACCTCATCGCTTCCTTAGCTTCCTTCACG. 5¢-GCGCTATACTACCTCATCGCTTCCTTAGCTTCCTTCACG (forward) 5¢-CGTGAAGGAAGCTAAGGAAGCGATGAGGTAGTATAGCGC (reverse) YM134–135FL FT to IN 5¢-CTACCTCATCTCCTCCATCAACGACCAGCTGCCCTGGAC (forward) 5¢-GTCCAGGGCAGCTGGTCGTTGATGGAGGAGATGAGGTAG