The Third Transmembrane Domain of the Serotonin Transporter Contains Residues Associated with Substrate and Cocaine Binding

• DOI: 10.1074/jbc.272.45.28321
• Publication Date: 2002-07-26T14:48:08Z
• Authors: Jie-Guang Chen; Aristidis Sachpat...; Gary Rudnick
• Keywords: Tropane; Alanine

Twenty residues in the third transmembrane domain of serotonin transporter (SERT) were mutated, one at a time, to cysteine. Almost all these mutants fully active for (5-HT) transport and insensitive inactivation by positively charged cysteine reagent [2-(trimethylammonium)ethyl]methanethiosul-fonate (MTSET). Two mutants, I172C I179C, sensitive rapid MTSET but relatively negatively (2-sulfonatoethyl)methanethiosulfonate (MTSES). Inactivation was blocked 5-HT cocaine, I179C not similarly protected. Replacement Tyr-175 with resulted mutant low activity, and, neighboring Tyr-176, replacement completely transport. The Y175C Y176C expressed on cell surface levels 84% 69%, respectively, that wild type (C109A) SERT. Mutants had lower cocaine affinity than C109A, as measured displacement high analog 2β-carbomethoxy-3β-(4-[125I]iodophenyl)tropane (β-CIT). For Y176C, also decreased. inactivated β-CIT binding only slightly inhibited C109A. sensitivity replacements positions 172, 176, 179 observed when replaced alanine, serine, or methionine. results suggest Ile-172, Tyr-176 Ile-179 are face an α-helical element, Ile-172 proximity site cocaine. 1The abbreviations used are: SERT, transporter; 5-HT, serotonin; DAT, dopamine TM, domain; EL, external loop; MTS, methanethiosulfonate; MTSET, [2-(trimethylammonium)ethyl]methanethiosulfonate; MTSES, (2-sulfonatoethyl)methanethiosulfonate; MTSEA, (2-aminoethyl)methanethiosulfonate; PBS, phosphate-buffered saline; β-CIT, 2β-carbomethoxy-3β- (4-[125I]iodophenyl)tropane. is responsible reuptake released during neurotransmission (1Rudnick G. Clark J. Biochim. Biophys. Acta. 1993; 1144: 249-263Crossref PubMed Scopus (366) Google Scholar). Inhibitors SERT clinically effective antidepressants (2Boyer W. Int. Clin. Psychopharm. 1992; 6: 5-12Crossref (51) Psychostimulants such amphetamine derivatives interact either inhibitors alternative substrates (3Wall S.C. Gu H.H. Rudnick Mol. Pharmacol. 1995; 47: 544-550PubMed which nontransported inhibitors, bind has been matter speculation. They could same be transported, they another, allosteric site. However, individual amino acid contribute this have identified. Competitive inhibition like most other biogenic amine transport, suggests it binds substrate, mutually exclusive possibility separate would justify searching drugs antagonize without inhibiting therapeutic agents abuse. Serotonin cotransported Na+ Cl−and countertransported K+ (4Rudnick Reith M. Neurotransmitter Transporters: Structure, Function, Regulation. Humana Press, Totowa, NJ1997: 73-100Crossref Previous work Cl− bound together translocated (5Humphreys C.J. Wall Biochemistry. 1994; 33: 9118-9125Crossref (55) After dissociation Na+, cytoplasm, internal thought medium (6Nelson P.J. Biol. Chem. 1979; 254: 10084-10089Abstract Full Text PDF translocation steps envisaged conformational changes expose substrate (formed domains) alternately side plasma membrane other. These triggered (5-HT, Cl−) member large gene family containing transporters many neurotransmitters substances (7Amara S. Kuhar Annu. Rev. Neurosci. 16: 73-93Crossref (991) Scholar, 8Uhl Johnson P. Exp. 196: 229-236PubMed 9Nelson N. Lill H. 213-228PubMed 10Borowsky B. Hoffman B.J. Neurobiol. 38: 139-199Crossref (97) 11Lester H.A. Cao Y.W. Mager Neuron. 1996; 17: 807-810Abstract (95) Each believed consist 12 domains connected hydrophilic loops cytoplasmic surfaces membrane, N C termini cytoplasm. Within closely related amines norepinephrine, 4Rudnick Mutagenesis identified some contributing (12Kitayama Shimada Xu Markham L. Donovan D. Uhl Proc. Natl. Acad. Sci. U. A. 89: 7782-7785Crossref (349) 13Kitayama Wang Synapse. 15: 58-62Crossref (91) 14Tate C. Blakely R. 269: 26303-26310Abstract 15Wang Moriwaki Neurochem. 64: 1416-1419Crossref (65) 16Barker E.L. R.D. 50: 957-965PubMed 17Lin F. Lester 71: 3126-3135Abstract (73) In (DAT), aspartate residue first (TM 1) found critical two serine TM 7 affect inhibitor Serine 11 DAT K m andV max 1-methyl-4-phenylpyridinium (13Kitayama Differences imipramine between rat human traced phenylalanine (16Barker removal consensus glycosylation sites second loop (EL 2), although expression level, did (14Tate et al. (15Wang Scholar) mutated three cysteines EL 1 2 DAT. single (equivalent Cys-109 SERT) alanine no effect function, (corresponding Cys-200 Cys-209 appearance immunofluorescence observed. We obtained similar using further concluded, from variety approaches, likely form disulfide native protein (18Chen J.G. Liu-Chen 1997; 36: 1479-1486Crossref (149) Neither nor reacted reagents, modification methanethiosulfonate (MTS) derivatives. A functional extracellular MTS reagents useful identifying exposed receptors channels (19Akabas Stauffer Karlin Science. 258: 307-310Crossref (595) 20Xu Akabas M.H. 268: 21505-21508Abstract 21Javitch Li X. Kaback 91: 10355-10359Crossref (131) 22Akabas Kaufmann Cook T.A. Archdeacon 14865-14868Abstract 23Kuner T. Wollmuth L.P. Seeburg P.H. Sakmann 343-352Abstract (192) derivatives, [2-(trimethylammonium)ethyl] (MTSET) (MTSES), membrane-impermeant, (2-aminoethyl)methanethiosulfonate (MTSEA), able permeate lipid bilayer membranes (24Holmgren Liu Y. Yellen Neuropharmacology. 35: 797-804Crossref (191) protein, surface, contact water-filled channel site, will react impermeant reagents. Site-directed if position crucial function. C109A provided good background insertions since activity cells expressing MTSEA Lin (17Lin mutations Asn-177 3 affected conductance mediated presence 5-HT. consistent lining pore through ions move while acts channel. Alternatively, may indirectly. To assess possibilities, examine whether we 20 3, studied reactivity resultant Mutant generated QuikChange™ site-directed mutagenesis kit (Stratagene, La Jolla, CA). region excised digestion NdeI BbsI then subcloned back into pCGT137, recombinant contains c-Myc FLAG epitope tags engineered termini, respectively Because A181C A173C introduced new unique restriction EcoRV RsrII pCGT137. All confirmed DNA sequencing. Confluent Intestine 407 (CCL-6, American Type Culture Collection, Rockville, MD) infected vTF-7 vaccinia virus transfected plasmids bearing cDNA under control T7 promoter described previously (25Blakely J.A. Amara S.G. Anal. Biochem. 1991; 194: 302-308Crossref (151) Transfected incubated 16–20 h 37 °C assay binding, expression. Cell determined membrane-impermeable biotinylation reagent, NHS-SS-biotin (Pierce) procedure Gottardi (26Gottardi Dunbar Caplan Am. Physiol. 37: F285-F295Crossref Briefly, labeled NHS-SS-biotin, excess quenched, solubilized. proteins isolated extract immobilized streptavidin, detected pool gel electrophoresis Western blotting anti-FLAG antibody (Kodak Scientific Imaging Systems, New Haven, CT). Immunoblots quantitated Alpha Innotech IS-1000. Transport adding 250 μl saline (PBS) 0.1 mm CaCl2 mmMgCl2 (PBS/CM) (unless indicated otherwise) 20.5 nm [3H]5-HT (3400 cpm/pmol, NEN Life Science Products) each well incubating 10 min 22 °C. Reactions terminated aspiration washing times ice-cold PBS. lysed 1% SDS contents transferred scintillation vials counting. Parallel wells measure content (27Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. 1951; 193: 265-275Abstract uptake measurements corrected subtracting blank values 100 Binding analog, (β-CIT), 24-well plates washed PBS/CM MTSES follows. Stock solutions H2O freshly prepared diluted 40-fold directly 400 achieve required final concentration. incubation period, twice before initiation uptake. effects after 30-min [125I]β-CIT Preincubation addition gave simultaneous β-CIT. Nonlinear regression fits experimental calculated data performed Origin (Microcal Software, Northampton, MA), uses Marquardt-Levenberg nonlinear least squares curve fitting algorithm. statistical analysis done experiments. experiments repeated total four cases essentially results. Unless otherwise indicated, error bars represent mean ± standard deviation duplicate samples. Using mutant, starting point, Ala-164 Leu-184 replaced, Cys-166 changed. This predicted 3) normal transient system Of seriously impaired (Fig.1, left panel). particular, indistinguishable background, transported 25–30% rate. consistently less ranging 50 70% measurements. investigate reason decreased Y175C, level (Fig. 2). Cells treated biotinylating streptavidin-agarose. fraction subjected immunoblotting against tag added terminus No significant differences 2, left) minor 2,right). Relative I172C, 100%, 84%, 75%, respectively. Decreased account small decrease significantly expected their levels. Incubation 5 insertion mutants. extremely 1, right, Fig. 3). more sensitive, t½ 4.5 1.2 min, 15 upper concentrations contrast much inactivating (Table I, lines about 10% reactive toward simple thiols (28Stauffer D.A. 6840-6849Crossref (256) Scholar), so 10-fold higher concentration relative lack due reaction rate, rapidly lead modified retains activity. test possibilities case, 10-min incubation. results, shown line Table indicate treatment protect MTSET. Thus, mutants.Table IRelative ability inactivate I179CI172CI179CMTSET (1 mm, min)16 2.819 5.3MTSES (10 min)63 5.777 10.9MTSES, MTSET13.5 2.110.7 4.5Cells followed min. assayed rates given percentage rate parallel incubation, contain MTSES. Open table tab Both ligands (Fig.4, protection half-maximal 1.3 0.3 μm 2.0 0.4 range affinities (29Rudnick 40: 421-426PubMed transporter. contrast, neither 4, Since sulfydryl group produces disulfide, free accessibility residue. MTSET-inactivated reactivated readily way resistant reactivation, suggesting access restricted (data shown). severely reduced retained analogue, (Fig.5). displace transporters. (upper panel) shows that, 8-fold, close normal. both (lower 2.5-fold 4.6-fold. By comparison, those control. Equilibrium constants (1.2 0.51 0.04 μm, respectively) afforded 4). transporters, tested binding. membranes, intact cells. Treatment led loss II, 1), II (lines 4), largely (line permeant addition, A169C, smaller penetrate along past Ile-172.Table IIInactivation substitution MTSETMutantRemaining activity%C109A70.3 7.3I172C5.8 1.7I179C59.7 21.3Y176C−3.0 2.5Membranes "Experimental Procedures." column, remaining shown, present. Membranes reversed centrifugation resuspension. TableIII extensively, still extent Consequently, larger initial recovered present III). greater compared (compare 94% 25 III 70–80% reason, membranes.Table IIIProtection bindingActivity (% control)MTSET alone+25 5-HT+25 cocaineI172C6.0 1.159.1 1.257.2 4.6Y176C27.3 4.353.6 2.852.5 12.0Membranes suspended HEPES, pH 8.0, 0.2 mg/ml. sample suspension (25 80 Y176C) ml buffer absence Control samples 4 Beckman Airfuge p.s.i. percent (66 fmol/mg 14 0.41 [125I]β-CIT). representative experiment, once Y176C. point contained 18 It possible changed structure allow endogenous possibility, TableIV functional, ranged 30% I172S full I172M (first column). Despite activities, (second Similar Ile-179. Although I179S inactive I179A I179M (third (fourth Only transporter.Table IVActivity Ile-179Ile-172Ile-179C109AMTSETC109AMTSET%%Ala70.5 1.6100.5 136.5 1.399 5.7Ser28.5 0.7106 1.41 0.1Met110 17104 368 1199 3.4The columns, assaying fourth columns show Tyr-176. Y176A Y176M Y176S. V demonstrates Y176S (rows roughly (row 1). rendered process 4).Table VActivity Tyr-176Tyr-176Binding MTSETTyrosine66 6.0Serine43 12.7Methionine48 5.7Cysteine2.3 3.2Membranes Y176S, Y176M, 30 amount presented here to, constitute part of, While association specific ligand One residues, highly conserved throughout NaCl-coupled pocket proteins. renders small, sulfhydryl (Figs. TableII). particular inserted must medium, detectable change fit criteria. spacing vertical patch structure. Fig.6 helical net representation 3. alignment (and somewhat 169) supports idea α-helix helix faces pocket. Aside increase therefore, interest N177C despite proposal lumen ion formed II), (TableIII). enough interferes residues. evidence When dramatically diminished 5). fact lends support essential agreement role Bismuth (30Bismuth Kavanaugh M.P. Kanner B.I. 272: 16096-16102Abstract (103) recently reported mutation corresponding Tyr-140 γ-aminobutyric transporter, GAT-1, disrupted proper SKF100330A. Other include Ile-168, Tyr-175, Ile-179, Ala-181, Tyr-182. reinforces nature its indicates prevented reaching Modification 172 productive interaction II). Taken together, observations strongly argues conclusion cysteine, methionine, disrupt where substitutions however, Furthermore, obviously fulfill different roles better isoleucine tyrosine. do rule out lie accessible occupied, protected partial Together result additional processes occur possible, example, cells, gains 176 ligand. facts decreases inactivation, overlap significantly. middle located protein. model deep within exterior. Substrate accomplished opened pathway block associated must, involved step follows gate closes prevent dissociating medium. introduces quaternary ammonium derivative might sterically electrostatically interfere closing gate. faster I). Some difference stem intrinsic thiolate anions even taking account, 4–6-fold positions. conceivable another influences electrostatic environment near domains. Asp-98 Glu-508 10. equivalent known function 31Blakely Moore K. Qian Soc. Gen. Ser. 48: 283-300PubMed proposed coordinate feature common members NaCl family, helps invariant influence Cys-172 Cys-179 I179C. Future studies thank Carl Richmond expert editorial assistance Dr. Elias Lolis reading manuscript.