Inhibition of the Norepinephrine Transporter by the Venom Peptide χ-MrIA
2003; Elsevier BV; Volume: 278; Issue: 41 Linguagem: Inglês
10.1074/jbc.m213030200
ISSN1083-351X
AutoresIain A. Sharpe, Elka Palant, Christina I. Schroeder, David M. Kaye, David J. Adams, Paul F. Alewood, Richard J. Lewis,
Tópico(s)Neuroscience and Neuropharmacology Research
Resumoχ-Conopeptide MrIA (χ-MrIA) is a 13-residue peptide contained in the venom of the predatory marine snail Conus marmoreus that has been found to inhibit the norepinephrine transporter (NET). We investigated whether χ-MrIA targeted the other members of the monoamine transporter family and found no effect of the peptide (100 μm) on the activity of the dopamine transporter and the serotonin transporter, indicating a high specificity of action. The binding of the NET inhibitors, [3H]nisoxetine and [3H]mazindol, to the expressed rat and human NET was inhibited by χ-MrIA with the conopeptide displaying a slight preference toward the rat isoform. For both radioligands, saturation binding studies showed that the inhibition by χ-MrIA was competitive in nature. It has previously been demonstrated that χ-MrIA does not compete with norepinephrine, unlike classically described NET inhibitors such as nisoxetine and mazindol that do. This pattern of behavior implies that the binding site for χ-MrIA on the NET overlaps the antidepressant binding site and is wholly distinct from the substrate binding site. The inhibitory effect of χ-MrIA was found to be dependent on Na+ with the conopeptide becoming a less effective blocker of [3H]norepinephrine by the NET under the conditions of reduced extracellular Na+. In this respect, χ-MrIA is similar to the antidepressant inhibitors of the NET. The structure-activity relationship of χ-MrIA was investigated by alanine scanning. Four residues in the first cysteine-bracketed loop of χ-MrIA and a His in loop 2 played a dominant role in the interaction between χ-MrIA and the NET. Hα chemical shift comparisons indicated that side-chain interactions at these key positions were structurally perturbed by the replacement of Gly-6. From these data, we present a model of the structure of χ-MrIA that shows the relative orientation of the key binding residues. This model provides a new molecular caliper for probing the structure of the NET. χ-Conopeptide MrIA (χ-MrIA) is a 13-residue peptide contained in the venom of the predatory marine snail Conus marmoreus that has been found to inhibit the norepinephrine transporter (NET). We investigated whether χ-MrIA targeted the other members of the monoamine transporter family and found no effect of the peptide (100 μm) on the activity of the dopamine transporter and the serotonin transporter, indicating a high specificity of action. The binding of the NET inhibitors, [3H]nisoxetine and [3H]mazindol, to the expressed rat and human NET was inhibited by χ-MrIA with the conopeptide displaying a slight preference toward the rat isoform. For both radioligands, saturation binding studies showed that the inhibition by χ-MrIA was competitive in nature. It has previously been demonstrated that χ-MrIA does not compete with norepinephrine, unlike classically described NET inhibitors such as nisoxetine and mazindol that do. This pattern of behavior implies that the binding site for χ-MrIA on the NET overlaps the antidepressant binding site and is wholly distinct from the substrate binding site. The inhibitory effect of χ-MrIA was found to be dependent on Na+ with the conopeptide becoming a less effective blocker of [3H]norepinephrine by the NET under the conditions of reduced extracellular Na+. In this respect, χ-MrIA is similar to the antidepressant inhibitors of the NET. The structure-activity relationship of χ-MrIA was investigated by alanine scanning. Four residues in the first cysteine-bracketed loop of χ-MrIA and a His in loop 2 played a dominant role in the interaction between χ-MrIA and the NET. Hα chemical shift comparisons indicated that side-chain interactions at these key positions were structurally perturbed by the replacement of Gly-6. From these data, we present a model of the structure of χ-MrIA that shows the relative orientation of the key binding residues. This model provides a new molecular caliper for probing the structure of the NET. Because of its poor lipid solubility and degree of ionization at physiological pH, norepinephrine crosses cell membranes poorly by diffusion (1Mack F. Bönisch H. Naunyn-Schmiedeberg's Arch. Pharmacol. 1979; 310: 1-9Crossref PubMed Scopus (150) Google Scholar) and so relies on the operation of the norepinephrine transporter (NET) 1The abbreviations used are: NET, norepinephrine transporter; DAT, dopamine transporter; SERT, serotonin transporter; 5-HT, 5-hydroxytryptamine (serotonin); NOESY, Nuclear Overhauser enhancement spectroscopy; TOCSY, total correlated spectroscopy; HBTU, N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uranium hexafluorophosphate); Fmoc, N-(9-fluorenyl)methoxycarbonyl; ANOVA, analysis of variance. for uptake into cells. Clearance by this integral membrane protein constitutes the major mechanism for the termination of action of this neurotransmitter at noradrenergic synapses (2Graefe K.H. Bönisch H. Trendelenburg U. Weiner N. Catecholamines I. Vol. 90. Springer-Verlag New York Inc., New, York1988: 193-245Google Scholar), and disturbances in the functioning of the NET are associated with pathological states including depression (3Klimek V. Stockmeier C. Overholser J. Meltzer H.Y. Kalka S. Dilley G. Ordway G.A. J. Neurosci. 1997; 17: 8451-8458Crossref PubMed Scopus (337) Google Scholar), congestive heart failure (4Eisenhofer G. Friberg P. Rundqvist B. Quyyumi A.A. Lambert G. Kaye D.M. Kopin I.J. Goldstein D.S. Esler M.D. Circulation. 1996; 93: 1667-1676Crossref PubMed Scopus (411) Google Scholar), and orthostatic intolerance, and tachycardia (5Shannon J.R. Flattem N.L. Jordan J. Jacob G. Black B.K. Biaggioni I. Blakely R.D. Robertson D. N. Engl. J. Med. 2000; 342: 541-549Crossref PubMed Scopus (488) Google Scholar). Known inhibitors of the NET include antidepressants (e.g. desipramine and nisoxetine), the appetite suppressant mazindol, and the abused drug cocaine (for review, see Ref. 6Bönisch H. Brüss M. Ann. N. Y. Acad. Sci. 1994; 733: 193-202Crossref PubMed Scopus (75) Google Scholar). The NET, together with the dopamine transporter (DAT) and the serotonin transporter (SERT), forms a family of Na+- and Cl--dependent monoamine transporters. A novel peptidic NET inhibitor, χ-MrIA, has been identified in cone snail venom (7Sharpe I.A. Gehrmann J. Loughnan M.L. Thomas L. Adams D.A. Atkins A. Palant E. Craik D.J. Adams D.J. Alewood P.F. Lewis R.J. Nature Neurosci. 2001; 4: 902-907Crossref PubMed Scopus (224) Google Scholar). Cone snails use a venom containing a mixture of bioactive peptides ("conopeptides") to capture their prey, and these are known to target an array of voltage-sensitive ion channels, ligand-gated ion channels, and G protein-coupled receptors (for review, see Ref. 8Olivera B.M. Cruz L.J. Toxicon. 2001; 39: 7-14Crossref PubMed Scopus (224) Google Scholar). Intrathecal injection of χ-MrIA has been found to be analgesic in hot plate and neuropathic pain models (9McIntosh J.M. Corpuz G.O. Layer R.T. Garett J.E. Wagstaff J.D. Bulaj G. Vyazovkina A. Yoshikami D. Cruz L.J. Olivera B.M. J. Biol. Chem. 2000; 275: 32391-32397Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 10Nielson C. Ross F. Lewis R. Drinkwater R. Smith M. 10th World Congress on Pain, San Diego, August 17-22, 2002. ISAP Press, San Diego, CA2002: 278Google Scholar). The inhibition of [3H]norepinephrine uptake by the NET caused by χ-MrIA was found to be non-competitive, reducing the maximum rate of transport and not affecting the affinity of the transporter for substrate (7Sharpe I.A. Gehrmann J. Loughnan M.L. Thomas L. Adams D.A. Atkins A. Palant E. Craik D.J. Adams D.J. Alewood P.F. Lewis R.J. Nature Neurosci. 2001; 4: 902-907Crossref PubMed Scopus (224) Google Scholar). The non-competitive mode of action of χ-MrIA distinguishes it from the majority of the classically described inhibitors of the NET that act in a competitive fashion. In this study, we explored the interaction of χ-MrIA with the monoamine transporters to gain an insight into the selectivity, Na+ dependence, site of action, and structure-activity relationship of the conopeptide. Peptide Synthesis—χ-MrIA and the singly substituted analogs, [N1A]MrIA, [G2A]MrIA, [V3A]MrIA, [G6A]MrIA, [Y7A]MrIA, [K8A]MrIA, [L9A]MrIA, [H11A]MrIA, [O12A]MrIA, [Y7F]MrIA, and [K8R]MrIA, were synthesized. The chain assembly of the peptides was performed on a manual shaker system using HBTU activation protocols (11Schnolzer M. Alewood P. Jones A. Alewood D. Kent S.B. Int. J. Pept. Protein Res. 1992; 40: 180-193Crossref PubMed Scopus (945) Google Scholar) to couple the Fmoc-protected amino acid to the resin. The Fmoc-protecting group was removed using 50% piperidine in dimethylformamide, and dimethylformamide was used as both the coupling solvent and for flow washes throughout the cycle. The progress of the assembly was monitored by quantitative ninhydrin monitoring (12Sarin V.K. Kent S.B. Tam J.P. Merrifield R.B. Anal. Biochem. 1981; 117: 147-157Crossref PubMed Scopus (1011) Google Scholar). Peptide was deprotected and cleaved from the resin by stirring at room temperature in trifluoroacetic acid:H2O:triisopropylsilane:ethanedithiol (90:5:2.5:2.5) for 2-3 h. Cold diethyl ether was then added to the mixture, and the peptide precipitated out. The precipitate was collected by centrifugation and subsequently washed with further cold diethyl ether to remove scavengers. The final product was dissolved in 50% aqueous acetonitrile and lyophilized to yield a fluffy white solid. The crude, reduced peptide was examined by reverse phase high performance liquid chromatography for purity, and the correct molecular weight was confirmed by electrospray mass spectrometry. Pure, reduced peptides were oxidized, and the major peak was purified to >95% purity and characterized by high performance liquid chromatography prior to further use. Cellular Uptake of [3H]Monoamines—COS-1 cells (ATCC, Manassas, VA) were grown in 24-well plates (Falcon, BC Biosciences) containing Dulbecco's modified Eagle medium (Invitrogen) and 10% fetal bovine serum (Invitrogen) at 37 °C in 5% CO2. Upon reaching ∼85% confluency, the cells were transiently transfected with plasmid DNA encoding the human NET (13Percy E. Kaye D.M. Lambert G.W. Gruskin S. Esler M.D. Du X.J. Br. J. Pharmacol. 1999; 128: 774-780Crossref PubMed Scopus (9) Google Scholar), the rat NET (14Brüss M. Pörzgen P. Bryan-Lluka L.J. Bönisch H. Mol. Brain Res. 1997; 52: 257-262Crossref PubMed Scopus (52) Google Scholar), the human DAT (15Giros B. el Mestikawy S. Godinot N. Zheng K. Han H. Yang Feng T. Caron M.G. Mol. Pharmacol. 1992; 42: 383-390PubMed Google Scholar), or the human SERT (16Ramamoorthy S. Bauman A.L. Moore K.R. Han H. Yang-Feng T. Chang A.S. Ganapathy V. Blakely R.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2542-2546Crossref PubMed Scopus (793) Google Scholar). Transfections were performed using LipofectAMINE 2000 reagent (Invitrogen) following the manufacturer's protocol using 800 ng of DNA/well. Assays measuring the cellular accumulation of the transporters' respective [3H]monoamine substrates were performed 24 h after transfection at room temperature in duplicate. The culture medium was removed, and the cells were washed three times with 500 μl of transport buffer containing 125 mm NaCl, 4.8 mm KCl, 1.2 mm MgSO4, 1.2 mm KH2PO4, 1.3 mm CaCl2, 25 mm HEPES, 5.55 mmd-(+)-glucose, 1.02 mm ascorbic acid, 10 μm U-0521 (to inhibit catechol-O-methyl transferase), and 100 μm pargyline (to inhibit monoamine oxidase), pH 7.4. In experiments examining the Na+ dependence of the NET inhibitors, the concentration of NaCl used in the transport buffer ranged from 25 to 125 mm with appropriate concentrations of LiCl added to retain equal osmolarity. Inhibitor drugs were preincubated with the cells for 15 min before the addition of 100 nm [3H]monoamine substrate (supplemented with unlabeled substrate as required). The final volume was 250 μl. Nonspecific uptake of [3H]norepinephrine by NET-transfected cells was defined by the accumulation occurring in the presence of 10-4m desipramine. Imipramine (10-6m) and GBR-12909 (10-6m) were used to determine the amount of nonspecific uptake of [3H]serotonin by SERT-transfected cells and [3H]dopamine uptake by DAT-transfected cells, respectively. Transfected cells were exposed to [3H]monoamine substrate for either 8 min (rat NET, human SERT) or 15 min (human NET, human DAT). The selection of these incubation times was based on the results of pilot studies that showed that the relationship between uptake and time was linear over these periods (data not shown). The solution containing unaccumulated 3H-substrate was then rapidly removed, and the cells were washed three times with 1 ml of ice-cold phosphate-buffered saline. The cells were lysed with 0.1% Triton X-100 in 10 mm Tris·HCl, pH 7.5, for 60 min at room temperature with gentle shaking. The level of radioactivity of the cell lysate was determined by liquid scintillation counting. Membrane Preparation—COS-7 cells (ECACC, Salisbury, Wiltshire, United Kingdom) were grown in 150-mm dishes and transiently transfected with 15 μg of plasmid DNA encoding the rat NET using the same method described for the uptake experiments. Membranes were prepared from cells 48 h after transfection for use in radioligand binding experiments. After washing the cells with warm phosphate-buffered saline, ice-cold TEM buffer (10 mm Tris·HCl, 1.4 mm EGTA, 12.5 mm MgCl2, pH 7.5) was added and the cells were scraped from the dish. Cells were then homogenized using a Polytron homogenizer (Brinkmann Instruments, Westbury, NY) and centrifuged at 1000 × g for 5 min at 4 °C to remove cellular debris then at 15,000 × g for 45 min at 4 °C. Pellets were washed with TEM buffer and recentrifuged. The resulting pellet was resuspended in TEM buffer containing 10% glycerol. Rat brain homogenates were prepared as described previously (17Lewis R.J. Nielsen K.J. Craik D.J. Loughnan M.L. Adams D.A. Sharpe I.A. Luchian T. Adams D.J. Bond T. Thomas L. Jones A. Matheson J. Drinkwater R. Andrews P.R. Alewood P.F. J. Biol. Chem. 2000; 275: 35335-35344Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Protein concentration was determined using the BCA protein assay kit (Pierce, Rockford, IL) following the manufacturer's protocol. Aliquots of membrane were stored at -80 °C until use. Radioligand Binding Assays—Binding reactions were set up in triplicate wells of 96-well plates. Membranes from COS-7 cells transfected with the rat or human NET (6 μg of protein/well) were incubated with either [3H]nisoxetine (4.3 nm) or [3H]mazindol (4 nm) in the absence or presence of χ-MrIA or one of its analogs (1 nm-100 μm) in buffer containing 20 mm Tris·HCl, 75 mm NaCl, 0.1 mm EDTA, 0.1 mm EGTA, 0.1% bovine serum albumin, pH 7.4, for 1 h at room temperature. The final assay volume was 150 μl. The amount of nonspecific binding was determined by the inclusion of desipramine (100 μm) in the reaction. Bound and free radioactivity were separated by rapid vacuum filtration onto GF/B filters (Wallac, Boston, MA) pretreated with 0.6% polyethylenimine. Filter mats were washed three times with ice-cold buffer containing 25 mm HEPES, 125 mm NaCl, pH 7.4, and allowed to dry. Filter-retained radioactivity was quantified by liquid scintillation counting. For saturation analysis experiments, the binding reactions contained 6 μg of membrane protein from rat NET-transfected COS-7 cells, either [3H]nisoxetine (4-100 nm) or [3H]mazindol (5-86 nm) and χ-MrIA (0, 2, or 20 μm). In other experiments, rat brain homogenates (equivalent to 20 μg of protein/well) and [3H]nisoxetine (4.3 nm) were incubated together in the absence and presence of unlabeled nisoxetine, desipramine, or χ-MrIA (1 pm-100 μm) in buffer containing 50 mm Tris·HCl, 300 mm NaCl, 5 mm KCl, pH 7.4, for 1 h at room temperature. The reactions were filtered, and the radioactivity counted as described above. 1H NMR Spectroscopy—All of the spectra were recorded on a Bruker ARX 500 spectrometer equipped with a z-gradient unit. Peptide concentrations were ∼2 mm. Each analog was examined in 95% H2O, 5% D2O, pH 3.0-3.5. The 1H NMR experiments recorded were NOESY (18Kumar A. Ernst R.R. Wuthrich K. Biochem. Biophys. Res. Commun. 1980; 95: 1-6Crossref PubMed Scopus (2050) Google Scholar, 19Jeener J. Meier B.H. Bachman P. Ernst R.R. J. Chem. Phys. 1979; 71: 4546-4553Crossref Scopus (4885) Google Scholar) with a mixing time of 400 ms and TOCSY (20Bax A. Davis D.G. J. Magn. Reson. 1985; 65: 355-360Google Scholar) with a mixing time of 65-120 ms. All of the spectra were recorded at 293 K and were run over 6024 Hz (500 MHz) with 4096 data points, 512 free induction decays, 16-80 scans, and a recycle delay of 1 s. The solvent was suppressed using the WATERGATE sequence (21Piotto M. Saudek V. Sklenar V. J. Biomol. NMR. 1992; 2: 661-665Crossref PubMed Scopus (3562) Google Scholar). Spectra were processed using XWINMR. Free induction decays were multiplied by a polynomial function and apodised using a 90° shifted sine-bell function in both dimensions prior to Fourier transformation. Base-line correction using a fifth order polynomial was applied. Chemical shift values were referenced internally to 2,2-dimethyl-2-silapetane-5-sulfonate at 0.00 ppm. The peptides were assigned according to the method of Wüthrich (22Wüthrich K. NMR of Proteins and Nucleic Acids, Wiley-Interscience. John Wiley & Sons, Inc., New York1986Google Scholar). Secondary Hα shifts were compared with the random coil shift values of Wishart et al. (23Wishart D.S. Bigam C.G. Holm A. Hodges R.S. Sykes B.D. J. Biomol. NMR. 1995; 5: 67-81Crossref PubMed Scopus (1441) Google Scholar). Materials—Desipramine hydrochloride, imipramine hydrochloride, mazindol, nisoxetine hydrochloride, (-)-norepinephrine bitartrate, and pargyline were obtained from Sigma. U-0521 and GBR-12909 dihydrochloride were from Biomol (Plymouth Meeting, PA). l-[7-3H]Norepinephrine (specific activity, 14.9 Ci/mmol), 5-[1,2-3H(N)]hydroxytryptamine creatinine sulfate ([3H]serotonin) (specific activity, 24.0 Ci/mmol), 3,4-[7-3H]dihydroxyphenylethylamine ([3H]dopamine) (specific activity, 27.5 Ci/mmol), [3H]mazindol (specific activity, 21 Ci/mmol), and [3H]nisoxetine (specific activity, 80 Ci/mmol) were obtained from PerkinElmer Life Sciences. Protected Fmoc-amino acid derivatives were from Novabiochem or Auspep (Melbourne, Australia). The following side-chain protected amino acids were used: Cys(tBu), Asn(Trt), His(Trt), Hyp(tBu), Tyr(tBu), and Lys(Boc). Dimethylformamide, dichloromethane, diisopropylethylamine, and trifluoroacetic acid, were all of peptide synthesis grade supplied by Auspep. HBTU was Fluka 12804 supplied by Sigma. High performance liquid chromatography grade acetonitrile and methanol was supplied by Sigma. Resin used was Fmoc-rink amide resin supplied by Polymer Laboratories. Triisopropylsilane was from Aldrich. Statistics and Data Analysis—Data are expressed as means ± S.E. of results obtained from 2 to 5 separate experiments. Student's two-tailed t test or, where appropriate, ANOVA with post hoc t tests performed by the Tukey method was used to evaluate the statistical significance of differences between groups. Values of p < 0.05 were considered significant. Curve fitting of concentration-response curves and radioligand binding data was performed by non-linear regression using individual data points with Prism 3.0 software for Macintosh (GraphPad, San Diego, CA). The equation of Cheng and Prusoff (24Cheng Y. Prusoff W.H. Biochem. Pharmacol. 1973; 22: 3099-3108Crossref PubMed Scopus (12526) Google Scholar) was used to convert IC50 values to K i values. Effect of χ-MrIA on the Cellular Uptake of [3H]Monoamines—COS-1 cells transfected with either the rat or human NET readily accumulated [3H]norepinephrine, and nonspecific uptake of [3H]norepinephrine was <2.5% of the total uptake. As shown in Fig. 1, the uptake of [3H]norepinephrine via the rat and human NET was sensitive to inhibition by χ-MrIA with pIC50 values of 6.21 ± 0.02 (rat; n = 3) and 5.90 ± 0.03 (human; n = 3). χ-MrIA acted as a full inhibitor of the NET of both species. For DAT- and SERT-transfected cells, nonspecific uptake represented 0.2 for each of the comparisons). The IC50 values for the inhibition were 1.1 nm (pIC50 = 8.9 ± 0.06) for desipramine, 6.2 nm (pIC50 = 8.2 ± 0.07) for nisoxetine, and 5.7 μm (pIC50 = 5.2 ± 0.22) for χ-MrIA. While desipramine and unlabeled nisoxetine inhibited the [3H]nisoxetine binding to the same extent (nonspecific binding of ∼43%), the estimated maximum extent of inhibition produced by χ-MrIA was significantly less (p < 0.001) with ∼32% of the nisoxetine- and desipramine-sensitive binding found to be insensitive to χ-MrIA. Sodium dependence of NET inhibition—The rate of uptake of [3H]norepinephrine by cells transfected with the human NET slowed substantially as the concentration of Na+ in the transport buffer was reduced. At the lowest Na+ concentration examined (25 mm), the rate of [3H]norepinephrine accumulation was approximately half of that observed at 125 mm Na+ (data not shown). Concentrations of desipramine and χ-MrIA that inhibited transport by 50% in assays where the buffer contained 125 mm Na+ (4.05 nm and 1.26 μm, respectively) were found to inhibit a progressively smaller proportion of the uptake in buffer containing less Na+ (Fig. 5). Effect of Residue Replacement on the Potency of χ-MrIA—Nine analogs of χ-MrIA in which the non-cysteine residues were systematically replaced with alanine were assayed for inhibition of [3H]nisoxetine binding to the expressed human NET, and their potency was compared (Fig. 6). The analogs with substitutions at N-terminal residues outside of the cysteine-bracketed loops ([N1A]MrIA, [G2A]MrIA, and [V3A]MrIA) displayed no significant change in potency compared with χ-MrIA. The replacement of any of the residues located in the first cysteine-bracketed loop, in contrast, had a severe impact on potency. No inhibition was observed with these analogs ([G6A]MrIA, [Y7A]MrIA, [K8A]MrIA, and [L9A]MrIA) at 100 μm, the highest concentration tested. Assuming that the Hill slope parameter for their inhibition remains unchanged compared with χ-MrIA, the IC50 concentrations of these peptides will be at least an order of magnitude greater still, yielding a conservative estimate of 10-3m. Alanine substitution of the first residue of the second cysteine-bracketed loop (analog [H11A]MrIA) caused a ∼60-fold reduction in potency. Replacement of the other residue in this loop (analog [O12A]MrIA) did not have a significant effect on potency. Two further analogs were assayed to investigate the effect of replacement with residues other than alanine at positions 7 and 8. The potency of [Y7F]MrIA was ∼3.8-fold lower (pIC50 = 5.2 ± 0.08) than χ-MrIA, and the potency of [K8R]MrIA was ∼6.8-fold lower (pIC50 = 4.9 ± 0.10) than χ-MrIA. Structural Effects of Alanine Substitutions—1D, TOCSY, and NOESY 1H NMR spectra of χ-MrIA and analogs were recorded at 500 MHz and subsequently assigned using the sequential assignment protocol (22Wüthrich K. NMR of Proteins and Nucleic Acids, Wiley-Interscience. John Wiley & Sons, Inc., New York1986Google Scholar). Secondary chemical shifts, i.e. Hα chemical shifts compared with random coil values (25Stein E.G. Rice L.M. Brunger A.T. J. Magn. Reson. 1997; 124: 154-164Crossref PubMed Scopus (283) Google Scholar), are a sensitive measure of backbone conformation (26Brooks B. Bruccoleri R. Olafson B.O. States D. Swaminathan S. Karplus M. J. Comput. Chem. 1983; 4: 187-217Crossref Scopus (14159) Google Scholar, 27Hyberts S.G. Goldberg M.S. Havel T.F. Wagner G. Protein Sci. 1992; 1: 736-751Crossref PubMed Scopus (400) Google Scholar, 28Gehrmann J. Alewood P.F. Craik D.J. J. Mol. Biol. 1998; 278: 401-415Crossref PubMed Scopus (152) Google Scholar) and can provide an indication whether the overall global fold of a series of a peptide is maintained (29Nielsen K.J. Skjaerbaek N. Dooley M. Adams D.A. Mortensen M. Dodd P.R. Craik D.J. Alewood P.F. Lewis R.J. J. Med. Chem. 1999; 42: 415-426Crossref PubMed Scopus (34) Google Scholar). For a series of structurally related peptides, secondary Hα chemical shifts can be used to identify the location but not the nature of local changes in conformation (29Nielsen K.J. Skjaerbaek N. Dooley M. Adams D.A. Mortensen M. Dodd P.R. Craik D.J. Alewood P.F. Lewis R.J. J. Med. Chem. 1999; 42: 415-426Crossref PubMed Scopus (34) Google Scholar). Secondary Hα chemical shifts were used in the first instance to compare χ-MrIA with its alanine-substituted analogs (Fig. 7). The results indicate that the overall global fold of the χ-MrIA analogs used in this study are conserved compared with native χ-MrIA with the exception of [G6A]MrIA where the overall fold of the peptide appears different. Small local changes are observed for [K8A]MrIA and [H11A]MrIA at the site of the altered residue. For [Y7A]MrIA, a small change in the secondary Hα chemical shift is seen at Lys-8. This is not surprising as Tyr-7 is a relatively large residue that, relative to Ala-7, could influence the chemical environment of Lys-8 and hence differentially influence its Hα chemical shift. In the case of [G6A]MrIA, a comparison of its secondary Hα chemical shifts with χ-MrIA indicates that replacement of this residue causes a significant structural perturbation. Interestingly, introduction of a stereocenter through substitution of Gly-6 with an alanine appears to alter the structural rigidity of [G6A]MrIA. This enhanced structural rigidity for [G6A]MrIA is supported by changes in secondary Hβ shifts for residue Cys-5 where the two Cys-5 β-protons are well separated in [G6A]MrIA. In contrast, the other χ-MrIA analogs investigated in this study all display degenerate β-protons (data not shown). The relative position of the side chains of Tyr-7, Lys-8, Leu-9, and His-11 of χ-MrIB (equivalent to χ-MrIA in structure (7Sharpe I.A. Gehrmann J. Loughnan M.L. Thomas L. Adams D.A. Atkins A. Palant E. Craik D.J. Adams D.J. Alewood P.F. Lewis R.J. Nature Neurosci. 2001; 4: 902-907Crossref PubMed Scopus (224) Google Scholar)) are shown in Fig. 8.Fig. 8Pharmacophore model of χ-MrIA. A, ribbon representation of χ-MrIA with residues determined to be important for interaction with the NET indicated as follows: Tyr-7 (pink), Lys-8 (blue), Leu-9, and His-11 (red). Disulfide connectivity is shown in orange. B, electrostatic surface of χ-MrIA with residues 7-9 and 11 and the N terminus labeled. Positively charged surface is shown in blue, and hydrophobic surface shown in white. The model of χ-MrIA was generated in Insight and was based on the solution structure of χ-MrIB (7Sharpe I.A. Gehrmann J. Loughnan M.L. Thomas L. Adams D.A. Atkins A. Palant E. Craik D.J. Adams D.J. Alewood P.F. Lewis R.J. Nature Neurosci. 2001; 4: 902-907Crossref PubMed Scopus (224) Google Scholar) (Protein Data Bank accession number 1IEO) using residue replacement of Val-1 in χ-MrIB to Asn-1 (the corresponding residue in χ-MrIA). The ribbon representation was generated using Insight 2000.1 (50Accelrys, Inc.Insight II Modeling Environment, version 97, 2000, 2000.1. Accelrys Inc, San Diego, CA2001Google Scholar), and the electrostatic surface was generated using GRASP (51Nicholls A. Sharp K. Honig B. Proteins, Structure, Function and Genetics. 1991; 11: 281ff-296ffCrossref Scopus (5324) Google Scholar) on a Silicon Graphics Octane computer.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The aim of the present study was to investigate the influence that the transporter identity, the co-substrate Na+, and in
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