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• W1964072600 abstract "Vc1.1 is a disulfide-rich peptide inhibitor of nicotinic acetylcholine receptors that has stimulated considerable interest in these receptors as potential therapeutic targets for the treatment of neuropathic pain. Here we present an extensive series of mutational studies in which all residues except the conserved cysteines were mutated separately to Ala, Asp, or Lys. The effect on acetylcholine (ACh)-evoked membrane currents at the α9α10 nicotinic acetylcholine receptor (nAChR), which has been implicated as a target in the alleviation of neuropathic pain, was then observed. The analogs were characterized by NMR spectroscopy to determine the effects of mutations on structure. The structural fold was found to be preserved in all peptides except where Pro was substituted. Electrophysiological studies showed that the key residues for functional activity are Asp5–Arg7 and Asp11–Ile15, because changes at these positions resulted in the loss of activity at the α9α10 nAChR. Interestingly, the S4K and N9A analogs were more potent than Vc1.1 itself. A second generation of mutants was synthesized, namely N9G, N9I, N9L, S4R, and S4K+N9A, all of which were more potent than Vc1.1 at both the rat α9α10 and the human α9/rat α10 hybrid receptor, providing a mechanistic insight into the key residues involved in eliciting the biological function of Vc1.1. The most potent analogs were also tested at the α3β2, α3β4, and α7 nAChR subtypes to determine their selectivity. All mutants tested were most selective for the α9α10 nAChR. These findings provide valuable insight into the interaction of Vc1.1 with the α9α10 nAChR subtype and will help in the further development of analogs of Vc1.1 as analgesic drugs. Vc1.1 is a disulfide-rich peptide inhibitor of nicotinic acetylcholine receptors that has stimulated considerable interest in these receptors as potential therapeutic targets for the treatment of neuropathic pain. Here we present an extensive series of mutational studies in which all residues except the conserved cysteines were mutated separately to Ala, Asp, or Lys. The effect on acetylcholine (ACh)-evoked membrane currents at the α9α10 nicotinic acetylcholine receptor (nAChR), which has been implicated as a target in the alleviation of neuropathic pain, was then observed. The analogs were characterized by NMR spectroscopy to determine the effects of mutations on structure. The structural fold was found to be preserved in all peptides except where Pro was substituted. Electrophysiological studies showed that the key residues for functional activity are Asp5–Arg7 and Asp11–Ile15, because changes at these positions resulted in the loss of activity at the α9α10 nAChR. Interestingly, the S4K and N9A analogs were more potent than Vc1.1 itself. A second generation of mutants was synthesized, namely N9G, N9I, N9L, S4R, and S4K+N9A, all of which were more potent than Vc1.1 at both the rat α9α10 and the human α9/rat α10 hybrid receptor, providing a mechanistic insight into the key residues involved in eliciting the biological function of Vc1.1. The most potent analogs were also tested at the α3β2, α3β4, and α7 nAChR subtypes to determine their selectivity. All mutants tested were most selective for the α9α10 nAChR. These findings provide valuable insight into the interaction of Vc1.1 with the α9α10 nAChR subtype and will help in the further development of analogs of Vc1.1 as analgesic drugs. Marine snails belonging to the Conus genus produce a variety of neurotoxic peptides in their venom glands that they use for the capture of prey (1Olivera B.M. Gray W.R. Zeikus R. McIntosh J.M. Varga J. Rivier J. de Santos V. Cruz L.J. Science. 1985; 230: 1338-1343Crossref PubMed Scopus (642) Google Scholar, 2Olivera B.M. Rivier J. Clark C. Ramilo C.A. Corpuz G.P. Abogadie F.C. Mena E.E. Woodward S.R. Hillyard D.R. Cruz L.J. Science. 1990; 249: 257-263Crossref PubMed Scopus (514) Google Scholar, 3Olivera B.M. Mol. Biol. Cell. 1997; 8: 2101-2109Crossref PubMed Scopus (333) Google Scholar). Within this repertoire of conopeptides, those that are disulfide-rich are referred to as conotoxins. Conotoxins typically range in size from 12 to 30 amino acids, contain 4 or more Cys residues, and exhibit high potency and selectivity toward a variety of membrane receptors and ion channels (4Adams D.J. Alewood P.F. Craik D.J. Drinkwater R.D. Lewis R.J. Drug. Dev. Res. 1999; 46: 219-234Crossref Scopus (109) Google Scholar, 5Terlau H. Olivera B.M. Physiol. Rev. 2004; 84: 41-68Crossref PubMed Scopus (807) Google Scholar). The α-conotoxin subfamily members typically range in size from 12 to 19 amino acids, contain 2 disulfide bonds in a CysI–CysIII and CysII–CysIV connectivity, and have an amidated C terminus, as depicted in Fig. 1. They interact with nicotinic acetylcholine receptors (nAChRs), 4The abbreviations used are: nAChRnicotinic acetylcholine receptorAChacetylcholinehα9rα10human α9 and rat α10 hydrid cloneHPLChigh-performance liquid chromatographyNOEnuclear Overhauser effectNOESYNOE spectroscopyTOCSYtotal correlation spectroscopy. of both the muscle and the neuronal type, which have been implicated in a range of neurological disorders varying from Alzheimer disease to addiction (6McIntosh J.M. Santos A.D. Olivera B.M. Annu. Rev. Biochem. 1999; 68: 59-88Crossref PubMed Scopus (276) Google Scholar, 7Dutton J.L. Craik D.J. Curr. Med. Chem. 2001; 8: 327-344Crossref PubMed Scopus (97) Google Scholar, 8Livett B.G. Gayler K.R. Khalil Z. Curr. Med. Chem. 2004; 11: 1715-1723Crossref PubMed Scopus (142) Google Scholar). nicotinic acetylcholine receptor acetylcholine human α9 and rat α10 hydrid clone high-performance liquid chromatography nuclear Overhauser effect NOE spectroscopy total correlation spectroscopy. The nAChRs are ligand-gated ion channels that respond to ACh, nicotine, and other competitive agonists/antagonists. They are composed of five subunits, with differing nAChR subunit composition according to the site of expression. The muscle-type nAChRs are composed of two α subunits, a β and δ subunit, and either an ϵ or a γ subunit (9Itier V. Bertrand D. FEBS Lett. 2001; 504: 118-125Crossref PubMed Scopus (156) Google Scholar, 10Cascio M. J. Biol. Chem. 2004; 279: 19383-19386Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 11Olivera B.M. Teichert R.W. Mol. Interv. 2007; 7: 251-260Crossref PubMed Scopus (153) Google Scholar, 12Olivera B.M. Quik M. Vincler M. McIntosh J.M. Channels. 2008; 2: 143-152Crossref PubMed Scopus (60) Google Scholar). The neuronal forms exist either as homomeric channels composed of α subunits alone or αβ heteromeric channels. The wide variety of possible subunit combinations has led to unique subtypes with distinct pharmacological properties. This makes α-conotoxins valuable neuropharmacological tools and drug leads, because they have the ability to distinguish between different nAChR subtypes. Effectively, they are small rigid scaffolds that display amino acids on their surface to selectively target their receptors (13Hu S.H. Gehrmann J. Alewood P.F. Craik D.J. Martin J.L. Biochemistry. 1997; 36: 11323-11330Crossref PubMed Scopus (80) Google Scholar). Of particular interest in this study is the α-conotoxin Vc1.1, a synthetic derivative of a naturally occurring peptide from the venom of the marine cone snail, Conus victoriae. It was discovered using PCR screening of cDNA extracted from the snail venom duct (14Sandall D.W. Satkunanathan N. Keays D.A. Polidano M.A. Liping X. Pham V. Down J.G. Khalil Z. Livett B.G. Gayler K.R. Biochemistry. 2003; 42: 6904-6911Crossref PubMed Scopus (171) Google Scholar). Fig. 1 depicts the sequences of selected α-conotoxins, including Vc1.1, which is 16 amino acids in length and displays the classic disulfide bond connectivity observed for α-conotoxins, together with a short helical segment as depicted in Fig. 1b. The conserved Cys framework of α-conotoxins defines two backbone loops, which vary in size and residue composition, and are classified by an n/m nomenclature to define subclasses of α-conotoxins. For example, Vc1.1 is a 4/7 subclass α-conotoxin, because it contains four residues in loop 1 and seven in loop 2. RgIA (15Ellison M. Haberlandt C. Gomez-Casati M.E. Watkins M. Elgoyhen A.B. McIntosh J.M. Olivera B.M. Biochemistry. 2006; 45: 1511-1517Crossref PubMed Scopus (139) Google Scholar, 16Clark R.J. Daly N.L. Halai R. Nevin S.T. Adams D.J. Craik D.J. FEBS Lett. 2008; 582: 597-602Crossref PubMed Scopus (31) Google Scholar, 17Ellison M. Feng Z.P. Park A.J. Zhang X. Olivera B.M. McIntosh J.M. Norton R.S. J. Mol. Biol. 2008; 377: 1216-1227Crossref PubMed Scopus (93) Google Scholar) is another conotoxin of interest in this study, because it is also selective for the α9α10 nAChR subtype, and has a 4/3 framework. Vc1.1 contains an amidated C terminus, a post-translational modification common to most α-conotoxins, but it is not present in RgIA. Vc1.1 lacks the post-translationally modified hydroxyproline and γ-carboxyglutamate residues present in the native peptide, vc1a, isolated from the venom duct of C. victoriae (18Jakubowski J.A. Keays D.A. Kelley W.P. Sandall D.W. Bingham J.P. Livett B.G. Gayler K.R. Sweedler J.V. J. Mass. Spectrom. 2004; 39: 548-557Crossref PubMed Scopus (54) Google Scholar). Vc1.1 has been under development as a drug lead for neuropathic pain (19Livett B.G. Sandall D.W. Keays D. Down J. Gayler K.R. Satkunanathan N. Khalil Z. Toxicon. 2006; 48: 810-829Crossref PubMed Scopus (127) Google Scholar). When tested in rat models of neuropathic pain, Vc1.1 induced analgesia when injected intramuscularly near the site of injury (20Satkunanathan N. Livett B. Gayler K. Sandall D. Down J. Khalil Z. Brain. Res. 2005; 1059: 149-158Crossref PubMed Scopus (161) Google Scholar). Initially, it was thought that α3-containing subtypes of nAChRs may be the target for Vc1.1 (21Clark R.J. Fischer H. Nevin S.T. Adams D.J. Craik D.J. J. Biol. Chem. 2006; 281: 23254-23263Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar); however, it was then reported that Vc1.1 has a 100-fold higher affinity at the α9α10 nAChR subtype (22Vincler M. Wittenauer S. Parker R. Ellison M. Olivera B.M. McIntosh J.M. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 17880-17884Crossref PubMed Scopus (205) Google Scholar, 23Nevin S.T. Clark R.J. Klimis H. Christie M.J. Craik D.J. Adams D.J. Mol. Pharmacol. 2007; 72: 1406-1410Crossref PubMed Scopus (93) Google Scholar). The α9α10 nAChR mediates synaptic transmission between efferent olivocochlear fibers and cochlear hair cells (24Elgoyhen A.B. Johnson D.S. Boulter J. Vetter D.E. Heinemann S. Cell. 1994; 79: 705-715Abstract Full Text PDF PubMed Scopus (749) Google Scholar, 25Elgoyhen A.B. Vetter D.E. Katz E. Rothlin C.V. Heinemann S.F. Boulter J. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 3501-3506Crossref PubMed Scopus (564) Google Scholar, 26Sgard F. Charpantier E. Bertrand S. Walker N. Caput D. Graham D. Bertrand D. Besnard F. Mol. Pharmacol. 2002; 61: 150-159Crossref PubMed Scopus (176) Google Scholar). The mRNA of these receptor subtypes is expressed in many different tissue types from the inner ear, dorsal root ganglion (27Lips K.S. Pfeil U. Kummer W. Neuroscience. 2002; 115: 1-5Crossref PubMed Scopus (101) Google Scholar), skin keratinocytes (28Nguyen V.T. Ndoye A. Grando S.A. Am. J. Pathol. 2000; 157: 1377-1391Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar), and lymphocytes (29Peng H. Ferris R.L. Matthews T. Hiel H. Lopez-Albaitero A. Lustig L.R. Life. Sci. 2004; 76: 263-280Crossref PubMed Scopus (115) Google Scholar) to the pituitary (26Sgard F. Charpantier E. Bertrand S. Walker N. Caput D. Graham D. Bertrand D. Besnard F. Mol. Pharmacol. 2002; 61: 150-159Crossref PubMed Scopus (176) Google Scholar). The α10 subunit has to be expressed with the α9 subunit to form a functional receptor. In the auditory system, the α9α10 nAChR plays an important role in hair cell development, but its role in other tissues is yet to be characterized (22Vincler M. Wittenauer S. Parker R. Ellison M. Olivera B.M. McIntosh J.M. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 17880-17884Crossref PubMed Scopus (205) Google Scholar, 26Sgard F. Charpantier E. Bertrand S. Walker N. Caput D. Graham D. Bertrand D. Besnard F. Mol. Pharmacol. 2002; 61: 150-159Crossref PubMed Scopus (176) Google Scholar, 30Baker E.R. Zwart R. Sher E. Millar N.S. Mol. Pharmacol. 2004; 65: 453-460Crossref PubMed Scopus (65) Google Scholar, 31Gómez-Casati M.E. Fuchs P.A. Elgoyhen A.B. Katz E. J. Physiol. 2005; 566: 103-118Crossref PubMed Scopus (73) Google Scholar). Owing to the promising antinociceptive effects of Vc1.1 in animals, its analogs are of interest as leads for the treatment of neuropathic pain (14Sandall D.W. Satkunanathan N. Keays D.A. Polidano M.A. Liping X. Pham V. Down J.G. Khalil Z. Livett B.G. Gayler K.R. Biochemistry. 2003; 42: 6904-6911Crossref PubMed Scopus (171) Google Scholar, 20Satkunanathan N. Livett B. Gayler K. Sandall D. Down J. Khalil Z. Brain. Res. 2005; 1059: 149-158Crossref PubMed Scopus (161) Google Scholar). To date, studies have predominantly focused on the α9α10 nAChR, but the very recent finding that Vc1.1 also targets the γ-aminobutyric acid, type B receptor (32Callaghan B. Haythornthwaite A. Berecki G. Clark R.J. Craik D.J. Adams D.J. J. Neurosci. 2008; 28: 10943-10951Crossref PubMed Scopus (151) Google Scholar) has raised interest in the molecular mode of action of Vc1.1 in analgesia. Hence there is a need to define structure-activity relationships of this peptide at several targets, including human and rat forms of the α9α10 nAChR. In particular, we were interested in analogs that maintain potency at the rat α9α10 nAChR but also show significant improvement in potency at human forms of the receptor, while maintaining selectivity over other nAChR subtypes. In this study we determined such structure-activity relationships for Vc1.1 at the α9α10 nAChR by successively mutating each non-Cys residue of Vc1.1 to either an “inert” residue (Ala), a negatively charged residue (Asp), or a positively charged residue (Lys) and observing the impact on the structure and functional activity of Vc1.1. Once the key residues had been identified, a second generation of analogs with new substitutions was synthesized and tested at the rat α9α10 nAChR. The analogs were also analyzed at the human α9/rat α10 (hα9rα10) hybrid clone, because a recent report 5Reported in an abstract by Azam and McIntosh from the “Neuroscience 2008” conference (November 15–19, 2008, Washington, D. C.). It can be found on the Society for Neuroscience website. suggested differences in the activity of Vc1.1 at the human and rat clones of the α9α10 nAChR. We also examined the effect of pH change on the structure of Vc1.1 using NMR αH chemical shift analysis. The results from this study provide valuable insight into the key residues involved in the interaction of Vc1.1 with the α9α10 nAChR subtype and have the potential to assist in the development of conotoxin analogs as drug leads for the treatment of neuropathic pain (4Adams D.J. Alewood P.F. Craik D.J. Drinkwater R.D. Lewis R.J. Drug. Dev. Res. 1999; 46: 219-234Crossref Scopus (109) Google Scholar, 33Miljanich G.P. Curr. Med. Chem. 2004; 11: 3029-3040Crossref PubMed Scopus (474) Google Scholar). All of the peptide mutants were assembled on rink amide methylbenzhydrylamine resin (Novabiochem) using manual solid-phase peptide synthesis with an in situ neutralization/2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate activation procedure for Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry. Cleavage of peptides from the resin was achieved by treatment with trifluoroacetic acid and triisopropylsilane and water as scavengers (9:0.5:0.5 trifluoroacetic acid:triisopropylsilane:water). The reaction was allowed to proceed at room temperature (20–23 °C) for 2.5 h. The trifluoroacetic acid was then evaporated, and the peptide was precipitated with ice-cold ether, filtered, dissolved in 50% buffer A/B (Buffer A:H2O/0.05% trifluoroacetic acid; Buffer B: 90% CH3CN/10%H2O/0.045% trifluoroacetic acid), and lyophilized. Crude peptides were purified by reversed phase-HPLC on a Phenomenex C18 column using a gradient of 0–80% B in 80 min, with the eluant monitored at 215/280 nm. These conditions were used in subsequent purification steps unless stated otherwise. Electrospray-mass spectroscopy confirmed the molecular mass of the fractions collected, and those displaying the correct molecular mass of linear peptide were pooled and lyophilized for oxidation. The linear peptides were oxidized by being dissolved in 0.1 m NH4HCO3 (pH 8.2) at a concentration of 0.3 mg/ml with stirring overnight at room temperature. The oxidized peptides were then purified by reversed phase-HPLC using a gradient of 0–80% buffer B over 160 min. Analytical reversed phase-HPLC and electrospray-mass spectroscopy confirmed the purity and molecular mass of the synthesized peptides. NMR data for all peptides were recorded on Bruker Avance 500- and 600-MHz spectrometers, with samples dissolved in 90% H2O/10% D2O. Two-dimensional NMR experiments included TOCSY and NOESY recorded at 280 K. Spectra were analyzed using Topspin 1.3 (Bruker) and Sparky software. Most spectra were recorded at pH 3.5, although a pH titration over the range 3–7 was done for Vc1.1. This involved adjusting the pH with NaOH and recording a series of TOCSY spectra. Most of the αH and amide NMR chemical shifts were unaffected by a change in pH from pH 3.03 to 5.77, suggesting that there are no pH-dependent conformational changes. Protonation effects were found for residues near His12. Although TOCSY spectra were collected up to pH 7, the spectra beyond pH 5.77 could not be fully assigned as they deteriorated in quality due to rapid amide exchange. Nevertheless, analysis of the data showed that the changes in αH and amide shifts reached a plateau near pH 4.41 and are likely to be unchanged at pH values higher than 5.77. RNA preparation, oocyte preparation, and expression of rat α9α10, human α9/rat α10 hybrid, rat α3β2, rat α3β4, and rat α7 nAChR subunits in Xenopus oocytes were performed as described previously (21Clark R.J. Fischer H. Nevin S.T. Adams D.J. Craik D.J. J. Biol. Chem. 2006; 281: 23254-23263Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 34Hogg R.C. Hopping G. Alewood P.F. Adams D.J. Bertrand D. J. Biol. Chem. 2003; 278: 26908-26914Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). We used the human α9/rat α10 hybrid as a surrogate for the human receptor because in our hands the human α9α10 nAChR expressed poorly. Azam and McIntosh5 earlier showed that the human α9/rat α10 hybrid receptor displayed the same sensitivity to the α-conotoxin RgIA, as human α9α10 and the sequence difference resides in the N-terminal binding region of α9 subunits. All oocytes were injected with 5 ng of RNA and kept at 18 °C in ND96 buffer (96 mm NaCl, 2 mm KCl, 1 mm CaCl2, 1 mm MgCl2, and 5 mm HEPES, at pH 7.4) supplemented with 50 mg/liter gentamicin and 5 mm pyruvic acid 2–5 days before recording. Membrane currents were recorded from Xenopus oocytes using a GeneClamp 500B amplifier (Molecular Devices), and an automated workstation with eight channels in parallel, including drug delivery and on-line analysis (OpusXpressTM 6000A workstation, Molecular Devices, Sunnyvale, CA). Both the voltage-recording and the current-injecting electrodes were pulled from borosilicate glass (Harvard Apparatus Ltd., Edenbridge, UK) and had resistances of 0.3–1.5 mΩ when filled with 3 m KCl. All recordings were conducted at room temperature using a bath solution of ND96, as described above. During recordings, the oocytes were perfused continuously at a rate of 1.5 ml/min, with 300-s incubation times for the conotoxin. Acetylcholine (10 μm unless otherwise specified) was applied for 2 s at 5 ml/min, with 180–300 s washout periods between applications. Vc1.1 and mutants were bath-applied and co-applied with the agonist. Oocytes were voltage clamped at a holding potential of −80 mV. Peak current amplitude was measured before and after incubation of the peptide (23Nevin S.T. Clark R.J. Klimis H. Christie M.J. Craik D.J. Adams D.J. Mol. Pharmacol. 2007; 72: 1406-1410Crossref PubMed Scopus (93) Google Scholar). Alanine scanning mutagenesis is a powerful technique for identifying residues important for the structure and activity of peptides. Here we combined it with Lys and Asp scanning to probe the role of neutral, positive, and negative charge substitutions at all non-Cys positions of Vc1.1. The Cys positions were not mutated because they are essential for the disulfide bonds that define the native structure. The suite of mutant peptides was assembled using solid-phase peptide synthesis, oxidized in ammonium bicarbonate buffer, purified using HPLC, and characterized by mass spectrometry and NMR. A sufficient quantity (>1 mg) of natively folded peptide was obtained for each of the 34 single point mutants for structural and electrophysiological studies. The native CysI–CysIII and CysII–CysIV disulfide connectivity is referred to as the globular isomer (35Gehrmann J. Alewood P.F. Craik D.J. J. Mol. Biol. 1998; 278: 401-415Crossref PubMed Scopus (150) Google Scholar) and was the predominant form observed after oxidation of most of the peptides. For some peptides a CysI–CysIV and CysII–CysIII disulfide connectivity, referred to as the ribbon isomer, was also formed. In these cases the isomers were separated by HPLC, and the globular form was used for further analysis. The sequences, molecular masses, disulfide isomers, and yields of the synthesized peptides are summarized in Table 1. The overall yield of globular folded peptide ranged from 30 to 70%, based on the quantity of crude peptide obtained after cleavage from the resin.TABLE 1Sequences synthesized, isomeric conformations formed, and the final yieldPeptideSequenceaAll sequences contain an amidated C terminus.Globular isomerbGlobular isomer has CysI–CysIII and CysII–CysIV disulfide connectivity.Ribbon isomercRibbon isomer has CysI–CysIV and CysII–CysIII disulfide connectivity.YielddYield of pure globular isomer formed from crude peptide.Molecular massVc1.1GCCSDPRCNYDHPEIC% 90% 10mg 15.8Da 1807First generation analogs [G1A] Vc1.1ACCSDPRCNYDHPEIC100010.01821 [S4A] Vc1.1GCCADPRCNYDHPEIC81193.91791 [D5A] Vc1.1GCCSAPRCNYDHPEIC60402.41763 [P6A] Vc1.1GCCSDARCNYDHPEIC10000.91781 [R7A] Vc1.1GCCSDPACNYDHPEIC10001.71722 [N9A] Vc1.1GCCSDPRCAYDHPEIC9191.41764 [Y10A] Vc1.1GCCSDPRCNADHPEIC87133.81715 [D11A] Vc1.1GCCSDPRCNYAHPEIC90103.11763 [H12A] Vc1.1GCCSDPRCNYDAPEIC80201.61741 [P13A] Vc1.1GCCSDPRCNYDHAEIC71293.71781 [E14A] Vc1.1GCCSDPRCNYDHPAIC10001.91749 [I15A] Vc1.1GCCSDPRCNYDHPEAC10003.61765 [G1K] Vc1.1KCCSDPRCNYDHPEIC93711.41878 [S4K] Vc1.1GCCKDPRCNYDHPEIC100013.41848 [D5K] Vc1.1GCCSKPRCNYDHPEIC87133.21820 [P6K] Vc1.1GCCSDKRCNYDHPEIC673315.01838 [R7K] Vc1.1GCCSDPKCNYDHPEIC100017.61779 [N9K] Vc1.1GCCSDPRCKYDHPEIC100011.21821 [Y10K] Vc1.1GCCSDPRCNKDHPEIC100012.11772 [D11K] Vc1.1GCCSDPRCNYKHPEIC100010.01820 [H12K] Vc1.1GCCSDPRCNYDKPEIC70303.01798 [P13K] Vc1.1GCCSDPRCNYDHKEIC10005.31838 [E14K] Vc1.1GCCSDPRCNYDHPKIC10003.81806 [I15K] Vc1.1GCCSDPRCNYDHPEKC653511.71822 [G1D] Vc1.1DCCSDPRCNYDHPEIC100010.01865 [S4D] Vc1.1GCCDDPRCNYDHPEIC100016.31835 [P6D] Vc1.1GCCSDDRCNYDHPEIC811910.21825 [R7D] Vc1.1GCCSDPDCNYDHPEIC93711.31766 [N9D] Vc1.1GCCSDPRCDYDHPEIC79214.31808 [Y10D] Vc1.1GCCSDPRCNDDHPEIC100012.41759 [H12D] Vc1.1GCCSDPRCNYDDPEIC811910.01785 [P13D] Vc1.1GCCSDPRCNYDHDEIC65355.31825 [E14D] Vc1.1GCCSDPRCNYDHPDIC9193.11793 [I15D] Vc1.1GCCSDPRCNYDHPEDC91913.31809Second generation analogs [N9I] Vc1.1GCCSDPRCIYDHPEIC100010.11806 [N9L] Vc1.1GCCSDPRCLYDHPEIC89119.81806 [N9G] Vc1.1GCCSDPRCGYDHPEIC100011.21750 [S4R] Vc1.1GCCRDPRCNYDHPEIC10007.21876 [S4K+N9A] Vc1.1GCCKDPRCAYDHPEIC100010.11805a All sequences contain an amidated C terminus.b Globular isomer has CysI–CysIII and CysII–CysIV disulfide connectivity.c Ribbon isomer has CysI–CysIV and CysII–CysIII disulfide connectivity.d Yield of pure globular isomer formed from crude peptide. Open table in a new tab Although our primary focus was on the globular isomer, it is of interest to note that in some cases the mutations caused changes in the relative distribution of globular versus ribbon isomer. For example, the replacement of Arg7 with an Ala or a Lys led to exclusive formation of the globular isomer, whereas substituting it with an Asp led to a small proportion of the ribbon isomer. In general, replacement of Pro residues led to a lower proportion of globular isomer, as did substitutions of Asp5 or His12. A sample analytical HPLC trace for mutant I15K is shown in Fig. 2. This mutant formed both the globular and the ribbon isomers, which were separated by preparative HPLC. NMR spectroscopy was used to confirm the native globular fold of the purified peptides. This analysis required assignment of the spectra, and for this task αHi − NHi+1 NOE connectivities observed in NOESY spectra were used in the sequential assignment of the individual spin systems determined from TOCSY spectra. For most of the mutants, sequential αHi − NHi+1 NOEs were observed for the entire peptide chain except, as expected, at Pro6 and Pro13 because these residues lack amide protons. In general, αH NMR chemical shifts can be used to indicate the nature of secondary structural elements present in peptides. Specifically, secondary αH chemical shifts represent the difference between an observed αH chemical shift and that for the corresponding residue in a random coil peptide and are strong indicators of the presence of secondary structure (36Wishart D.S. Bigam C.G. Holm A. Hodges R.S. Sykes B.D. J. Biomol. NMR. 1995; 5: 67-81Crossref PubMed Scopus (1418) Google Scholar). Analysis of the measured chemical shift data indicated that, for most of the mutants, residues Cys2–Ser4, Pro6–Asp11, and Pro13–Cys16 have negative αH secondary shifts (Fig. 3). As reported previously (21Clark R.J. Fischer H. Nevin S.T. Adams D.J. Craik D.J. J. Biol. Chem. 2006; 281: 23254-23263Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), the negative αH secondary shifts between Pro6–Asp11 indicate the conservation of the α-helix as seen in the parent peptide. This predominance of helical secondary structure is characteristic of other α-conotoxin structures reported to date (7Dutton J.L. Craik D.J. Curr. Med. Chem. 2001; 8: 327-344Crossref PubMed Scopus (97) Google Scholar). The trends in shifts for most of the mutants are almost identical to those for Vc1.1, illustrating the high degree of structural similarity between the mutants. In a few cases the change in residue type causes a structural change. This is particularly so with the mutations of the Pro residues. The P6K mutant folded to yield two isomers, both of which displayed similar secondary αH shifts. Fig. 3 shows the shifts of what is believed to be the globular isomer and clearly illustrates how they are different from the shifts of the other mutants, suggesting a change in structure. Likewise, P13K, P13D, and P13A have significant changes in αH secondary shifts of loop 2 relative to Vc1.1, suggesting a perturbation in the core of the structure. Small localized differences were seen in the secondary αH shifts for N9I, N9G, S4R, and S4K+N9A, as shown in the lower panel of Fig. 3. Aside from the exceptions noted above, most mutations did not lead to perturbations of chemical shifts. αH shifts are sensitive to the backbone conformation and, with most shifts superimposing well, any differences in the biological activities between the mutant peptides can therefore be attributed to changes in the nature of the side-chain interactions with the receptor rather than structural perturbations of the conotoxin ligands. It has been reported that Vc1.1 has increased potency at lower pH values, presumably as a result of His12 protonation (21Clark R.J. Fischer H. Nevin S.T. Adams D.J. Craik D.J. J. Biol. Chem. 2006; 281: 23254-23263Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Because His12 is unlikely to be protonated at physiological pH, we conducted a titration to determine the effect of pH on the structure of Vc1.1. No global change in the structure was apparent with changes in pH, as judged from the lack of change in the chemical shifts for most residues. There were some localized changes in the αH and amide shifts of Tyr10, Asp11, and His12 with changes in pH from 5.2 to 3.0, with His12 αH shifts being the most affected; however, these reflect local charge-state effects rather than a change in the overall fold of the peptide. Fig. 4 shows the backbone and side-chain orientation of Asp11 and His12 in a structure determined at pH 3.5, and highlights the proximity of His12 to Asp11. The largest observed shift with pH was for the amide of Glu14. Fig. 4 shows that the backbone amide proton of Glu14 and its carboxyl side chain form a hydrogen bond. Because Glu14 is a surface-exposed residue, it is not surprising that it is susceptible to changes in pH, but again the effect is only local. Overall, we saw no change in the structure of Vc1.1 at the pH at which the NMR data were collected and at the pH used for the electrophysiological experiments to functionally characterize the mutants. The functional activity of Vc1.1 and analogs was investigated via its effects on ACh-evoked membrane currents in Xenopus oocytes. The EC50 for activation of rat α9α10 nAChR by ACh was determined to be ∼10 μm, similar to that reported previously (37Plazas P.V. Katz E. Gomez-Casati M.E. Bouzat C. Elgoyhen A.B. J. Neurosci. 2005; 25: 10905-10912Crossref PubMed Scopus (84) Google Scholar). The IC50 for inhibition of ACh-evoked current by Vc1.1 was 109 nm. All Vc1.1 analog" @default.
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• W1964072600 title "Scanning Mutagenesis of α-Conotoxin Vc1.1 Reveals Residues Crucial for Activity at the α9α10 Nicotinic Acetylcholine Receptor" @default.
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