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α-Conotoxin GIC from Conus geographus, a Novel Peptide Antagonist of Nicotinic Acetylcholine Receptors

2002; Elsevier BV; Volume: 277; Issue: 37 Linguagem: Inglês

10.1074/jbc.m205102200

1083-351X

J. Michael McIntosh, Cheryl Dowell, Maren Watkins, James E. Garrett, Doju Yoshikami, Baldomero M. Olivera,

Receptor Mechanisms and Signaling

Many venomous organisms produce toxins that disrupt neuromuscular communication to paralyze their prey. One common class of such toxins comprises nicotinic acetylcholine receptor antagonists (nAChRs). Thus, most toxins that act on nAChRs are targeted to the neuromuscular subtype. The toxin characterized in this report, α-conotoxin GIC, is a most striking exception. The 16-amino acid peptide was identified from a genomic DNA clone from Conus geographus. The predicted mature toxin was synthesized, and synthetic toxin was used in all studies described. α-Conotoxin GIC shows no paralytic activity in fish or mice. Furthermore, even at concentrations up to 100 μm, the peptide has no detectable effect on the human muscle nicotinic receptor subtype heterologously expressed in Xenopus oocytes. In contrast, the toxin has high affinity (IC50 ≈1.1 nm) for the human α3β2 subunit combination, making it the most neuronally selective nicotinic antagonist characterized thus far. Although α-conotoxin GIC shares some sequence similarity with α-conotoxin MII, which is also a potent α3β2 nicotinic antagonist, it is much less hydrophobic, and the kinetics of channel block are substantially different. It is noteworthy that the nicotinic ligands in C. geographus venom fit an emerging pattern in venomous predators, with one nicotinic antagonist targeted to the muscle subtype (thereby causing paralysis) and a second nicotinic antagonist targeted to the α3β2 nAChR subtype (possibly inhibiting the fight-or-flight response). Many venomous organisms produce toxins that disrupt neuromuscular communication to paralyze their prey. One common class of such toxins comprises nicotinic acetylcholine receptor antagonists (nAChRs). Thus, most toxins that act on nAChRs are targeted to the neuromuscular subtype. The toxin characterized in this report, α-conotoxin GIC, is a most striking exception. The 16-amino acid peptide was identified from a genomic DNA clone from Conus geographus. The predicted mature toxin was synthesized, and synthetic toxin was used in all studies described. α-Conotoxin GIC shows no paralytic activity in fish or mice. Furthermore, even at concentrations up to 100 μm, the peptide has no detectable effect on the human muscle nicotinic receptor subtype heterologously expressed in Xenopus oocytes. In contrast, the toxin has high affinity (IC50 ≈1.1 nm) for the human α3β2 subunit combination, making it the most neuronally selective nicotinic antagonist characterized thus far. Although α-conotoxin GIC shares some sequence similarity with α-conotoxin MII, which is also a potent α3β2 nicotinic antagonist, it is much less hydrophobic, and the kinetics of channel block are substantially different. It is noteworthy that the nicotinic ligands in C. geographus venom fit an emerging pattern in venomous predators, with one nicotinic antagonist targeted to the muscle subtype (thereby causing paralysis) and a second nicotinic antagonist targeted to the α3β2 nAChR subtype (possibly inhibiting the fight-or-flight response). nicotinic acetylcholine receptor acetylcholine N-(9-fluorenyl)methoxycarboxyl high performance liquid chromatography Many organisms employ toxins that act on nAChRs1 to defend against predators or to facilitate prey capture. Numerous low molecular weight toxins, characterized from a variety of biological sources, are likely used to discourage consumption by predators. Nicotine, an alkaloid from the tobacco plant, causes paralysis when ingested by insects. Nicotine extracts have been used since the 1900s as a natural insecticide (1McIndoo N.E. J. Agric. Res. 1916; 7: 89-122Google Scholar). The harvesting of tobacco plants by humans commonly leads to a syndrome known as green tobacco sickness. This occurs when nicotine is absorbed through the skin, leading to weakness, nausea, vomiting, dizziness, abdominal cramps, headache, and difficulty breathing (2Ballard T. Ehlers J. Freund E. Auslander M. Brandt V. Halperin W. Arch. Environ. Health. 1995; 50: 384-389Crossref PubMed Scopus (54) Google Scholar, 3Arcury T.A. Quandt S.A. Preisser J.S. Norton D. J. Occup. Environ. Med. 2001; 43: 601-609Crossref PubMed Scopus (50) Google Scholar, 4D'Alessandro A. Benowitz N.L. Muzi G. Eisner M.D. Filberto S. Fantozzi P. Montanari L. Abbritti G. Arch. Environ. Health. 2001; 56: 257-263Crossref PubMed Scopus (19) Google Scholar).d-Tubocurarine is isolated from the Chondodendron tomentosum bush. Arrows tipped with curare have been used for centuries to hunt wild game in South America (5Taylor P. Gilman A.G. Rall T.W. Nies A.S. Taylor P. Goodman and Gilman's The Pharmacological Basis of Therapeutics. 8th Ed. Pergamon Press, New York1990: 166-186Google Scholar). Death of the prey results from paralysis of skeletal muscles. Lophotoxins are isolated from Lophogorgia pseudopterogorgia (a species of soft coral); lophotoxins bind irreversibly to the muscle nAChR by forming a covalent bond with a tyrosine residue in the α-subunit of the receptor (6Abramson S.N. Culver P. Kline T., Li, Y. Guest P. Gutman L. Taylor P. J. Biol. Chem. 1988; 263: 18568-18573Abstract Full Text PDF PubMed Google Scholar, 7Abramson S.N. Li Y. Culver P. Taylor P. J. Biol. Chem. 1989; 264: 12666-12672Abstract Full Text PDF PubMed Google Scholar). Erythroidine is an alkaloid with curare-like activity that is isolated from the seeds of the trees and shrubs of the genus Erythrina. Methyllycaconitine is a tertiary diterpenoid isolated from the seeds of Delphinium brownii(the larkspur plant). Larkspurs are widely distributed in western North America and they kill more cattle on range lands than any other poisonous plant (8Majak W. McDiarmid R.E. Hall J.W. Willms W. J. Range Mgmt. 2000; 53: 207-210Crossref Scopus (8) Google Scholar). Neosurugatoxin is the primary toxin component of the Japanese ivory mollusc, Babylonia japonica (9Hayashi E. Isogai M. Kagawa Y. Takayanagi N. Yamada S. J. Neurochem. 1984; 42: 1491-1494Crossref PubMed Scopus (51) Google Scholar). Among toxins used by venomous organisms to capture prey, the most well known examples are the snake venom polypeptides. More than 90 neurotoxin antagonists of nAChRs have been isolated from dozens of species of land and sea snakes of the Elapidae and Hydrophidae families. The α-neurotoxins are a dominant component of snake venoms. The most extensively characterized α-neurotoxin is α-bungarotoxin from the Taiwanese banded krait. α-Bungarotoxin causes paralysis and death in prey by binding (with near irreversibility) to the neuromuscular junction nAChR (for a review, see Ref. 10Chiappinelli V.A. Harvey A. Natural and Synthetic Neurotoxins. Academic Press, Harcourt Brace Jovanovich, London1993: 65-109Google Scholar). α-Conotoxins are small disulfide-rich peptides that have been isolated from Conus. Conus is a large genus of predatory marine snails, some of which feed on fish. A major component of the complex venomous arsenal that the fish-eating Conusemploy are toxins that act at the muscle nicotinic receptor subtype. Examples include the α-, αA- and ψ-conotoxins (11McIntosh J.M. Santos A.D. Olivera B.M. Annu. Rev. Biochem. 1999; 68: 59-88Crossref PubMed Scopus (276) Google Scholar). In this report, we describe the cloning and synthesis of a novel α-conotoxin from the fish-eating cone snail Conus geographus. In contrast to previously isolated α-conotoxins from this species, the new α-conotoxin has no detectable activity at the muscle subtype of receptor, but instead, it potently targets neuronal nAChRs. DNA from C. geographushepatopancreas was isolated using the Puregene DNA Isolation Kit (Gentra Systems, Minneapolis, MN). Frozen tissue was placed in 600 μl of lysis buffer, homogenized with a disposable microcentrifuge pestle, and digested overnight with 60 μg of proteinase K at 55 °C. The remainder of the procedure followed the kit manufacturer's suggested protocol for marine invertebrates. The resulting genomic DNA was used as a template for PCR using oligonucleotides, tailed for cloning, corresponding to the 3′-end of the intron preceding the toxin region of α-conotoxin prepropeptides and the 3′-UTR (untranslated region) sequence of the α prepropeptides (12Schoenfeld R.A. The Genomic Structure of Delta-contoxins and other O-superfamily Conotoxins.Ph.D. thesis. University of Utah, 1999Google Scholar). The resulting PCR product was purified using the High Pure PCR product purification kit (Roche Molecular Biochemicals) following the manufacturer's suggested protocol. The eluted DNA fragment was annealed to pAMP1 vector, and the resulting product was used to transfect competent DH5α cells with the CloneAmp pAMP system for rapid cloning of amplification products (Invitrogen) following the manufacturer's suggested protocols. The nucleic acid sequences of the resulting clones were determined according to the standard protocol for the Sequenase version 2.0 DNA sequencing kit as described previously (13Jimenez E.C. Craig A.G. Watkins M. Hillyard D.R. Gray W.R. Gulyas J. Rivier J.E. Cruz L.J. Olivera B.M. Biochemistry. 1997; 36: 989-994Crossref PubMed Scopus (118) Google Scholar). Peptide was synthesized, 0.45 mmol/g, on a Fmoc amide resin (Applied Biosystems, catalog No. 401435) using Fmoc chemistry and standard side chain protection except on cysteine residues. Cys residues were protected in pairs with eitherS-trityl on Cys2 and Cys8 orS-acetamidomethyl on Cys3 and Cys16. Amino acid derivatives were from Bachem (Torrance, CA). The peptides were removed from the resin and precipitated; a two-step oxidation protocol was used to selectively fold the peptides as described previously (14Walker C. Steel D. Jacobsen R.B. Lirazan M.B. Cruz L.J. Hooper D. Shetty R. DelaCruz R.C. Nielsen J.S. Zhou L. Bandyopadhyay P. Craig A. Olivera B.M. J. Biol. Chem. 1999; 274: 30664-30671Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Briefly, the first disulfide bridge was closed by dripping the peptide into an equal volume of 20 mmpotassium ferricyanide, 0.1 m Tris, pH 7.5. The solution was allowed to react for 30 min, and the monocyclic peptide was purified by reverse phase HPLC. Simultaneous removal of theS-acetamidomethyl groups and closure of the second disulfide bridge was carried out by iodine oxidation. The monocyclic peptide and HPLC eluent was dripped into an equal volume of iodine (10 mm) in H2O:trifluoroacetic acid:acetonitrile (78:2:20 by volume) and allowed to react for 10 min. The reaction was terminated by the addition of ascorbic acid diluted 20-fold with 0.1% trifluoroacetic acid, and the bicyclic was purified by HPLC. Clones of human nAChR subunits were used to produce cRNA for injection into Xenopus oocytes as described previously (15McIntosh J.M. Gardner S. Luo S. Garrett J.E. Yoshikami D. Eur. J. Pharmacol. 2000; 393: 205-208Crossref PubMed Scopus (43) Google Scholar). A 30-μl cylindrical oocyte recording chamber fabricated from Sylgard was gravity-perfused with ND96A (96.0 mm NaCl, 2.0 mm KCl, 1.8 mmCaCl2, 1.0 mm MgCl2, 1 μm atropine, 5 mm HEPES, pH 7.1–7.5) at a rate of ∼2/min (16Luo S. Kulak J.M. Cartier G.E. Jacobsen R.B. Yoshikami D. Olivera B.M. McIntosh J.M. J. Neurosci. 1998; 18: 8571-8579Crossref PubMed Google Scholar). All toxin solutions also contained 0.1 mg/ml bovine serum albumin to reduce nonspecific adsorption of peptide. The perfusion medium could be switched to one containing peptide or ACh by utilizing a series of three-way solenoid valves. ACh-gated currents were obtained with a two-electrode voltage clamp amplifier (model OC-725B, Warner Instrument, Hamden, CT) and data captured as previously described (16Luo S. Kulak J.M. Cartier G.E. Jacobsen R.B. Yoshikami D. Olivera B.M. McIntosh J.M. J. Neurosci. 1998; 18: 8571-8579Crossref PubMed Google Scholar). The membrane potential of the oocytes was clamped at −70 mV. To apply a pulse of ACh to the oocytes, the perfusion fluid was switched to one containing ACh for 1 s. This was done automatically at intervals of 1–5 min. The shortest time interval was chosen such that reproducible control responses were obtained with no observable desensitization. The concentration of ACh was 10 μm for trials with α1β1δε and 50 μmfor all other nAChRs. For responses in toxin (test responses), the perfusion solution was switched to ND96A and toxin. Toxin was perfused until the maximum block was obtained. Thereafter, toxin was washed away and ACh pulses were given. All ACh pulses contain no toxin, for it was assumed that little if any bound toxin washed away in the brief time ( 5 orders-of-magnitude selectivity for the α3β2 subunit combinationversus the muscle subtype. The pharmacological profile of α-conotoxin GIC is generally similar to that of α-conotoxin MII; both have high potency for the α3β2 subunit combination. However, the kinetics of block by these toxins are substantially different. As shown in Fig. 5, block by α-conotoxin GIC is reversed relatively rapidly (>50% recovery in 1 min) upon toxin washout. In contrast, block by α-conotoxin MII is only slowly reversed, with ∼50% recovery after 19 min.Table IIC50s of α-conotoxin GIC on nAChRs expressed in Xenopus oocytesnAChRIC50nmhα1β1δɛ>100,000hα3β21.1hα3β4755hα4β2309h, human. Open table in a new tab Figure 5Wash-out kinetics of α-conotoxins GIC and MII.A, nAChR block by α-conotoxin GIC is rapidly reversible. After control responses were obtained, 100 nm α-conotoxin GIC was applied to human α3β2-expressing oocytes for 10 min. The oocyte was then continuously perfused with buffer without toxin while responses to ACh were obtained. Similar results were obtained in two other experiments. B, the same protocol was used for application of 100 nm α-conotoxin MII. The recovery from block was ∼20-fold slower than in the case of α-conotoxin GIC.View Large Image Figure ViewerDownload Hi-res image Download (PPT) h, human. We have characterized a 16-amino acid conotoxin with four Cys residues. The homology of the encoding gene with other conotoxin genes reveals that the peptide belongs to the A-superfamily ofConus toxins. The A-superfamily comprises a variety of peptides, most of which act on nAChRs, although others act at potassium or sodium channels (18McIntosh J.M. Olivera B.M. Cruz L.J. Methods Enzymol. 1998; 294: 605-624Crossref Scopus (77) Google Scholar). The sequence and activity of the newly characterized toxin relegates it to the α-conotoxin family, members of which act on nAChRs. The total chemical synthesis of the new peptide, α-conotoxin GIC, was carried out using orthogonal protection of Cys residues to direct disulfide bond formation in the previously characterized pattern of α-conotoxins. We note that α-conotoxin GIC has not yet been isolated from venom. It is possible that there are post-translational modifications present in the native peptide that could influence the properties reported for the synthetic peptide described in this report. Previously isolated α-conotoxins from the fish-hunting speciesC. geographus (see Table II) have what is referred to in the literature as a 3/5 spacing, indicating that there are respectively three and five amino acids in between Cys residues in the two loops of the toxin. The newly characterized toxin has a 4/7 spacing. The α3/5 toxins, isolated based on their ability to cause paralysis in mice, previously have been shown to potently inhibit the muscle nicotinic receptor subtype. Likewise, the major α3/5 conotoxin from C. geographus, α-conotoxin GI, is a potent paralytic in fish. In contrast, α-conotoxin GIC fails to produce paralysis in either mouse or fish. Electrophysiological testing of α-conotoxin GIC on the human muscle subtype of receptor expressed in Xenopus oocytes is consistent with lack of paralytic activity. Little or no block of ACh-induced current was produced at concentrations up to 100 μm toxin.Table IIα-Conotoxinsα-ConotoxinSpeciesSequencenAChR targetGICC. geographusGCCSHPACAGNNQHIC#NeuronalMIIC. magusGCCSNPVCHLEHSNLC#NeuronalGIC. geographusECCNPACGRHYSC#MuscleGIAC. geographusECCNPACGRHYSCGK#MuscleGIBC. geographusECCNPACGRHYSCKG#MuscleMIC. magusGRCCHPACGKNYSC#MuscleMIAC. magusDGRCCHPACAKHFNC#MuscleMIBC. magusNGRCCHPACARKYNC#Muscle#, C-terminal amidation. Open table in a new tab #, C-terminal amidation. On the other hand, α-conotoxin GIC potently blocks the α3β2 subunit combination of receptor with an IC50 of ∼1 nm (Fig. 3 and Table I). It also has a modest amount of activity at α3β4 and α4β2 receptor subtypes. With respect to its pharmacological profile of activity, α-conotoxin GIC is similar to α-conotoxin MII, which also has high affinity for α3β2 receptors, and is structurally similar in that both have an α4/7 spacing. The two α-conotoxins differ in a number of respects, however. Although α-conotoxins MII and GIC share six of the eight N-terminal amino acids, they differ in seven of the eight C-terminal amino acids (Table II). α-Conotoxin GIC is much more hydrophilic than α-conotoxin MII (Fig. 2). Particularly striking is the difference in the kinetics of block between the two toxins. We previously demonstrated that block of rat α3β2 receptors by α-conotoxin MII is only slowly reversible (t12∼7.7 min; (19Cartier G.E. Yoshikami D. Gray W.R. Luo S. Olivera B.M. McIntosh J.M. J. Biol. Chem. 1996; 271: 7522-7528Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar). In this report, we demonstrate that the off-time of α-conotoxin MII is even slower at human α3β2 receptors (t12 ∼19 min). In contrast, block by α-conotoxin GIC is much more rapidly reversed (∼20-fold faster; Fig. 5), making α-conotoxin GIC a potentially useful ligand for electrophysiological experiments, in which high potency and rapid reversibility are desired characteristics. α-Conotoxin GIC has the highest known selectivity for neuronal (e.g. the α3β2 subtype) versus muscle subtype of any nicotinic ligand characterized thus far. Although α-conotoxin MII itself is already quite selective (>1,000-fold selectivityversus the muscle subtype), α-conotoxin GIC has >100,000-fold selectivity for α3β2 versus the muscle receptor. Why does C. geographus, a mollusc that depends on paralyzing its prey, produce a nonparalytic toxin targeted to neuronal nicotinic acetylcholine receptors? The present findings do not answer this question, but it is worth noting compounds with similar pharmacological specificity reported from other venomous organisms. Although the major component in Bungarus multicinctus venom is α-bungarotoxin, this snake also produces a minor component known as κ-bungarotoxin, which potently blocks α3β2 receptors expressed in Xenopus oocytes (IC50 ∼1 nm). The mechanism of action is consistent with competitive blockade (20Harvey S.C. Luetje C.W. J. Neurosci. 1996; 16: 3798-3806Crossref PubMed Google Scholar,21Luetje C.W. Maddox F.N. Harvey S.C. Mol. Pharmacol. 1998; 53: 1112-1119PubMed Google Scholar). Additional subtypes of nicotinic receptors are also blocked by κ-bungarotoxin, including the muscle subtype. The kinetics of block, however, are significantly different, and an α3β2-like receptor seems to be the primary target (20Harvey S.C. Luetje C.W. J. Neurosci. 1996; 16: 3798-3806Crossref PubMed Google Scholar, 22Papke R.L. Duvoisin R.M. Heinemann S.F. Proc. R. Soc. Lond. B Biol. Sci. 1993; 252: 141-148Crossref PubMed Scopus (45) Google Scholar). C. magus produces peptides that are highly selective for the muscle subtype of nicotinic receptor and that are relatively inactive on the α3β2 nAChRs (e.g. α-conotoxin MI) (17Johnson D.S. Martinez J. Elgoyhen A.B. Heinemann S.F. McIntosh J.M. Mol. Pharmacol. 1995; 48: 194-199PubMed Google Scholar,23McIntosh J.M. Cruz L.J. Hunkapiller M.W. Gray W.R. Olivera B.M. Arch. Biochem. Biophys. 1982; 218: 329-334Crossref PubMed Scopus (158) Google Scholar). However, C. magus also produces α-conotoxin MII, which has only very weak activity at the muscle subtype (19Cartier G.E. Yoshikami D. Gray W.R. Luo S. Olivera B.M. McIntosh J.M. J. Biol. Chem. 1996; 271: 7522-7528Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar). We establish in this report that C. geographus produces, in addition to its muscle-targeted toxins, α-conotoxin GIC, which has potent neuronal activity and a striking lack of activity on the muscle receptor. Perhaps it is not coincidental that these polypeptides, κ-bungarotoxin, α-conotoxin MII, and α-conotoxin GIC, have weak or no activity on the muscle receptor, and yet each has high affinity for α3β2-like receptors. It seems striking that two very distinct predatory venomous organisms (snakes and snails) appear to have independently evolved not only toxins that target neuromuscular nAChRs but also those that specifically target α3β2-like receptors. The biological utility of the latter type of toxins is unknown; however, α3β2-like receptors exist in autonomic ganglia, where they in part modulate the fight-or-flight response (24Tavazoie S.F. Tavazoie M.F. McIntosh J.M. Olivera B.M. Yoshikami D. Br. J. Pharmacol. 1997; 120: 995-1000Crossref PubMed Scopus (20) Google Scholar). One possibility is that the function of the toxins that target α3β2-like receptors is to suppress this response rather than to block neuromuscular activity. Alexandria Vyazovkina assisted with the synthesis of α-conotoxin GIC.