A Noncompetitive Peptide Inhibitor of the Nicotinic Acetylcholine Receptor from Conus purpurascens Venom † Ki-Joon Shon, ‡,§ Michelle Grilley, ‡ Richard Jacobsen, ‡ G. Edward Cartier, ‡ Chris Hopkins, ‡ William R. Gray, ‡ Maren Watkins, | David R. Hillyard, | Jean Rivier, ⊥ Josep Torres, ⊥ Doju Yoshikami, ‡ and Baldomero M. Olivera* ,‡ Department of Biology and Pathology, UniVersity of Utah, Salt Lake City, Utah, 84112, and The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, California 92037 ReceiVed January 31, 1997; ReVised Manuscript ReceiVed April 28, 1997 X ABSTRACT: A paralytic peptide, ψ-conotoxin PIIIE has been purified and characterized from Conus purpurascens venom. Electrophysiological studies indicate that the peptide inhibits the nicotinic acetylcholine receptor (nAChR). However, the peptide does not block the binding of R-bungarotoxin, a competitive nAChR antagonist. Thus, ψ-conotoxin PIIIE appears to inhibit the receptor at a site other than the acetylcholine-binding site. As ascertained by sequence analysis, mass spectrometry, and chemical synthesis, the peptide has the following covalent structure: HOOCCLYGKCRRYOGCSSASCCQR* (O ) 4-trans hydroxyproline; * indicates an amidated C-terminus). The disulfide connectivity of the toxin is unrelated to the R- or the RA-conotoxins, the Conus peptide families that are competitive inhibitors of the nAChR, but shows homology to the µ-conotoxins (which are Na + channel blockers). Marine snails of the genus Conus paralyze their prey by injecting venom through a hollow harpoon-like tooth. The active paralytic agents in these venoms, the conotoxins, are small, multiply disulfide-bonded peptides. Most of the 500 cone snails prey on various invertebrates; however, there are ca. 50-70 Conus species which prey exclusively on fish. In the highly venomous fish-hunting Conus geographus (the geography cone), the major paralytic conotoxins fall into three distinct classes (for reviews, see refs 1 and 2). The µ-conotoxins, 22 amino acids in length, have three disulfide bonds and inhibit skeletal muscle voltage-gated sodium channels at site I, the tetrodotoxin-saxitoxin binding site. Another class of C. geographus paralytic conotoxins are the ω-conotoxins, 25-29 amino acids in length, with three disulfide bonds, which specifically inhibit presynaptic volt- age-sensitive calcium channels. Finally, several R-conotox- ins have been characterized; these peptides are 13-15 amino acids in length and block the acetylcholine binding sites of nicotinic acetylcholine receptors. These are the same sites to which R-neurotoxins from snakes, and curare, the South American Indian poison arrow toxin, bind to manifest their neurotoxicity. We have recently been investigating the venom of another Conus species, the purple cone Conus purpurascens (3-5). It is found in the eastern Pacific, from the Gulf of California to Peru, including outlying islands such as the Galapagos and Clipperton Atoll, and is the only fish-hunting Conus species known from the eastern Pacific marine province. C. purpurascens is believed to have long been geographically isolated from the major line of fish-hunting Conus species found in the Indo-Pacific, such as C. geographus. Previous analysis of the venom of C. purpurascens revealed that this species simultaneously uses two broad physiological strategies for prey immobilization: excitotoxic shock and neuromuscular block (5). Among the toxins in C. purpurascens venom which effect neuromuscular block is a peptide that inhibits the acetylcholine receptor at the ligand binding site, RA-conotoxin PIVA (3). Although this peptide is structurally distinct from the R-conotoxins from C. geographus and other piscivorous Indo-Pacific Conus species, it nevertheless appears to target the same ligand- binding site on the acetylcholine receptor complex. Recently, a µ-conotoxin in C. purpurascens venom with significant homology to the C. geographus µ-conotoxin has been characterized (K.-J. Shon et al., manuscript in preparation). We have not yet identified an ω-conotoxin from C. pur- purascens venom. In this report, we describe the isolation and characterization of a third type of paralytic peptide in C. purpurascens venom. It is distinct in its physiological activity, pharmacology, and specificity from previous classes of paralytic conotoxins characterized from fish-hunting cone snails. The peptide therefore defines a novel family of conotoxins, which we designate ψ-conotoxins. METHODS C. purpurascens Specimen Collection and Venom Extrac- tion. Specimens of the purple cone C. purpurascens were collected from the Gulf of California. The venom was acquired by milking the snails as previously described (3). The snails were milked twice a week, and an average-sized C. purpurascens (∼4 cm) yielded approximately 10-15 µL of venom from each milking. Milked venom stored at -70 °C was pooled. Most large-scale purification runs were carried out with ca. 0.5 mL of milked venom per run. Peptide Purification by HPLC. Preparative scale reversed- phase HPLC was used as the first step in the purification of the milked venom. The venom was diluted with 0.1% trifluoroacetic acid (TFA), spun for a few minutes with a † This research was supported by P01 GM48677. * To whom all correspondence should be addressed. ‡ Department of Biology, University of Utah. § Current address: Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH 44106. | Department of Pathology, University of Utah. ⊥ The Salk Institute. X Abstract published in AdVance ACS Abstracts, July 1, 1997. 9581 Biochemistry 1997, 36, 9581-9587 S0006-2960(97)00235-3 CCC: $14.00 © 1997 American Chemical Society