A new family of Conus peptides targeted to the nicotinic acetylcholine receptor.

In this work, a new family of Conus peptides, the αA-conotoxins, which target the nicotinic acetylcholine receptor, is defined. The first members of this family have been characterized from the eastern Pacific species, Conus purpurascens (the purple cone); three peptides that cause paralysis in fish were purified and characterized from milked venom. The sequence and disulfide bonding pattern of one of these, αA-conotoxin PIVA, is as follows: where O represents trans-4-hydroxyproline. The two other peptides purified from C. purpurascens venom are the under-hydroxylated derivatives, αA-conotoxin PIVA and [Pro]αA-conotoxin PIVA. The peptides have been chemically synthesized in a biologically active form. Both electrophysiological experiments and competition binding with α-bungarotox- in demonstrate that αA-PIVA acts as an antagonist of the nicotinic acetylcholine receptor at the postsynaptic membrane.

All of the ␣-conotoxins from fish-hunting Indo-Pacific cone snails show high structural homology and a conserved sequence motif. The seven peptides that have been purified share the consensus sequence XCC(H/N)PACGXX(Y/F)XC. All of these peptides appear to be potent blockers of neuromuscular transmission in teleosts but differ significantly in their potency when tested in other vertebrates; thus, ␣-conotoxin GI appears to be very potent in blocking the nicotinic acetylcholine receptor at the neuromuscular junction of all vertebrates tested, while ␣-conotoxins SI and SII are highly potent in teleosts, but not mammals (3,4). In addition to the ␣-conotoxins from Indo-Pacific fish-hunting Conus, two ␣-conotoxins have been purified from non-fish-hunting Indo-Pacific species (5,6). Although the peptides purified differ significantly from the consensus sequence given above, they do retain the same pattern of Cys residues (the "Cys framework"), i.e. X n CCX n CX n CX n .
Because this basic motif was found in both fish-hunting and non-fish-hunting Conus species, it seemed reasonable to expect that all nAChR-targeted peptides in Conus venoms would share the same conserved ␣-conotoxin Cys framework. In this report, we describe a novel nAChR-targeted conotoxin from C. purpurascens, the purple cone (Fig. 1). C. purpurascens is definitely a piscivorous snail, and it was therefore a surprise to find that in this species, the group of peptides that blocks nicotinic acetylcholine receptors does not have a Cys framework typical of ␣-conotoxins.
A unique feature of the study described below is that in contrast to all previous biochemical studies on Conus venoms, which were carried out with dissected venom ducts, the venom used here was milked from living animals. The milking procedure we have developed allows us to harvest much larger amounts of venom from relatively few cone snail specimens.

MATERIALS AND METHODS
Venom Collection and Preparation-C. purpurascens specimens (3-5 cm in length) were collected from the Gulf of California. Because a sufficient number of C. purpurascens specimens to prepare venom from dissected ducts for biochemical purification was not available, C. purpurascens were milked approximately twice a week. A goldfish was placed in front of the snail to be milked and in most instances, this elicited full extension of the proboscis (see Fig. 1B). A surrogate fish consisting of a microcentrifuge tube covered with a latex membrane, cut out from a condom or a glove and overlaid with a fresh goldfish fin, was then substituted for the goldfish at the tip of the proboscis (see Fig. 2). The snail would harpoon the surrogate and typically eject about 5 l of venom; the harpoon was cut with scissors after ejection of the venom. The goldfish was fed to the snail, and the snail was returned to its aquarium. The venom was centrifuged to the bottom of the tube and pooled together with previous milkings from the same snail. For the general purification described below, crude milked venom from many snails was combined.
There seemed to be considerable variation in how often it was possible to milk individual snails. Some specimens seemed unable or unwilling to harpoon the surrogate at all under these conditions, but the majority of the specimens of C. purpurascens could be milked regularly twice a week. Most of the specimens of C. purpurascens could be milked on this schedule for 6 months to a year. Among the other Conus species milked by this procedure are: C. striatus, Conus obscurus, Conus stercusmuscarum, Conus ermineus, Conus monachus and Conus catus.
Peptide Purification by HPLC-A preparative scale reversed-phase HPLC was used for the first line purification of the milked venom. One-half ml of milked venom was diluted with 10 ml of 0.1% trifluoroacetic acid solution and spun for a few minutes in a bench top microfuge. The supernatant was applied to a C 18 Vydac preparative column (22 ϫ 250 mm; 20 ml/min) with a guard column (22 ϫ 50.8 mm). As a secondary purification, a C 18 Vydac analytical column (218TP54, 4.6 ϫ 250 mm;1 ml/min) was used. HPLC buffers were 0.1% trifluoroacetic acid in water (buffer A) and 0.085% trifluoroacetic acid in 90% CH 3 CN (buffer B) for preparative runs and 0.092% trifluoroacetic acid in 60% acetonitrile for analytical runs. For both preparative and analytical runs, the peptides were eluted with a linear gradient of 1% buffer B increase/min. The C 18 analytical column was also used for purifying alkylated peptides for amino acid sequence analysis.
Analysis of Covalent Structure-Three closely related peptides were isolated, which differ solely in hydroxylation of their proline residues; fully hydroxylated peptide is designated ␣A-conotoxin PIVA, with the others being assigned derivative status.
Primary Structure-Disulfides were reduced by TCEP, and the linear peptide was purified and alkylated with 4-vinylpyridine as described by Gray (7). The pyridylethylated peptide was repurified by HPLC, and a sample was analyzed in an ABI model 477A sequencer. A second sample was digested with endoproteinase Lys-C (18 h, 37°C); the fragments were isolated by HPLC and subjected to sequencing and/or mass analysis.
Disulfide Connectivity-Natural ␣A-conotoxin PIVA was analyzed by the partial reduction method of Gray (7,8), suitable reduction conditions being established by small scale trials. To obtain partially reduced intermediates, 10 nmol of peptide in HPLC effluent and 20 mM TCEP in 0.17 M sodium citrate (pH 3) were preequilibrated to 61°C for 5 min. Equal volumes of the reactants were mixed and incubated a further 2 min before injection onto the HPLC column. Eluted peptides were immediately frozen (pH 2.0) at Ϫ20°C to minimize disulfide exchange. They were individually thawed, repurified, and immediately alkylated with 2.2 M iodoacetamide using the rapid procedure of Gray (7). The carboxyamidomethylated peptides were desalted, and their residual disulfides were reduced and alkylated with 4-vinylpyridine. Sequencer analysis was then used to determine the positions of Cys(cam) and Cys(pe) residues, thus establishing which residues had been free or bridged. Analysis of two intermediates was sufficient to deduce the disulfide connectivity.
Mass Spectrometry-Positive ion LSIMS spectra were obtained using a Jeol JMS HX110 double-focusing spectrometer, fitted with a Cs ϩ ion gun operated at ϩ30 kV.
Solid Phase Peptide Synthesis-␣A-Conotoxin PIVA was built using standard Fmoc chemistry on an ABI model 430A peptide synthesizer, couplings were carried out with equimolar amounts of amino acid FIG. 1. A, a specimen of the purple cone, C. purpurascens, from the Cocos Islands, in the Eastern Pacific marine province. B, C. purpurascens with its proboscis extended, in response to the presence of a fish. A dose of venom (about 5 l) has been transferred from the venom duct to the proboscis. If the snail is able to strike the fish, it will inject the venom through a disposable harpoon-like tooth, which it has already transferred from a quiverlike organ, the radula sac, to the tip of the proboscis. Photograph by Alex Kerstitch.

FIG. 2. Milking procedure.
A, a fish is placed in front of the snail until it extends its proboscis. The collection tube (surrogate fish) is quickly substituted for the fish as the snail is about to strike. B, once the snail has harpooned the collection tube, the harpoon is cut off. Right panel shows the collection tube assembly. The cap of a microcentrifuge tube is hollowed out and placed over a membrane with a cut up fish tail to seal the top of the tube. C. purpurascens will generally be reluctant to strike unless the tip of its proboscis actually contacts fish tissue; the fish tail suffices for this purpose. Once the venom has been collected, the microcentrifuge tube is resealed with its own cap and some parafilm, and the venom is collected by centrifugation. derivative, dicyclohexylcarbodiimide, and hydroxybenzotriazole. All amino acids were from Bachem (Torrance, CA) with side-chain protection: pentamethylchromansulfonyl (Arg); trityl (Asn, Cys, Gln, His); t-butyl (Asp, Hyp, Ser, Tyr); t-butyloxycarbonyl (Lys). At the completion of synthesis, the terminal Fmoc group was removed by a standard machine cycle.
Peptide was removed from the resin and deprotected by treatment (2 h, 20°C) with trifluoroacetic acid/H 2 O/ethanedithiol/phenol/thioanisole (36/2/1/3/2 by volume), and the whole mixture was filtered rapidly into t-butyl methyl ether at Ϫ10°C. The precipitate was collected by centrifugation, dissolved in 60% CH 3 CN containing 0.092% trifluoroacetic acid, and diluted 10-fold with 0.1% trifluoroacetic acid in water. Linear peptide was purified by reversed-phase HPLC on a C 18 preparative column (Vydac 218TP1022, 20 ml/min), with a gradient of CH 3 CN (6 -30%) in 0.1% trifluoroacetic acid. The peptide solution was diluted to approximately 20 M, glutathione was added to give final concentrations of 1.0 mM reduced and 0.5 mM oxidized, and the pH was adjusted to 7.5 with NaOH.
Purification was carried out on a Vydac C 18 column (5 ϫ 30 cm) using a gradient of CH 3 CN in TEAP buffer at pH 5.30 (6 -21%/60 min; 100 ml/min). Fractions were collected and screened for purity by analytical HPLC on a Vydac C 18 column (0.46 ϫ 25 cm) eluted isocratically with 16.2% CH 3 CN in 0.1% trifluoroacetic acid (2 ml/min). Samples were also analyzed by capillary electrophoresis at 20 kV, using 0.1 M sodium phosphate at pH 2.50 and a fused silica capillary (50 cm ϫ 75 m). Fractions of the highest purity were pooled and repurified/desalted on the same preparative column, using a gradient of CH 3 CN in 0.1% trifluoroacetic acid (6 -36%/60 min; 100 ml/min).
Electrophysiology-Wire electrodes were used to record extracellular synaptic currents from populations of endplates in frog cutaneus pectoris preparations that had been treated with ␣-bungarotoxin to irreversibly attenuate the synaptic response to a subthreshold level as described previously (10).
Biological Assays-Intraperitoneal injections into goldfish and 10 -14-day-old Swiss Webster mice, and intracranial injection into mice were performed as described previously (11).
Binding Assays-Binding assays were performed using a membrane fraction enriched in nAChR prepared from Torpedo californica electroplax (Pacific Bio-Marine, Venice, CA). All procedures were performed at 0 -4°C. Fifty ml of buffer (0.25 M NaCl, 0.02 M HEPES, 2 mM EDTA, pH 7.4) containing 1 mM phenylmethylsulfonyl fluoride, 1 M pepstatin, and 2 M leupeptin were added to 50 g of frozen Torpedo tissue, followed by processing in a Sorvall Omnimixer (30 s on highest setting, 30 s off, for a total of 6 min) and 5 stroke cycles in a Dounce homogenizer. Large particulate matter was removed by centrifugation at 3000 ϫ g for 10 min. Membrane fragments were collected by centrifugation of the supernatant at 17000 ϫ g for 50 min. The resulting pellet was resuspended in 10 ml of buffer and stored in small aliquots at Ϫ70°C. Torpedo receptors (0.38 pmol of ␣-bungarotoxin binding sites/mg of protein, 2 g protein/0.1 ml assay) were diluted into 0.02 M HEPES, pH 7.4, 5 mM EDTA, 0.5 mg/ml bovine serum albumin, and 0.5 mg/ml lysozyme and preincubated for 30 min at room temperature with either unlabeled ␣-bungarotoxin or with various concentrations of synthetic PIVA, followed by a 15-min incubation with ␣-[ 125 I]bungarotoxin (Amersham Corp., 2000 Ci/mmol, 12.5 pmol/assay). Unbound ␣-bungarotoxin was separated from receptor by centrifugation in a microcentrifuge for 3 min at ϳ15,000 ϫ g. The pellet was washed with 0.1 ml of 0.02 M HEPES, pH 7.4, and 5 mM EDTA, followed by centrifugation. Radioactivity in pellet and supernatant was determined using a Packard Multi-Prias ␥ counter. Nonspecific binding was typically less than 2.5% of total binding, determined by preincubation with 0.01 mM unlabeled ␣-bungarotoxin. Fig. 3A. Arrows indicate three fractions that caused paralysis upon injection into goldfish, and that proved to be proline-hydroxylation isoforms of a single peptide. Further purification of peptide A, in which all three proline residues were hydroxylated, is shown in Fig. 3, B and C. Peptides B and C were similarly purified.

Toxin Purification and Characterization-A chromatographic profile of crude milked venom is shown in
Amino Acid Sequences-Sequencer analysis of reduced and alkylated peptide A is reported in Table I. A single unambiguous sequence was obtained for 25 cycles. Attempts to obtain LSIMS mass spectra were initially unsuccessful because of limited amounts of material; all of these peptides have consistently given a 20 -50-fold lower sensitivity than is typical for peptides in this size range. Lys-C digestion of pyridylethylated toxin yielded two peptides. One of these corresponded in mass to that expected for the amidated C-terminal fragment (resi- After completion of the synthesis of peptides A and C (below), and completion of the biological characterization, more snails became available, allowing reisolation of all three isoforms. LSIMS analysis was successfully carried out on intact peptides, and the monoisotopic molecular ions MH ϩ were in agreement with those predicted from the above sequences: A (observed, 2648.0; theoretical, 2647.93); B (observed, 2631.9; theoretical, 2631.93); C (observed, 2615.9; theoretical, 2615.94).
Disulfide Bridge Analysis-This was carried out on 10 nmol of native peptide isolated from venom. Reaction with 10 mM TCEP at pH 3.0 (2 min, 61°C) gave a suitable distribution of partially reduced peptides (Fig. 4A). Three major intermediates (intermediates 1-3) are evident, and others are indicated by broadening of the original peptide peak N. Intermediates 1 and 3 were purified and alkylated with iodoacetamide to label free thiols; residual bridges were then reduced and alkylated with 4-vinylpyridine. Sequencer analysis of intermediate 1 released Cys(cam) at cycles 3 and 11, while Cys(pe) was released at cycles 2, 14, 16, and 23. Analysis of intermediate 3 released Cys(cam) at cycles 2, 3, 11, and 16 and Cys(pe) at cycles 14 and 23. All other amino acids were identical to those shown in Table  I, fully confirming the original sequence assignment. Thus the bridging pattern is [Cys 2 -Cys 16 ; Cys 3 -Cys 11 ; Cys 14 -Cys 23 ], and the two intermediates lie on the reduction pathway shown in Fig. 4B. This particular disulfide topology has not previously been found in conotoxins.
Peptide Synthesis-Peptides A and C were chosen as synthetic targets to provide sufficient material for more detailed biological study. Peptide A was constructed using Fmoc chemistry, linear peptide was purified by HPLC, and disulfide bridges were allowed to form in the presence of a glutathione redox buffer. Under these conditions greater than 50% of the product eluted at the position corresponding to natural peptide. The misfolded material was recycled to obtain more of the correct form. After further HPLC purification in the trifluoroacetic acid system the peptide was judged to be 97-98% pure by analytical HPLC in TEAP buffers. Co-elution experiments confirmed identical chromatographic behavior of natural and synthetic products. LSIMS analysis of synthetic peptide indicated the correct monoisotopic MH ϩ (observed, 2647.9; theoretical, 2647.93). As with the natural peptide, ionization was very inefficient, and relatively large amounts of sample were required.
Peptide C was constructed using t-butyloxycarbonyl chemistry. Crude linear peptide was air-oxidized by stirring at pH 7.0; the reaction was judged complete after 4 days. Purification was carried out by adsorption onto Bio-Rex 70 cation exchanger, followed by preparative HPLC using TEAP buffers, with a final desalting and purification by HPLC in the trifluoroacetic acid system. TEAP at pH 5.30 was found to be preferable to the usual TEAP, pH 2.25, since the latter did not yield high purity fractions or a good yield of purified peptide. The advantage of using TEAP at various pH values has been recognized previously in the purification of synthetic peptides (12). The resulting peptide, which co-eluted with material isolated from venom, appeared to be 99% pure as analyzed by analytical HPLC and capillary electrophoresis. LSIMS showed the expected molecular ion MH ϩ (observed, 2616.0; theoretical, 2615.94).
The major refolded material for both synthetic peaks A and C proved to be identical to their native counterparts; a mixture of native and synthetic peptide in each case gave a single sharp peak upon HPLC analysis (see Fig. 5). Both synthetic peptides were found to be biologically active by the fish paralysis assay. The chemical synthesis of these two peptides therefore confirms the sequence assignments given above, including the C-terminal amidation.
Because these peptides, like the ␣-conotoxins, inhibit the nicotinic acetylcholine receptor (see below), we have designated peak A ␣A-conotoxin PIVA, where the Roman numeral indi- FIG. 4. A, reversed-phase HPLC chromatogram of ␣A-conotoxin PIVA, partially reduced by TCEP (10 mM, pH 3, 5 min at 61°C). The absorbance peaks labeled 1, 2, and 3 correspond to the partially reduced peptides PR1, PR2, and PR3; N and R correspond to the native and completely reduced peptides, respectively. An analytical C 18 Vydac column was used for separating individual peaks using a gradient of CH 3 CN (6 -30%) in trifluoroacetic acid (see "Materials and Methods"). B, disulfide bridge arrangements in native and partially reduced peptides. However, the proportion of the under-hydroxylated forms (peaks B and C) relative to the fully hydroxylated toxin (peak A) varies from one milked venom preparation to the next. Although we previously detected under-hydroxylated forms of other conotoxins (see, for example Ref. 13), they have never been major species in any venom previously characterized. The under-hydroxylated forms may arise as a consequence of artificial aquarium conditions. ␣A-conotoxin PIVA Blocks the Nicotinic Acetylcholine Receptor-The basis for the paralytic activity of ␣A-conotoxin PIVA was investigated by electrophysiological tests and binding experiments. The effect of the toxin on the frog neuromuscular junction is shown in Fig. 6A. The peptide clearly blocks the excitatory postsynaptic response. There was no difference between the time course of the partially blocked response and the control (Fig. 6B). The peptide blocked spontaneous miniature end plate potentials as well as the response to iontophoretically applied carbamylcholine without affecting the resting potential (results not shown), indicating that the peptide acts postsynaptically. These electrophysiological results strongly suggest that the peptide directly inhibits nicotinic acetylcholine receptors.
This possibility was supported by competition binding experiments with ␣-[ 125 I]bungarotoxin used as a reporter for receptor occupancy of high affinity sites in the Torpedo electric organ, which are well-established to be on the nicotinic acetylcholine receptor. The results of binding experiments with ␣Aconotoxin PIVA and the Pro 7,13 derivative are shown in Fig. 7. Both peptides competitively inhibit ␣-bungarotoxin binding, indicating that these peptides target the macrosite that ␣-bungarotoxin binds to on the Torpedo receptor.
A survey of the in vivo biological activity of these peptides is shown in Table II. DISCUSSION The results described above establish that the major paralytic toxin in C. purpurascens venom targeted to nAChRs, ␣A-conotoxin PIVA, has a strikingly different amino acid se- 2 The peptides in this report define both a new Conus peptide family (␣A), as well as a novel structural class. As we will detail elsewhere, we use the Roman numeral IV for all Conus peptides with the Cys framework -CCX n CX 2 CXCX n C-. We have characterized other conotoxin families of the same structural class but with different pharmacological specificities; these have the same disulfide framework but are not targeted to nicotinic acetylcholine receptors. All members of a given Conus peptide family share both a conserved Cys framework as well as a common pharmacological mechanism. We retain the Greek letter ␣ for all Conus peptide families that inhibit the nAChR by binding to the ligand site ␣; to distinguish the new peptides from the ␣-conotoxins, we designate the former as ␣A-conotoxins. An additional distinction is that the distance between the first pair of cysteines and the next cysteine residue in the primary sequence is 7 amino acids in ␣A-conotoxin PIVA but 3-4 amino acids in all ␣-conotoxins.

FIG. 5.
A, reversed-phase HPLC chromatogram of synthetic ␣A-conotoxin PIVA (fully hydroxylated peptide). The peptide was eluted from a C 18 Vydac analytical column with a gradient of CH 3 CN (6 -24%) in trifluoroacetic acid (see "Materials and Methods"). B, co-elution experiment. Equal amounts of the synthetic and native peptides were mixed and co-eluted using the same column, gradient, flow rate, and buffers as in panel A. quence from all other nAChR-targeted peptides previously characterized from Conus venoms. Despite the striking structural divergence, the mechanism by which this peptide causes paralysis is nevertheless similar to the well-characterized ␣-conotoxins from other fish-hunting Conus species, i.e. the peptide blocks the ACh binding site of the nAChR at the neuromuscular junction.
Comparison of the sequence of this new toxin, ␣A-conotoxin PIVA, with previously characterized ␣-conotoxins is shown in Table III. With one exception (␣-conotoxin SII), the latter toxins have two disulfide bonds; in contrast, ␣A-PIVA has three. Furthermore, all paralytic ␣-conotoxins from fish-hunting Conus share the following conserved patterns of Cys and non-Cys amino acids, i.e. CCX 3 CX 5 C. The ␣-conotoxins from non-fishhunting Conus species that have been described so far (5,6), while having spacing different from that for fish-hunters as indicated above, nevertheless have the same Cys framework. In contrast, the new peptide, ␣A-PIVA, not only has a different Cys framework, but the spacing between the first pair of Cys residues and the third Cys is 7 amino acids instead of the 3 or 4 amino acids found in the nine characterized ␣-conotoxins. Thus, the new peptide is the first member of a new family of nAChR-targeted Conus peptides. Homologs of the C. purpurascens peptide will be designated ␣A-conotoxins (because of the divergence in the Cys framework, peptides belonging to this structural class will be given Roman numeral IV, as opposed to the regular ␣-conotoxins, which are always numbered I or II). In addition, Conus peptides with a Cys framework similar to that of PIVA but which are not targeted to the nAChR (and are therefore not ␣A-conotoxins) have also been characterized. 3 Thus, ␣A-PIVA is both the first member of a new family of Conus peptides, the ␣A-conotoxins, and the first representative of a new structural class of Conus peptides. The availability of a new group of nAChR-targeted Conus peptides that have significantly diverged from the ␣-conotoxin series provides new opportunities for probing the nAChR. Previously, reporter groups were attached to ␣-conotoxins at specific loci (14); such an approach can in principle be used to map the topology of the nAChR (15). Since Conus peptides are extensively cross-linked by disulfide bonds and are, therefore, fairly rigid, they provide a structurally discrete probe, which can be used to pinpoint the locations of residues in the receptor. Some ␣-conotoxins have been analyzed by multidimensional NMR techniques (16,17); sufficient amounts of ␣A-conotoxin PIVA have been synthesized, and a structural analysis of these peptides by NMR methods is presently being carried out. Once the structural work is complete, ␣A-PIVA could be a useful probe for the nAChR based on an entirely different structural framework from that of the ␣-conotoxins.
The degree of under-hydroxylation of proline residues in ␣A-conotoxin PIVA deserves comment. Different samples of milked C. purpurascens venom show considerable variation in the degree of under-hydroxylation; in some venom samples, the two under-hydroxylated species described above are present at higher levels than the completely hydroxylated ␣A-PIVA (in contrast to the venom sample shown in Fig. 3A). Small differences in IC 50 s shown in Fig. 7 are seen reproducibly, raising the possibility that the different hydroxylated forms have functional biological significance. We think that at least some of the under-hydroxylation observed may be an artifact of maintaining C. purpurascens in aquaria for extended periods of time. We have observed that several different Conus species become increasingly susceptible to pathology both in the periostracum and in laying down new shell if kept in aquaria in artificial sea water. Thus, it is possible that some under-hydroxylated forms observed in the milked venom are present at lower levels in C. purpurascens under natural conditions and that the underhydroxylation observed may be a biochemical manifestation of a progressive pathology that occurs in aquaria. It is possible that some factors necessary for proline hydroxylation may be-  come limiting. The pattern of under-hydroxylation suggests that the Pro residues are not equivalent as substrates for the hydroxylation enzyme, and that the ease of hydroxylation is in the order: Pro 20 Ͼ Pro 7 Ͼ Pro 13 . In any case, the results in Fig.  7 were unexpected and surprising, and further studies investigating functional effects of proline hydroxylation are clearly desirable. C. purpurascens is believed to be the only fish-hunting Conus species in the eastern Pacific marine geographic province. It has probably been isolated from fish-hunting Indo-Pacific species for an extended period of time. Its closest relative is thought to be C. ermineus, the major fish-hunting Conus species in the Atlantic marine province. An ␣A-conotoxin that differs significantly in sequence from ␣A-conotoxin PIVA has recently been identified in this species. 4 The discovery of the nAChR-targeted peptides in C. purpurascens that are so divergent from those found in other Conus species raises the intriguing possibility that fish-hunting may have evolved more than once in the Conidae. It will be of interest to determine which Conus species use the ␣A-conotoxin family as nAChR ligands instead of members of the ␣-conotoxin family.