A Novel α-Conotoxin, PeIA, Cloned from Conus pergrandis, Discriminates between Rat α9α10 and α7 Nicotinic Cholinergic Receptors*

The α9 and α10 nicotinic cholinergic subunits assemble to form the receptor believed to mediate synaptic transmission between efferent olivocochlear fibers and hair cells of the cochlea, one of the few examples of postsynaptic function for a non-muscle nicotinic acetylcholine receptor (nAChR). However, it has been suggested that the expression profile of α9 and α10 overlaps with that of α7 in the cochlea and in sites such as dorsal root ganglion neurons, peripheral blood lymphocytes, developing thymocytes, and skin. We now report the cloning, total synthesis, and characterization of a novel toxin α-conotoxin PeIA that discriminates between α9α10 and α7 nAChRs. This is the first toxin to be identified from Conus pergrandis, a species found in deep waters of the Western Pacific. α-Conotoxin PeIA displayed a 260-fold higher selectivity for α-bungarotoxin-sensitive α9α10 nAChRs compared with α-bungarotoxin-sensitive α7 receptors. The IC50 of the toxin was 6.9 ± 0.5 nm and 4.4 ± 0.5 nm for recombinant α9α10 and wild-type hair cell nAChRs, respectively. α-Conotoxin PeIA bears high resemblance to α-conotoxins MII and GIC isolated from Conus magus and Conus geographus, respectively. However, neither α-conotoxin MII nor α-conotoxin GIC at concentrations of 10 μm blocked acetylcholine responses elicited in Xenopus oocytes injected with the α9 and α10 subunits. Among neuronal non-α-bungarotoxin-sensitive receptors, α-conotoxin PeIA was also active at α3β2 receptors and chimeric α6/α3β2β3 receptors. α-Conotoxin PeIA represents a novel probe to differentiate responses mediated either through α9α10 or α7 nAChRs in those tissues where both receptors are expressed.

The venoms of predatory cone snails (Conus) represent a rich combinatorial-like library of evolutionarily selected, neuropharmacologically active peptides (18). There are more than 500 species of these snails. Each Conus venom appears to contain a unique set of 50 -200 small disulfide-bonded peptides that target receptors and ion channels in a highly subtype-selective manner. For peptides where function has been determined, three classes of targets have been elucidated: voltage-gated ion channels, G protein-coupled receptors, and ligand-gated ion channels (19). Perhaps the most conserved feature of cone snail venom is the ␣-conotoxins; these are a series of structurally and functionally related peptides that target nAChRs. Every venom examined thus far has its own distinct complement of nicotinic receptor antagonists, suggesting that, within the genus, there are literally thousands of novel peptides that act on nAChRs. A major advance in recent years in the neuropharmacology of nAChRs has been the ability to characterize particular neuronal subtypes more readily by using specific conotoxins.
We now report the cloning of a gene encoding a novel peptide of the ␣-conotoxin family from Conus pergrandis, ␣-conotoxin PeIA (␣-CTx PeIA). As far as we are aware, this is the first toxin to be characterized from this species found in deep waters (50 -530 meters) of the Western Pacific. We show that the peptide has unusual targeting specificity; it has high affinity for recombinant ␣9␣10-containing nAChR receptors and can readily discriminate this ␣-bungarotoxin-sensitive receptor from the neuronal ␣7 ␣-bungarotoxin-sensitive receptor. In addition, ␣-CTx PeIA blocks native cochlear hair cell nAChRs with a high potency, demonstrating an in vivo target for the peptide. Thus, ␣-CTx PeIA can be used to discern selectively between ␣9␣10and ␣7-mediated functions at those sites where both types of receptors are expressed. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Identification and Sequencing of Genomic Clones Encoding ␣-CTx
PeIA-Genomic DNA was prepared from 50 mg of C. pergrandis hepatopancreas using a Gentra PUREGENE DNA Isolation Kit (Gentra Systems, Minneapolis, MN) according to the manufacturer's standard protocol. It was used as a template for PCR with oligonucleotides corresponding to the conserved intron and 3Ј-untranslated region sequences of ␣-conotoxin prepropeptides. The resulting PCR products were purified using a High Pure PCR Product Purification Kit (Roche Applied Science). The eluted DNA fragments were annealed to pAMP1 vector, and the resulting products were transformed into competent DH5␣ cells using the CloneAmp pAMP System for rapid cloning of amplification products (Invitrogen). The nucleic acid sequences of the resulting ␣-CTx-encoding clones were determined according to the standard protocol for automated sequencing.
Chemical Synthesis-␣-CTx PeIA (0.45 mmol/g) was synthesized on an amide resin using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry and standard side protection, except on cysteine residues. Cys residues were protected in pairs with either S-trityl on Cys 2 and Cys 8 , or Sacetamidomethyl on Cys 3 and Cys 16 . The peptide was removed from the resin and precipitated. A two-step oxidation protocol was used to fold the peptides selectively as described previously (20). Briefly, the disulfide bridge between Cys 2 and Cys 8 was closed by dripping the peptide into an equal volume of 20 mM potassium 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 the S-acetamidomethyl groups and closure of the disulfide bridge between Cys 3 and Cys 16 were carried out by iodine oxidation. The monocyclic peptide and HPLC eluent were dripped into an equal volume of iodine (10 mM) in H 2 O: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 peptide was purified by HPLC on a reverse-phase C 18 Vydac column using a linear gradient of 0.1% trifluoroacetic acid, 0.092% trifluoroacetic acid, 60% acetonitrile, remainder H 2 O.
Mass Spectrometry-Matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry was utilized.
Expression of Recombinant Receptors in Xenopus laevis Oocytes-Capped cRNAs were in vitro transcribed from linearized rat plasmid DNA templates using the mMessage mMachine Transcription Kit (Ambion Corporation, Austin, TX). The maintenance of X. laevis as well as the preparation and cRNA injection of stage V and VI oocytes has been described in detail elsewhere (21). Typically, oocytes were injected with 50 nl of RNase-free water containing 0.01-1.0 ng of cRNAs (at a 1:1 molar ratio when pairwise combined) and maintained in Barth's solution at 17°C.
Electrophysiological recordings were performed 2-6 days after cRNA injection under two-electrode voltage clamp with an OC-725B oocyte clamp (Warner Instruments, Wamden, CT). Both voltage and current electrodes were filled with 3 M KCl and had resistances of ϳ1-2 megohms. Data were digitized, stored on a PC, and analyzed using Clamp Fit from the pClamp 6 software (Axon Instruments Corp., Union City, CA). During electrophysiological recordings, oocytes were superfused continuously (ϳ10 ml/min) with normal frog saline (comprising, in mM) 115 NaCl, 2.5 KCl, 1.8 CaCl 2 , and 10 HEPES buffer, pH 7.2, and voltageclamped at Ϫ70 mV. To follow the same conditions we have employed previously to analyze the properties of ␣9␣10 nAChRs (7), when expressing ␣9␣10, experiments were performed in oocytes incubated with the Ca 2ϩ chelator BAPTA-AM (100 M) for 3-4 h prior to electrophysiological recordings. Drugs were applied in the perfusion solution of the oocyte chamber. All toxin solutions also contained 0.1 mg/ml bovine serum albumin to reduce nonspecific adsorption of peptide. The toxin was preapplied for 10 min prior to the addition of the agonist. For the construction of the inhibition curves, the concentration of acetylcholine (ACh) used was near the corresponding EC 50 for each receptor; i.e. Recordings from Inner Hair Cells-Apical turns of the organ of Corti were excised from Sprague-Dawley rats at postnatal ages 8 -10 days. Cochlear preparations were mounted under an Axioskope microscope (Zeiss, Oberkochem, Germany), and viewed with differential interference contrast using a 63 ϫ water immersion objective and a camera with contrast enhancement (Hamamatsu C2400-07, Hamamatsu City, Japan). Methods to record from inner hair cells were essentially as described previously (22). Briefly, inner hair cells were identified visually, by the size of their capacitance (7-12 picofarads) and by their characteristic voltage-dependent Na ϩ and K ϩ currents, including at older ages a fast activating K ϩ -conductance (23). Some cells were re-moved to access inner hair cells, but mostly the pipette moved through the tissue using positive fluid flow to clear the tip. The extracellular solution was as follows (in mM): 155 NaCl, 5.8 KCl, 1.3 CaCl 2 , 0.9 MgCl 2 , 0.7 NaH 2 PO 4 , 5.6 D-glucose, and 10 HEPES, pH 7.4. The pipette solution was (in mM): 150 KCl, 3.5 MgCl 2 , 0.1 CaCl 2 , 10 BAPTA, 5 HEPES, 2.5 Na 2 ATP, pH 7.2. Glass pipettes (1.2-mm inner diameter) had resistances of 7-10 megohms. Cells were held at a holding potential of Ϫ90 mV. Postsynaptic currents caused by the spontaneous release of ACh from efferent synaptic terminals contacting inner hair cells were occasionally observed. Therefore, to study the effect of the toxin on synaptic currents in these cells, transmitter release from efferent endings was accelerated by depolarization using 25 mM external potassium saline. In this case, the pipette solution was (in mM): 150 KCl, 3.5 MgCl 2 , 0.1 CaCl 2 , 5 EGTA, 5 HEPES, 2.5 Na 2 ATP, pH 7.2. Solutions containing 60 M ACh (the EC 50 in this preparation) or elevated potassium (25 mM K ϩ ) and the toxin were applied by a gravity-fed multichannel glass pipette (ϳ150-m tip diameter) positioned about 300 m from the recorded inner hair cell. All toxin solutions also contained 0.1 mg/ml bovine serum albumin to reduce nonspecific adsorption of peptide. The extracellular solution containing the drugs was similar to that described above, except that Mg 2ϩ was omitted, and the Ca 2ϩ concentration was lowered to 0.5 mM to optimize the experimental conditions for measuring currents flowing through the ␣9␣10 receptors (24,25). To minimize the contribution of SK channel currents, 1 nM apamin, a specific SK channel blocker, was added to the external working solutions. Currents in inner hair cells were recorded in the whole-cell patch clamp mode using (Axopatch 200B amplifier) low pass filtered at 2-10 kHz and digitized at 5-20 kHz with a Digidata 1200 board (Axon Instruments, Union City, CA). Recordings were made at room temperature (22-25°C). Voltages were not corrected for the voltage drop across the uncompensated series resistance.
For constructing the inhibition curves, both in oocytes and the cochlear preparation, the average peak amplitude of three control responses just before the exposure to the toxin was used to normalize the amplitude of each test response in the presence of the toxin. Each data point of the inhibition curves represents the average value Ϯ S.E. of measurements from at least three experiments. Curves were fitted with Equation 1, where n H is the Hill coefficient, [T] is the concentration of toxin, and IC 50 is the concentration of toxin which reduces to 50% the maximal response to ACh. Statistical Analysis-Statistical significance was evaluated by Student's t test (two-tailed, unpaired samples). A p Ͻ 0.05 was considered significant.

RESULTS
Cloning of ␣-CTx PeIA-Although the mature ␣-CTx sequences are highly variable, the organization of their encoding genes is constant across species. The ␣-CTxs are proteolytically cleaved from a larger precursor protein. This prepropeptide is ϳ40 amino acids long, with the mature ␣-CTx moiety of ϳ13-18 amino acids located at the carboxyl terminus of the precursor. A basic amino acid, immediately preceding the mature toxin in the precursor sequence, acts as a processing site. In contrast to the highly variable sequence of the mature toxins, the precursor proteins of ␣-CTxs are highly conserved. The signal sequence region is practically invariant among the different ␣-CTx precursors, even in distantly related Conus species. Also, sequence segments in the 3Ј-untranslated region of the ␣-CTx mRNA and in the intron immediately preceding the toxin sequence are similarly conserved (18). We utilized conserved intronic and 3Ј-untranslated region sequences of the ␣-CTx gene structure to design oligonucleotide primers for PCR amplification of the ␣-CTx-coding region. The resulting sequence from C. pergrandis is shown in Fig. 1. The toxin was named ␣-CTx PeIA ("Pe" designating the species, pergrandis; Roman numeral "I" to designate the canonical ␣-CTx disulfide bond pattern; and "A" to indicate that it is the first ␣-CTx reported from this species).
Chemical Synthesis of ␣-CTx PeIA-Solid phase chemical synthesis of the predicted mature toxin was performed. In synthesizing the peptide, it was assumed that the disulfide bridging pattern of ␣-CTx PeIA was the same as all previously characterized ␣-CTxs, that is, Cys 2 to Cys 8 and Cys 3 to Cys 16 (18). The glycine at the carboxyl terminus was assumed to be post-translationally modified to a carboxyl-terminal amide. Cys groups were orthogonally protected in pairs to direct disulfide bond formation. Acid-labile S-trityl groups were removed simultaneously with peptide cleavage from the resin, and closure of the disulfide bridge between these Cys residues was accomplished with FeCN. The single-bridge peptide was purified by HPLC, and the acid-stable acetomidomethyl groups were removed; the disulfide bridges formed by iodine oxidation. The bicyclic peptide was subsequently purified by HPLC and analyzed with MALDI mass spectrometry. The mass of the synthetic peptide was consistent with the amidated sequence (monoisotopic MH ϩ : calculated, 1651.62; observed, 1651.6). This synthesized toxin was utilized in all subsequent experiments.
Effect of ␣-CTx PeIA on ACh-evoked Currents through nAChRs-Conotoxins have been used widely as a pharmacological tool to characterize neuronal nAChRs (26). We therefore decided to analyze the effects of ␣-CTx PeIA on different nAChRs. Fig. 2A shows representative responses of X. laevis oocytes expressing either the ␣-bungarotoxin-sensitive ␣9␣10 or ␣7 nAChRs to ACh and blockage of responses in the presence of ␣-CTx PeIA. Complete block of ACh-evoked currents was obtained with 0.3 M ␣-CTx PeIA in the case of ␣9␣10 compared with 100 M in the case of ␣7 nAChRs. As derived from the inhibition curves in Fig. 2B, currents elicited by ACh in ␣9␣10 nAChRs were potently blocked by ␣-CTx PeIA with a mean IC 50 and S.E. of 6.9 Ϯ 0.5 nM (n ϭ 6). In the case of ␣7, the IC 50 value, 1.8 Ϯ 0.1 M (n ϭ 5), was 260-fold higher than that obtained for ␣9␣10, thus indicating a high degree of selectivity of the toxin for ␣9␣10. The blockage produced by ␣-CTx PeIA on both types of receptors was reversible after washing with saline solution. Fig. 3 shows the washout kinetics in the case of ␣9␣10. The effect of ␣-CTx PeIA was reversed relatively rapidly: Ͼ50% recovery in 3 min, and total recovery after 12-15 min.
Cochlear outer and developing inner hair cells are the main targets of descending cholinergic olivocochlear efferent fibers (27). The efferent fibers-hair cell synapse is most likely mediated by ␣9␣10 nAChRs (7,28,29), providing one of the few postsynaptic functions for non-muscle nAChRs. However, ␣7 transcripts as well as ␣7 immunostaining have been reported in the mammalian organ of Corti (30, 31). Recordings from inner hair cells are an excellent tool to evaluate the effects of the toxin in native hair cell nAChRs. As shown in Fig. 4A, ␣-CTx PeIA potently blocked ACh-evoked responses in inner hair cells. The IC 50 value obtained, 4.4 Ϯ 0.5 nM, was similar to that found in recombinant ␣9␣10 receptors, thus confirming the ␣9␣10 identity of this nAChR.
The physiologic conditions of neurotransmission in vivo, for instance a synaptic regime, differ notably from the conditions of the oocyte recording or of a bath application of ACh to the organ of Corti. Transmitter released in a synaptic cleft, in close proximity to postsynaptic receptors, reaches millimolar ranges, sufficient to activate in the millisecond range receptors with a low affinity active state and a fast desensitization rate (32). We therefore studied the effect of ␣-CTx PeIA on synaptic currents evoked by the release of presynaptic ACh in the presence of 25 mM KCl. As observed in Fig. 5, 30 nM ␣-CTx PeIA also blocked responses to synaptically released ACh, thus indicating that the toxin is a valuable tool to examine in vivo responses mediated through ␣9␣10 receptors. Fig. 5A shows K ϩ -evoked synaptic currents either in the absence or presence of ␣-CTx PeIA. The blocking effect of the toxin was rapidly reversible upon washing the preparation. The effect of ␣-CTx PeIA was a reduction of the amplitude (Fig. 5B) of the synaptic currents (from 57.0 Ϯ 0.7 pA with 997 events and 3 cells, to 37.5 Ϯ 0.7 pA with 766 events and 3 cells, p Ͻ 0.0001). This result confirms a postsynaptic effect of the toxin on the nAChRs present in cochlear hair cells. A reduction in the frequency of events (from 2.2 Ϯ 0.5 to 1.2 Ϯ 0.2 Hz, data not shown) was observed. This could be either because the blocking action of the toxin on the synaptic events of small amplitudes could not be resolved within the noise of the recordings or because of an additional presynaptic effect of the toxin. The latter was not investigated further.
Nicotinic AChRs are members of the "Cys-loop" family of neurotransmitter-gated ion channels, which also includes ␥-aminobutyric acid types A and C, glycine, 5 erotonin type 3, and some invertebrate anionic glutamate receptors (34). To assess the selectivity of ␣-CTx PeIA for nAChRs, we tested the effect of the toxin on some other members of the family, such as the ␥-aminobutyric acid type A and 5-serotonin receptors. Concentrations of ␣-CTx PeIA as high as 10 M did not modify responses to 30 M ␥-aminobutyric acid or 10 M serotonin in oocytes injected with the respective recombinant receptors (n ϭ 4), thus confirming the selectivity of this ␣-CTx from C. pergrandis on nAChRs.
The mature toxin sequence of ␣-CTx PeIA bears high resemblance to ␣-CTxs MII (␣-CTx MII) and GIC (␣-CTx GIC) isolated from Conus magus and Conus geographus, respectively (Table II). Although ␣-CTx MII potently targets ␣3and ␣6containing neuronal nAChRs, ␣-CTx GIC has a higher selectivity for ␣6-containing receptors (35). We therefore examined the effect of these toxins in ␣9␣10-expressing oocytes. At a 10 M concentration, neither ␣-CTx MII nor ␣-CTx GIC blocked ␣9␣10 nAChRs (n ϭ 3). DISCUSSION By utilizing conserved sequences in CTx genes, we have cloned a gene encoding a novel peptide of the ␣-CTx family from C. pergrandis. This species was only very rarely collected and once regarded among the 10 most valuable Conus species for shell collectors (36). In recent years, however, commercial col-lectors in the Central Philippines have collected a moderate number of specimens. ␣-CTx PeIA is the first toxin to be characterized from this species and adds to the arsenal of peptides active at nAChRs that have been isolated from Conus. Thus, C. pergrandis represents a new source of useful pharmacological probes to characterize nAChRs. We note, however, that the peptide has not yet been isolated from the venom of C. pergrandis, and it is possible that the native venom-derived peptide has post-translational modifications not evident from inspection of the genetic sequence. In this report, we describe the properties of a synthetic version of the putative CTx. The peptide is 16 residues in length, with two disulfide bonds. ␣-CTx PeIA belongs to the A superfamily, as do the majority of Conus peptides that are known to affect the function of nAChRs (37). The spacing between Cys residues, four amino acids in the first loop and seven in the second loop, is typical of several previously isolated ␣-CTxs of the ␣4/7 family such as MII and GIC, which preferentially target non-muscle nAChRs (18,35).
Current data support the notion that a receptor assembled from both ␣9 and ␣10 nAChR subunits mediates synaptic transmission between efferent olivocochlear fibers and cochlear hair cells (3,7,28,29,41). However, ␣7 transcripts as well as ␣7 immunostaining have been reported in the mammalian organ of Corti (30,31). The fact that native hair cell nAChRs were potently blocked by ␣-CTx PeIA precludes the participation of ␣7 nAChRs in mediating ACh-evoked responses in inner hair cells. The situation for other tissues remains to be determined. Dorsal root ganglion neurons express multiple nAChR subtypes, including ␣7-like, ␣3␤4-like, and ␣4␤2-like (42). They also coexpress both ␣9 and ␣10 subunits (43,44). In addition, peripheral blood lymphocytes and developing thymocytes have been shown to express cholinergic receptors, including the nAChR subunits ␣2-5, ␣7, ␣9, ␣10, and ␤4, which could participate at different steps in the regulation of the immune response (45)(46)(47). Finally, ␣7, ␣9, and ␣10 are expressed in skin keratinocytes and might be involved at different steps in the regulation of skin homeostasis (48 -51). The observation that ␣-CTx PeIA has a 260-fold selectivity for ␣9␣10 compared with ␣7 indicates that it is a useful probe to differentiate responses mediated either through ␣9␣10 or ␣7 nAChRs in the above mentioned tissues where both receptors are expressed.
The carnivorous marine snails of the genus Conus are a rich source of peptides targeted to nAChRs. Major components of the complex venomous arsenal that the fish-eating Conus employ are toxins that act at the muscle nicotinic receptor type (18). Why might these snails have evolved a toxin with high affinity for ␣9␣10 receptors? C. pergrandis is found in deep waters (50 -530 meters) of the Western Pacific. The feeding habits of C. pergrandis are unknown, but Conus species in general prey upon fish, mollusks, and/or worms. Little is known about the nAChR subtypes found in these prey. However, the Fugu rubripes (pufferfish) genome contains three candidate ␣7, two ␣8, and four ␣9 subunit genes. Moreover, it has been described that this fish genome contains the largest family of vertebrate nAChR subunits reported to date (52). In addition three ␣9 subunit genes have been described in the rainbow trout (Oncorhynchus mykiss) (53). Although none of the Fugu nAChR sequences show close identity to the mammalian or avian ␣10 subunits, three of the ␣9-like subunits possess glycosylation sites also found in the higher vertebrate ␣10 subunit (52), suggesting that these ␣9-like subunits might coassemble to form a functional nAChR, much like the higher vertebrate homomeric ␣9 and/or heteromeric ␣9␣10 nAChRs. Higher vertebrate ␣9 and ␣9␣10 nAChRs mediate efferent cholinergic inhibition at cochlear and vestibular hair cells (3,7,28,29,41). The cholinergic pharmacology of efferent block in the fish lateral line organ is similar to that in hair cells of the cochlea, indicating that the same nicotinic cholinergic receptor is likely involved (54,55). The proper orientation of mechanosensory hair cells along the lateral line organ of a fish is essential for the ability of the animal to sense directional water movements. This sensory system appears to be important in many behavioral tasks such as prey capture, orientation with respect to external environmental cues, navigation in low light conditions, and mediation of interactions with nearby animals (56 -59). Thus, block of efferent modulation of the lateral line activity by Conus toxins could facilitate prey capture by these predatory snails.
␣-CTx PeIA shows considerable sequence similarity to the ␣4/7 ␣-CTxs MII and GIC (26). The comparison of the structure of ␣-CTx PeIA, MII, and GIC indicates that the four Cys residues are placed identically, having the same disulfide connectivity. Moreover, a conserved proline and the identical placement of a histidine and an asparagine that are known to either initiate or immediately precede an ␣-helix in GIC and MII indicate that the peptide backbone topology of ␣-CTx PeIA is likely similar to that of the other two ␣-CTxs. Indeed, compared as a group, there are only four nonconservative amino acid   substitutions among the three peptides (see bold residues in Table II). The ␣4/7 toxins have a common structural scaffold. Their polypeptide backbones appear to be virtually identical: the canonical ␣ helical structure and two ␤ turns are prominent structural features of the family. Given the near identity of the peptide backbones the ability of the different ␣4/7 Conus peptides to discriminate among different neuronal nAChR subtypes must clearly be mediated through their divergent side chain groups (18). Although all three toxins, MII, GIC, and PeIA, have high affinity for the ␣3␤2 nAChR (35,60), only ␣-CTx PeIA has high affinity for ␣9␣10. Thus, the amino acids shown in bold in Table II are likely structural determinants of the high selectivity of ␣-CTx PeIA for ␣9␣10 nAChRs.
Conclusion-A remarkable accomplishment of the Conus genus has been the evolution of nAChR antagonist of diverse subtype specificities. The diversity of their venom products is likely a consequence of the complex marine environment in which these slow moving and otherwise unarmed predators must compete. Conus hunt a broad range of organisms (five different phyla) and also must defend themselves against crustaceans and other predators. We have now identified a novel peptide, ␣-CTx PeIA, which selectively targets the most recently identified nAChR subunits, ␣9 and ␣10. Thus, isolation of peptides from Conus venoms continues to prove useful to identify novel pharmacological tools to characterize the nAChR family.