The µ-conotoxin KIIIA is a three disulfide-bridged blocker of voltage-gated sodium channels (VGSCs). The Lys7 residue in KIIIA is an attractive target for manipulating the selectivity and efficacy of this peptide. Here, we report the design and chemical synthesis of µ-conopeptoid analogues (peptomers) in which we replaced Lys7 with peptoid monomers of increasing side-chain size: N-methylglycine, N-butylglycine and N-octylglycine. In the first series of analogues, the peptide core contained all three disulfide bridges; whereas in the second series, a disulfide-depleted selenoconopeptide core was used to simplify oxidative folding. The analogues were tested for functional activity in blocking the Nav1.2 subtype of mammalian VGSCs exogenously expressed in Xenopus oocytes. All six analogues were active, with the N-methylglycine analogue, [Sar7]KIIIA, the most potent in blocking the channels while favoring lower efficacy. Our findings demonstrate that the use of N-substituted Gly residues in conotoxins show promise as a tool to optimize their pharmacological properties as potential analgesic drug leads.
µ-conotoxins; KIIIA, peptomers; selenocysteine; diselenide; sodium channels; electrophysiology
µ-SIIIA, a novel µ-conotoxin from Conus striatus, appeared to be a selective blocker of tetrodotoxin-sensitive sodium channels in frog preparations. It also exhibited potent analgesic activity in mice, although its selectivity profile against mammalian sodium channels remained unknown. We have determined the structure of µ-SIIIA in aqueous solution and characterized its backbone dynamics by NMR and its functional properties electrophysiologically. Consistent with the absence of hydroxyprolines, µ-SIIIA adopts a single conformation with all peptide bonds in the trans conformation. The C-terminal region contains a well-defined helix encompassing residues 11–16, while residues 3–5 in the N-terminal region form a helix-like turn resembling 310 helix. The Trp12 and His16 side chains are in close proximity, as in the related conotoxin µ-SmIIIA, but Asn2 is further away. Dynamics measurements show that the N-terminus and Ser9 have larger magnitude motions on the sub-ns timescale, while the C-terminus is more rigid. Cys4, Trp12 and Cys13 undergo significant conformational exchange on µs - ms timescales. µ-SIIIA is a potent, nearly irreversible blocker of NaV1.2, but also blocks NaV1.4 and NaV1.6 with submicromolar potency. The selectivity profile of µ-SIIIA, including poor activity against the cardiac sodium channel, NaV1.5, is similar to that of the closely related µ-KIIIA, suggesting that the C-terminal regions of both are critical for blocking neuronal NaV1.2. The structural and functional characterization described in this paper of an analgesic µ-conotoxin that targets neuronal subtypes of mammalian sodium channels provides a basis for the design of novel analogues with an improved selectivity profile.
Among the μ-conotoxins that block vertebrate voltage-gated sodium channels (VGSCs), some have been shown to be potent analgesics following systemic administration in mice. We have determined the solution structure of a new representative of this family, μ-BuIIIB, and established its disulfide connectivities by direct mass spectrometric collision induced dissociation fragmentation of the peptide with disulfides intact. The major oxidative folding product adopts a 1-4/2-5/3-6 pattern with the following disulfide bridges: Cys5-Cys17, Cys6-Cys23 and Cys13-Cys24. The solution structure reveals that the unique N-terminal extension in μ-BuIIIB, which is also present in μ-BuIIIA and μ-BuIIIC but absent in other μ-conotoxins, forms part of a short α-helix encompassing Glu3 to Asn8. This helix is packed against the rest of the toxin and stabilized by the Cys5-Cys17 and Cys6-Cys23 disulfide bonds. As such, the side chain of Val1 is located close to the aromatic rings of Trp16 and His20, which are located on the canonical helix that displays several residues found to be essential for VGSC blockade in related μ-conotoxins. Mutations of residues 2 and 3 in the N-terminal extension enhanced the potency of μ-BuIIIB for NaV1.3. One analog, [d-Ala2]BuIIIB, showed a 40-fold increase, making it the most potent peptide blocker of this channel characterized to date and thus a useful new tool with which to characterize this channel. Based on previous results for related μ-conotoxins, the dramatic effects of mutations at the N-terminus were unanticipated, and suggest that further gains in potency might be achieved by additional modifications of this region.
The μ-conotoxin μ-KIIIA, from Conus kinoshitai, blocks mammalian neuronal voltage-gated sodium channels (VGSCs) and is a potent analgesic following systemic administration in mice. We have determined its solution structure using NMR spectroscopy. Key residues identified previously as being important for activity against VGSCs (Lys7, Trp8, Arg10, Asp11, His12 and Arg14) all reside on an α-helix with the exception of Arg14. To further probe structure-activity relationships of this toxin against VGSC subtypes, we have characterised the analogue μ-KIIIA[C1A,C9A], in which the Cys residues involved in one of the three disulfides in μ-KIIIA were replaced with Ala. Its structure is quite similar to that of μ-KIIIA, indicating that the Cys1-Cys9 disulfide bond could be removed without any significant distortion of the α-helix bearing the key residues. Consistent with this, μ-KIIIA[C1A,C9A] retained activity against VGSCs, with its rank order of potency being essentially the same as that of μ-KIIIA, namely, NaV1.2 > NaV1.4 > NaV1.7 ≥ NaV1.1 > NaV1.3 > NaV1.5. Kinetics of block were obtained for NaV1.2, NaV1.4 and NaV1.7, and in each case both kon and koff values of μ-KIIIA[C1A,C9A] were larger than those of μ-KIIIA. Our results show that the key residues for VGSC binding lie mostly on an α-helix and that the first disulfide bond can be removed without significantly affecting the structure of this helix, although the modification accelerates the on- and off-rates of the peptide against all tested VGSC subtypes. These findings lay the groundwork for the design of minimized peptides and helical mimetics as novel analgesics.
In the preparation of synthetic conotoxins containing multiple disulfide bonds, oxidative folding can produce numerous permutations of disulfide bond connectivities. Establishing the native disulfide connectivities thus presents a significant challenge when the venom-derived peptide is not available, as is increasingly the case when conotoxins are identified from cDNA sequences. Here, we investigate the disulfide connectivity of μ-conotoxin KIIIA, which was predicted originally to have a [C1-C9,C2-C15,C4-C16] disulfide pattern based on homology with closely-related μ-conotoxins. The two major isomers of synthetic μ-KIIIA formed during oxidative folding were purified and their disulfide connectivities mapped by direct mass spectrometric CID fragmentation of the disulfide-bonded polypeptides. Our results show that the major oxidative folding product adopts a [C1-C15,C2-C9,C4-C16] disulfide connectivity, while the minor product adopts a [C1-C16,C2-C9,C4-C15] connectivity. Both of these peptides were potent blockers of NaV1.2 (Kd 5 and 230 nM, respectively). The solution structure for μ-KIIIA based on NMR data was recalculated with the [C1-C15,C2-C9,C4-C16] disulfide pattern; its structure was very similar to the μ-KIIIA structure calculated with the incorrect [C1-C9,C2-C15,C4-C16] disulfide pattern, with an α-helix spanning residues 7–12. In addition, the major folding isomers of μ-KIIIB, an N-terminally extended isoform of μ-KIIIA identified from its cDNA sequence, were isolated. These folding products had the same disulfide connectivities as for μ-KIIIA, and both blocked NaV1.2 (Kd 470 and 26 nM, respectively). Our results establish that the preferred disulfide pattern of synthetic μ-KIIIA/μ-KIIIB folded in vitro is 1-5/2-4/3-6 but that other disulfide isomers are also potent sodium channel blockers. These findings raise questions about the disulfide pattern(s) of μ-KIIIA in the venom of Conus kinoshitai; indeed, the presence of multiple disulfide isomers in the venom could provide a means to further expand the snail's repertoire of active peptides.
Disulfide bridges, which stabilize the native conformation of conotoxins impose a challenge in the synthesis of smaller analogs. In this work, we describe the synthesis of a minimized analog of the analgesic μ-conotoxin KIIIA that blocks two sodium channel subtypes, the neuronal NaV1.2 and skeletal muscle NaV1.4. Three disulfide-deficient analogs of KIIIA were initially synthesized in which the native disulfide bridge formed between either C1-C9, C2-C15 or C4-C16 was removed. Deletion of the first bridge only slightly affected the peptide’s bioactivity. To further minimize this analog, the N-terminal residue was removed and two non-essential Ser residues were replaced by a single 5-amino-3-oxapentanoic acid residue. The resulting “polytide” analog retained the ability to block sodium channels and to produce analgesia. Until now, the peptidomimetic approach applied to conotoxins has progressed only modestly at best; thus, the disulfide-deficient analogs containing backbone spacers provide an alternative advance toward the development of conopeptide-based therapeutics.
conopeptide; conotoxin; sodium channels; backbone spacers; disulfide bridges
μ-Conotoxin KIIIA (μ-KIIIA) blocks mammalian voltage-gated sodium channels (VGSCs) and is a potent analgesic following systemic administration in mice. Previous structure-activity studies of μ-KIIIA identified a helical pharmacophore for VGSC blockade. This suggested a route for designing truncated analogues of μ-KIIIA by incorporating the key residues into an α-helical scaffold. As (i, i+4) lactam bridges constitute a proven approach for stabilizing α-helices, we designed and synthesized six truncated analogues of μ-KIIIA containing single lactam bridges at various locations. The helicity of these lactam analogues was analysed by NMR spectroscopy, and their activities were tested against mammalian VGSC subtypes NaV1.1 through 1.7. Two of the analogues, Ac-cyclo9/13[Asp9,Lys13]KIIIA7–14 and Ac-cyclo9/13[Lys9,Asp13]KIIIA7–14, displayed µM activity against VGSC subtypes NaV1.2 and NaV1.6; importantly, the subtype selectivity profile for these peptides matched that of μ-KIIIA. Our study highlights structure-activity relationships within these helical mimetics and provides a basis for the design of additional truncated peptides as potential analgesics.
The structure, assembly, and function of the bacterial flagellum involves about 60 different proteins, many of which are selectively secreted via a specific type III secretion system (T3SS) (J. Frye et al., J. Bacteriol. 188:2233–2243, 2006). The T3SS is reported to secrete proteins at rates of up to 10,000 amino acid residues per second. In this work, we showed that the flagellar T3SS of Salmonella enterica serovar Typhimurium could be manipulated to export recombinant nonflagellar proteins through the flagellum and into the surrounding medium. We translationally fused various neuroactive peptides and proteins from snails, spiders, snakes, sea anemone, and bacteria to the flagellar secretion substrate FlgM. We found that all tested peptides of various sizes were secreted via the bacterial flagellar T3SS. We subsequently purified the recombinant μ-conotoxin SIIIA (rSIIIA) from Conus striatus by affinity chromatography and confirmed that T3SS-derived rSIIIA inhibited mammalian voltage-gated sodium channel NaV1.2 comparably to chemically synthesized SIIIA.
Manipulation of the flagellar secretion system bypasses the problems of inclusion body formation and cellular degradation that occur during conventional recombinant protein expression. This work serves as a proof of principle for the use of engineered bacterial cells for rapid purification of recombinant neuroactive peptides and proteins by exploiting secretion via the well-characterized flagellar type III secretion system.
Despite the therapeutic promise of disulfide-rich, peptidic natural products, their discovery and structure/function studies have been hampered by inefficient oxidative folding methods for their synthesis. Here we report that converting the three disulfide-bridged μ-conopeptide KIIIA into a disulfide-depleted selenoconopeptide (by removal of a noncritical disulfide bridge and substitution of another disulfide bridge with a diselenide bridge) dramatically simplified its oxidative folding while preserving the peptide’s ability to block voltage-gated sodium channels. The simplicity of synthesizing disulfide-depleted selenopeptide analogues containing a single disulfide bridge allowed rapid positional scanning at Lys7 of μ-KIIIA, resulting in the identification of K7L as a mutation that improved the peptide’s selectivity in blocking a neuronal (Nav1.2) over a muscle (Nav1.4) subtype of sodium channel. The disulfide-depleted selenopeptide strategy offers regioselective folding compatible with high-throughput chemical synthesis and on-resin oxidation methods, and thus shows great promise to accelerate the use of disulfide-rich peptides as research tools and drugs.
Conotoxins; diselenide bridges; selenocysteines; oxidative folding; disulfide-rich peptides
The possibility of independently manipulating the affinity and efficacy of pore-blocking ligands of sodium channels is of interest for the development of new drugs for the treatment of pain. The analgesic µ-conotoxin KIIIA, a 16-residue peptide with three disulfide bridges, is a pore-blocker of voltage-gated sodium channels, including the neuronal subtype NaV1.2 (Kd of 5 nM). At saturating concentrations, µ-KIIIA incompletely blocks the sodium current of NaV1.2, leaving a 5% residual current (rINa). Lys7 is an important residue: the mutation K7A decreases both the efficacy (i.e., increases rINa to 23%) and the affinity of the peptide (Kd, 115 nM). In this report, various replacements of residue 7 were examined to determine whether affinity and efficacy were inexorably linked. Because of their facile chemical synthesis, KIIIA analogs were used that had as a core structure the disulfide-depleted KIIIA[C1A,C2U,C9A,C5U] (where U is selenocysteine) or ddKIIIA. The analogs ddKIIIA and ddKIIIA[K7X], where X represents one of nine different amino acids, were tested on voltage-clamped Xenopus oocytes expressing rat NaV1.2 or NaV1.4. Their affinities ranged from 0.01 to 36 µM and rINa's from 2 to 42%, and these two variables appeared uncorrelated. Instead, rINa varied inversely with side chain size, and remarkably charge and hydrophobicity appeared inconsequential. The ability to manipulate a µ-conopeptide's affinity and efficacy, as well as its capacity to interfere with subsequent tetrodotoxin-binding, greatly expands its scope as a reagent to probe sodium channel structure and function, and may also lead to the development of µ-conotoxins as safe analgesics.
Despite the therapeutic promise of disulfide-rich, peptidic natural products, their discovery and structure/function studies have been hampered by inefficient oxidative folding methods for their synthesis. Here we report that converting the three disulfide-bridged μ-conopeptide KIIIA into a disulfide-depleted selenoconopeptide (by removal of a noncritical disulfide bridge and substitution of a disulfide- with a diselenide-bridge) dramatically simplified its oxidative folding while preserving the peptide’s ability to block voltage-gated sodium channels. The simplicity of synthesizing disulfide-depleted selenopeptide analogs containing a single disulfide bridge allowed rapid positional scanning at Lys7 of μ-KIIIA, resulting in the identification of K7L as a mutation that improved the peptide’s selectivity in blocking a neuronal (Nav1.2) over a muscle (Nav1.4) subtype of sodium channel. The disulfide-depleted selenopeptide strategy offers regioselective folding compatible with high throughput chemical synthesis and on-resin oxidation methods, and thus shows great promise to accelerate the use of disulfide-rich peptides as research tools and drugs.
conotoxins; diselenide bridges; selenocysteines; oxidative folding; disulfide-rich peptides
Chemical synthesis of disulfide-rich peptides requires improvements in oxidative folding and disulfide mapping. To address these challenges, we combined the use of diselenide and selectively (15N/ 13C)-labeled disulfide bridges. Conotoxin analogs, each with a pair of selenocysteines and labeled cysteines, exhibited significantly improved folding while the labeled cysteines allowed correctly folded species to be rapidly identified by NMR.
diselenide; disulfide; oxidative folding; NMR; conotoxin
Tetrodotoxin (TTX) is the quintessential ligand of voltage-gated sodium channels (NaVs). Like TTX, μ-conotoxin peptides are pore blockers, and both toxins have helped to define the properties of neurotoxin receptor Site 1 of NaVs. Here, we report unexpected results showing that the recently discovered μ-conotoxin KIIIA and TTX can simultaneously bind to Site 1 and act in concert. Results with saturating concentrations of peptide applied to voltage-clamped Xenopus oocytes expressing brain NaV1.2, and single-channel recordings from brain channels in lipid bilayers, show that KIIIA or its analog, KIIIA[K7A], block partially, with a residual current that can be completely blocked by TTX. In addition, the kinetics of block by TTX and peptide are each affected by the prior presence of the other toxin. For example, bound peptide slows subsequent binding of TTX (an antagonistic interaction) and slows TTX dissociation when both toxins are bound (a synergistic effect on block). The overall functional consequence resulting from the combined action of the toxins depends on the quantitative balance between these opposing actions. The results lead us to postulate that in the bi-liganded NaV complex, TTX is bound between the peptide and the selectivity filter. These observations refine our view of Site 1 and open new possibilities in NaV pharmacology.
conotoxin; contratoxin; NaV1.2; oocyte; sodium channel; site 1; syntoxin; tetrodotoxin; voltage clamp
Described herein is a general approach to identify novel compounds using the biodiversity of a megadiverse group of animals; specifically, the phylogenetic lineage of the venomous gastropods that belong to the genus Conus (“cone snails”). Cone snail biodiversity was exploited to identify three new μ-conotoxins, BuIIIA, BuIIIB and BuIIIC, encoded by the fish-hunting species Conus bullatus. BuIIIA, BuIIIB and BuIIIC are strikingly divergent in their amino acid composition compared to previous μ-conotoxins known to target the voltage-gated Na channel skeletal muscle subtype Nav1.4. Our preliminary results indicate that BuIIIB and BuIIIC are potent inhibitors of Nav1.4 (average block ~96%, at a 1 μM concentration of peptide), displaying a very slow off-rate not seen in previously characterized μ-conotoxins that block Nav1.4. In addition, the three new Conus bullatus μ-conopeptides help to define a new branch of the M-superfamily of conotoxins, namely M-5. The exogene strategy used to discover these Na channel-inhibiting peptides was based on both understanding the phylogeny of Conus, as well as the molecular genetics of venom μ-conotoxin peptides previously shown to generally target voltage-gated Na channels. The discovery of BuIIIA, BuIIIB and BuIIIC Na channel blockers expands the diversity of ligands useful in determining the structure-activity relationship of voltage-gated sodium channels.
Biodiversity-derived compounds; Sodium channel ligands; exogenes
Neurotoxin receptor site 1, in the outer vestibule of the conducting pore of voltage-gated sodium channels (VGSCs), was first functionally defined by its ability to bind the guanidinium-containing agents, tetrodotoxin (TTX) and saxitoxin (STX). Subsequent studies showed that peptide μ-conotoxins competed for binding at site 1. All of these natural inhibitors block single sodium channels in an all-or-none manner on binding. With the discovery of an increasing variety of μ-conotoxins, and the synthesis of numerous derivatives, observed interactions between the channel and these different ligands have become more complex. Certain μ-conotoxin derivatives block single-channel currents partially, rather than completely, thus enabling the demonstration of interactions between the bound toxin and the channel’s voltage sensor. Most recently, the relatively small μ-conotoxin KIIIA (16 amino acids) and its variants have been shown to bind simultaneously with TTX and exhibit both synergistic and antagonistic interactions with TTX. These interactions raise new pharmacological possibilities and place new constraints on the possible structures of the bound complexes of VGSCs with these toxins.
guanidinium toxins; conopeptides; pore block
Disulfide-rich peptides represent a megadiverse group of natural products with very promising therapeutic potential. To accelerate their functional characterization, high-throughput chemical synthesis and folding methods are required, including efficient mapping of multiple disulfide bridges. Here, we describe a novel approach for such mapping and apply it to a three-disulfide bridged conotoxin, μ-SxIIIA (from the venom of Conus striolatus) whose discovery is also reported here for the first time. μ-SxIIIA was chemically synthesized with three cysteine residues labeled 100% with 15N/13C, while the remaining three cysteine residues were incorporated using a mixture of 70%:30% unlabeled:labeled Fmoc-protected residues. After oxidative folding, the major product was analyzed by NMR spectroscopy. Sequence-specific resonance assignments for the isotope-enriched Cys residues were determined with 2D versions of standard triple resonance (1H,13C,15N) NMR experiments and 2D [13C,1H] HSQC. Disulfide patterns were directly determined with cross-disulfide NOEs confirming that the oxidation product had the disulfide connectivities characteristic of μ-conotoxins. μ-SxIIIA was found to be a potent blocker of the sodium channel subtype NaV1.4 (IC50 = 7 nM). These results suggest that differential incorporation of isotope-labeled cysteine residues is an efficient strategy to map disulfides and should facilitate the discovery and structure-function studies of many bioactive peptides.
The excitotoxic conopeptide ι-RXIA induces repetitive action potentials in frog motor axons and seizures upon intracranial injection into mice. We recently discovered that ι-RXIA shifts the voltage-dependence of activation of voltage-gated sodium channel NaV1.6 to a more hyperpolarized level. Here, we performed voltage-clamp experiments to examine its activity against rodent NaV1.1 through NaV1.7 co-expressed with the β1 subunit in Xenopus oocytes and NaV1.8 in dissociated mouse DRG neurons. The order of sensitivity to ι-RXIA was NaV1.6 > 1.2 > 1.7, and the remaining subtypes were insensitive. The time course of ι-RXIA-activity on NaV1.6 during exposure to different peptide concentrations were well fit by single-exponential curves that provided kobs. The plot of kobs versus [ι-RXIA] was linear, consistent with a bimolecular reaction with a Kd of ~3 μM, close to the steady-state EC50 of ~2 μM. ι-RXIA has an unusual residue, D-Phe, and the analog with an L-Phe instead, ι-RXIA[L-Phe44], had a two-fold lower affinity and two-fold faster off-rate than ι-RXIA on NaV1.6 and furthermore was inactive on NaV1.2. ι-RXIA induced repetitive action potentials in mouse sciatic nerve with conduction velocities of both A- and C-fibers, consistent with the presence of NaV1.6 at nodes of Ranvier as well as in unmyelinated axons. Sixteen peptides homologous to ι-RXIA have been identified from a single species of Conus, so these peptides represent a rich family of novel sodium channel-targeting ligands.
channel-activation; conopeptide; excitotoxin; iota-conotoxin RXIA; neurotoxin; voltage-gated sodium channel
Striatal dopamine plays a major role in the regulation of motor coordination and in the processing of salient information. We used voltammetry to monitor dopamine-release evoked by electrical stimulation in striatal slices, where interneurons continuously release acetylcholine. Use of the α6-selective antagonist α-conotoxin MII[E11A] and α4 knockout mice enabled identification of two populations of dopaminergic fibers. The first population had a low action potential threshold, and action potential-evoked dopamine-release from these fibers was modulated by α6. The second population had a higher action potential threshold, and only α4(non-α6) modulated action potential-evoked dopamine-release. Striatal dopaminergic neurons fire in both tonic and phasic patterns. When stimuli were applied in a train to mimic phasic firing, more dopamine-released was observed in α4 knockout vs. wildtype mice. Furthermore, block of α4(non-α6), but not of α6, increased dopamine release evoked by a train. These results indicate that there are different classes of striatal dopaminergic fibers that express different subtypes of nicotinic receptors.
voltammetry; nicotine; knockout mice; alpha-conotoxin; striatum
Conotoxin ι-RXIA, from the fish-hunting species Conus radiatus, is a member of the recently characterized I1-superfamily, which contains eight cysteine residues arranged in a −C-C-CC-CC-C-C- pattern. ι-RXIA (formerly designated r11a) is one of three characterized I1 peptides in which the third last residue is post-translationally isomerized to the d- configuration. Naturally occurring ι-RXIA with d-Phe44 is significantly more active as an excitotoxin than the l-Phe analogue both in vitro and in vivo. We have determined the solution structures of both forms by NMR spectroscopy, the first for an I1-superfamily member. The disulfide connectivities were determined from structure calculations and confirmed chemically as 5-19, 12-22, 18-27, and 21-38, suggesting that ι-RXIA has an ICK structural motif with one additional disulfide (21-38). Indeed, apart from the first few residues, the structure is well defined up to around residue 35 and does adopt an ICK structure. The C-terminal region, including Phe44, is disordered. Comparison of the d-Phe44 and l-Phe44 forms indicates that the switch from one enantiomer to the other has very little effect on the structure, even though it is clearly important for receptor interaction based on activity data. Finally, we identify the target of ι-RXIA as a voltage-gated sodium channel; ι-RXIA is an agonist, shifting the voltage dependence of activation of mouse NaV1.6 expressed in Xenopus oocytes to more hyperpolarized potentials. Thus, there is a convergence of structure and function in ι-RXIA, as its disulfide pairing and structure resemble those of funnel web spider toxins that also target sodium channels.
We have characterized the defining members of a novel subfamily of excitatory conotoxins, the short κA-conotoxins (κAS-conotoxins). κA-conotoxins PIVE and PIVF (κA-PIVE and κA-PIVF) were purified from Conus purpurascens venom. Both peptides elicited excitatory activity upon injection into fish. κA-PIVE was synthesized for further characterization. The excitatory effects of κA-PIVE in vivo were dose dependent, causing hyperactivity at low doses and rapid immobilization at high doses, symptomatic of a type of excitotoxic shock. Consistent with these observations, κA-PIVE caused repetitive action potentials in frog motor axons in vitro. Similar results have been reported for other structurally distinct conotoxin families; such peptides appear to be required by most fish-hunting cone snails for the rapid immobilization of prey. Unexpected structure-function relationships were revealed between these peptides and two families of homologous conotoxins: the αA-conotoxins (muscle nAChR antagonists) and κA-conotoxins (excitotoxins), which all share a common arrangement of cysteine residues (CC--C--C--C--C). Biochemically, the κAS-conotoxins more closely resemble the αAS-conotoxins than the other κA-conotoxin subfamily, the long κA-conotoxins (κAL-conotoxins); however, κAS- and αAS-conotoxins produce different physiological effects. In contrast, the κAS-and κAL-conotoxins that diverge in several biochemical characteristics are clearly more similar in their physiological effects.
Conus venom; Conus peptide; conotoxin; toxin; antagonist; excitatory