Peptide neurotoxins from cone snails continue to supply compounds with therapeutic potential. Although several analgesic conotoxins have already reached human clinical trials, a continuing need exists for the discovery and development of novel nonopioid analgesics, such as subtype-selective sodium channel blockers. -Conotoxin KIIIA is representative of -conopeptides previously characterized as inhibitors of tetrodotoxin (TTX)-resistant sodium channels in amphibian dorsal root ganglion neurons. Here, we show that KIIIA has potent analgesic activity in the mouse pain model. Surprisingly, KIIIA was found to block most (>80%) of the TTX-sensitive, but only ϳ20% of the TTX-resistant, sodium current in mouse dorsal root ganglion neurons. KIIIA was tested on cloned mammalian channels expressed in Xenopus oocytes. Both Na V 1.2 and Na V 1.6 were strongly blocked; within experimental wash times of 40 -60 min, block was reversed very little for Na V 1.2 and only partially for Na V 1.6. Other isoforms were blocked reversibly: Na V 1.3 (IC 50 8 M), Na V 1.5 (IC 50 284 M), and Na V 1.4 (IC 50 80 nM). "Alanine-walk" and related analogs were synthesized and tested against both Na V 1.2 and Na V 1.4; replacement of Trp-8 resulted in reversible block of Na V 1.2, whereas replacement of Lys-7, Trp-8, or Asp-11 yielded a more profound effect on the block of Na V 1.4 than of Na V 1.2. Taken together, these data suggest that KIIIA is an effective tool to study structure and function of Na V 1.2 and that further engineering of -conopeptides belonging to the KIIIA group may provide subtype-selective pharmacological compounds for mammalian neuronal sodium channels and potential therapeutics for the treatment of pain.Venoms are a rich source of neuroactive compounds that target various ion channels and receptors with exquisite potency and selectivity (1-4). There is a continuing need for more subtype-selective pharmacological agents against sodium channels (5), and cone snail venoms provide a unique pharmacopoeia of diverse sodium channel-targeting toxins, including channel blockers as well as inhibitors of channel inactivation (6 -18). -Conotoxins are short peptides that potently block sodium channels (Table 1). The first -conotoxins to be discovered from venom of Conus snails, GIIIA, GIIIB, GIIIC, and PIIIA, were paralytic in fish and potently inhibited skeletal muscle sodium channels in amphibian and mammalian systems.Recently, a second group of -conotoxins has been identified that, in contrast to previously characterized peptides that targeted the skeletal muscle sodium channels, inhibited TTX-resistant (TTX-r) 4 sodium channels when screened on amphibian neuronal preparations (19 -21). This group of conotoxins includes -conotoxin SmIIIA from Conus stercusmuscarum and -conotoxin KIIIA from Conus kinoshitai (Fig. 1). Structural and functional studies on peptides in this group to date suggest that amino acid residues in the C-terminal region of these peptides, including Trp and His (see Table 1), are important for function (19,22).It ...

Voltage-gated sodium channels (VGSCs) are important for action potentials. There are seven major isoforms of the pore-forming and gate-bearing α-subunit (Na V 1) of VGSCs in mammalian neurons, and a given neuron can express more than one isoform. Five of the neuronal isoforms, Na V 1.1, 1.2, 1.3, 1.6, and 1.7, are exquisitely sensitive to tetrodotoxin (TTX), and a functional differentiation of these presents a serious challenge. Here, we examined a panel of 11 μ-conopeptides for their ability to block rodent Na V 1.1 through 1.8 expressed in Xenopus oocytes. Although none blocked Na V 1.8, a TTX-resistant isoform, the resulting "activity matrix" revealed that the panel could readily discriminate between the members of all pair-wise combinations of the tested isoforms. To examine the identities of endogenous VGSCs, a subset of the panel was tested on A-and C-compound action potentials recorded from isolated preparations of rat sciatic nerve. The results show that the major subtypes in the corresponding A-and C-fibers were Na V 1.6 and 1.7, respectively. Ruled out as major players in both fiber types were Na V 1.1, 1.2, and 1.3. These results are consistent with immunohistochemical findings of others. To our awareness this is the first report describing a qualitative pharmacological survey of TTX-sensitive Na V 1 isoforms responsible for propagating action potentials in peripheral nerve. The panel of μ-conopeptides should be useful in identifying the functional contributions of Na V 1 isoforms in other preparations. Hodgkin and Huxley developed a quantitative theory for the ionic basis of the action potential (1)-the molecular correlates of their pioneering efforts are the voltage-gated sodium channel (VGSC) and the voltage-gated potassium channel. Critical for the appreciation of the discrete biochemical nature of VGSCs were the investigations of the mechanism of action of the alkaloids tetrodotoxin (TTX) and saxitoxin (2-4). In the half century since these classic experiments, our understanding of the molecular structure of VGSCs has advanced dramatically (5). We now recognize that mammals have nine isoforms of the α-subunit of VGSCs, or Na V 1, the subunit containing both the Na + -conducting pore and voltage-sensing gate (5-7). Meanwhile, progress in the characterization of ligands that target VGSCs has also progressed (8, 9), although TTX remains a major pharmacological investigative tool for VGSCs.The nine mammalian Na V 1 isoforms can be categorized into those that are TTX-sensitive versus those that are resistant, with K d or IC 50 values in the nM versus μM ranges, respectively (7). Two, Na V 1.8 and Na V 1.9, are found in primary sensory neurons and are highly resistant to TTX (10-12). One, Na V 1.5, found in cardiac muscle, is moderately TTX resistant (13), and the remaining six Na V 1 isoforms are exquisitely sensitive to TTX. One of these, Na V 1.4 is found exclusively in skeletal muscle. Presently, the five neuronal subtypes that are TTX-sensitive (i.e., Na V 1.1, 1.2, 1.3, 1.6, and 1.7) cannot be re...

A cone snail venom peptide, μO §-conotoxin GVIIJ from Conus geographus, has a unique posttranslational modification, S-cysteinylated cysteine, which makes possible formation of a covalent tether of peptide to its target Na channels at a distinct ligandbinding site. μO §-conotoxin GVIIJ is a 35-aa peptide, with 7 cysteine residues; six of the cysteines form 3 disulfide cross-links, and one (Cys24) is S-cysteinylated. Due to limited availability of native GVIIJ, we primarily used a synthetic analog whose Cys24 was S-glutathionylated (abbreviated GVIIJ SSG ). The peptide-channel complex is stabilized by a disulfide tether between Cys24 of the peptide and Cys910 of rat (r) Na V 1.2. A mutant channel of rNa V 1.2 lacking a cysteine near the pore loop of domain II (C910L), was >10 3 -fold less sensitive to GVIIJ SSG than was wild-type rNa V 1.2. In contrast, although rNa V 1.5 was >10 4 -fold less sensitive to GVIIJ SSG than Na V 1.2, an rNa V 1.5 mutant with a cysteine in the homologous location, rNa V 1.5[L869C], was >10 3 -fold more sensitive than wildtype rNa V 1.5. The susceptibility of rNa V 1.2 to GVIIJ SSG was significantly altered by treating the channels with thiol-oxidizing or disulfide-reducing agents. Furthermore, coexpression of rNa V β2 or rNa V β4, but not that of rNa V β1 or rNa V β3, protected rNa V 1.1 to -1.7 (excluding Na V 1.5) against block by GVIIJ SSG . Thus, GVIIJrelated peptides may serve as probes for both the redox state of extracellular cysteines and for assessing which Na V β-and Na V α-subunits are present in native neurons.oltage-gated sodium channels (VGSCs) are responsible for the upstroke of action potentials in excitable tissues. Each VGSC is composed of a pore-and voltage sensor-bearing α-subunit and one or more auxiliary β-subunits. Mammals have nine α-subunit isoforms (Na V 1.1 to -1.9) and four β-subunit isoforms (Na V β1 to -β4) (1). An Na V 1 has about 2,000-aa residues arranged in four homologous domains, where each domain has six transmembrane spanning segments with an extracellular "pore" loop between segments 5 and 6 (1, 2); furthermore, each Na V 1 has about a dozen extracellular cysteine residues, all located in or near the pore loops. For the most part, not much is known about these cysteines (including whether they are disulfide bonded).Na V β-subunits can affect the function and cellular localization of Na V 1s (1, 3-5). Each Na V β-subunit has some 200-aa residues and consists of a single transmembrane segment with a large extracellular domain and a smaller intracellular domain (1). Na V β2-and Na V β4-subunits, unlike Na V β1-and Na V β3-subunits, are disulfide bonded to α-subunits (1, 6). A given neuron can have multiple isoforms of these subunits whose identities are challenging to appraise pharmacologically (7).Toxins that target VGSCs have been invaluable for probing the structure and function of these channels. Venoms are a rich source of such toxins. For example, in Conus snails, four families of neuroactive peptides have been characterized that target VGSCs:...

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.

Disulfide-rich neurotoxins from venomous animals continue to provide compounds with therapeutic potential. Minimizing neurotoxins often results in removal of disulfide bridges or critical amino acids. To address this drug-design challenge, we explored the concept of disulfide-rich scaffolds consisting of isostere polymers and peptidic pharmacophores. Flexible spacers, such as amino-3-oxapentanoic or 6-aminohexanoic acids, were used to replace conformationally constrained parts of a three-disulfide-bridged conotoxin, SIIIA. The peptide-polymer hybrids, polytides, were designed based on cladistic identification of nonconserved loci in related peptides. After oxidative folding, the polytides appeared to be better inhibitors of sodium currents in dorsal root ganglia and sciatic nerves in mice. Moreover, the polytides appeared to be significantly more potent and longer-lasting analgesics in the inflammatory pain model in mice, when compared to SIIIA. The resulting polytides provide a promising strategy for transforming disulfide-rich peptides into therapeutics.