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 are important in initiating and propagating nerve impulses in various tissues, including cardiac muscle, skeletal muscle, the brain, and the peripheral nerves. Hyperexcitability of these channels leads to such disorders as cardiac arrhythmias

(2009) Unexpected synergism between Tetrodotoxin and µ-conotoxin in blocking voltage-gated sodium channels, Channels, 3:1, 32-38,

Conotoxins have emerged as useful leads for the development of novel therapeutic analgesics. These peptides, isolated from marine molluscs of the genus Conus, have evolved exquisite selectivity for receptors and ion channels of excitable tissue. One such peptide, α-conotoxin Vc1.1, is a 16-mer possessing an interlocked disulfide framework. Despite its emergence as a potent analgesic lead, the molecular target and mechanism of action of Vc1.1 have not been elucidated to date. In this paper we describe the regioselective synthesis of dicarba analogues of Vc1.1 using olefin metathesis. The ability of these peptides to inhibit acetylcholine-evoked current at rat α9α10 and α3β4 nicotinic acetylcholine receptors (nAChR) expressed in Xenopus oocytes has been assessed in addition to their ability to inhibit high voltage-activated (HVA) calcium channel current in isolated rat DRG neurons. Their solution structures were determined by NMR spectroscopy. Significantly, we have found that regioselective replacement of the native cystine framework with a dicarba bridge can be used to selectively tune the cyclic peptide's innate biological activity for one receptor over another. The 2,8-dicarba Vc1.1 isomer retains activity at γ-aminobutyric acid (GABAB) G protein-coupled receptors, whereas the isomeric 3,16-dicarba Vc1.1 peptide retains activity at the α9α10 nAChR subtype. These singularly acting analogues will enable the elucidation of the biological target responsible for the peptide's potent analgesic activity.

Eukaryotic, voltage-gated sodium (Na V ) channels are large membrane proteins which underlie generation and propagation of rapid electrical signals in nerve, muscle and heart. Nine different Na V receptor sites, for natural ligands and/or drugs, have been identified, based on functional analyses and site-directed mutagenesis. In the marine ecosystem, numerous toxins have evolved to disrupt Na V channel function, either by inhibition of current flow through the channels, or by modifying the activation and inactivation gating processes by which the channels open and close. These toxins function in their native environment as offensive or defensive weapons in prey capture or deterrence of predators. In composition, they range from organic molecules of varying size and complexity to peptides consisting of ~10-70 amino acids. We review the variety of known Na V -targeted marine toxins, outlining, where known, their sites of interaction with the channel protein and their functional effects. In a number of cases, these natural ligands have the potential applications as drugs in clinical settings, or as models for drug development.