Structure/Function Characterization of μ-Conotoxin KIIIA, an Analgesic, Nearly Irreversible Blocker of Mammalian Neuronal Sodium Channels

Abstract

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 ...

Mapping of voltage sensor positions in resting and inactivated mammalian sodium channels by LRET

“…Conversely, KIIIA is a shorter peptide of only 16 aa that also blocks Na + ion conductance through Nav1.4. However, the blockage by KIIIA is incomplete, allowing guanidinium toxins like tetrodotoxin (TTX) access to a deeper pocket of site 1 of most Nav isoforms, as indicated by experiments where complete block is achieved when TTX and KIIIA are applied simultaneously (21,22,30,32).…”

Neuronally Selective μ-Conotoxins from Conus striatus Utilize an α-Helical Motif to Target Mammalian Sodium Channels

“…In contrast, SIIIA and KIIIA have a much shorter loop 2 and a lysine instead of an arginine in the corresponding position. Recent structure-activity studies on TIIIA (15) and KIIIA (20) have confirmed that the arginine in loop 2 is crucial for TIIIA affinity at both neuronal and skeletal muscle sodium channels, whereas the structurally equivalent lysine was only important for affinity at the skeletal muscle VGSC. Three-dimensional structures of -conotoxins GIIIA, GIIIB, PIIIA, TIIIA, and SmIIIA have been determined by NMR (14,15,17,26,27).…”

“…-Conotoxins GIIIA, GIIIB, and GIIIC from Conus geographus selectively target the skeletal muscle Na v 1.4, PIIIA from Conus purpurasence (12)(13)(14) and TIIIA from Conus tulipa (15) target Na v 1.2 and 1.4, and -conotoxins SmIIIA from Conus stercusmuscsarum (16,17), SIIIA from Conus striatus (18,19), and KIIIA from Conus kinoshitai (18) target amphibian TTX-R sodium channels. More recently, KIIIA has also been shown to inhibit TTX-S sodium channels in mouse DRG and rat TTX-S sodium channels expressed in Xenopus oocytes (20). Lastly, -conotoxins CnIIIA, CnIIIB from Conus consors, CIIIA from Conus catus, and MIIIA from Conus magus were found to inhibit TTX-R and TTX-S sodium channels in frog DRG but not rat or mouse DRG neurons (21).…”

“…These structures reveal a similar fold for the -conotoxins despite high sequence divergence. At present, structures of -conotoxins SIIIA and KIIIA are modeled from SmIIIA (18,20,21).…”

Conotoxins: from the biodiversity of gastropods to new drugs

“…Ещё более универсальное действие на ТТХ-чувствительные натриевые каналы характерно для недавно описанного m-конотоксина KIIIA [62]. Тестирование на периферических нейронах мыши показало, что этот токсин необратимо ингибирует 80% ТТХ-чувствительных каналов и лишь 20% ТТХ-устойчивых.…”