AA sequence: CCNCSSKWCRDHSRCC-NH2
Disulfide bonds: Cys1-Cys9, Cys2-Cys15 and Cys4-Cys16
Length (aa): 16
Molecular Weight: 1884.16 Da
Purity rate: > 95 %
µ-conotoxin KIIIA is a potent Nav1.2 blocker
µ-conotoxin KIIIA was originally identified within the venom of the marine fish-hunting cone snail Conus kinoshitai1. The peptide has 16 amino acid residues and possesses 3 disulfide bridges originally assumed to be connected along the following pattern: Cys1-Cys9, Cys2-Cys15, and Cys4-Cys16. Upon chemical synthesis, the major isoform possesses in fact the Cys1-Cys15, Cys2-Cys9 and Cys4-Cys16 connectivity, the one with the most potent pharmacological activity2. The peptide was found to possess potent analgesic activity inflammatory pain (formalin test) without producing motor impairment3. ED50 was 0.1 mg/kg by intraperitoneal injection. In mouse DRG neurons, 5 mM m-conotoxin KIIIA blocks over 50% TTX-sensitive voltage-gated sodium currents, while marginally affecting 20% of the TTX-resistant current. The toxin blocked the various Nav isoforms with the following rank of potencies: rat Nav1.2 (Kd of 3 nM) > rat Nav1.4 (Kd of 50 nM) > rat Nav1.1 (Kd of 290 nM) = rat Nav1.7 >> rat Nav1.3 (Kd of 8 mM). Rat Nav1.5 and Nav1.8 seemed not affected by the peptide. A particularity of the Nav1.2 block is that it is irreversible contrary to other Nav isoform blockades. This irreversible block relies on Trp8. The mature peptide sequence of m-conotoxin KIIIA produced in the venom of Conus kinoshitai possesses two additional residues preceding the N-terminus (Asn1 and Gly2) forming an extended N-terminal isoform that was termed m-conotoxin KIIIB. m-conotoxin KIIIA interacts with the extracellular segments in repeats I, II and III of Nav1.2 with Lys7 blocking Na+ entry to the Na+ binding site within the selectivity filter vestibule4.
AA sequence: CCNCSSKWCRDHSRCC-NH2
The peptides isolated from venoms of predatory marine Conus snails (“conotoxins”) are well-known to be highly potent and selective pharmacological agents for voltage-gated ion channels and receptors. We report the discovery of two novel TTX-resistant sodium channel blockers, mu-conotoxins SIIIA and KIIIA, from two species of cone snails. The two toxins were identified and characterized by combining molecular techniques and chemical synthesis. Both peptides inhibit TTX-resistant sodium currents in neurons of frog sympathetic and dorsal root ganglia but poorly block action potentials in frog skeletal muscle, which are mediated by TTX-sensitive sodium channels. The amino acid sequences in the C-terminal region of the two peptides and of the previously characterized mu-conotoxin SmIIIA (which also blocks TTX-resistant channels) are similar, but the three peptides differ in the length of their first N-terminal loop. We used molecular dynamics simulations to analyze how altering the number of residues in the first loop affects the overall structure of mu-conotoxins. Our results suggest that the naturally occurring truncations do not affect the conformation of the C-terminal loops. Taken together, structural and functional differences among mu-conotoxins SmIIIA, SIIIA, and KIIIA offer a unique insight into the “evolutionary engineering” of conotoxin activity.
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 collision-induced dissociation 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 Na(V)1.2 (K(d) values of 5 and 230 nM, respectively). The solution structure for μ-KIIIA based on nuclear magnetic resonance 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 μ-KIIIA, and both blocked Na(V)1.2 (K(d) values of 470 and 26 nM, respectively). Our results establish that the preferred disulfide pattern of synthetic μ-KIIIA and 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 of further expanding the snail’s repertoire of active peptides.
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 non-opioid analgesics, such as subtype-selective sodium channel blockers. Micro-conotoxin KIIIA is representative of micro-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 approximately 20% 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 (IC50 8 microM), Na(V)1.5 (IC50 284 microM), and Na(V)1.4 (IC50 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 micro-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.
X Pan, Z Li, X Huang, G Huang, S Gao, H Shen, L Liu, J Lei and N Yan. Molecular basis for pore blockade of human Na+ channel Nav1.2 by the µ-conotoxin KIIIA. Science 363, 1309-1313 (2019).
The voltage-gated sodium channel Nav1.2 is responsible for the initiation and propagation of action potentials in the central nervous system. We report the cryo–electron microscopy structure of human Nav1.2 bound to a peptidic pore blocker, the μ-conotoxin KIIIA, in the presence of an auxiliary subunit, β2, to an overall resolution of 3.0 angstroms. The immunoglobulin domain of β2 interacts with the shoulder of the pore domain through a disulfide bond. The 16-residue KIIIA interacts with the extracellular segments in repeats I to III, placing Lys7 at the entrance to the selectivity filter. Many interacting residues are specific to Nav1.2, revealing a molecular basis for KIIIA specificity. The structure establishes a framework for the rational design of subtype-specific blockers for Nav channels.