Identification of sodium channel toxins from marine cone snails of the subgenera Textilia and Afonsoconus

Abstract

Voltage-gated sodium (NaV) channels are transmembrane proteins that play a critical role in electrical signaling in the nervous system and other excitable tissues. µ-Conotoxins are peptide toxins from the venoms of marine cone snails (genus Conus) that block NaV channels with nanomolar potency. Most species of the subgenera Textilia and Afonsoconus are difficult to acquire; therefore, their venoms have yet to be comprehensively interrogated for µ-conotoxins. The goal of this study was to find new µ-conotoxins from species of the subgenera Textilia and Afonsoconus and investigate their selectivity at human NaV channels. Using RNA-seq of the venom gland of Conus (Textilia) bullatus, we identified 12 µ-conotoxin (or µ-conotoxin-like) sequences. Based on these sequences we designed primers which we used to identify additional µ-conotoxin sequences from DNA extracted from historical specimens of species from Textilia and Afonsoconus. We synthesized six of these µ-conotoxins and tested their activity on human NaV1.1–NaV1.8. Five of the six synthetic peptides were potent blockers of human NaV channels. Of these, two peptides (BuIIIB and BuIIIE) were potent blockers of hNaV1.3. Three of the peptides (BuIIIB, BuIIIE and AdIIIA) had submicromolar activity at hNaV1.7. This study serves as an example of the identification of new peptide toxins from historical DNA and provides new insights into structure–activity relationships of µ-conotoxins with activity at hNaV1.3 and hNaV1.7.

Voltage-Gated Sodium Channel Inhibition by µ-Conotoxins

“…Considering that over 750+ Conus species have been identified to date, including recent work extracting new µ-conotoxins sequences from historical specimens, and that only a small handful of µ-conotoxins have been functionally characterised, it is likely that a large number of µ-conotoxins remain unexplored [ 32 , 101 ]. Given the advances in structure–function technology, particularly landmark structural studies which provide new insights into channel blockage mechanisms in humans, the field of µ-conotoxin research has much more to offer and offers great potential to develop subtype selective Na V channel inhibitors.…”

“…These findings are in agreement with earlier work that showed an [Arg13Lys]GIIIA analogue inhibiting skeletal muscle subtypes with ten-fold lower potency (IC 50 1.12 µM), compared to native GIIIA (IC 50 0.10 µM) [ 71 ]. Similarly, the presence of a negatively charged amino acid at position 21 (Glu21) may explain why the recently characterised µ-conotoxin BuIIID had no effect on hNa V 1.4 and hNa V 1.7 current in whole-cell patch-clamp experiments up to 10 µM, despite sharing high sequence homology with all M-5 branch µ-conotoxins described to date ( Table 1 ) [ 32 ]. These results further emphasise the importance of a positively charged residue at this position for µ-conotoxin inhibition of Na V channels.…”

“…However, this observation did not extend to mouse and rat TTX-r isoforms, where these µ-conotoxins were active only at selected TTX-s Na V channels [ 26 , 40 , 66 ]. A recent study reporting BuIIIB activity at hNa V 1.1–1.8 using whole-cell patch-clamp electrophysiology [ 32 ] showed that the selectivity profile of 1.3 ≈ 1.4 < 1.7 ≈ 1.1 ≈ 1.2 ≈ 1.6 varied from that previously determined for rNa V channels using TEVC experiments (1.4 < 1.2 < 1.3 < 1.1 < 1.6 < 1.5) [ 26 ]. While differences between techniques could possibly explain the differences in Na V channel activity, it is likely that µ-conotoxin binding is different between homologous channels of different species.…”

“…These different KIIIA isomers have subsequently been shown to possess different potencies at a number of hNa V channel subtypes [ 43 ]. Likewise, BIIIA with cystine connectivity 1/5, 2/4 and 3/6 showed reduced potency at hNa V 1.2 and increased potency at hNa V 1.4, compared to BIIIA, with canonical cysteine connectivity (1/4, 2/5 and 3/6) in whole-cell patch-clamp experiments [ 32 ]. The data indicate that some μ-conotoxins can not only tolerate different disulfide connectivity, but that this can lead to subtle but important changes in their selectivity profiles.…”

“… * C-terminal amidation; # The Na V subtype at which the individual peptide is the most active. a [ 26 ] b [ 41 ] c [ 42 ] d [ 32 ] e [ 43 ] f [ 44 ] represent selectivity data reported independent of discovery. …”