Novel Conotoxins from Conus striatus and Conus kinoshitai Selectively Block TTX-Resistant Sodium Channels

Abstract

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.

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

“…Both SIIIA and SIIIB were full inhibitors of 125 I-TIIIA binding to rat brain or skeletal muscle sodium channels. Interestingly, removing the pyroglutamate residue at the N terminus (SIIIA- (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) and SIIIB-(2-20)) enhanced SIIIA and decreased SIIIB activity at Na v 1.2, while the reverse effect was observed at Na v 1.4 (Fig. 4).…”

“…SIIIA- (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) had decreased affinity at Na v 1.4, but increased binding affinity at Na v 1.2 sodium channel, whereas SIIIB-(2-20) had increased affinity at both subtypes. In an attempt to identify residues contributing to the neuronal selectivity, we produced an alanine scan of SIIIA- (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), except for the small Asn, Gly, and Ser residues in loops 1 and 2. Surprisingly, [K11A]SIIIA- (2-20) had only a small 6-fold reduction in affinity at Na v 1.2 and Na v 1.4, compared with the ϳ100 -300-fold reduction in affinity for the equivalent substitution in Of the mutants tested, [H16A]SIIIA- (2)(3)(4)(5)(6)(7)(8)(9)(10)…”

“…In an attempt to identify residues contributing to the neuronal selectivity, we produced an alanine scan of SIIIA- (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), except for the small Asn, Gly, and Ser residues in loops 1 and 2. Surprisingly, [K11A]SIIIA- (2-20) had only a small 6-fold reduction in affinity at Na v 1.2 and Na v 1.4, compared with the ϳ100 -300-fold reduction in affinity for the equivalent substitution in Of the mutants tested, [H16A]SIIIA- (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) showed the largest drop in affinity at both Na v 1.2 and Na v 1.4, confirming the results reported for KIIIA (20) To identify the structural determinants underlying these shifts in affinity and selectivity, the three-dimensional solution structure of SIIIA was calculated using 1 H NMR spectroscopy. In addition, H␣ chemical shifts were determined to establish that each analogue adopted the native fold found in SIIIA.…”

“…Currently 12 -conotoxins have been identified from nine species of fish hunting cone snails (Table 1). -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).…”

“…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).…”