N‐ and c‐terminal extensions of μ‐conotoxins increase potency and selectivity for neuronal sodium channels

Abstract

μ-Conotoxins are peptide blockers of voltage-gated sodium channels (sodium channels), inhibiting tetrodotoxin-sensitive neuronal (Na(v) 1.2) and skeletal (Na(v) 1.4) subtypes with highest affinity. Structure-activity relationship studies of μ-conotoxins SIIIA, TIIIA, and KIIIA have shown that it is mainly the C-terminal part of the three-loop peptide that is involved in binding to the sodium channel. In this study, we characterize the effect of N- and C-terminal extensions of μ-conotoxins SIIIA, SIIIB, and TIIIA on their potency and selectivity for neuronal versus muscle sodium channels. Interestingly, extending the N- or C-terminal of the peptide by introducing neutral, positive, and/or negatively charged residues, the selectivity of the native peptide can be altered from neuronal to skeletal and the other way around. The results from this study provide further insight into the binding profile of μ-conotoxins at voltage-gated sodium channels, revealing that binding interactions outside the cysteine-stablilized loops can contribute to μ-conotoxin affinity and sodium channel selectivity.

Where cone snails and spiders meet: design of small cyclic sodium‐channel inhibitors

“…To improve the potency of Pn, we designed a first series of mutants (Series 1) using data available from structurefunction studies on KIIIA and other m-conotoxins ( Fig. 1D) (19,20,(32)(33)(34)(35)(36). From this first series of mutants, PnM2 showed the most promising increase in activity when tested on Na V 1.2-Na V 1.6 ( Table 2).…”

Structure and Function of μ-Conotoxins, Peptide-Based Sodium Channel Blockers with Analgesic Activity

“…It was suggested that the negative charge at this position created an unfavorable interaction with the binding site within the sodium channel [66]. More recent work has shown that addition of either a neutral residue (μ-TIIIA[E15A, 23A]) or basic residue (μ-TIIIA[E15A, 23K]) to the C-terminus decreased overall potency for all subtypes, but increased preference for Na V 1.2 [67]. Based on these findings, further positional scanning of Glu15 would be of interest to explore whether the selectivity profiles of μ-TIIIA could be shifted in favor of pain-relevant Na V 1-subtypes.…”

“…These data were interesting in light of functional data which suggested that the N-terminus was important for subtype selectivity, while the C-terminus contributed mainly to neuronal subtype interactions [78]. Schroeder et al explored the structural and functional consequences of N-terminal modification of μ-SIIIA/B [67]. Replacement of the N-terminal pyroglutamate (Pyr1 or single letter code Z) with charged residues (i.e., μ-SIIIA[Z1R] and μ-SIIIB[Z1E]) either decreased (μ-SIIIA) or increased (μ-SIIIB) preference for Na V 1.2.…”

“…The role of negatively-charged residues at the N-terminus for Na V 1.2 subtype selectivity was further demonstrated by the μ-SIIIB analogs μ-SIIIB[E0, Z1R] and μ-SIIIB(22–0)[N2E], respectively. Extension of the C-terminus of μ-SIIIA[D15A], using charged amino acids, afforded greater selectivity for the neuronal subtype [67]; specifically, the μ-SIIIA analogs μ-SIIIA(2-21)[D15A, 21K] and μ-SIIIA(2-21) [D15A, 21D] showed improved discrimination between Na V 1.2 and Na V 1.4 [67]. …”

Conotoxins Targeting Neuronal Voltage-Gated Sodium Channel Subtypes: Potential Analgesics?

“…In contrast with these two studies, adding residues to the N-terminus of SIIIA, SIIIB and TIIIA has been shown to change their selectivity preference from rat muscle channels to rat neuronal VGSCs [121]. This suggests that N-terminal residues also contribute to the toxins’ affinity and selectivity.…”