µ-Conotoxins Targeting the Human Voltage-Gated Sodium Channel Subtype NaV1.7

Abstract

µ-Conotoxins are small, potent, peptide voltage-gated sodium (NaV) channel inhibitors characterised by a conserved cysteine framework. Despite promising in vivo studies indicating analgesic potential of these compounds, selectivity towards the therapeutically relevant subtype NaV1.7 has so far been limited. We recently identified a novel µ-conotoxin, SxIIIC, which potently inhibits human NaV1.7 (hNaV1.7). SxIIIC has high sequence homology with other µ-conotoxins, including SmIIIA and KIIIA, yet shows different NaV channel selectivity for mammalian subtypes. Here, we evaluated and compared the inhibitory potency of µ-conotoxins SxIIIC, SmIIIA and KIIIA at hNaV channels by whole-cell patch-clamp electrophysiology and discovered that these three closely related µ-conotoxins display unique selectivity profiles with significant variations in inhibitory potency at hNaV1.7. Analysis of other µ-conotoxins at hNaV1.7 shows that only a limited number are capable of inhibition at this subtype and that differences between the number of residues in loop 3 appear to influence the ability of µ-conotoxins to inhibit hNaV1.7. Through mutagenesis studies, we confirmed that charged residues in this region also affect the selectivity for hNaV1.4. Comparison of µ-conotoxin NMR solution structures identified differences that may contribute to the variance in hNaV1.7 inhibition and validated the role of the loop 1 extension in SxIIIC for improving potency at hNaV1.7, when compared to KIIIA. This work could assist in designing µ-conotoxin derivatives specific for hNaV1.7.

Voltage-Gated Sodium Channel Inhibition by µ-Conotoxins

“…Interestingly, under identical electrophysiology assay conditions at hNa V 1.7, these IC 50 values were similar to KIIIA (IC 50 383.5 ± 15.2 nM) [ 44 ]. As KIIIA has a similarly truncated loop 1, it was proposed that the additional loop-one residues in SxIIIC are required for increased inhibition of hNa V 1.7, when compared to KIIIA [ 44 ].…”

“…Replacement of residues in loop one (between Cys2 and Cys3) in SIIIA with non-natural backbone spacers produced longer-lasting analgesic effects in a murine model of inflammatory pain than native SIIIA [ 79 ]. In the case of SxIIIC, analogues with a loop-one truncation ([∆7,8]SxIIIC) reduced potency at hNa V 1.7 (IC 50 313.6 ± 36.6 nM), compared to native SxIIIC (IC 50 152.2 ± 26.8 nM) [ 17 , 44 ]. Interestingly, under identical electrophysiology assay conditions at hNa V 1.7, these IC 50 values were similar to KIIIA (IC 50 383.5 ± 15.2 nM) [ 44 ].…”

“…In the case of SxIIIC, analogues with a loop-one truncation ([∆7,8]SxIIIC) reduced potency at hNa V 1.7 (IC 50 313.6 ± 36.6 nM), compared to native SxIIIC (IC 50 152.2 ± 26.8 nM) [ 17 , 44 ]. Interestingly, under identical electrophysiology assay conditions at hNa V 1.7, these IC 50 values were similar to KIIIA (IC 50 383.5 ± 15.2 nM) [ 44 ]. As KIIIA has a similarly truncated loop 1, it was proposed that the additional loop-one residues in SxIIIC are required for increased inhibition of hNa V 1.7, when compared to KIIIA [ 44 ].…”

Marine natural products

“…577 Comparative evaluation of μ-conotoxins SxIIIC, SmIIIA, and KIIIA in patch-clamp and receptor-based studies has identified that the number of residues in loop 3 can influence the peptides ability to inhibit human voltage-gated sodium hNa V 1.7 channels. 578 Five peptides were purified from the venom of Conus marmoreus – notably, none of the peptides contained disulfide bonds, and they were inactive towards a panel of rat nAChR subtypes. 579…”

Historical Perspective of the Characterization of Conotoxins Targeting Voltage-Gated Sodium Channels

“…SxIIIC was isolated from Conus striolatus [172], and its characterization revealed its block of several neuronal sodium channel isoforms including Na V 1.3, Na V 1.1, Na V 1.6, Na V 1.7, and Na V 1.2 in order of potency. A subsequent comparison of relative potencies of µ-conotoxins on Na V 1.7 showed that three µ-conotoxins with reported block of Na V 1.7, SxIIIC, KIIIA, and SmIIIA blocked that sodium channel isoform with the greatest variability compared to actions on other neuronal channels [192]. That study explored structural differences in KIIIA, SmIIIA, and SxIIIC, focused on N-terminal residue extension in SmIIIA and SxIIIC compared to KIIIA, number of residues in loop 1, and charged residues in loop 3.…”