Structural and functional insights into the inhibition of human voltage-gated sodium channels by μ-conotoxin KIIIA disulfide isomers

Abstract

μ-Conotoxins are components of cone snail venom, well-known for their analgesic activity through potent inhibition of voltage-gated sodium channel (Na_V) subtypes, including Na_V1.7. These small, disulfide-rich peptides are typically stabilized by three disulfide bonds arranged in a 'native' CysI-CysIV, CysII-CysV, CysIII-CysVI pattern of disulfide connectivity. However, μ-conotoxin KIIIA, the smallest and most studied μ-conotoxin with inhibitory activity at Na_V1.7, forms two distinct disulfide bond isomers during thermodynamic oxidative folding, including Isomer 1 (CysI-CysV, CysII-CysIV, CysIII-CysVI) and Isomer 2 (CysI-CysVI, CysII-CysIV, CysIII-CysV), but not the native μ-conotoxin arrangement. To date, there has been no study on the structure and activity of KIIIA comprising the native μ-conotoxin disulfide bond arrangement. Here, we evaluated the synthesis, potency, sodium channel subtype selectivity, and 3D structure of the three isomers of KIIIA. Using a regioselective disulfide bond-forming strategy, we synthetically produced the three μ-conotoxin KIIIA isomers displaying distinct bioactivity and Na_V subtype selectivity across human Na_V channel subtypes 1.2, 1.4, and 1.7. We show that Isomer 1 inhibits Na_V subtypes with a rank order of potency of Na_V1.4 > 1.2 > 1.7 and Isomer 2 in the order of Na_V1.4≈1.2 > 1.7, while the native isomer inhibited Na_V1.4 > 1.7≈1.2. The three KIIIA isomers were further evaluated by NMR solution structure analysis and molecular docking with hNa_V1.2. Our study highlights the importance of investigating alternate disulfide isomers, as disulfide connectivity affects not only the overall structure of the peptides but also the potency and subtype selectivity of μ-conotoxins targeting therapeutically relevant Na_V subtypes.

Keywords: KIIIA; Na(V)1.7; disulfide bond isomers; voltage-gated sodium channels; μ-conotoxin.

Figure 1

Analytical RP-HPLC traces showing disulfide bond isomers of KIIIA obtained through thermodynamic oxidation and isomers obtained through step-wise directed folding conditions.A, Isomer 1-T and Isomer 2-T formed in thermodynamic condition (black), directed Isomer 1-R (blue), Isomer 2-R (green), and Native (red) conformation. B, Isomer 1-T (black) coeluted with Isomer 1-R (CysI-CysV, CysII-CysIV, CysIII-CysVI) (blue). C, Isomer 2-T coeluted with both directed Isomer 2-R (CysI-CysVI, CysII-CysIV, CysIII-CysV) (green) and Native (CysI-CysIV, CysII-CysV, CysIII-CysVI) isomer (red) and was ultimately assigned as Isomer 2-R based on NMR. RP-HPLC, reverse-phase HPLC.

Figure 2

Regioselective synthesis of Native KIIIA (CysI-CysIV, CysII-CysV, CysIII-CysVI), forming disulfide bonds in the order of CysIII-CysVI, CysII-CysV, and CysI-CysIV.A, synthetic regioselective oxidation scheme showing reaction conditions for each step. B, analytical RP-HPLC traces corresponding to the product obtained by each step of the synthesis including the linear starting peptide, following the formation of first, second, and third disulfide bond. C, observed mass fragmentation corresponding to each folding step acquired by LC-MS. ∗ denotes peptides of interest. RP-HPLC, reverse-phase HPLC.

Figure 3

Pharmacology of KIIIA isomers at hNa_Vchannels.A, representative current trace before and after the addition of Isomer 1 (blue), Isomer 2 (green), and Native KIIIA (red) at hNa_V1.2, hNa_V1.4, and hNa_V1.7. B–D, concentration-response curve showing inhibitory activity of KIIIA isomers at hNav1.2, hNa_V1.4, and hNa_V1.7 respectively, the data are presented as mean ± SEM. E, selectivity profile of KIIIA Isomer, Isomer 2, and Native acquired using automated whole-cell patch-clamp electrophysiology on HEK293 cells overexpressing hNa_V1.2, hNa_V1.4, or hNa_V1.7 in combination with β1-subunits. The data are presented as mean ± SD, with n = 5 cells per data point. 2-way ANOVA significance ∗ p value < 0.0332, ∗∗ p value < 0.0021, ∗∗∗ p value < 0.0002, ∗∗∗∗ p value < 0.0001. NaV, voltage-gated sodium channel.

Figure 4

Three-dimensional structure of KIIIA isomers used in this study and docking studies with KIIIA-hNa_V1.2 complex (Pan et al. (PDB6J8E) (50)).A, KIIIA Isomer 1 (PDB ID: 2XLG) (blue). B, KIIIA Isomer 2 (green) (this study). C, KIIIA Native (red) (this study). D, docked KIIIA Isomer 1 (blue) and Native KIIIA (red) compared to cryo-EM of KIIIA (Isomer 1, teal) in complex with hNa_V1.2. E, docked Isomer 2 (green) of KIIIA compared to cryo-EM of KIIIA (Isomer 1, teal) in complex with hNa_V1.2. F, putative interactions between KIIIA isomers and hNa_V1.2 compared to cryo-EM of KIIIA (Isomer 1) in complex with hNa_V1.2 (50). NaV, voltage-gated sodium channel.