1. Introduction
Voltage-gated sodium (Na
V) channels are highly conserved pore-forming proteins permeable to sodium ions and are important for the initiation and propagation of action potentials [
1,
2]. Nine different subtypes are expressed in humans (Na
V1.1–1.9) and are encoded by the
SCNxA genes (x = 1–5, 8–11) [
3]. Genetic mutations in human Na
V channels give rise to a number of neurological disorders, for example, epilepsy (Na
V1.1), myotonia (Na
V1.4) and cardiac arrhythmia (Na
V1.5) [
4,
5,
6]. Moreover, patients with loss of function mutations in Na
V1.7 present with congenital insensitivity to pain and aside from anosmia (loss of smell) have no serious physiological deficits, highlighting the potential of Na
V1.7 as a drug target for pain therapeutics [
7]. Despite compelling genetic evidence and studies implicating several additional subtypes, including Na
V1.1, 1.3, 1.6, 1.8, and 1.9, as potential targets for pain therapies [
8], the clinical utility of Na
V channel inhibitors has had limited success due to high sequence homology between Na
V channel subtypes, particularly within the pore domain [
9], as off-target Na
V inhibition could lead to undesired side-effects. Therefore, a thorough understanding of subtype selectivity is essential for exploiting Na
V channel inhibitors as therapeutics. Toxins from venomous creatures, such as snakes, spiders, and cone snails, provide excellent pharmacological tools to study Na
V channels [
10,
11].
The venom of predatory marine cone snails represents a complex source of disulfide-rich bioactive peptides that modulate ion channels, called conotoxins [
12]. One of the most numerous and best-characterised conotoxin classes are the µ-conotoxins, which have a distinctive type III cysteine framework (CC–C–C–CC). They are potent and selective Na
V channel inhibitors and µ-conotoxin research has led to many advances in the understanding of Na
V channel function. In 2001, Li et al. established the clockwise arrangement of the four domains of Na
V channels using μ-conotoxin GIIIA as a probe [
13] and more recently, the structure of human Na
V1.2 bound to μ-conotoxin KIIIA was solved by cryogenic electron microscopy (cryo-EM), confirming for the first time interactions between µ-conotoxins and the Na
V channel pore [
14]. These recent developments have reinvigorated efforts to exploit µ-conotoxins as drug leads.
µ-Conotoxins represent favourable drug leads due to two key characteristics. First, they are rich in cysteines which form disulfide bonds to afford structural integrity and secondly, their small molecular size (typically 16–26 residues) presents an advantage over larger biologics as they are easily synthesised and amenable to chemical modifications to improve pharmacological traits [
15]. Conversely, they present as favourable molecules over similarly acting small molecule Na
V inhibitors including anesthetics [
16], as the increased surface area provides greater contacts with the Na
V channel pore, which can result in an increased subtype selectivity. To date, 22 µ-conotoxins have been described from 13 different
Conus species [
17]. µ-Conotoxins typically show a preference for Na
V1.2 and Na
V1.4 when evaluated in rat homologues, with the exception of BuIIIB, which shows a preference for Na
V1.3 [
18]. To date only a small number of µ-conotoxins have been found to inhibit human (h)Na
V1.7, including KIIIA (IC
50 147 nm) [
19] and CnIIIC (IC
50 485 ± 94 nM) [
20]. One species receiving relatively little attention is the piscivorous (fish-hunting)
Conus striolatus. Until recently only four peptide inhibitors (Sx11.2, Sx4.1, and µ-conotoxins SxIIIA, and SxIIIB) were reported from this species, with SxIIIA potently inhibiting Na
V1.4 (IC
50 7 nM), but not Na
V1.7 [
21]. The activity of the remaining peptides has not been assessed to date.
In the current study, we present the discovery of a novel µ-conotoxin from C. striolatus, named SxIIIC. We have characterised its pharmacological activity as a NaV channel inhibitor and evaluated the NMR solution structure. Our research discovered that SxIIIC displays a unique NaV channel subtype selectivity profile when compared to other highly-homologous µ-conotoxins. Interestingly, at the therapeutically relevant subtype NaV1.7, SxIIIC displays nanomolar potency and near irreversible inhibition. To date, SxIIIC represents one of the most potent µ-conotoxin putative pore blocker inhibitors of hNaV1.7. Structural differences between SxIIIC and other µ-conotoxins may provide insight into residues driving potent NaV channel inhibition. Future investigation of structure-activity relationships of these pore blockers may open avenues for the successful development of therapeutic NaV channel inhibitors.
2. Experimental Section
2.1. Chemicals
All chemicals were purchased from Sigma Aldrich (Sigma Aldrich, St. Louis, MO, USA) and amino acids were purchased from Chem-Impex International (Chem-Impex International, Inc., Wood Dale, IL, USA) unless otherwise stated.
2.2. Activity-Guided Isolation of SxIIIC
Inhibition of Na
V-mediated responses by crude and fractionated venom from
C. striolatus was assessed using a FLIPR
TETRA (Molecular Devices, Sunnyvale, CA, USA) high-throughput assay, as previously described [
22]. In brief, SH-SY5Y neuroblastoma cells were plated at a density of 120,000 cells/well on 96-well black-walled imaging plates (Corning Inc., Corning, NY, USA) in growth medium (RPMI medium: 15% fetal bovine serum and 1 mM L-glutamine) and cultured for 48 h. Cells were loaded with Calcium 4 No-wash dye (Molecular Devices) diluted in physiological salt solution (PSS; composition (in mM) 140 NaCl, 11.5 glucose, 5.9 KCl, 1.4 MgCl
2, 1.2 NaH
2PO
4, 5 NaHCO
3, 1.8 CaCl
2, and 10 HEPES) by replacing growth medium with dye solution and incubating at 37 °C for 30 min. Fluorescence responses to the addition of crude venom or venom fractions were recorded every second for 300 s (excitation, 470–495 nm; emission, 515–575 nm) and responses to the addition of veratridine (50 µM) were recorded at 1 s intervals for a further 300 s.
Crude venom was isolated from one specimen of C. striolatus by stripping the venom duct contents. The crude venom was dissolved in 30% acetonitrile/0.1% formic acid, vortexed, and centrifuged at 10,000× g for 5 min to remove insoluble components. Crude venom (200 µg) was fractionated into 45 × 0.7 mL fractions (1 per min) using a Vydac, 5 µm C18 218TP, 250 × 4.6 mm column (Grace Davison Discovery Sciences, Columbia, MD, USA) eluted at a flow rate of 0.7 mL/min with 5–45% solvent B over 45 min (solvent A, water (H2O)/0.1% formic acid; solvent B, 90% acetonitrile/0.1% formic acid), with detection at 214 nm. Activity-guided fractionation was performed using the FLIPRTetra high-throughput NaV assay described above. Further purification was carried out by size-exclusion chromatography (Superdex Peptide, HR 10/30; Amersham Biosciences, Little Chalfont, UK) using elution with 30% acetonitrile, 0.1% formic acid, aqueous at a flow rate of 0.5 mL/min, with detection at 214 nm. The resulting purified fraction was then analysed on a MALDI-TOF mass spectrometer (4700 Proteomics Analyzer; Applied Biosystems, Mulgrave, VIC, Australia) using α-cyano-4-hydroxycinnamic acid (5 mg/mL) as the matrix.
2.3. Peptide Sequencing
The purified peptide was sequenced using Edman degradation at the Australian Proteome Research Facility. Briefly, the peptide was dissolved in 4 M urea in 50 mM ammonium bicarbonate and reduced with 100 nM dithiothreitol at 56 °C for 1 h under argon. Subsequently, the sample was alkylated using 220 nM acrylamide for 0.5 h in the dark. The reaction was quenched by addition of excess dithiothreitol. Following desalting by reversed-phase high-performance liquid chromatography (RP-HPLC), the collected fraction was loaded onto pre-cycled bioprene discs and subjected to 35 cycles of Edman N-terminal sequencing using an Applied Biosystems 494 Procise Protein Sequencing System.
2.4. Peptide Synthesis
Synthetic SxIIIC (RGCCNGRGGCSSRWCRDHARCC*) was assembled by solid-phase peptide synthesis on a Liberty Prime automatic synthesiser (CEM, Matthews, NC, USA), on rink amide-AM resin (0.139 mmol/g) at a 0.1 mmol scale to produce C-terminal amidated peptide (*) using 9-fluorenylmethoxycarbonyl (Fmoc) methodology. Side-chain protecting groups used were Arg(Pbf), Asn(Trt), Cys(Trt), His(Trt), Ser(tBu), and Trp(Boc). The peptide was cleaved from resin and side chains simultaneously removed following a 2 h incubation with 92.5% trifluoroacetic acid (TFA)/2.5% anisole/2.5% triisopropylsilane (TIPS)/2.5% H2O. Excess TFA was evaporated by N2 flow and reaction precipitated with ice-cold diethyl ether. Precipitant was collected via gravity filtration on a 20 µM frit (Phenomenix, Torrance, CA, USA) and crude peptide dissolved in 50% solvent B and lyophilised. Linear SxIIIC was dissolved in <2% solvent B and loaded onto a Gemini, 5 µm C18 110 Å, 250 × 21.2 mm column (Phenomenex, Torrance, CA, USA). Following column equilibration (0–5% solvent B over 5 min), synthetic SxIIIC was purified by RP-HPLC using a linear gradient between 5–25% over 40 min at 8 mL/min. Fractions containing the desired product, identified by electrospray mass spectrometry (ESI-MS), were pooled, lyophilised, and stored at −20 °C.
KIIIA (CCNCSSKWCRDHSRCC*) with disulfide connectivity Cys1/Cys15, Cys2/Cys9, and Cys4/Cys16 was synthesised as previously described [
23]. Briefly, peptide was assembled on Liberty Prime automatic synthesiser (CEM, Matthews, NC, USA) using rink amide-AM resin. Peptide was cleaved from solid support and side chains simultaneously deprotection in 92.5% TFA/2.5% TIPS/2.5% H
2O/2.5% 3,6-dioxa-1,8-octanedithiol (DODT) for 2 h at room temperature. Excess TFA was evaporated by N
2 flow, followed by peptide precipitation in ice-cold diethyl ether and centrifugation. Peptide was redissolved in 50% solvent B and lyophilised.
2.5. Oxidation
Reduced SxIIIC peptide (0.05 mg/mL) was oxidised at room temperature for 1 h in 0.1 M Tris-HCl pH 7.5, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM glutathione oxidised, and 1 mM glutathione reduced. The reaction was quenched by adding formic acid to a final concentration of 5%. The final SxIIIC product was purified by RP-HPLC on a Gemini, 5 µm C18 110 Å, 250 × 10 mm column (Phenomenex, Torrance, CA, USA) using a linear gradient between 0–20% over 40 min at 3 mL/min. Fractions containing the desired product eluted at 10% solvent B and were pooled, lyophilised, and stored at −20 °C.
Reduced KIIIA peptide (0.3 mg/mL) was oxidised at room temperature for 24 h in 0.1 M NH
4HCO
3 pH 8.0, 0.81 mM glutathione oxidised, and 0.81 mM glutathione reduced. The KIIIA reaction was quenched with 1% TFA. The final KIIIA product was purified, as previously described [
23].
2.6. Whole-Cell Patch-Clamp Electrophysiology
Automated whole-cell patch-clamp recordings were performed with a QPatch-16 automated electrophysiology platform (Sophion Bioscience, Ballerup, Denmark) using single hole (QPlate 16 with a standard resistance of 2 ± 0.4 MΩ) or multi-hole (QPlate 16X with a standard resistance 0.2 ± 0.04 MΩ–NaV1.8 only) planar chips on HEK293 cells heterologously expressing hNaV1.1–1.7/β1 (SB Drug Discovery, Glasgow, UK) or CHO cells heterologously expressing hNaV1.8/β3 in a tetracycline-inducible system (ChanTest, Cleveland, OH, USA). Recordings were acquired at 25 kHz and filtered with a Bessel filter at 8 kHz, and the linear leak was corrected by P/4 subtraction. Recordings were excluded from analysis if series resistance >10 MΩ.
Cells were maintained in Minimum Essential Medium Eagle (M5650) supplemented with 10% v/v fetal bovine serum, 2 mM L-glutamine, and the selection antibiotics geneticin, blasticidin and zeocin as recommended by the manufacturer. Cells were incubated at 37 °C with 5% CO2 and split every 3–4 days when reaching 70–80% confluency using the dissociation reagent TrypLE Express (Thermo Fisher Scientific, Scoresby, VIC, Australia). Cells were passaged 48 h prior to patch-clamp assay in a T-175 flask and cultured at 37 °C. Expression of hNaV1.8 was induced by the addition of tetracycline (1 μg/mL) for 48 h prior to assays. Cells were harvested at 80% confluence and dissociated with TrypLE Express and resuspended in Ham’s F-12 medium (Gibco) supplemented with 25 mM HEPES (Sigma Aldrich, St. Louis, MI, USA), 100 U/mL Penicillin-Streptomycin (Gibco) and 40 µg/mL trypsin inhibitor from Glycine max (soybean) (Sigma Aldrich), and allowed to recover with stirring for 30 min.
The extracellular solution consisted of (in mM) 145 NaCl, 4 KCl, 2 CaCl
2, 1 MgCl
2, 10 HEPES, and 10 glucose, pH to 7.4 with NaOH (adjusted to 305 mOsm/L with sucrose). The intracellular solution consisted of (in mM) 140 CsF, 1 EGTA, 5 CsOH, 10 HEPES, and 10 NaCl, pH to 7.3 with CsOH (adjusted to 320 mOsm/L with sucrose). SxIIIC was diluted in extracellular solution with 0.1% bovine serum albumin. Na
V1.1–1.7 currents were elicited by a 50 ms pulse to −20 mV from a holding potential of −90 mV (repetition interval 20 s). Na
V1.8 currents were elicited by a 50 ms pulse to +10 mV from a holding potential of −90 mV (repetition interval 20 s) in the presence of TTX (1 µM), which fully inhibits TTX-sensitive channels but not Na
V1.8 at this concentration. Recordings were taken at ambient room temperature (22 °C) after 5 min incubation of each concentration. Peak current post-SxIIIC addition (I) was normalised to buffer control (I
0). IC
50s were determined by plotting difference in peak current (I/I
0) and log peptide concentration. Concentration-response curves were fitted using the log (inhibitor) vs. response-variable slope (four parameters) Equation (1).
Calculated IC50 were compared across subtypes and statistical differences determined by ordinary one-way ANOVA.
Current-voltage (I-V) curves were generated from a holding potential of −90 mV using a series of 500 ms step pulses ranging from −100 to +55 mV in 5 mV increments (repetition interval 5 s). Conductance-voltage (G-V) curves were generated by calculating the conductance (G) at each voltage (V) using the equation G = I/(V − Vrev), where Vrev is the reversal potential, and were fitted with a Boltzmann Equation (2).
Voltage dependence of steady-state fast inactivation was assessed using a 10 ms pulse of −20 mV immediately after the 500 ms step (described above) to assess the availability of non-inactivated channels. Off rate data were acquired as previously described [
22]. Briefly, using a holding potential of −90 mV and a 50 ms pulse to −20 mV every 20 s (0.05 Hz) peptides at IC
90 concentration, SxIIIC (1 µM) and KIIIA (3 µM; determined in previous studies [
23]), were incubated for 3 min before step-wise wash out with extracellular solution every 3 min for 35 min.
2.7. NMR Experiments
NMR experiments were carried out on a Bruker Avance III equipped with a cryoprobe (Bruker, Sydney, NSW, Australia). A sample of SxIIIC was dissolved in 90% H2O/10% D2O (v/v) at 1 mg/mL. All NMR experiments were conducted at pH 4.0, and one-dimensional (1D) 1H spectra, two-dimensional (2D) total correlated spectroscopy (TOCSY) and nuclear Overhauser effect spectroscopy (NOESY) experiments were run at 298 K. In addition, experiments including 1H–15N HSQC spectra and variable temperature 2D TOCSY (283–303 K) in 90% H2O/10% D2O (v/v) and 1H–13C HSQC in 100% D2O (v/v) at 298 K were also run.
2.8. NMR Structure Calculations
TopSpin 3.5 (Bruker, Sydney, NSW, Australia) was used to process all NMR spectra. Following the sequential assignment of SxIIIC using CCPNMR Analysis 2.4 [
24], a list of inter-proton distances was generated. By comparing measured Hα chemical shifts to reported random coil Hα shifts [
25], the secondary structure of SxIIIC was predicted. Hα, Cα, Cβ, HN chemical shifts derived from TOCSY, NOESY,
1H–
13C HSQC, and
1H–
15N HSQC spectra were used to calculate backbone (Φ and φ) dihedral angles constraints in TALOS-N [
26]. Additional χ1 and χ2 side-chain dihedral angle restraints were derived using DISH [
27]. Peak assignments were refined following several rounds using the ANNEAL function in CYANA v3.97 [
28]. Additional H-bond restraints were included derived from temperature coefficient experiments in combination with D
2O exchange data [
29]. Finally, protocols in the RECOORD database were used in CNS to calculate 50 structures. These 50 structures were further refined in a water shell using CNS [
30]. Based on lowest energy, fewest violations, and the best MolProbity scores [
31], a final set of 20 structures were chosen to represent SxIIIC. The final 20 structure of SxIIIC have been deposited in the Protein Data Bank (PDB ID: 6X8R) as well as the Biological Magnetic Resonance Bank (BMRB ID: 30758).
4. Discussion
NaV1.7, along with NaV1.1, 1.3, 1.6, 1.8, and 1.9, plays an important role in pain signalling and has shown promise as a drug target for pain therapies. Toxins isolated from highly venomous creatures have evolved to potently and specifically target NaV channels and offer a great starting point as drug leads. However, a major limitation in their success has been the high sequence homology of the NaV channel pore, which could lead to undesirable side effects caused by off-target inhibition. As most toxins modulators that target the therapeutically relevant subtype NaV1.7 act as gating modifiers and bind to the voltage-sensing domain, they offer limited insights into the channel pore interactions. Despite µ-conotoxins NaV1.7 pore blockers being less potent than gating modifiers, they may be used to further our understanding of potency and selectivity for NaV channel. In this body of work, we have isolated a novel µ-conotoxin, SxIIIC, from C. striolatus, characterised the selectivity profile against hNaV channel subtypes and determined the NMR solution structure.
We first used activity-guided fractionation to isolate native SxIIIC from crude venom and subsequently determined the peptide sequence by Edman degradation. This sequence was identical to STRIO_41, recently reported from a
C. striolatus transcriptome [
40]. The unusual initial hydrophilic elution profile of native SxIIIC may be explained by the preparation of crude venom in 30% acetonitrile which artificially attributed a higher gradient and may account for lack of identification in earlier proteomic studies [
40]. When we synthetically produced SxIIIC the peptide eluted at ~10% acetonitrile on a Gemini, 5 µm C18 column. We next evaluated the pharmacological inhibition of hNa
V channel subtypes by automated whole-cell patch-clamp electrophysiology. SxIIIC displayed potent inhibition of hNa
V1.4 (IC
50 15.11 ± 10.7 nM) which is characteristic of most other µ-conotoxins, and may provide an evolutionary advantage as Na
V1.4 is expressed exclusively in skeletal muscle and cone snail venom acts rapidly to immobilise prey [
41,
42]. Interestingly, when compared to hNa
V1.4, synthetic SxIIIC was significantly less potent against hNa
V1.2 (IC
50 363.8 ± 53.8 nM). This was surprising as many other µ-conotoxins preferentially target Na
V1.4 and Na
V1.2 [
37], with the exception of BuIIIB which preferences Na
V1.3, as mentioned earlier [
18]. However, many previous studies used rat and mouse isoforms as opposed to human, and species specificity may explain this variation. Another explanation may be the disulfide connectivity. Due to limited quantity of native material, we were unable to unambiguously determine disulfide connectivity. However, when we produced synthetic SxIIIC we observed a single major peak following thermodynamic oxidation of synthetic SxIIIC. Moreover, the biological activity of synthetic peptide at Na
V1.7 is consistent with that of the native material, which inhibited Na
V1.7 expressed in SH-SY5Y cells. Biological activity of the alternate folding arrangements remains to be determined.
Although SxIIIC was not selective for hNa
V1.7, potent inhibition of this isoform is nonetheless unusual for the µ-conotoxins. Only two µ-conotoxins have reported nanomolar activity against hNa
V1.7 isoforms: KIIIA and CnIIIC (485 nM) [
20]. In 2011, McArthur et al. showed KIIIA inhibited hNa
V1.7 with an IC
50 of 147 nM [
19]. However, as these channel isoforms lacked β subunits it is difficult to directly compare results. In our hands, in a comparable study by Tran et al. [
23], KIIIA with native connectivity exhibited an IC
50 of 363 nM which is approximately two-fold less active than SxIIIC. At hNa
V1.7, the wash-off rate of SxIIIC was markedly slower than that of KIIIA assessed under identical experimental conditions. Notably, previous reports of a more readily reversible KIIIA [
19] could be due to the step-wise, rather than continuous, solution exchange necessitated by using automated patch-clamp, and the relative lack of control of seal quality in these systems. In addition, the aforementioned co-expression with the β1 subunit, as well as use of KIIIA isomer 1 with disulfide connectivity (Cys1/Cys15, Cys2/Cys9, and Cys4/Cys16) may contribute to these seemingly disparate results. However, given that TTX is readily reversible under the same conditions [
23], and we do not observe any accumulation of inactivated channels with our pulse protocol, it is likely that a comparably slower off-rate of SxIIIC contributes to its greater potency at hNa
V1.7. Notably, the protocols used to compute IC
50 values at each subtype do not directly permit calculation of on-rates, although these appeared comparatively slower at Na
V1.4 and Na
V1.7 (
Supplementary Material, Figure S1). Furthermore, the relatively slow on-rate of SxIIIC resulted in steady-state inhibition only being achieved at higher concentrations. While this may be circumvented by substantially longer incubation times, this is difficult to achieve experimentally. As such, it is possible that our calculations are an under-estimate of the true IC
50 values.
Given the high sequence homology between µ-conotoxins (
Figure 1d) and the highly conserved nature of Na
V channel subtypes, particularly within the pore region, these findings may indicate subtle differences that could be exploited to further our understanding of potent Na
V1.7 inhibition. Compared to KIIIA, the N-terminal tail of SxIIIC is extended by two residues, while the peptide sequence between the second and third cysteines (referred to as loop 1) is extended by four residues. It is possible that the extended loops of SxIIIC permit more interactions with Na
V channels than KIIIA, and we thus sought to determine the NMR structure of SxIIIC by 2D homonuclear NMR for comparison to other μ-conotoxins and to provide a foundation for future structure-activity relationship studies. SxIIIC structure is composed of a C-terminal α-helix and is stabilised by three disulfide bonds. A marginally shorter α-helix seen in the final structure compared to the earlier predicted structure is likely explained by additional restraints applied during refinement.
Recent landmark cryo-EM studies of hNa
V channels provide insights into the molecular basis of µ-conotoxin binding [
14,
43]. The calculated SxIIIC structure shared the characteristic α-helix of other M-5 branch µ-conotoxins KIIIA, BuIIIB, and SIIIA [
18,
35,
38]. Residues extending from this helix have been shown to play an important role in Na
V channel inhibition, including Lys7, Trp8, Arg10, Asp11, and His12 (KIIIA numbering) [
38,
44,
45]. SxIIIC is almost identical to KIIIA in this region, with the exception of Arg13 (SxIIIC) which has a conservative substitution from Lys7 in KIIIA. However, this residue is either an Arg or Lys at this position in all other µ-conotoxins and it is unsurprising that the cryo-EM structure has shown Lys7 in KIIIA to play an important role in channel binding, with the side chain extending down into the pore and acting to occlude the outer channel mouth [
14]. It is likely that the analogous residue in SxIIIC similarly contributes to activity, although this remains to be experimentally confirmed. Compared to KIIIA, both the N-terminus and loop 1 of SxIIIC are extended. When SxIIIC is superimposed with the KIIIA/Na
V1.2 structure (
Supplementary Material, Figure S2), residues in loop 1 of SxIIIC appear to clash with the extracellular pore loop of domain I. As these are dynamic environments, it is likely that SxIIIC shifts from this proposed binding mode to a more energetically favourable position, and in doing so may contribute additional contacts to the channel that could contribute to its irreversible inhibition and increase in potency at Na
V1.7 compared to KIIIA. Furthermore, the N-terminal extension has been shown to influence µ-conotoxin selectivity for neuronal (Na
V1.2) and skeletal subtypes (Na
V1.4) and may have influenced the observation of decreased potency at Na
V1.2 [
46]. Interestingly, µ-conotoxin SmIIIA, which shares 90% sequence identity with SxIIIC (
Figure 1d), has an extended N-terminal (Pyroglutamate1 and Gly2) and an additional charged residue in loop 1 compared to SxIIIC, but only weakly inhibits mammalian Na
V1.7 (IC
50 1.3 ± 0.23 µM) [
37]. Further investigation of the structure-activity relationships of these peptides may help us understand the role of these extensions in Na
V channel inhibition and selectivity.
In conclusion, we discovered a novel µ-conotoxin SxIIIC, produced the peptide synthetically and evaluated its pharmacological and structure characteristics. To date, SxIIIC is one of the most potent µ-conotoxin hNaV1.7 inhibitors, acting as a presumptive pore blockers, and presents as a promising peptide for NaV channel inhibitor development.