The E15R point mutation in toxin Cn2 uncouples its depres- sant and excitatory activities on human NaV1.6 Mathilde R. Israel,†,# Panumart Thongyoo,†,# Jennifer R. Deuis,† David J. Craik,† Irina Vetter,†,‡,* and Thomas Durek†,*

†Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia ‡School of Pharmacy, The University of Queensland, Woolloongabba, QLD 4102, Australia KEYWORDS: native chemical ligation, peptide synthesis, analgesic scorpion toxin, NMR, voltage-gated

ABSTRACT: We report the chemical synthesis of scorpion toxin Cn2; a potent and highly-selective activator of the human voltage-gated sodium channel NaV1.6. In an attempt to decouple channel activation from channel binding, we also synthe- sised the first analogue of this toxin, Cn2[E15R]. This mutation caused uncoupling of the toxin’s excitatory and depressant activities, effectively resulting in a NaV1.6 inhibitor. In agreement with the in vitro observations, Cn2[E15R] is anti-nocicep- tive in mouse models of NaV1.6-mediated pain.

INTRODUCTION: vitro.5 In the above-mentioned studies, the long-chain scor- pion -toxin Cn2 from noxious proved espe- The generation and propagation of action potentials in cially useful in dissecting the roles of the individual NaV neurons is realized through a well-orchestrated interplay of isoforms in vivo because of its potent4 and highly selective ion channels in which the family of voltage-gated sodium activation of NaV1.6.6 These pharmacological properties channels (NaV) plays a central role.1-2 Humans have at least make Cn2 a promising candidate for development of ad- nine functional NaV  subunits (NaV1.1–NaV1.9) that medi- vanced NaV1.6-selective molecular probes with altered ate specialized physiological processes in different tissues mechanism of action to explore their effect on NaV1.6 phys- and electrically excitable cell types. Dissecting the physio- iology. logical roles of individual subtypes has been hampered in the past because multiple individual NaV subtypes often co- localize and their central  subunits are structurally similar. The latter aspect in particular has also made it difficult to develop highly NaV–specific and subtype–selective modula- tors that could be used as molecular probes or as potential therapeutics in NaV -related diseases.

Molecules modulating the activity of NaVs are abundant in the venoms of cone snails, and and are noteworthy for their high potency and selectivity for indi- vidual Nav isoforms.3 These small disulfide-rich peptides bind to NaVs with high affinity and can interfere with spe- cific steps of the NaV gating mechanism; for example by oc- cluding the central pore or by interacting with one or more of the four voltage-sensing domains (VSDs) that control channel opening and closing. Scheme 1: Chemical synthesis of Cn2 by native chemical liga- tion. (i) native chemical ligation; (ii) folding and disulfide for- Using a range of NaV modulators and knockout , mation. The inset shows the primary structure of Cn2, with cys- we recently demonstrated an important role for NaV1.6 in teines in bold and the ligation site (Glu28-Cys29) underlined. multiple peripheral pain pathways, particularly those in- The inverted ‘E’ highlights the location of the point mutation volved in mediating cold and mechanical allodynia.4 Further (E15R). exploration of the role of NaV1.6 in pain pathways and eval- uation of NaV1.6 as a potential therapeutic target hinge on RESULTS AND DISCUSSION: the availability of advanced molecular tools or model sys- Scorpion -toxin Cn2 is a polypeptide of 66 amino acid tems such as NaV1.6 knock-out animals (which are not via- residues that is C-terminally carboxamidated and adopts a ble) and/or NaV1.6-selective molecular probes, such as 4,9- cysteine-stabilized  fold supported by four disulfide anhydro-tetrodotoxin that shows some NaV1.6 selectivity in 1 bonds.7-8 C-terminal carboxamidation is a posttranslational for high-affinity NaV1.4 binding of the related long-chain modification that is difficult to reproduce in bacterial ex- scorpion toxin Ts3.9 pression systems and was recently shown to be important

Figure 1: Characterization of synthetic Cn2. a) uHPLC and MALDI-MS (inset) of synthetic wild-type Cn2 (calculated MW: 7584.49 Da (M+H+, monoisotopic composition). b) NMR H-chemical shift analysis derived from 2D TOCSY and NOESY spectra indicates correct folding of all synthetic analogs relative to native, venom derived Cn2 (PDB ID: 1CN2).7

Thus, to realize the envisioned analogues, we developed effect of synthetically produced Cn2 on VGSC isoforms a total chemical synthesis approach that permits milligram NaV1.1-1.8, a high-throughput fluorescence imaging plate scale production of the relatively complex wild-type Cn2 reader (FLIPRTETRA) assay was used as previously de- and variants thereof. We initially attempted chemical syn- scribed.12 Briefly, HEK293 cells expressing NaV1.1-1.8 in a thesis of full-length wild-type Cn2 by Fmoc solid-phase pep- monolayer were incubated with membrane potential dye tide synthesis. A trial TFA deprotection with concomitant (Molecular Devices, Sunnyvale, CA) and the change in fluo- peptide cleavage from the resin was conducted after cou- rescence was continuously measured prior to and during pling of Cys29 to evaluate peptide assembly. HPLC-MS anal- compound addition (FLIPRTETRA). Native and synthetic Cn2 ysis of the crude cleavage product indicated significant concentration-dependently activated NaV1.6-expressing amounts of truncation and deletion side products, which HEK293 cells with EC50s of 72 nM (pEC50 7.14 ± 0.91) and prompted us to revise our plan to a fragment-based ligation 15.8 nM (pEC50 7.81 ± 0.20), respectively (Fig. 2a). In line strategy. The full-length sequence was divided into two seg- with native Cn2 previously reported, synthetic Cn2 (hence- ments, Cn2(1-28) and Cn2(29-66), which were joined by forth termed Cn2) at a concentration of up to 100 nM had native chemical ligation at Glu28 and Cys29 (Scheme 1).10-11 no significant effect on sodium channel isoforms NaV1.1- Synthesis of the required Cn2(1-28)-−thioester fragment NaV1.3, NaV1.5, NaV1.7 or NaV1.8 (Fig. 2b). Activity at the via Boc-SPPS was straightforward and the ligation with muscle specific NaV subtype, NaV1.4, has not been observed Cn2(29-66) proceeded smoothly, producing the full-length for native Cn2 at concentrations of 300 nM.6 However, our polypeptide in 44.8% yield. Folding and disulfide formation data show that synthetic Cn2 is a weak agonist for NaV1.4 in of the polypeptide required extensive optimization; guani- the FLIPR assay. Collectively, these data suggest that syn- dinium HCl at high concentrations (1.5 M) was essential thetic and venom-derived wild-type Cn2 are structurally during folding to prevent toxin from aggregating and pre- and pharmacologically identical. cipitating, and relatively long folding times (72 h) were nec- Having established synthetic access to the wild-type essary to favour the native disulfide isomer and improve toxin, we turned our attention to the design of Cn2 ana- yields (SI Fig. 5). The synthetic toxin was isolated by RP- logues with altered activity. Gurevitz and colleagues have HPLC in acceptable yield (30.8%) and its monoisotopic mo- shown that substituting a conserved glutamate in -scor- lecular weight, determined by high resolution MALDI-MS pion toxins Css4 or Bj-xtrIT with arginine (E15R) results in (7584.4 ± 0.2 Da, Fig. 1a), was in excellent agreement with uncoupling of NaV-binding and NaV-activation.13-14 A similar the theoretical value (7584.5 Da; taking into account C-ter- strategy was recently adopted to -scorpion toxin Ts1 to ob- minal amidation and formation of four disulfide bonds). tain NaV1.4 probes with reduced NaV excitatory activity.15 HPLC comparison of the purified synthetic Cn2 toxin with The corresponding Cn2 mutant, Cn2[E15R], was prepared native toxin isolated from C. noxious venom under identical using the same synthetic strategy used for wild-type toxin conditions revealed excellent agreement, suggesting that by replacing the N-terminal ligation fragment with Cn2(1- the correct disulfide isomer had been obtained (SI Fig. 8). 28, E15R)-thioester and folded using the protocol estab- The structural integrity of synthetic Cn2 was further con- lished for wildtype toxin. firmed by 2D TOCSY and NOESY NMR spectroscopy, which allowed complete assignment of backbone proton reso- nances. Comparison of the H-chemical shifts to published values for venom-derived Cn2 indicated excellent agree- ment, suggesting that the synthetic and venom-derived Cn2 molecules are structurally identical (Fig. 1b).7 To assess the 2 Figure 2: Native and synthetic Cn2 activate NaV1.6 in the FLIPRTETRA membrane potential assay. a) Synthetic or venom-derived Cn2 elicited Nav1.6-mediated increase in fluo- rescence normalised to baseline vehicle control (% Control) with EC50s of 15.8 nM and 72 nM, respectively. b) Cn2 had little effect on sodium channel isoforms NaV1.1–1.5, 1.7 and 1.8 at concentrations up to 100 nM but only elicited concentration- dependent changes in membrane potential in HEK293 cells ex- pressing NaV1.6.

Figure 3: Effect of synthetic Cn2 (a, b) and Cn2[E15R] (c, d) on the electrophysiological properties of hNaV1.6 expressed in HEK293 cells. a, c) Current-voltage relationship in the absence (black circles) and presence of 10 nM Cn2 (grey squares) or c) 200 nM Cn2[E15R] (grey squares). All data presented as mean ± SEM. Inset, Cn2, but not Cn2[E15R], significantly increased current at membrane potentials of -60 to -40 mV (*= P < 0.05, paired t-test) (n=5). b, d) Voltage dependence of activation (G/V) and steady- state fast inactivation (I/Imax) of hNaV1.6 in the absence (black circles) and presence of 10 nM Cn2 (grey squares) or d) 200 nM Cn2[E15R] (grey squares), fitted with single Boltzmann relationship (solid line).

NMR confirmed that the tertiary structure of this mutant ± 0.1) (SI Fig. 10). This value is in agreement with the pre- is virtually unchanged from wild-type toxin (Fig. 1b). With dicted EC50 from Schiavon et al.,6 39.2 ± 3.7 nM. To test for the two analogues in hand, we examined their electrophys- the typical shift in the voltage dependence of activation as- iological properties at human NaV1.6. sociated with β scorpion toxins, current-voltage (I-V) curves were generated. This protocol includes a depolariz- -Scorpion toxins bind to site-4 on NaVs, which has been ing prepulse to 0 mV in order to facilitate channel opening mapped to the S3-S4 linker of domain II of the NaV -subu- nit.2 Binding causes a hyperpolarizing shift in the voltage and toxin binding as described by the voltage sensor trap- ping model.16 Indeed, in the presence of 10 nM Cn2, sodium dependence of channel activation while NaV inactivation ki- netics are not affected.6, 16-18 To assess whether syntheti- current was observed at previously prohibitive potentials cally produced Cn2 retains these properties we obtained (Fig. 3a), with significantly increased current at pulses be- whole-cell patch clamp recordings from HEK293 cells ex- tween -60 to -40 mV (P< 0.05; paired t-test). In contrast, Cn2 (10 nM) decreased current between potentials -10 mV and pressing hNaV1.6. Cn2 caused a concentration-dependent decrease in peak current (obtained by recording resultant +50 mV, with peak whole cell current elicited by a pulse of 0 mV decreased by 47.3 ± 0.1%. The V1/2 of NaV1.6 activation currents at pulses to 0 mV) with an EC50 of 6.2 nM (pEC50 8.2 3 for Cn2 treated cells (-24.1 ± 0.8 mV) was not significantly different from control (-24.4 ± 0.3 mV) (Fig. 3b). However, Cn2 significantly increased the slope factor at NaV1.6 (con- trol: 4.8 ± 0.3; Cn2: 8.2 ± 0.8, p < 0.05) leading to a higher conductance at more hyperpolarised membrane potentials. Cn2 (10 nM) also caused a depolarizing shift in the V1/2 of NaV1.6 steady-state fast inactivation (ΔV1/2: +10.2 mV) (Fig. 3b), which in combination with a shallower activation slope, led to an increase in window current. In effect, there was a 20% increase in channel availability at -35 mV in the pres- ence of Cn2 compared with control. This excitatory (activat- ing) Cn2 effect is coupled to a depressant activity as evi- denced by the concomitant reduction in peak sodium con- ductance.

The electrophysiological effects of Cn2[E15R] on hNaV1.6 were evaluated at a toxin concentration of 200 nM, because preliminary studies indicated that at this concentration hNaV1.6 sodium current is inhibited by approximately 50%. In contrast to Cn2, 200 nM Cn2[E15R] did not elicit current at previously prohibitive potentials (Fig. 3c), but led to a de- polarizing shift in the voltage-dependence of activation (Fig. 3d, V1/2: control -17.4 ± 0.2 mV; 200 nM Cn2[E15R], -11.4 ± 0.6 mV). In addition, Cn2[E15R] significantly increased the slope factor at NaV1.6 (control: 5.0 ± 0.2; Cn2 7.9 ± 0.6, p < 0.05), reducing conductance at more depolarised mem- brane potentials without changing conductance at hyperpo- larized potentials. In line with these observations, Cn2[E15R] at 200 nM decreased peak current by 42.4 ± 1.5%. The V1/2 of steady-state fast inactivation of NaV1.6 was not significantly changed in the presence of 200 nM Cn2[E15R], but a proportion (~25%) of the Cn2[E15R] treated channels resisted inactivation altogether as evi- Figure 4: Cn2[E15R] causes NaV1.6 mediated analgesia in denced by the current elicited at pulses between -40 and 0 in vivo spontaneous pain models. a) Cn2 causes non-stimu- mV (Fig. 3d). lus evoked pain behaviour when injected intraplantar, whereas Cn2[E15R] does not. b) Co-administration of Cn2[E15R] re- duces pain behaviour caused by 10 nM Cn2 in a concentration dependent manner. c) Intraplantar injection of NaV activator CTX (5 nM) causes non-stimulus evoked pain behaviours that are significantly reduced by Cn2[E15R] treatment (**** = P < 0.001, unpaired t test, n=6-7) d) Effect of Cn2[E15R] on forma- lin induced pain behaviours; phase II pain behaviours were sig- nificantly reduced with intraplantar injection of 10 μM Cn2[E15R] (*= P < 0.05, unpaired t test, n = 3-13). Thus, some (but not all) of the effects seen in wild-type Cn2 are significantly altered in the Cn2[E15R] mutant. First, Cn2[E15R] does not activate NaV1.6 at otherwise prohibi- tive membrane potentials whereas Cn2 does (Fig. 3a, c). Second, Cn2[E15R] causes a significant depolarizing (right- ward) shift in the steady state activation curves, indicating that channels are more difficult to open (Fig. 3b, d). Third, Cn2[E15R] does not shift the V1/2 of Nav1.6 channel inacti- vation (whereas Cn2 does). In contrast, both Cn2 and Cn2[E15R] significantly reduce NaV1.6 peak current at nM concentrations (Fig. 3a, c). These data collectively show that the single amino acid (E15R) substitution not only causes an uncoupling of NaV binding and activation in scorpion - toxins as previously reported,13-14 but, at least in the Cn2:hNaV1.6 studied here, also an uncoupling of excitatory (activating) and depressant (inhibitory) effects.

Although the excitatory -toxin effects on NaV gating have been satisfactorily explained by the voltage sensor trapping

4 model,16 our findings are better rationalized by a ‘two-state to Cn2[1−28]-α-thioester (KEGYLVDKNTGCKYECLKLGDND- voltage sensor trapping’ model proposed recently by Heine- YCLRE-[S-CH2-CH2-CO]F), Cn2[1−28,E15R]-α-thioester (KEGYL- mann and colleagues to take into account the depressant ef- VDKNTGCKYRCLKLGDNDYCLRE-[S-CH2-CH2-CO]L) were assem- fects observed for many of these toxins.19 In this model, Cn2 bled by manual in situ neutralization Boc SPPS on pre-loaded PAM polystyrene resins as described recently.22 Cn2[29-66] binds with similar affinities both resting and activated con- (CKQQYGKGAGGYCYAFACW-CTHLYEQAIVWPLPNKRCS-NH2) was formations of VSDII, macroscopically giving rise to the ob- assembled by automated Boc SPPS on a CSBio peptide synthesizer served excitatory and depressant effects. Our data for using MBHA polystyrene resin and standard protocols. The follow- Cn2[E15R] demonstrate uncoupling of these activities, sug- ing side chain protecting groups were employed: Arg(Tos), gesting that at the molecular level the mutation destabilizes Asp(OcHex), Asn(Xan), Cys(4-MeBzl), Glu(OcHex), Gln(Xan), the activated VSDII:toxin complex while not significantly af- His(Bom), Lys(2Cl-Z), Ser(Bzl), Thr(Bzl), Trp(Hoc) and Tyr(2Br-Z). fecting the resting VSD-II:toxin complex. After chain assembly, peptides were side chain-deprotected and simultaneously cleaved from the resin by treatment with anhy- Given that Cn2[E15R] only retains NaV1.6 depressant ac- drous HF containing 10% (v/v) p-cresol and 5 eq. of Cys-OEt (per tivity, we hypothesized that it would show antinociceptive equivalent of His(Bom) residues) for 1 h at 0ºC. HF was evaporated properties in mouse models of NaV1.6-mediated pain.4 In- under reduced pressure. The crude product was precipitated and traplantar injection of Cn2 (10 nM) caused nocifensive be- washed with chilled diethylether, then dissolved in 50 % (v/v) haviours in mice including flinching, lifting and shaking of aqueous acetonitrile containing 0.1 % trifluoroacetic acid (TFA) the hindpaw (Fig. 4a). In contrast, 10 μM Cn2[E15R] failed (v/v) and lyophilized. to cause spontaneous pain behaviours when administered Peptides were purified by reversed-phase high-performance liq- uid chromatography (RP-HPLC) using preparative Vydac C8 (22 × alone (Fig. 4a). Co-administration of Cn2 with Cn2[E15R] 250 mm), semi-preparative Zorbax C3 (10 × 250 mm) and analyti- concentration-dependently reduced spontaneous pain be- cal Zorbax C18 (4.6 × 50 mm) columns. Preparative and semi-pre- haviours evoked by Cn2 (pain behaviours/5 minutes, con- parative peptide purifications were performed on Shimadzu Prom- trol (Cn2 alone): 73 ± 10; Cn2[E15R]: 52 ± 11 (0.2 μM) or 1 inence LC-30AT HPLC system using Phenomenex C18 RP-HPLC ± 1 (2 μM)) (Fig. 4b) suggesting that Cn2 and Cn2[E15R] (21.2×250 mm, 15 μm, 300 Å) or Agilent C18 RP-HPLC (9.4×250, 5 compete for the same NaV1.6 binding site in vivo. μm, 300 Å) columns. Crude peptides were dissolved in a 10 % (v/v) acetonitrile/water mixture containing 0.05 % (v/v) TFA, before Ciguatoxins (CTX) are non-selective sodium channel acti- being loaded onto the column pre-equilibrated with 10 % of sol- 12, 20-21 vators responsible for ciguatera fish poisoning. De- vent B (90:10:0.1 – acetonitrile:water:TFA) in solvent A (0.1 % spite being non-selective, blocking either NaV1.6 or NaV1.7 (v/v) TFA in water). Peptides were eluted using linear gradients of abolishes the spontaneous pain induced by intraplantar in- solvent B in solvent A, and fractions were collected across the ex- jection of CTX.12 Cn2[E15R] significantly reduced nocifen- pected elution time. sive behaviours associated with CTX (pain behaviours: con- Peptide purity and identity were assessed by HPLC on a Shi- trol 350 ± 35, Cn2[E15R] (10 µM) 63 ± 14; P < 0.001 un- madzu Nexera LC-30AD uHPLC system equipped with an Agilent 300SB-C18 column (2.1×50 mm, 1.8 µm, 300 Å) by using a linear paired t-test), confirming a distinct role for NaV1.6 in cigua- toxin-induced spontaneous pain (Fig. 4c). Finally, gradient of 15-45% of buffer B in buffer A over 12 min at 50C. Electrospray ionization mass spectrometry (ESI-MS) was per- Cn2[E15R] was tested in the formalin mouse model, in formed on Shimadzu LCMS-2020 system or on a Shimadzu Nexera which phase I is caused by direct activation of nociceptors uHPLC coupled with an AB SCIEX 5600 mass spectrometer and phase II is caused by inflammatory sensitization. equipped with a Turbo V ion source. Matrix-Assisted Laser-De- Cn2[E15R] significantly reduced flinching associated with sorption Ionization-Time of Flight (MALDI)-TOF was conducted on phase II (pain behaviours: control 232 ± 18, Cn2[E15R] (10 an Applied Biosystems 4700 TOF/TOF Proteomics Analyzer using µM) 138 ± 15; P < 0.05 two-way ANOVA) (Fig. 4d). Thus, α-cyano-4-hydroxycinnamic acid (CHCA) as a matrix (8 mg/mL in Cn2[E15R] not only competes with Cn2, but is a functional 75% (v/v) acetonitrile, 0.05% (v/v) formic acid in water. inhibitor of hNav1.6 in vivo in its own right. More broadly, Cn2[1−28]-α-thioester observed mass 3505.6 ± 0.4 Da, calculated our data also confirms an important, previously underap- mass (average isotope composition) 3505.9 Da; Cn2[1−28, E15R]- α-thioester observed mass 3498.8 ± 0.4 Da, calculated mass 3498.9 preciated role for NaV1.6 in peripheral nociception. Da; Cn2[29-66] observed mass 4343.4 ± 0.5 Da, calculated mass 4344.0 Da. Fractions containing the desired product were pooled, CONCLUSIONS: lyophilized and stored at –20ºC. In summary, we successfully applied a native chemical li- Peptide ligation and folding: We dissolved 25 mg of Cn2[29- 66] MW: 4344.0, 5.8 μmol) and 22.9 mg of Cn2[1-28]-α-thioester, gation synthetic strategy to gain access to the highly NaV1.6- MW: 3505.9, 6.9 μmol) in 4 mL of ligation buffer (6M guanidinium selective scorpion toxin Cn2 and a pharmacologically HCl (GdmHCl), 200 mM Na-phosphate, 50 mM 4-mercaptophenyla- unique analogue. Our data demonstrate that the excitatory cetic acid (MPAA), 50 mM tris(2-carboxyethyl)phosphine (TCEP), and depressant activities in scorpion -toxins can be uncou- pH 7.1). After dissolution, the pH was re-adjusted to 6.9–7.0 and pled, suggesting that these activities are governed by dis- the mixture stirred under an argon atmosphere for 18 h. The reac- tinct toxin pharmacophore residues. This work paves the tion was quenched by adding TFA to a final concentration of 2% way for rational design of NaV inhibitors based on -scor- (v/v) and the product isolated by preparative HPLC (isolated yield: pion toxins, which so far have been limited to a role as mo- 20.0 mg, 2.6 μmol (44.8%); Cn2[1-66] observed mass 7596.0 ± 1.0 Da, calculated mass 7596.7 Da (average isotope composition, ESI- lecular tools due to their potent NaV-excitatory activity. We MS). expect these molecules to be powerful probes for studying 20 mg (2.6 μmol) of reduced Cn2 was dissolved in 5 mL of 6 M the physiological roles of NaV1.6 in health and disease. GdmHCl (pH ~ 5) to give a final peptide concentration of 4 mg/mL. The peptide solution was then added to 100 mL of oxidation buffer EXPERIMENTAL SECTION: consisting of 100 mM Tris, 1.5 M GdmHCl, 10 mM reduced L-gluta- Peptide synthesis: Peptides were synthesized by solid-phase thione (GSH), 1 mM oxidized L-glutathione (GSSG), pH 8.1. Oxida- peptide synthesis (SPPS). Peptide α-thioalkylesters corresponding tion was performed at 22°C, monitored by HPLC and found to reach 5 equilibrium after 72 hours. 2 mL of TFA was added to the oxidation Electrophysiology: The effect of Cn2 and analogues was as- mixture and the folded Cn2 purified by semi-preparative HPLC. sessed in whole cell recordings under voltage-clamp conditions us- Isolated yield: 6.5 mg, 0.8 μmol (30.8%). Cn2 observed mass ing a QPatch 16-well automated electrophysiology platform (Soph- 7588.3 ± 1.0 Da, calculated mass 7588.7 Da (average isotope com- ion Bioscience, Ballerup, Denmark) and 16-well planar chip plates position). High-resolution MALDI-MS: Cn2 observed MW: 7584.41 (Qplates, Sophion) with resistance of 2 ± 0.02 MΩ and diameter of ± 0.2 Da (M+H+); calculated MW: 7584.49 Da (M+H+, most abun- 1 μm. HEK293 cells expressing human NaV1.6 were expanded in dant isotope composition). normal growth media into a T175 flask 48hrs prior to experiment Cn2 (E15R) was prepared via the same protocol used for wild and maintained at 37°C in humidified 5% CO2. At 70-80% conflu- type toxin and afforded the analogue in similar yields (30-40% iso- ence, cells were washed twice with Dulbecco’s phosphate-buffered lated yield for final folding step). High resolution ESI-MS: Cn2 saline prior to harvesting with 1 mL DetachinTM. Finally, cells were (E15R) observed MW: 7610.62 ± 0.2 Da; calculated MW: 7610.56 resuspended in 3 ml Ex-Cell ACF CHO media supplemented with 25 Da (most abundant isotope composition, SI Fig. 7). All final prod- mM HEPES (Sigma-Aldrich, NSW, Australia) and placed on the ucts had a purity of 95% based on analytical uHPLC-MS analysis Qpatch’s Qstirrer for 30 minutes prior to the assay. Positioning (Agilent 300SB-C18 column (2.1×50 mm, 1.8 µm, 300 Å), linear pressure was set at -60 mBar, holding potential -100 mV, minimum gradient of 15-45% of buffer B in buffer A over 12 min, 50C, de- seal resistance 0.1 GΩ, holding pressure -20 mBar and currents tection at 214/280 nm). were filtered at 25 kHz (8th order Bessel, cut off 5 kHz). NMR spectroscopy: Lyophilized Cn2 samples were dissolved in Extracellular solution contained (in mM): NaCl 145, KCl 4, CaCl2 90% H2O/10% D2O at a concentration of 0.2 mM and a pH of 3.6. 2, MgCl2 1, HEPES 10 and glucose 10; pH 7.4; osmolarity 305 Spectra were recorded at 303 K on a Bruker Avance 600 MHz spec- mOsm. The intracellular solution contained (in mM): CsF 140, trometer equipped with a cryogenically-cooled probe. Phase-sen- EGTA/CsOH 1/5, HEPES 10 and NaCl 10; pH 7.3 with CsOH; osmo- sitive mode using time-proportional phase incrementation for larity 320 mOsm. Cells were incubated for 5 minutes before voltage quadrature detection in the t1 dimension was used for all two-di- protocols with working concentrations of peptides which were di- mensional spectra. Excitation sculpting with gradients was used to luted in ECS containing 0.1% bovine serum albumin (BSA). achieve water suppression. NMR experiments included TOCSY us- All protocols contained a pre-pulse to 0 mV and 120 ms recovery ing a MLEV-17 spin lock sequence with a 80 ms mixing time, and at the holding potential to allow toxin binding and voltage sensor NOESY with a 200 ms mixing time. Spectra were recorded with trapping consistent with previously described protocols.16 Current 4096 data points in the F2 dimension and 512 increments in the F1 (I) – voltage (V) relationship before and after toxin application was dimension. The t1 dimension was zero-filled to 1024 real data assessed as follows, cells were held at -100 mV, (50 ms recovery) points, and the F1 and F2 dimensions were multiplied by a sine- test pulses were recorded for 50 ms between -90 mV and +55 mV squared function prior to Fourier transformation. The spectra in 5 mV increments. Conductance-voltage relationships were ob- were referenced to water at 4.715 ppm (303 K). All spectra were tained for voltage sweeps using G = I/(V-Vrev), Vrev being the rever- processed using TopSpin (Bruker) and manually assigned with sal potential. Resulting data was fitted using a Boltzmann equation: CCPNMR using the sequential assignment protocol.23-24 GNa = GNa,max/1 + exp [(Vm – V1/2) / k] in GraphPad Prism; GNa is the Cn2 reference material, isolated from the venom of C. noxius voltage dependent sodium conductance, GNa,max is the maximal so- Hoffmann, was a generous gift from Lourival Possani (Institute of dium conductance, Vm the membrane potential and k is the slope Biotechnology, National Autonomous University of Mexico). factor. Cell culture: Human embryonic kidney (HEK) cells with consti- behavioural testing: Animal ethics approval was ob- tutive expression of NaV isoforms 1.1-1.8 were purchased from tained from The University of Queensland Animal ethics commit- Scottish Biomedical (Glasgow, Scotland). Cells were maintained in tee. All experiments were conducted in accordance with local and minimal essential media (MEM) (Sigma-Aldrich, Australia) supple- national regulations and the International Associations for the mented with 2mM L-glutamine and 10 % foetal bovine serum Study of Pain guidelines for the use of animals. In order to assess (FBS) (Assay Matrix, Ivanhoe, Australia). Cells lines were cultured evoked pain, Cn2, Pacific Ciguatoxin-1 (P-CTX-1) and formalin with appropriate selection antibiotics; blasticidin and G418 for were used as previously described.4, 12, 26-28 Briefly, Cn2 was diluted NaV1.1, 1.2, 1.3, 1.5, 1.6, 1.7 and 1.8; blasticidin, G418 and zeocin in phosphate-buffered saline with 0.1% bovine serum albumin for NaV1.4. Each cell line was grown in 37°C in a humidified 5% CO2 (BSA) to give final concentrations of 10 nM. Under isofluorane incubator to 70-80% confluence, passaged every 3-4 days with (3%) anaesthesia, 40 µL Cn2 was administered via shallow subcu- TrypLE Express (Invitrogen). taneous (intraplantar i.pl.) injection into the hindpaw of male Fluorescent membrane potential assay: The activity of Cn2 at C57BL/6 mice. Mice were allowed to recover in clear polyvinyl NaV1.1- NaV1.8 was assessed using a FLIPRTetra fluorescent mem- boxes and recorded for 20 minutes post injection. Following this, brane potential assay as previously described.12, 25 In brief, HEK293 nocifensive behaviours including flinches, lifts and shakes were cells stably expressing human NaV isoforms NaV1.1-NaV1.8 were counted every 5-minute interval from a video recording by a plated on 384-well plates at a density of 10 000 – 15 0000 cells per blinded observer. In order to test the effect of the pharmacological well in normal growth medium (Minimal Essential Medium (MEM; modulator Cn2[E15R] (0.2 µM, 2 µM, 10 µM) on the induction of Sigma-Aldrich, Australia). After 48 h culture at 37oC/5% CO2, NaV1.6 spontaneous pain, 40 µL Cn2[E15R] was injected alone or growth medium was replaced with 20 uL of red membrane poten- co-administered with spontaneous pain inducing compounds (Cn2 tial dye (Molecular Devices, Sunnyvale, CA) prepared in physiolog- (10 nM), P-CTX-1 (10 nM) and formalin (1%; v/v)). ical salt solution (composition (in mM): NaCl (140), glucose (11.5), KCl (5.9), MgCl2 (1.4), NaH2PO4 (1.2), NaHCO3 (5), CaCl2 (1.8), 4-(2- ASSOCIATED CONTENT hydroxyethyl)-1-piperazineethane-sulfonic acid (HEPES) (10), pH 7.4) according to the manufacturer’s instructions and cells were in- Supporting Information o cubated for 30 minutes at 37 C. Changes in fluorescence (excita- Analytical data for all peptides and supporting electrophysio- tion: 510-545 nm, emission: 565-625 nm) after addition of native logical characterization material are available in pdf format. and synthetic Cn2 were measured every second for 300 s using a The Supporting Information is available free of charge on the FLIPRTetra fluorescent plate reader (Molecular Devices, Sunnyvale, CA) and analysed using Screenworks 3.1.1.4 (Molecular Devices). ACS Publications website. GraphPad Prism (Version 4.00, San Diego, California) was used to fit a 4-parameter Hill equation with variable Hill slope to the data. AUTHOR INFORMATION All data, unless otherwise stated, are expressed as the mean ± standard error of the mean (SEM). Corresponding Author 6 * [email protected] or [email protected] 10. Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. Synthesis of Proteins by Native Chemical Ligation. Science 1994, Author Contributions 266 (5186), 776-779. The manuscript was written through contributions of all au- 11. Dang, B. B.; Kubota, T.; Mandal, K.; Bezanilla, F.; Kent, S. thors. All authors have given approval to the final version of B. H. Native Chemical Ligation at Asx-Cys, Glx-Cys: Chemical the manuscript. #: These authors contributed equally. Synthesis and High-Resolution X-Ray Structure of Shk Toxin by Racemic Protein Crystallography. J. Am. Chem. Soc. 2013, 135 (32), Funding Sources 11911-11919. 12. Inserra, M. C.; Israel, M. R.; Caldwell, A.; Castro, J.; Deuis, This work was supported by an Australian Research Council J. R.; Harrington, A. M.; Keramidas, A.; Garcia-Caraballo, S.; (ARC) Future Fellowship awarded to IV (FT130101215) and Maddern, J.; Erickson, A.; Grundy, L.; Rychkov, G. Y.; Zimmermann, an ARC Laureate Fellowship to DJC (FL150100146). MRI is K.; Lewis, R. J.; Brierley, S. M.; Vetter, I. Multiple Sodium Channel supported by an Australian Government Research Training Isoforms Mediate the Pathological Effects of Pacific Ciguatoxin-1. Program Scholarship. Sci. Rep. 2017, 7, 42810. 13. Karbat, I.; Ilan, N.; Zhang, J. Z.; Cohen, L.; Kahn, R.; ACKNOWLEDGMENT Benveniste, M.; Scheuer, T.; Catterall, W. A.; Gordon, D.; Gurevitz, M. Partial Agonist and Antagonist Activities of a Mutant Scorpion - We thank Lourival Possani for providing a sample of Cn2 iso- Toxin on Sodium Channels. J. Biol. Chem. 2010, 285 (40), 30531- lated from C. noxius venom and Richard Lewis for providing 30538. CTX. 14. Karbat, I.; Cohen, L.; Gilles, N.; Gordon, D.; Gurevitz, M. Conversion of a Scorpion Toxin Agonist into an Antagonist ABBREVIATIONS Highlights an Acidic Residue Involved in Voltage Sensor Trapping Cn2, Centruroides noxius toxin 2; CTX, Ciguatoxin; GdmHCl, During Activation of Neuronal Na+ Channels. FASEB J. 2004, 18 (6), guanidinium hydrochloride; HEPES, 4-(2-hydroxyethyl)-1-pi- 683-689. perazineethanesulfonic acid; MBHA, 4-methylbenzhydryla- 15. Kubota, T.; Dang, B. B.; Carvalho-de-Souza, J. L.; Correa, A. mine; MEM, minimal essential medium; MPAA, 4-mercapto- M.; Bezanilla, F. NaV Channel Binder Containing a Specific Conjugation-Site Based on a Low Toxicity -Scorpion Toxin. Sci. phenylacetic acid; NaV1.X, voltage-gated sodium channel; TCEP, Rep. 2017, 7, 16329. tris(2-carboxyethyl)phosphine; VSD, voltage-sensing domain 16. Cestele, S.; Qu, Y.; Rogers, J. C.; Rochat, H.; Scheuer, T.; Catterall, W. A. Voltage Sensor-Trapping: Enhanced Activation of REFERENCES Sodium Channels by -Scorpion Toxin Bound to the S3-S4 Loop in 1. Catterall, W. A. Voltage-Gated Sodium Channels at 60: Domain II. Neuron 1998, 21 (4), 919-931. Structure, Function and Pathophysiology. J. Physiol. 2012, 590 17. Escalona, M. P.; Possani, L. D. Scorpion -Toxins and (11), 2577-2589. 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