Addition of K22 Converts Spider Venom Peptide Pme2a from an Activator to an Inhibitor of Na_V1.7

Abstract

Spider venom is a novel source of disulfide-rich peptides with potent and selective activity at voltage-gated sodium channels (Na_V). Here, we describe the discovery of μ-theraphotoxin-Pme1a and μ/δ-theraphotoxin-Pme2a, two novel peptides from the venom of the Gooty Ornamental tarantula Poecilotheria metallica that modulate Na_V channels. Pme1a is a 35 residue peptide that inhibits Na_V1.7 peak current (IC₅₀ 334 ± 114 nM) and shifts the voltage dependence of activation to more depolarised membrane potentials (V_1/2 activation: Δ = +11.6 mV). Pme2a is a 33 residue peptide that delays fast inactivation and inhibits Na_V1.7 peak current (EC₅₀ > 10 μM). Synthesis of a [+22K]Pme2a analogue increased potency at Na_V1.7 (IC₅₀ 5.6 ± 1.1 μM) and removed the effect of the native peptide on fast inactivation, indicating that a lysine at position 22 (Pme2a numbering) is important for inhibitory activity. Results from this study may be used to guide the rational design of spider venom-derived peptides with improved potency and selectivity at Na_V channels in the future.

1. Introduction

Voltage-gated sodium channels (Na_V) are pore-forming transmembrane proteins that regulate the influx of Na⁺ ions across excitable cell membranes, making them essential for the initiation and propagation of action potentials. In humans, nine different subtypes have been described (Na_V1.1-1.9), each with unique biophysical properties and tissue-specific expression profiles [1]. Several subtypes, including Na_V1.7 and Na_V1.8, are highly expressed in peripheral sensory neurons and are therefore critical for somatosensation and nociception.

Na_V1.7 and Na_V1.8 are almost exclusively expressed in the peripheral nervous system, with preferential expression in small-diameter unmyelinated nociceptive or “pain-sensing” neurons [2,3,4]. In humans, loss-of-function mutations in SCN9A, the gene encoding Na_V1.7, leads to congenital insensitivity to pain, while several gain-of-function mutations in SCN9A and SCN10A (the gene encoding Na_V1.8) are associated with painful peripheral neuropathies [5,6,7]. This is consistent with studies in rodents, whereby knockout of Scn9a or Scn10a leads to deficits in mechanical, thermal and inflammatory pain [8,9], making both Na_V1.7 and Na_V1.8 promising therapeutic targets of interest for the treatment of pain.

While many small-molecule Na_V inhibitors are used in the clinic, non-selective activity over other Na_V subtypes, including the central isoforms Na_V1.1 and Na_V1.2 and the cardiac specific isoform Na_V1.5 [10,11], limits their widespread use as analgesics. Therefore, there has been a push to develop novel Na_V inhibitors with improved subtype selectivity that target the less conserved voltage-sensing domains of the channel [12]. One source of novel Na_V modulators is venoms from spiders, cone snails and scorpions, from which many peptides with exquisite Na_V subtype selectivity have been described [13,14]. However, compared to tetrodotoxin (TTX)-sensitive Na_V subtypes, relatively few venom-derived peptides with sub-micromolar potency at Na_V1.8 have been characterised [15].

Therefore, the aim of this study was to use a high-throughput screen at Na_V1.8 to identify novel bioactives from spider venom. Here, we describe the isolation and characterisation of two novel peptides from the Gooty Ornamental tarantula Poecilotheria metallica that modulate sodium channels.

2. Experimental Section

2.1. Cell Culture

Human Embryonic Kidney (HEK) 293 cells stably expressing human Na_V1.7/β1 (SB Drug Discovery, Glasgow, UK) were cultured in Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, and the selection antibiotics G-418 (0.6 mg/mL) and blasticidin (4 μg/mL), as recommended by the manufacturer. Chinese Hamster Ovary (CHO) cells stably expressing human Na_V1.8/β3 in a tetracycline-inducible system (ChanTest, Cleveland, OH, USA) were cultured in MEM supplemented with 10% FBS and 2 mM l-glutamine. Expression of hNa_V1.8 was induced by the addition of tetracycline (1 μg/mL) for 48–72 h prior to assays. HEK293 cells stably expressing rat transient receptor potential vanilloid 1 (TRPV1) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% FBS under selection with hygromycin B (100 μg/mL), generated as previously described [16]. Cells were grown in an incubator at 37 °C with 5% CO₂ and passaged every 3–4 days (at 70%–80% confluency) using TrypLE Express (Thermo Fisher Scientific, Scoresby, VIC, Australia).

2.2. Venom Collection

Venom of female P. metallica spiders was extracted by weak electrical stimulation as previously described [17] and dried and stored at −20 °C, before being pooled and redissolved in milliQ water for further analysis.

2.3. Membrane Potential Assay at Na_V1.8

CHO cells stably expressing hNa_V1.8/β3 were plated 48 h before the assay on 384-well black-walled imaging plates coated with CellBIND (Corning, MA, USA) at a density of 10,000–15,000 cells/well and loaded with red membrane potential dye (Molecular Devices, Sunnyvale, CA, USA) plus TTX (1 μM) diluted in physiological salt solution (PSS; 140 mM NaCl, 11.5 mM glucose, 5.9 mM KCl, 1.4 mM MgCl₂, 1.2 mM NaH₂PO₄, 5 mM NaHCO₃, 1.8 mM CaCl₂, 10 mM HEPES) and incubated for 30 min at 37 °C. Crude dried venom (10 μg/well) was diluted in PSS with 0.1% bovine serum albumin (BSA) and added using the FLIPR^TETRA (Molecular Devices) and incubated for 5 min before activation of Na_V1.8 by addition of deltamethrin (150 μM). Changes in membrane potential were assessed using the FLIPR^TETRA (excitation, 515–545 nm; emission, 565–625 nm) every 2 s for 25 min after adding the agonist. To quantify the activity of crude venom at Na_V1.8, the area under the curve (AUC) after the addition of deltamethrin was computed using ScreenWorks (Molecular Devices, Version 3.2.0.14).

2.4. Isolation of Pme1a and Pme2a

Crude P. metallica venom (1 mg dried mass) was dissolved in 5% acetonitrile (ACN)/0.1% trifluoroacetic acid (TFA) and loaded onto an analytical C₁₈ Reversed-Phase (RP) High-Pressure Liquid Chromatography (HPLC) column (Vydac 4.6 × 250 mm, 5 μm; Grace, Columbia, MD, USA) attached to an UltiMate 3000 HPLC system (Dionex, Sunnyvale, CA, USA). Venom fractions were collected in 1 min intervals eluting at a flow rate of 0.7 mL/min with solvent A (0.1% formic acid in H₂O) and solvent B (90% ACN, 0.1% formic acid in H₂O) using the gradient: 5% solvent B over 5 min, followed by 5%–50% solvent B over 45 min followed by 50%–100% solvent B over 25 min.

Venom fractions were assessed for activity at hNa_V1.8 using the FLIPR^TETRA membrane potential assay as described above. The active fraction was further fractionated to near-purity and peptide masses were determined using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) using a Model 4700 Proteomics Analyser (Applied Biosystems, Foster City, CA, USA) with α-cyano-4-hydroxycinnamic acid (7 mg/mL in 50% ACN + 5% formic acid in H₂O) as the matrix. Peptide sequences were determined by Edman degradation performed by the Australian Proteome Analysis Facility (Macquarie University, NSW, Australia). Sequence ambiguity was clarified by MS/MS sequencing.

2.5. Peptide Synthesis

Peptides (Pme1a, Pme2a and [+K22]Pme2a) were assembled using a Symphony (Protein Technologies Inc., Tuscon, AZ, USA) automated peptide synthesizer on Fmoc-Rink-amide polystyrene resin on 0.1 mmol scale. Amino acid sidechains were protected as Asn(Trt), Arg(Pbf), Asp(OtBu), Cys(Trt), Gln(Trt), Glu(OtBu), His(Trt), Lys(Boc), Ser(tBu), Thr(tBu), Trp(Boc) and Tyr(tBu). Fluorenylmethyloxycarbonyl (Fmoc) removal was achieved using successive treatments with 30% piperidine/dimethylformamide (DMF) of 1 min then 3 min. Couplings were performed using 5 equivalents of HCTU/Fmoc-amino acid/N,N-diisopropylethylamine (DIEA) (1:1:1) in DMF, repeated twice (4 min then 8 min). Peptide-resins were cleaved using 3% triisopropylsilane (TIPS)/3% H₂O/TFA for 2 h. Following evaporation of TFA under a stream of nitrogen, peptides were precipitated and washed with cold diethyl ether, dissolved in 50% ACN/0.1 % TFA/H₂O and lyophilised. Crude peptides were purified by preparative RP-HPLC. Oxidative folding was performed in the presence of oxidised and reduced glutathione for 2 day at 4 °C, and the major folded products of correct mass were isolated by preparative RP-HPLC.

2.6. Calcium Responses in TRPV1-HEK Cells

HEK cells stably expressing rTRPV1 were plated 48 h before the assay on 384-well black-walled imaging plates coated with poly d-lysine (ViewPlate-384, Perkin Elmer, Victoria, Australia) at a density of 10,000–15,000 cells/well and loaded with Calcium 4 no-wash dye (Molecular Devices) diluted in PSS and incubated for 30 min at 37 °C. Capsaicin and Pme1a were diluted in PSS with 0.1% bovine serum albumin (BSA) at the concentrations stated and added using the FLIPR^TETRA. Changes in fluorescence were assessed using the FLIPR^TETRA (excitation 470–495 nm, emission 515–575 nm) every 1 s for 300 s after the addition of compounds. Fluorescence responses were quantified by the maximum increase in fluorescence calculated using ScreenWorks 3.2.0.14.

2.7. Electrophysiology

Whole-cell patch-clamp experiments in Na_V1.7-HEK cells and Na_V1.8-CHO cells were performed on a QPatch-16 automated electrophysiology platform (Sophion Bioscience, Ballerup, Denmark) as previously described [18]. The extracellular solution contained in mM: NaCl 140, KCl 4, CaCl₂ 2, MgCl₂ 1, HEPES 10 and glucose 10; pH 7.4; osmolarity 305 mOsm. The intracellular solution contained in mM: CsF 140, EGTA/CsOH 1/5, HEPES 10 and NaCl 10; pH 7.3 with CsOH; osmolarity 320 mOsm. Concentration–response curves were acquired using a holding potential of −90 mV and a 50 ms pulse to −20 mV (for Na_V1.7) or +10 mV (for Na_V1.8) every 20 s (0.05 Hz). Peptides were diluted in extracellular solution with 0.1% BSA and each peptide concentration was incubated for 5 min. Peak current was normalized to buffer control. The time constant of fast inactivation (τ) was computed by fitting the current decay traces with a single exponential function using the QPatch Assay Software 5.6 (Sophion). I-V curves were obtained with a holding potential of −90 mV followed by a series of 500 ms step pulses that ranged from −110 to +55 mV in 5 mV increments (repetition interval 5 s) before and after 5 min incubation or Pme1a (1 μM). Conductance-voltage curves were obtained by calculating the conductance (G) at each voltage (V) using the equation G = I/(V − V_rev), where V_rev is the reversal potential and were fitted with a Boltzmann equation.

2.8. Data Analysis

Data were plotted and analysed using GraphPad Prism, version 8.2.0. For concentration–response curves, a four-parameter Hill equation with variable Hill coefficient was fitted to the data. Data are presented as the mean ± SEM.

3. Results

3.1. Isolation of the Novel Spider Venom Peptides μ-TRTX-Pme1a and μ/δ-TRTX-Pme2a from P. metallica

Crude venom isolated from P. metallica (Figure 1A) inhibited deltamethrin-induced membrane potential changes in CHO cells stably expressing hNa_V1.8, with activity guided fractionation isolating this activity to a single peak eluting at ~35% solvent B (Figure 1A). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) indicated that this fraction was dominated by two masses (M + H)⁺ of 3911.6 m/z and 3808.5 m/z (Figure 1B). N-terminal sequencing revealed two novel 35 and 33 residue peptides that we named μ-TRTX-Pme1a (hereafter Pme1a) and μ/δ-TRTX-Pme2a (hereafter Pme2a) based on the rational nomenclature for peptide toxins (Figure 1C) [19]. The calculated masses and observed masses differed by –1 Da, indicating both Pme1a and Pme2a have an amidated C-terminus. Synthetic Pme1a and Pme2a with C-terminal amidation were used for all further experiments.

3.2. Pharmacological Activity of Pme1a

Alignment of Pme1a to peptide sequences from the Universal Protein Resource (www.uniprot.org) revealed that the peptide shares high sequence homology (56%–80%) with the vanillotoxins (Figure 2), a family of peptides from Psalmopoeus cambridgei that activate TRPV1 with EC₅₀s ranging between 0.32 and 12 μM [20]. We therefore tested the activity of Pme1a on TRPV1 heterologously expressed in HEK293 cells. In comparison to the TRPV1 activator capsaicin (EC₅₀ 83 nM), Pme1a (up to 30 μM) had no activity on TRPV1, suggesting that amino acid residue(s) crucial for TRPV1 activity are missing from this peptide (Figure 3A). As Pme1a was discovered using a Na_V channel screen, we next characterised its activity on Na_V channels using automated whole-cell patch-clamp electrophysiology.

Despite being isolated as an inhibitor of Na_V1.8 using a fluorescent screen, Pme1a only had modest activity on Nav1.8, inhibiting 21 ± 2 % of the peak current at a concentration of 10 μM (Figure 3B). In contrast, Pme1a was more potent at Na_V1.7, inhibiting peak current with an IC₅₀ of 334 ± 114 nM (Figure 3C,D). Activity at other Na_V channel subtypes is not unexpected, given the high-sequence homology between Na_V subtypes [12]. To examine the mechanism of channel block, we next assessed the effect of Pme1a on the voltage-current relationship at Na_V1.7 (Figure 3E). Pme1a (1 μM) shifted the voltage dependence of activation at Na_V1.7 to more depolarised membrane potentials (V_1/2 activation: Δ = +11.6 mV), confirming it binds to the voltage-sensing domain(s) to modify channel gating (Figure 3F).

3.3. Pharmacological Activity of Pme2a

Alignment of Pme2a to peptide sequences from the Universal Protein Resource (www.uniprot.org) revealed that the peptide shares sequence homology (<65%) to Pme1a and other peptides with activity at Na_V channels (Figure 2). Similar to Pme1a, Pme2a had no activity at TRPV1 (data not shown), and only modest activity at Nav1.8, with the highest concentration tested (10 μM) inhibiting 22 ± 4 % of peak current (Figure 4A). Pme2a also had modest activity at Na_V1.7, concentration-dependently delaying-fast inactivation, albeit not potently, with an estimated EC₅₀ > 10 μM (Figure 4B,C). Due to low potency at Na_V1.7, no further pharmacological characterisation was performed.

Sequence alignment revealed that loop 4 of Pme2a is one amino acid residue shorter compared to others in the family, and peptides with activity on TTX-sensitive Na_V channels generally contain a positively charged amino acid at the equivalent position, most often a lysine (Figure 2). We therefore synthesised a [+22K]Pme2a mutant and assessed how the addition of a lysine affected activity at Na_V1.7. [+22K]Pme2a concentration-dependently inhibited Na_V1.7 peak current, without the delay in fast activation seen with the native peptide, with a more potent IC₅₀ of 5.6 ± 1.1 μM (Figure 4D,E).

4. Discussion

Here, we describe the isolation and Na_V activity of two novel spider venom-derived peptides, which are the first peptides to be isolated and functionally characterised from the venom of the species P. metallica, as well as the entire Poecilotheria genus [21]. A transcriptome and proteome from the venom gland of the related species P. formosa identified two related, but not identical peptide sequences (Pf29 and Pf32) [22], indicating that the entire Poecilotheria genus is likely to be a rich source of novel Na_V modulators (Figure 2). Indeed, crude venom from several species of the Poecilotheria genus, including P. metallica, has previously been shown to modulate Na_V1.7 [23], consistent with activity of Pme1a and Pme2a at both Na_V1.7 and Na_V1.8 described here. The two peptides isolated here were only moderately potent at Na_V1.7 and Na_V1.8, indicating that there may still be additional peptides in the crude venom with more potent activity.

Specifically, both Pme1a and Pme2a align with NaSpTx family 2, which is a large family of spider venom peptides that have promiscuous activity on Na_V, K_V and Ca_V channels [24]. Interestingly, this family also contains members that have high selectivity for Na_V channels of therapeutic interest, including the Na_V1.7 inhibitor μ-TRTX-Pn3a and the Na_V1.1 activator δ-TRTX-Hm1a. However, little is known about the structure–activity relationships that define the pharmacology of this family [25,26]. Here, we describe the importance of a lysine (K) at position “22” (using Pme2a numbering) for Na_V channel activity. The presence of the positively charged amino acid residue lysine is often important for facilitating membrane interactions in venom-based peptides [18,27]. Addition of K22 altered the pharmacology of Pme2a, converting the native peptide from being a Na_V1.7 channel activator to a more potent Na_V1.7 channel inhibitor. This is a significant finding, indicating that the amino acid residue(s) present at or around this site can dictate whether family 2 peptides function as Na_V activators or inhibitors. It is also possible that extending loop 4 of Pme2a by one amino acid residue contributed to the change in activity by slightly altering the overall structure of the peptide. Future studies elucidating the full Na_V selectivity of Pme1a and Pme2a will further define which amino acid residues are important for Na_V channel potency and subtype selectivity.

In conclusion, we have identified two novel spider venom-derived peptides that modulate Na_V channels. Results from this study provide structure–activity relationship information that may guide the future rational design of spider venom-derived peptides with improved activity and selectivity at Na_V channels.