Molecular Surface of JZTX-V (β-Theraphotoxin-Cj2a) Interacting with Voltage-Gated Sodium Channel Subtype NaV1.4

Abstract

Voltage-gated sodium channels (VGSCs; NaV1.1–NaV1.9) have been proven to be critical in controlling the function of excitable cells, and human genetic evidence shows that aberrant function of these channels causes channelopathies, including epilepsy, arrhythmia, paralytic myotonia, and pain. The effects of peptide toxins, especially those isolated from spider venom, have shed light on the structure–function relationship of these channels. However, most of these toxins have not been analyzed in detail. In particular, the bioactive faces of these toxins have not been determined. Jingzhaotoxin (JZTX)-V (also known as β-theraphotoxin-Cj2a) is a 29-amino acid peptide toxin isolated from the venom of the spider Chilobrachys jingzhao. JZTX-V adopts an inhibitory cysteine knot (ICK) motif and has an inhibitory effect on voltage-gated sodium and potassium channels. Previous experiments have shown that JZTX-V has an inhibitory effect on TTX-S and TTX-R sodium currents on rat DRG cells with IC50 values of 27.6 and 30.2 nM, respectively, and is able to shift the activation and inactivation curves to the depolarizing and the hyperpolarizing direction, respectively. Here, we show that JZTX-V has a much stronger inhibitory effect on NaV1.4, the isoform of voltage-gated sodium channels predominantly expressed in skeletal muscle cells, with an IC50 value of 5.12 nM, compared with IC50 values of 61.7–2700 nM for other heterologously expressed NaV1 subtypes. Furthermore, we investigated the bioactive surface of JZTX-V by alanine-scanning the effect of toxin on NaV1.4 and demonstrate that the bioactive face of JZTX-V is composed of three hydrophobic (W5, M6, and W7) and two cationic (R20 and K22) residues. Our results establish that, consistent with previous assumptions, JZTX-V is a Janus-faced toxin which may be a useful tool for the further investigation of the structure and function of sodium channels.

Pharmacological characterization of potent and selective NaV1.7 inhibitors engineered from Chilobrachys jingzhao tarantula venom peptide JzTx-V

“…JzTx-V was originally described as an inhibitor of both TTX-S (IC 50 = 28 nM) and TTX-R (IC 50 = 30 nM) sodium channels in rat DRG neurons with additional inhibitory activity against the voltage-gated potassium channels K V 4.1, K V 4.2 and K V 2.1 [ 25 ] [ 34 ]. Consistent with our findings, JzTx-V exhibited weak inhibition of Na V 1.5 and had nominal selectivity over Na V 1.4 when evaluated in heterologous HEK293 cells [ 26 ]. In order to identify a better compound for further interrogation of Na V 1.7 biology and encouraged by the inherent potency of the parent JzTx-V peptide, we sought to engineer improved selectivity against Na V 1.4 using the JzTx-V scaffold.…”

“…Given the lack of structural information of human Na V channels at the time of this work from X-ray crystallography or cryo-electron microscopy needed to elucidate atomic-level structural differences between the homologous TTX-S sodium channels Na V 1.7 and Na V 1.4, we focused on an empirical attribute-based positional scan analoging strategy previously successfully employed for K V 1.3 and Na V 1.7 [ 22 , 27 ]. The effect of a JzTx-V Ala-scan on rat Na V 1.4 block has been reported [ 26 ], and our Ala-mutant data validated the importance of residues Trp5, Trp24 and Arg26 in peptide block of human TTX-S channels. However, we did not observe any indication of differential peptide activity against Na V 1.7 and Na V 1.4 with Ala scan mutants.…”

“…To identify peptides with improved Na V 1.7 inhibitory potency compared to GpTx-1 analogs, we embarked on another fractionated tarantula venom electrophysiology screen and identified the 29-residue inhibitory cystine knot (ICK) peptide JzTx-V (β/κ-theraphotoxin-Cg2a also known as β-theraphotoxin-Cj2a) with sub-nM Na V 1.7 IC 50 from the Chinese earth tiger tarantula Chilobrachys jingzhao . JzTx-V was originally described as an inhibitor of native Na V channels in rat DRG neurons with no selectivity over Na V 1.4 in HEK293 cells [ 25 , 26 ]. Given the lack of high-resolution co-crystal structures of Na V channels with inhibitory peptides to enable rational design of selectivity, we applied the attribute-based positional scan analoging strategy previously employed for K V 1.3 and Na V 1.7 inhibition [ 22 , 27 ] to engineer selectivity-enhancing JzTx-V modifications that retain Na V 1.7 potency.…”

Antimicrobial Activity of Purified Toxins from <i>Crossopriza lyoni</i> (Spider) against Certain Bacteria and Fungi

“…Spider toxins show diverse biological effects in animals such as muscle and respiratory paralysis, integumentary, neuro-inflammatory and neuro-pathological effects in prey. Spider toxins (JZ TX-V) inhibit ion channels present on nerve cell membrane [2] and show platelet aggregation in haemostatic system [3]. These toxins are either acylpolyamine or polypeptides which cause irreversible paralysis in lepidopteran insects by massive transmitter release.…”

“…Spider venom toxins generate necrotizing skin lesions, cell necrosis, and show systemic reactions and impose death in animals [4]. Venom toxins show analgesic effect, do inhibition of voltage gated ion channels [5] and generate various channelopathies including epilepsy, arrhythmia, and paralytic myotonic effect with severe pain [2]. Toxin peptides can also become a good source of strong antimicrobials and can replace broad spectrum antibiotics which are highly toxic and show multiple biological effects and responsible for drug resistance in microbes [6].…”