Purification and Characterization of JZTx-14, a Potent Antagonist of Mammalian and Prokaryotic Voltage-Gated Sodium Channels

Abstract

Exploring the interaction of ligands with voltage-gated sodium channels (NaVs) has advanced our understanding of their pharmacology. Herein, we report the purification and characterization of a novel non-selective mammalian and bacterial NaVs toxin, JZTx-14, from the venom of the spider Chilobrachys jingzhao. This toxin potently inhibited the peak currents of mammalian NaV1.2–1.8 channels and the bacterial NaChBac channel with low IC50 values (<1 µM), and it mainly inhibited the fast inactivation of the NaV1.9 channel. Analysis of NaV1.5/NaV1.9 chimeric channel showed that the NaV1.5 domain II S3–4 loop is involved in toxin association. Kinetics data obtained from studying toxin–NaV1.2 channel interaction showed that JZTx-14 was a gating modifier that possibly trapped the channel in resting state; however, it differed from site 4 toxin HNTx-III by irreversibly blocking NaV currents and showing state-independent binding with the channel. JZTx-14 might stably bind to a conserved toxin pocket deep within the NaV1.2–1.8 domain II voltage sensor regardless of channel conformation change, and its effect on NaVs requires the toxin to trap the S3–4 loop in its resting state. For the NaChBac channel, JZTx-14 positively shifted its conductance-voltage (G–V) and steady-state inactivation relationships. An alanine scan analysis of the NaChBac S3–4 loop revealed that the 108th phenylalanine (F108) was the key residue determining the JZTx-14–NaChBac interaction. In summary, this study provided JZTx-14 with potent but promiscuous inhibitory activity on both the ancestor bacterial NaVs and the highly evolved descendant mammalian NaVs, and it is a useful probe to understand the pharmacology of NaVs.

Spider Knottin Pharmacology at Voltage-Gated Sodium Channels and Their Potential to Modulate Pain Pathways

“…Furthermore, the toxin delayed the fast inactivation along with the reduction of peak currents in hNa V 1.1, hNa V 1.3 and hNa V 1.5 [92]. Such subtype-varying profiles have also recently been reported for Pn3a ( Pamphobeteus nigricolor ) [61], Pre1a ( Psalmopoeus reduncus) [111] and JzTx-14 ( C. jingzhao ) [125]. These multi-site effects of spider knottins on Na V channels need to be carefully considered when establishing their pharmacological profiles.…”

“…Spider knottins include a growing number of depressant toxins, including JzTx-V ( C. jingzhao ), Df1a ( D. fasciatus ), Pre1a ( P. reduncus ), PnTx4(5-5) from P. nigriventer , CcoTx-1 ( Ceratogyrus cornuatus ), PaurTx ( Phrixotrichus auratus) and Cd1a ( Ceratogyrus darlingi ) which induce a depolarizing shift in voltage-dependence of activation at specific Na V subtypes [73,79,92,107,110,111]. However, a number of spider depressant knottins reduced the sodium currents without changing the activation or inactivation kinetics (e.g., HwTx-IV, HnTx-III, HnTx-IV) [114,116,129], by shifting activation and inactivation to hyperpolarizing potentials (e.g., Df1a) [92] or by inhibiting both activation and inactivation (e.g., JzTx-14) [125]. Spider knottins (e.g., JzTx-V) also altered the slope factor of activation and inactivation associated to shifts in their voltage dependence [79,91] suggesting cooperativity between the four S4 segments or multiple binding sites [9,91].…”

“…In addition, the ProTx-II–hNa V 1.7 cryo-EM structure confirmed a key role of the C-terminal basic residues capped by hydrophobic residues in anchoring the toxin into the membrane [58]. In contrast, JzTx-14 belonging to NaSpTx7 has a loop 4 that lacks positively charged residues and Arg serving as a C-terminal cap, but still inhibits eight out of nine Na V channel subtypes at nanomolar concentrations [125]. It has additional hydrophobic residues in each of the four loops with only one acidic residue that suggest an alternative binding mode.…”

“…The NaSpTx family 1–3 generally target Site 4 to inhibit channel activity, except Hm1a which targets Site 3 to excite the channel [20]. JzTx-14 [125] from NaSpTx7 targets Site 4 and inhibits the channel, whereas JzTx-I [100] and JzTx-II [98] from NaSpTx7 targets Site 3 to excite the channel. Yellow highlights conserved cysteines, green highlights hydrophobic residues, cyan indicates positively charged residues, red indicates negatively charged residues and bold letter indicates the aromatic residues.…”

Molecular mechanism of the spider toxin κ-LhTx-I acting on the bacterial voltage-gated sodium channel NaChBac

“…We previously purified two potent gating-modifier toxins, JZTx-27 and JZTx-14, from the venom of spider Chilobrachys jingzhao . These toxins interact with the S3–S4 loop region of NaChBac and inhibit the channel activity by impeding voltage-sensor activation ( Tang et al, 2017 ; Zhang et al, 2018 ). A recent study testing the activity of several known peptide modulators of mammalian Na V channels showed that the site 4 toxins GsAF-I, GrTx1, and GsAF-II also effectively inhibit NaChBac currents, possibly by affecting the voltage-dependent gating of the channel ( Zhu et al, 2020 ).…”