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

Exploring the interaction of ligands with voltage-gated sodium channels (Na(V)s) has advanced our understanding of their pharmacology. Herein, we report the purification and characterization of a novel non-selective mammalian and bacterial Na(V)s toxin, JZTx-14, from the venom of the spider Chilobrachys jingzhao. This toxin potently inhibited the peak currents of mammalian Na(V)1.2–1.8 channels and the bacterial NaChBac channel with low IC(50) values (<1 µM), and it mainly inhibited the fast inactivation of the Na(V)1.9 channel. Analysis of Na(V)1.5/Na(V)1.9 chimeric channel showed that the Na(V)1.5 domain II S3–4 loop is involved in toxin association. Kinetics data obtained from studying toxin–Na(V)1.2 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 Na(V) currents and showing state-independent binding with the channel. JZTx-14 might stably bind to a conserved toxin pocket deep within the Na(V)1.2–1.8 domain II voltage sensor regardless of channel conformation change, and its effect on Na(V)s 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 Na(V)s and the highly evolved descendant mammalian Na(V)s, and it is a useful probe to understand the pharmacology of Na(V)s.