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 (Nas) has advanced our understanding of their pharmacology. Herein, we report the purification and characterization of a novel non-selective mammalian and bacterial Nas toxin, JZTx-14, from the venom of the spider . This toxin potently inhibited the peak currents of mammalian Na1.2⁻1.8 channels and the bacterial NaChBac channel with low IC values (<1 µM), and it mainly inhibited the fast inactivation of the Na1.9 channel. Analysis of Na1.5/Na1.9 chimeric channel showed that the Na1.5 domain II S3⁻4 loop is involved in toxin association. Kinetics data obtained from studying toxin⁻Na1.2 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 currents and showing state-independent binding with the channel. JZTx-14 might stably bind to a conserved toxin pocket deep within the Na1.2⁻1.8 domain II voltage sensor regardless of channel conformation change, and its effect on Nas 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 Nas and the highly evolved descendant mammalian Nas, and it is a useful probe to understand the pharmacology of Nas.