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

摘要

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.