Structural and Functional Characterization of a Novel Scorpion Toxin that Inhibits Na_V1.8 via Interactions With the DI Voltage Sensor and DII Pore Module

Voltage-gated sodium channel Na_V1.8 regulates transmission of pain signals to the brain. While Na_V1.8 has the potential to serve as a drug target, the molecular mechanisms that shape Na_V1.8 gating are not completely understood, particularly mechanisms that couple activation to inactivation. Interactions between toxin producing animals and their predators provide a novel approach for investigating Na_V structure-function relationships. Arizona bark scorpions produce Na⁺ channel toxins that initiate pain signaling. However, in predatory grasshopper mice, toxins inhibit Na_V1.8 currents and block pain signals. A screen of synthetic peptide toxins predicted from bark scorpion venom showed that peptide NaTx36 inhibited Na⁺ current recorded from a recombinant grasshopper mouse Na_V1.8 channel (OtNa_V1.8). Toxin NaTx36 hyperpolarized OtNa_V1.8 activation, steady-state fast inactivation, and slow inactivation. Mutagenesis revealed that the first gating charge in the domain I (DI) S4 voltage sensor and an acidic amino acid (E) in the DII SS2 - S6 pore loop are critical for the inhibitory effects of NaTx36. Computational modeling showed that a DI S1 - S2 asparagine (N) stabilizes the NaTx36 - OtNa_V1.8 complex while residues in the DI S3 - S4 linker and S4 voltage sensor form electrostatic interactions that allow a toxin glutamine (Q) to contact the first S4 gating charge. Surprisingly, the models predicted that NaTx36 contacts amino acids in the DII S5 - SS1 pore loop instead of the SS2 - S6 loop; the DII SS2 - S6 loop motif (QVSE) alters the conformation of the DII S5 - SS1 pore loop, enhancing allosteric interactions between toxin and the DII S5 - SS1 pore loop. Few toxins have been identified that modify Na_V1.8 gating. Moreover, few toxins have been described that modify sodium channel gating via the DI S4 voltage sensor. Thus, NaTx36 and OtNa_V1.8 provide tools for investigating the structure-activity relationship between channel activation and inactivation gating, and the connection to alternative pain phenotypes.