ProTxII Interacts Specifically with the Domain II Voltage Sensor of NaV1.4 Modifying Gating Without Immobilization

ProTxII, a peptide extracted from the venom of the tarantula Thrixopelma pruriens, binds to multiple voltage-gated sodium channel isoforms. In NaV1.2 ProTxII reduces the sodium conductance and decreases total gating charge (Sokolov et al., Mol. Pharm. 73:1020). In this isoform the toxin's effect could be partially reversed with strong depolarization indicating interactions with one or more of the voltage sensors. Mutagenesis data further implicated the domain II voltage sensor in the toxin-channel interaction in NaV1.2. Here we have shown that, as was seen for NaV1.2, ProTxII (2-5μM) produced a decrease in maximum conductance (∼60%) and a decrease in total gating charge (∼20%) in the NaV1.4 isoform expressed in Xenopus oocytes. Unlike NaV1.2, however, these effects on NaV1.4 could not be reversed with strong depolarization. We also discovered a single residue, S660, located at the S3-S4 linker/S4 boundary of DII, which when mutated, renders this channel insensitive to toxin. We used site-specific fluorescent measurements to determine the effect of ProTxII on the movement of individual voltage sensors. In the presence of ProTxII the voltage dependence of the fluorescent signal of DIIS4 was modified, but not eliminated, indicating that this voltage sensor is not completely immobilized upon toxin binding. The fluorescent signals measured from domains I and III were not significantly affected by ProTxII implicating a specific interaction with DII in producing the effect of ProTxII on NaV1.4. Supported by GM30376 (FB) and NS061535-01(GBE).