Evidence for multiple effects of ProTxII on activation gating in Na_V1.5

The peptide toxin ProTxII, recently isolated from the venom of the tarantula spider Thrixopelma pruriens, modifies gating in voltage-gated Na⁺ and Ca²⁺ channels. ProTxII is distinct from other known Na⁺ channel gating modifier toxins in that it affects activation, but not inactivation. It shifts activation gating positively and decreases current magnitude such that the dose-dependence of toxin action measured at a single potential reflects both effects. To test the extent to which these effects were independent, we tracked several different measures of current amplitude, voltage-dependent activation, and current kinetics in Na_V1.5 in a range of toxin concentrations. Changes in voltage dependence and a decrease in G_max appeared at relatively low concentrations (40-100 nM) while a positive shift in the voltage range of activation was apparent at higher toxin concentrations (≥500 nM). Because ProTxII carries a net +4 charge we tested whether electrostatic interactions contributed to toxin action. We examined the effects of ProTxII in the presence of high extracellular Ba²⁺, known to screen and/or bind to surface charge. Some, but not all aspects of ProTxII modification were sensitive to the presence of Ba²⁺ indicating the contribution of an electrostatic, surface charge-like mechanism and supporting the idea of a multi-faceted toxin-channel interaction.