GrTX-SIA inhibition of Na V 1.6 currents. GrTX-SIA application (1 µM) leads to a quick initial inhibition (red traces) followed by a somewhat slower and smaller inhibitory step (grey/blue). This effect was variable between measurements and only served as a rationale to investigate the potential presence of multiple toxin binding sites within NaV1.6. (A and B) Panel A shows a representative trace, whereas panel B displays a typical NaV1.6 current that is inhibited (Y-axis: percentage of current remaining assessed with 5 s pulses to −25 mV) in two phases during a 4-min application of 1 µM GrTX-SIA. The second phase is not related to channel current rundown. Current amplitude partially recovered during a 5-min wash-off period (black circles in B). (C) The normalized G–V (G/Gmax) and channel availability (I/Imax) relationships are shown for the starting condition without toxin (black), after the first inhibitory phase (red) and after the remaining slow step (grey/blue). Currents were recorded by applying a depolarizing voltage-step protocol from −100 to +20 mV from a holding potential of −90 mV with 5 s between depolarizing pulses. Circles represent the mean ± SEM with n = 6 oocytes.