Mutations in Na _V 1.6 VSDII and VSDIV influence GrTX-SIA binding. The effect of 1 µM GrTX-SIA on Na_V1.6 VSDIV^mut is drastically reduced compared to the wild-type channel. The top row shows a representative trace before (black) and after (red) toxin application for 4 min, the normalized G-V (G/G_max) and channel availability (I/I_max) relationships under these conditions, and a representative example of partial Na_V1.6 VSDIV^mut inhibition by 1 µM GrTX-SIA followed by complete wash-off with ND-100 after 4 min. The bottom row displays the virtual lack of effect of 1 µM GrTX-SIA on the Na_V1.6 VSDII^mut–VSDIV^mut construct. In both panels, circles represent the mean ± SEM of n = 8 oocytes for Na_V1.6 VSDIV^mut and 5 oocytes for Na_V1.6 VSDII^mut–VSDIV^mut, with n = 3 for toxin wash-in/wash-off experiments.