(A) Predicted topology of Nav1.6 with mutations in domains I, III, and IV. Sequence alignment of Nav1.6 pore-loop motifs revealed three amino acid differences in newts from Oregon or Idaho populations. (B) Representative currents from wild-type mouse Nav1.6 or Nav1.6 with newt substitutions Y371A, V1407I, and I1699V treated with 1 µM (blue) or 10 µM (orange) TTX. (C) Current-voltage (I–V) relationships showing normalized currents for wild-type (n = 21) and mutant Nav1.6 (n = 20) channels. Wild-type Nav1.6 was blocked by TTX (Tukey’s multiple comparisons test with Bonferroni correction: control vs. 1 µM, p<0.0001; control vs. 10 µM, p<0.0001), while mutated Nav1.6 was unaffected (repeated measures ANOVA, p=0.879). (D) Dose-response curves showing the proportion of Na+ current elicited during a step depolarization from −100 to −20 mV for wild-type, individual mutants, and triple-mutant Nav1.6 channels exposed to increasing concentrations of TTX. Sample sizes are provided in Table 2. Data were fit with a Hill equation to estimate IC50 values.