The varied outcomes of DIVS6 N→T on BTX sensitivity among the poison bird, human, rat, and poison frog NaVs highlight the importance of context in determining the functional consequences of mutations. Because the equivalent residue is conserved in all four S6 helices (Figs. S1 and S2), we systematically introduced S6 N→T into each of the Pum NaV1.4 S6 segments and measured channel properties and BTX responses to investigate the question of context-dependent effects further (Fig. 4). Whole-cell patch-clamp recordings from HEK293 cells transfected with these mutant channels revealed clear, domain-specific differences. Contrasting the effect of DIVS6 N→T (Fig. S6 b), voltage-dependent activation of channels having the N→T mutation in DI, DII, or DIII (Pum NaV1.4 N432T [DI], Pum NaV1.4 N830T [DII], and Pum NaV1.4 N1306T [DIII]) was unchanged relative to WT (V1/2 = −20.2 ± 1.9, −22.4 ± 1.0, −26.7 ± 1.0, and −23.4 ± 1.0 mV for Pum NaV1.4 N432T [DI], Pum NaV1.4 N830T [DII], Pum NaV1.4 N1306T [DIII], and Pum NaV1.4, respectively; Fig. 4, a–j; Fig. S8, a and b; and Tables 1 and 2). By contrast, we found varied effects on steady-state inactivation. DI and DIII changes showed WT-like behavior, whereas the DII mutant had an ∼10-mV hyperpolarizing shift (V1/2 inact = −61.2 ± 1.6, −75.0 ± 0.9, −65.3 ± 1.0, and −64.2 ± 1.3 mV for Pum NaV1.4 N432T [DI], Pum NaV1.4 N830T [DII], Pum NaV1.4 N1306T [DIII], and Pum NaV1.4, respectively; Fig. S8 c, Table 2, and Table S1). All three had strong BTX responses similar to WT (ΔV1/2 BTX = −30.8 ± 2.1, −31.6 ± 2.0, −36.9 ± 1.4, and −33.6 ± 1.2 mV for Pum NaV1.4 N432T [DI], Pum NaV1.4 N830T [DII], Pum NaV1.4 N1306T [DIII], and Pum NaV1.4, respectively; Fig. 4 and Tables 1 and 2). Thus, the only site where the conserved S6 N→T change affects BTX responses is in DIVS6, in line with its proposed contribution to the BTX binding site (Wang and Wang, 2017).

As with the biophysical changes, the effects on current density from placing the N→T change in different channel domains were not uniform. The DIS6 and DIIIS6 N→T mutants had current densities matching WT (Fig. 4, a, c, and g; Fig. S8, a and d; and Tables 1 and 2), whereas, DIIS6 N→T lowered the current density and was more detrimental to channel activity than DIVS6 N1609T or N1609A (Fig. 4, a and e; Fig. S8, a and d; and Tables 1 and 2). Together, these data show that there is no correlation between changes in channel biophysical properties and the acquisition of BTX resistance and are in line with the results from DIVS6 N→T and N→A mutants (Figs. 3, S6, and S7, and Table 2).

Consideration of the conserved S6 asparagine structural locale provides insight into the context-dependent effects. The two S6 sites where N→T has no impact on channel biophysics, BTX responses, or current density, DIS6 and DIIIS6, occupy positions that are partially exposed to the channel inner pore (Fig. S8, e and f). By contrast, the two positions that affect channel biophysics and current density, DIIS6 and DIVS6, interact with the S4–S5 linkers (Pan et al., 2018; Fig. S8, e and f), and altering these buried sites comes with substantial functional costs. Hence, DIVS6 N→T carries major disadvantages for protecting animals such as Pitohui and poison frogs against BTX autointoxication.