Xenopus oocyte recordings (BgNav1)

All Blattella germanica Na_v (BgNa_v1) mutants were generated using the Gibson assembly method. For each mutant, four linear DNA fragments with approximately 20-bp homology arms were generated using PCR (Phusion High-Fidelity DNA Polymerase, New England Biolabs, USA). Fragments were purified (MinElute PCR Purification Kit, Qiagen, USA) and subsequently incubated for 2 hours at 55°C with Gibson Assembly Master Mix (New England Biolabs, USA). “Assembled” plasmids were then transformed (CopyCutter EPI400, Epicentre, USA) and mutagenesis was confirmed by automated Sanger sequencing. cRNA was synthesized using T7 polymerase (mMessage mMachine kit, Life Technologies, USA) after linearizing the DNA with appropriate restriction enzymes.

BgNa_v1 control and mutant constructs were expressed in Xenopus laevis oocytes (sourced from Xenopus one, USA), and electrophysiological recordings were taken after 2 to 4 days post cRNA injection. Oocytes were incubated at 17°C in Barth’s medium (96 mM NaCl, 2 mM KCl, 5 mM HEPES, 1 mM MgCl₂, and 1.8 mM CaCl₂, 50 μg/ml gentamycin, pH 7.6 with NaOH) and studied using the two-electrode voltage-clamp recording technique (OC-725C, Warner Instruments) with a 150-μl recording chamber. All data were filtered at 4 kHz and digitized at 20 kHz using pClamp 10 software (Molecular Devices, USA). The external recording solution used was ND-100 (100 mM NaCl, 5 mM HEPES, 1 mM MgCl₂, and 1.8 mM CaCl₂, pH 7.6 with NaOH) and microelectrode resistances were 0.5 to 1.0 MΩ when filled with 3 M KCl. All experiments were performed at room temperature (~22°C), and AaH2 samples were diluted in ND-100 with 0.1% BSA. Leak and background conductances were identified and subtracted by blocking the channel with TTX (Alomone Labs, Israel). After adding toxin to the recording chamber, equilibration between channel and toxin was monitored using weak depolarizations (~20) at 5-s intervals unless otherwise noted. Typically, voltage-activation relationships were recorded before and after toxin addition. Off-line data analysis was performed using Clampfit 10 (Molecular Devices, USA), Microsoft Excel (Microsoft, USA) and Prism 7 (GraphPad, USA). For data presented in figs. S8 and S9, significance of all normalized conductance-voltage (G-V) and steady-state inactivation (I-V) relationships was analyzed using two-way analysis of variance (ANOVA) with post hoc Tukey’s test. Individual time point values for fast-inactivation time constants (t), persistent current, peak current, and recovery from fast inactivation (RFI) were analyzed using Student’s t test. Values in all cases reflect the mean, and error bars reflect SEM, P < 0.05 (*) or 0.001 (**). For all data presented in table S2, Student’s t (unpaired) against wild-type was used.