Fig. 1. Human codon optimization of BacNa_v gene improves expression of functional channels.

a–d Representative images of HEK293 cells transduced with bicistronic lentiviruses in which GFP gene was linked via T2A peptide with non-optimized (bacterial) Na_vSheP D60A sequence (bSheP, a) or Na_vSheP D60A sequences codon-optimized using Genscript (hSheP, b) or ATUM (h2SheP, c) algorithms and corresponding quantification by flow cytometry (d). e, f Representative current traces (e) and corresponding quantifications of peak I_Na–V curves (f) recorded in bSheP, hSheP, or h2SheP-expressing HEK293 cells using whole-cell voltage clamp at 25 °C (n = 6). g–j Representative action potential (AP) traces (g) measured via current clamp in BacNa_v-transduced K_ir2.1-expressing HEK293 cells and corresponding quantifications of maximum upstroke velocity (h, AP upstroke; n = 5), AP duration at 80% repolarization (i, APD80; n = 5), and resting membrane potential (j, RMP; n = 5), all recorded at 37 °C. ^#P < 0.05 among all three groups and ^{^}P < 0.05 for h2SheP vs. bSheP in f, exact P-values for all groups are included in Source Data. *P = 0.0403, **P = 0.0073 vs. bSheP in h. Error bars indicate s.e.m; statistical significance was determined by two-way ANOVA in f and one-way ANOVA in h, followed by Tukey’s post-hoc test to calculate P-values. Source data are provided as a Source Data file.