Voltage dependence of the conductance in wild-type Nav and Nav-LBTs after pretreatment with Ts1-W50Pra or Ts1-S14R. (a) Sequence alignment of the S3-S4 linkers of wild-type Nav1.4 in DI, DIII and DIV, compared to the respective S3-S4 linkers of the LBT constructs: DI-3GLBT-R(-5), DIII-3GLBT-R(-5) and DIV-3GLBT-R(-5), (adopted from Kubota T. et al.21). The underlined amino acids are overlapping amino acids with those prior to LBT insertions. (b–e) Normalized conductance of wild- type Nav channel (WT in b), DI-3GLBT-R(-5) (DI in c), DIII-3GLBT-R(-5) (DIII in d), and DIV-3GLBT-R(-5) (DIV in e). In each plot, blue squares are data obtained after pretreatment with 1 µM Ts1-S14R (WT: n = 5; DI: n = 4; DIII: n = 4; and, DIV: n = 5). Data plots before treatment (black squares) and after pretreatment with 1 µM Ts1-W50Pra (red squares) are adapted from Kubota T et al.21 (Black squares: WT, n = 5; DI, n = 4; DIII, n = 5; and, DIV, n = 5. Red squares; WT, n = 4; DI, n = 4; DIII, n = 4; and, DIV, n = 4). Error-bars are standard error of mean (SEM). The inset in (d) indicates the pulse protocol used for (b,d), and the inset in (e), that for (c,e).