Figure 2. Customization of E-Fib phenotype via mutagenesis.

(a) BacNa_v-based strategy for E-Fib customization. (b) Experimental (n=8) and modelling peak I_Na–V curves. Cell membrane was held at −80 mV before stepping the voltage from −50 to 60 mV in 10 mV increments. (c) Experimental (n=21) and modelling steady-state I_K–V curves. Cell membrane was held at −40 mV before stepping the voltage from −90 to 50 mV in 10 mV increments. (d,e) AP initiation and conduction characteristics of BacNa_v mutant expressing E-HDFs obtained by computational modeling: upstroke velocity (d) and CV (e). For modelling, parameters derived from voltage-clamp measurements at 25 °C were scaled for the temperature of 37 °C. Highly hyperpolarized inactivation of WT and D60E channels prevented AP initiation. (f,g) Time constants of activation (f) and inactivation (g) of selected Na_vSheP D60X mutants compared with Na_vRosD G217A (RosDm), recorded in E-HEK293 cells at 25 °C (n=5). (h–j) When co-expressed with Kir2.1 in E-HEK293 cells, Na_vSheP mutants give rise to APs (h) with faster upstroke (i; n=10–18) and shorter duration (j; n=10–18) than Na_vRosD G217A (n=11 for i,j). (k) Na_vSheP D60X expressions in anisotropic E-HDF monolayers yield faster CV than RosDm expression (n=5–10). (l) Representative isochrone map of AP conduction in an electrically stimulated anisotropic monolayer of E-HDFs stably co-expressing Kir2.1, Na_vSheP D60N and Cx43 (compare also with Fig. 1j). *P<0.001 versus RosDm (i–k). All electrophysiological data obtained at 37 °C, unless otherwise specified. Error bars indicate s.e.m; statistical significance was determined by one-way analysis of variance, followed by Tukey's post-hoc test to calculate P-values.