Figure 5
From: Identifying mutation hotspots reveals pathogenetic mechanisms of KCNQ2 epileptic encephalopathy
The L268F and K552T mutations decreased current expression of heteromeric Kv7 channels whereas all selected EE mutations disrupted their current potentiation by diC8-PIP2 inclusion. Whole cell patch clamp recordings were measured using the voltage clamp protocol described in Fig. 3 from GFP-positive CHOhm1 cells cotransfected with Kv7.3 and Kv7.2 WT (1:1 ratio) or Kv7.3, Kv7.2 WT, Kv7.2 mutant (2:1:1 ratio). The raw current traces and data are shown in Supplementary Figs. S6 and S7. (a) Representative leak-subtracted current traces. (b) Average leak-subtracted peak current densities at all voltage steps. *p < 0.05, ***p < 0.005 based on one-way ANOVA Fisher’s test. (c) Average leak-subtracted peak current densities at -20 mV (top) and at + 20 mV (bottom). p values are computed from one-way ANOVA Tukey test. (d) Normalized conductance (G/Gmax) at all voltage steps. (e) Activation time constant (τ) at + 20 mV. The number of GFP-cotransfected cells that were recorded without diC8-PIP2: Kv7.2 WT (n = 14), L203P (n = 15), L268F (n = 18), K552T (n = 15), or R553L (n = 15). The number of GFP-cotransfected cells that were recorded with diC8-PIP2: Kv7.2 WT (n = 12), L203P (n = 12), L268F (n = 16), K552T (n = 14), or R553L (n = 15). (f) Immunoblot analyses of CHOhm1 cells co-transfected with Kv7.2 wild-type or mutant and Kv7.3 reveal both monomeric bands (around 90 kD) and multimeric bands (around 180 kD, 270 kD, and 370 kD) of Kv7.2 proteins. For clarity, cropped gel images are shown. Full-length gels can be found in Supplementary Fig. S8,b. Data represent the Ave ± SEM. ***p < 0.005.
