Fig 2: CBD reverses the voltage-dependent shift induced by Kv7.2mut. Shown are conductance plots to test the effects of bath applied 1 μM CBD on the indicated combinations of Kv7 subunits expressed in tsA-201 cells. The mean values of Va are indicated in the inset bar plots(A and B) CBD applied to a combination of Kv7.2 wt + Kv7.3 (A) or Kv7.2 wt + Kv7.2mut + Kv7.3 (B) invokes a significant negative shift in Va.(C) CBD (1 μM) induces a similar magnitude negative shift of Va for Kv7.2 wt + Kv7.2mut + Kv7.3 in the presence of the CB1/CB2 receptor blockers 0.5 μM AM-251 and 0.5 μM SR144528.All average values are mean ± SEM and sample numbers provided in brackets. ∗∗p < 0.01, ∗∗∗p < 0.001, Paired sample Student’s t test. Number of cells was shown in brackets.

Fig 3: A Kv7.2 mutation reduces current amplitude of Kv7.2 + Kv7.3 channels by shifting voltage dependence and reducing channel conductance(A and B) Schematic diagram of the Kv7.2 channel (A, Uniprot O43526) with 6 transmembrane domains (1–6) and the N- and C-termini positioned in the cytosol. A region of interest in (A) (box) is expanded in (B) to illustrate the position of a H260del mutation in the external membrane loop between transmembrane domains 5 and 6.(C) Representative whole-cell recordings from tsA-201 cells with the indicated Kv7 channel subunits transiently expressed and depolarized from −80 mV to 40 mV in 10 mV steps followed by a step to 0 mV.(D) Mean current amplitudes of the indicated Kv7 subunits when evoked with the protocol shown in (C). Sample numbers are shown in brackets.(E) Mean-dot plots of current density (pA/pF measured at 10 mV) for whole-cell recordings of Kv7.2 wt or Kv7.2mut when expressed in tsA-201 cells alone (p = 0.0311) or when coexpressed with Kv7.3 (p < 0.0001, post hoc Tukey’s test following two-way ANOVA).(F and G) Conductance plots for the indicated expressed Kv7 channel subunits, with bar plots of mean Va (insets). The Kv7.2mut significantly shifts the channel activation curves in the depolarizing direction (F, p = 0.007; G, p = 0.00017, two sample Student’s t test).(H) Mean-dot plots for the indicated expressed Kv7 channel subunits show a reduction in maximal conductance when Kv7.2mut is coexpressed with either Kv7.2 wt (p = 0.0323) or with Kv7.2 wt + Kv7.3 (p < 0.0001, post hoc Tukey’s test following two-way ANOVA).All average values are mean ± SEM and number of cells provided in brackets. ∗p < 0.05, ∗∗∗p < 0.001. Images in (A and B) were constructed with the online Protter tool (http://wlab.ethz.ch/protter/) based on UniProt entry number O43526. The C-terminus in (A) is truncated for display purposes.

Fig 4: A Kv7.2H260del mutant changes the balance of surface expression of Kv7.2 and Kv7.3 subunits(A-D) Transfected tsA201 cells were lysed after treatment with Sulfo-NHS-SS-biotin to identify surface proteins by immunocytochemical detection. Western blots of Kv7.2. (A and B) and Kv7.3 (C and D) in the total lysate and the biotinylated (cell membrane) fraction were probed with rabbit anti-Kv7.2 antibody and rabbit anti-Kv7.3 antibody, respectively. A clean biotinylated fraction of the lysate was confirmed with the absence of GAPDH in cell membrane samples. Bar graphs of mean values (B) for surface Kv7.2 presented as the ratio of biotinylated Kv7.2 to total Kv7.2 in different expression conditions reveals more Kv7.2 protein at the membrane in the Kv7.2mut transfected cells (p = 0.021, Mann-Whitney test, n = 4). D) Membrane Kv7.3 expression is decreased upon coexpression with Kv7.2mut (p = 0.021, Mann-Whitney test, n = 4).(E and F) Total expression of Kv7.2mut was significantly increased when coexpressed with Kv7.3 (E) (p = 0.0072, Mann-Whitney test, n = 5), while the total level of Kv7.3 is not affected (F) (p = 0.6558, Mann-Whitney test, n = 5).Data in bar plots of (E and F) are expressed in relation to GAPDH. Average values are mean ± SEM. ∗p < 0.05, Mann-Whitney test. N represents trials repeated.

Fig 5: The Kv7.2mut subunit associates with Kv7.2 wt and Kv7.3 with nanometer proximities(A) Confocal images of tsA-201 cells expressed with Kv7.2wt-EGFP + Kv7.2wt-mKate for detection of FRET. Only cells with both EGFP and mKate2 fluorescence are measured for the intensity of the EGFP channel after dye unmixing (see STAR Methods).(B) Spectral images excited at 488 nm from two representative groups before and after photobleaching with 552 nm excitation. The intensity-wavelength plots of the ROI (circled) before and after photobleaching are presented on the right, with the insets showing the intensity scale for green fluorescence. An increase in the intensity of EGFP after photobleaching mKate in the cells expressing Kv7.2wt-EGFP + Kv7.2wt-mKate identifies FRET between EGFP and mKate.(C) Quantification of the change of EGFP intensity under different expression combinations shows Kv7.2mut-EGFP FRETs with Kv7.2wt-mKate or Kv7.3-mKate, indicating a closeness, proximity of these channel subunits. Cells expressing Kv7.2wt-EGFP and mKate2 serve as a non-FRET control which exhibited a decrease of ∼5% EGFP intensity after photobleaching.Scale bars in (A), 20 μm. All average values are mean ± SEM, ∗p < 0.05; ∗∗p < 0.01, ∗∗∗p < 0.001, Student’s t test. The sample sizes in the bar graphs represent the number of cells analyzed.