Figure 3

All selected EE mutations variably alter voltage-dependent activation of homomeric K_v7.2 channels and disrupt their current enhancement upon diC8-PIP₂ inclusion. (a) Sites of selected EE mutations (L203P, L268F, K552T, and R553L) characterized in this study. These mutations are highlighted with red spheres on the C-alpha atoms of one subunit on the modeled tetrameric human K_v7.2 structure (ribbons) in complex with four CaM subunits (transparent green surfaces). (b) Localization of selected EE mutations are shown in red in the amino acid sequence of K_v7.2 (NP_742105.1). The EE mutations are shown in bold. The critical residues in S4 and the selectivity filter in the pore are underlined. (c–h) Whole cell voltage clamp recordings of macroscopic K⁺ currents in CHO hm1 cells transfected with GFP and K_v7.2 WT or EE mutants. Cells were held at -80 mV. Currents were evoked by depolarization for 1.5 s from −100 mV to +20 mV in 10 mV increments, followed by a step to 0 mV for 300 ms. To examine PIP₂ sensitivity of K_v7.2 channels, the recording was repeated with internal patch pipette solution containing diC8-PIP₂ (100 μM) which also contained EGTA to sequester free Ca²⁺. The raw current traces and data are shown in Supplementary Figs. S1–S2. (c) Immunoblot analyses of CHOhm1 cells reveal both monomeric bands (around 90 kD) and multimeric bands (around 180 and 270 kD) of K_v7.2 proteins. For clarity, cropped gel images are shown. Full-length gels can be found in Supplementary Fig. S7,a. (d) Representative recordings after subtraction of leak currents. Leak current was defined as non-voltage-dependent current from GFP-transfected cells. (e) Average peak current densities at all voltage steps. *p < 0.05, ***p < 0.005 based on one-way ANOVA Fisher’s test. (f) Average peak current densities at -20 mV (left) and + 20 mV (right). p values are computed from one-way ANOVA Tukey test. (g) Normalized conductance (G/Gmax) at all voltage steps. (h) Activation time constant (τ) at + 20 mV. The number of GFP-cotransfected cells that were recorded without diC8-PIP₂: K_v7.2 WT (n = 12), L₂03P (n = 17), L268F (n = 17), K552T (n = 13), or R553L (n = 13). The number of GFP-cotransfected cells that were recorded with diC8-PIP₂: K_v7.2 WT (n = 11), L₂03P (n = 14), L268F (n = 13), K552T (n = 11), or R553L (n = 11). Data shown represent the Ave ± SEM.