A novel loss-of-function mutation of the voltage-gated potassium channel Kv10.2 involved in epilepsy and autism

Figures

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  Fig. 1. Electrophysiological properties of WT and N856H Kv10.2 channels expressed in COS7 cells. A Current recordings obtained in COS7 cells transfected with WT or N856H Kv10.2, as indicated, after applying the pulse protocol shown on the top. B Current–voltage relationship obtained after plotting the amplitude of the current measured at the end of the 3-s pulses versus membrane potential, as in A. C Normalised current recordings obtained after depolarising cells expressing WT or N856H from −80 mV to + 50 mV. D Time constant of activation of WT or N856H at + 50 mV. E Voltage dependence of the time constant of activation of WT or N856H. n = 12 for WT Kv10.2, n = 11 for N856H Kv10.2. *p < 0.05, **p < 0.01, ***p < 0.001

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  Fig. 2. N856H Kv10.2 mutant primary cells isolated from the patient display a strong growth defect and enhanced terminal differentiation. Healthy keratinocytes from two healthy infantile donors were used as control (KCT) compared with mut keratinocytes containing the mutation (Kmut). A Representative clonal expansion, as monitored by clonogenicity assays (n = 3 triplicates per essay, 2 essays per healthy strain). B Number of cells harvested in A at confluence of healthy strains, relative to KCT (n = 2, 2 essays per healthy strain). C Representative phase contrast microphotograph of KCT or Kmut cells as indicated. Dotted lines enclose keratinocyte colonies, surrounded by a fibroblastic feeder layer (see “Materials and methods”). Black arrow points to a typical detaching differentiated cell that were very frequent in the mutant cultures. Scale bar, 50 μm. D Bar histogram shows the percentage of cells with high size and complexity, by light scattering parameters (HS), typical of differentiated keratinocytes, as quantified by flow-cytometry (n = 2, as in A). E Expression of the epidermoid differentiation marker involucrin n KCT or Kmut, as indicated, measured by immunodetection and flow…

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  Fig. 3. Pharmacological treatments with CAIs recover expansion capacity of the mutant primary cells isolated from the patient without significantly altered the cell cycle. A Representative flow cytometry histograms for the epidermoid differentiation marker involucrin of KCT or mutant Kmut cells as indicated. Cells were treated with 10 μM brinzolamide (BZA) for 6 days as indicated. B Bar histogram shows percentage of KCT or Kmut involucrin positive cells, as quantified by flow cytometry (n = 2). ns: non-significant. C Clonal expansion capacity of KCT upon 10 μM AZA or RGB 7 days treatments. Cells plated at high density, Kmut double number of cells than KCT, colonies in pink (n = 3 in each essay, four essays, with KCT cells from two different individuals). D Number of cells in C, harvested at confluence of KCT. E cell cycle analyses of cells in D, as determined by DNA content stained with Propidium Iodide. *p < 0.05, **p < 0.01

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  Fig. 4. Electrophysiological measurements of N856H Kv10.2 channel expressed in COS7 cells, in presence or absence of AZA or RTG drugs. A Original current recordings of N856H Kv10.2 channels in the absence or presence of AZA, as indicated, after applying the pulse protocol shown on the top. Current traces are shown in 20-mV increments for clarity. B Current–voltage relationship obtained after plotting the amplitude of the current measured at the end of the 3-s pulses versus membrane potential in the absence and in the presence of AZA. C Time constant of activation of N856H Kv10.2 mutant channels at + 50 mV cells in the absence or in the presence of AZA. D–F As in A–C but with RTG instead of AZA. G For comparison, WT (wild type protein) Kv10.2 currents with the exponential fit of activation. H Current recordings elicited after the activation of N856H Kv10.2 mutant channels in the absence or presence of AZA. I Same as in H but with RTG instead of AZA. Data are expressed as the mean ± SEM of n = 4 (for AZA) and n = 5 (for RTG). *p < 0.05, ***p < 0.001