Cameron JM , Maljevic S , Nair U , Aung YH , Cogné B , Bézieau S , Blair E , Isidor B , Zweier C , Reis A , Koenig MK , Maarup T , Sarco D , Afenjar A , Huq AHMM , Kukolich M , Billette de Villemeur T , Nava C , Héron B , Petrou S , Berkovic SF .

1091593 National Health and Medical Research Council

	Figure 1. Facial features of patient 3 showing a large mouth, smooth philtrum, up‐slanting palpebral fissure, and dental enlargement.
	Figure 2. Functional expression of Kv3.1 variants in Xenopus laevis oocytes (A) Schematic of the Kv3.1 channel with putative positions of variants reported in this study (Orange ‐ Developmental and Epileptic Encephalopathy [DEE] variant; Green ‐ Developmental Encephalopathy variants [DE]; Light blue ‐ variant of uncertain significance). The dashed line shows the distal part of the C‐terminus that is present only in the longer transcript variant (Kv3.1b) indicating that the A513V variant is only found in this transcript. (B) Representative traces of whole‐cell currents recorded from Xenopus laevis oocytes injected with the same amount of cRNA encoding Kv3.1 wild‐type (Kv3.1a and Kv3.1b) and different variants during 0.5 sec voltage steps ranging from − 60 mV to + 60 mV. Insets show blown up tail currents, which were analyzed to generate conductance‐voltage relationships for WT and mutant channels shown in E. (C, D) Current amplitudes analyzed at the end of the voltage step to + 60 mV and normalized to the mean current amplitude of the corresponding WT recorded on the same day; Kv3.1a (n = 136), A421V (n = 62), R317H (n = 32), Q492X (n = 30), R339X (n = 48), and water (n = 35); Kv3.1b (n = 15), A513V (n = 17). ****P < 0.0001, using one‐way ANOVA with Dunnett’s multiple comparisons test (C). Mann–Whitney non‐parametric test (D) revealed P = 0.5. (E) Conductance‐voltage relationships for the WT transcripts and variants showing current amplitudes above the background level. V0.5 and k values were as follows: for Kv3.1a 23 ± 2 mV, 12.8 ± 0.6 (n = 37), for Kv3.1b 33.8 ± 1.1 mV, 12.80 ± 1.04 (n = 18), for Q492X 15 ± 3 mV, 12.7 ± 0.8 (n = 19), and for A513V 29 ± 2 mV, 11.6 ± 0.6 (n = 21); V0.5 Kv3.1a vs V0.5 Kv3.1b, ANOVA with Tukey’s multiple comparisons test.
	Figure 3. Dominant‐negative effect of Kv3.1 variants. (A) Representative current traces recorded from Xenopus laevis oocytes injected with the same amount of cRNA encoding Kv3.1a wild‐type with addition of either H2O or the same amount cRNA encoding Kv3.1 variants in a 1:1 ratio. (B) Current amplitudes analyzed at the end of the voltage step to + 60 mV and normalized to the mean current amplitude of WT + H2O recorded on the same day revealed a significant reduction for R317H and R339X but not for the A421V coexpression, ***P < 0.001, **P < 0.01 using one‐way ANOVA with Dunnett’s multiple comparisons test; WT + H2O (n = 111), WT + A421V (n = 62); WT + R317H (n = 11); WT + Q492X (n = 21); WT + R339X (n = 17). (C) Conductance‐voltage relationships of the WT and its coexpressions with A421V, R317H and R339X. V0.5 and slope factor (k) values were as follows: for WT + H2O 18.2 ± 1.4 mV, 12.4 ± 0.4 (n = 27), for WT + A421V 16.6 ± 1.8, 15.2 ± 0.4 (n = 36), for WT + R317H 21.1 ± 4.5 mV, 17.8 ± 1.9 (n = 8), for WT + Q492X 25 ± 3 mV, 13.9 ± 0.5 (n = 12), and for WT + R339X 15.8 ± 3.1 mV, 12.2 ± 1.1 (n = 15).

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