Figure 7
From: Identifying mutation hotspots reveals pathogenetic mechanisms of KCNQ2 epileptic encephalopathy
L268F, K552T, and R553L mutations decreased enrichment of heteromeric Kv7 channels at the axonal surface in cultured hippocampal neurons. Immunostaining of surface Kv7.3 and total Kv7.2 containing an extracellular hemagglutinin epitope (HA-Kv7.3) in healthy hippocampal neurons cotransfected with Kv7.2 WT or Kv7.2 with EE mutations L203P, L268F (a–e), K552T, and R553L (f–j). (a,f) Representative images of surface HA-Kv7.3 (Upper) as pseudo-color that display differences in the surface HA intensities from high (red) to low (blue). Total Kv7.2 (Middle-inverted gray) and the AIS identified by antibodies for phospho IκBα-Ser32 (14D4) (Lower-fluorescence) are shown in same neurons. Arrows mark the AIS. Scale bars: 25 μm. (b,g) Normalized background-subtracted mean intensities of surface HA fluorescence from neurons expressing WT and EE mutant HA-Kv7.3/Kv7.2 channels. The number of transfected neurons that were analyzed in Fig. 7b: WT (n = 41), L203P (n = 39), L268F (n = 34), untransfected (UT) (n = 20). The number of transfected neurons that were analyzed in Fig. 7g: WT (n = 42), K552T (n = 34), R553L (n = 21), untransfected (UT) (n = 19). The raw data from 3 independent experiments are shown in Supplementary Fig. S11. (c,h) Surface HA intensity ratio at distal axon over dendrite. (d,i) Background-subtracted mean intensities of total Kv7.2 fluorescence in Kv7.2-transfected neurons and untransfected neurons (UT, blue dotted lines). The number of transfected neurons that were analyzed in Fig. 7d: Kv7.2 WT (n = 14), L203P (n = 15), L268F (n = 14), UT (n = 14). The number of transfected neurons that were analyzed in Fig. 7i: Kv7.2 WT (n = 17), K552T (n = 23), R553L (n = 14), UT (n = 13). (e,j) Background-subtracted mean intensities of surface HA fluorescence from the transfected neurons treated with vehicle (CTL) control or dynamin inhibitory peptide (DIP). The number of transfected neurons that were analyzed in Fig. 7e: WT + CTL (n = 14), WT + DIP (n = 13), L268F + CTL (n = 8), L268F + DIP (n = 8). The number of transfected neurons that were analyzed in Fig. 7j: WT + CTL (n = 8), WT + DIP (n = 13), L268F + CTL (n = 6), L268F + DIP (n = 7). Data represent the Ave ± SEM (*p < 0.05, **p < 0.01, ***p < 0.005 against WT channels).
