Figure 6
From: Identifying mutation hotspots reveals pathogenetic mechanisms of KCNQ2 epileptic encephalopathy
The K552T and R553L mutations reduced CaM binding to Kv7.2 whereas none of the tested EE mutations affected Kv7.3 interaction with Kv7.2. (a,b) Co-immunoprecipitation of YFP-CaM with wild-type Kv7.2 (WT) or Kv7.2 containing EE mutations in the presence of EGTA. (a) Representative immunoblots of HEK293T cells expressing Kv7.2 and YFP-CaM. For clarity, cropped gel images are shown. Full-length gels can be found in Supplementary Fig. S9. (b) Quantification of immunoblots: untransfected/None (n = 6), YFP-CaM (n = 6), YFP-CaM cotransfection with Kv7.2 WT (n = 6), L203P (n = 3), L268F (n = 3), K552T (n = 3), or R553L (n = 3). (c,d) Co-immunoprecipitation of HA-Kv7.3 with wild-type Kv7.2 (WT) or Kv7.2 containing EE mutations in the presence of EGTA. (c) Representative immunoblots of HEK293T cells expressing Kv7.2 and Kv7.3. For clarity, cropped gel images are shown. Full-length gels can be found in Supplementary Figs. S9–10. (d) Quantification of immunoblots: untransfected cells (None: n = 3), or cells transfected with HA-Kv7.3 (n = 4), HA-Kv7.3 and Kv7.2 WT (n = 4), L203P (n = 3), L268F (n = 3), K552T (n = 4), or R553L (n = 3). GAPDH served as a loading control. Both monomeric Kv7.2 bands (around 90kD, arrows) and multimeric Kv7.2 bands (around 180 kD, 270 kD, and 370 kD) are observed in the IP samples and lysate in (a,c). Data represent the Ave ± SEM (*p < 0.05, ***p < 0.005 against Kv7.2 WT).
