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. 2021 Apr 29;10:e68274. doi: 10.7554/eLife.68274

Figure 2. KCTD subtype expression in the IPN and interaction of Cav2.3 with KCTD8 and KCTD12b in vitro.

(A–C) Confocal images of immunofluorescence signals of KCTD8, KCTD12, and KCTD12b in the IPN of WT (upper panels) and respective KO mice (lower panels). KCTD8 immunofluorescence was present in all IPN subnuclei, whereas KCTD12 and KCTD12b signals were observed only in the rostral/central but not the lateral IPN subnuclei. Scale bars: 100 µm. (D) Co-immunoprecipitation from total cell lysates of HEK293T cells transfected with Flag-tagged KCTDs and Cav2.3. Immunoprecipitation of Cav2.3 co-precipitated KCTD8 and KCTD12b, but not KCTD12. Input lanes (bottom) indicate expression of the tagged proteins in the cell lysates. (E) KCTDs are co-localized and interact with Cav2.3 at the cell surface of HEK293T cells. The three input lanes to the right show expression of Flag-tagged KCTD8 (top) and Flag-tagged KCTD12b (bottom) in the cytosol, the total membrane fraction (‘total membrane’) and the plasma membrane fraction, from left to right. The two IP lanes to the left show that immunoprecipitation of Cav2.3 in the total membrane fraction (‘total membranes’) and the plasma membrane fraction co-precipitated KCTD8 (top) and KCTD12b (bottom), from left to right. Membrane-bound Cav2.3 (bottom lanes) is expressed in the total membrane fraction and the plasma membrane fraction, but absent from the cytosol fraction. (F) Whole-cell recordings from HEK293 cells stably expressing Cav2.3. Ba2+ current densities measured in response to a single depolarizing voltage step from −80 to 10 mV were significantly increased in KCTD8 co-transfected cells. *p<0.05, **p<0.01 one-way ANOVA with Tukey post hoc test. (G) Current density-to-voltage relationship demonstrating higher current densities in KCTD8-transfected cells compared with Control- and KCTD12b-transfected cells. ****p<0.0001 two-way ANOVA with Tukey post hoc test. (H) Activation and inactivation curves in Control-, KCTD8-, and KCTD12b-transfected cells. **p<0.01, two-way ANOVA with Tukey post hoc test. See also Figure 2—figure supplement 1.

Figure 2—source data 1. KCTD subtype expression in the IPN and interaction of Cav2.3 with KCTD8 and KCTD12b in vitro.

Figure 2.

Figure 2—figure supplement 1. Quantification of KCTDs at the plasma and total membrane in HEK cells co-expressing Cav2.3, auxiliary β3 and α2δ1 subunits.

Figure 2—figure supplement 1.

Fold increase of KCTD signal at the plasma membrane (PM) compared to the averaged KCTD signal in total membrane (TM).
Figure 2—figure supplement 1—source data 1. Quantification of KCTDs at the plasma and total membrane in HEK cells co-expressing Cav2.3, auxiliary β3 and α2δ1 subunits.