Fig. 5: The G592R Kv3.3 channel causes trafficking of Hax-1 into inclusions within lysosomes. | Nature Communications

Fig. 5: The G592R Kv3.3 channel causes trafficking of Hax-1 into inclusions within lysosomes.

From: Cerebellar Kv3.3 potassium channels activate TANK-binding kinase 1 to regulate trafficking of the cell survival protein Hax-1

Fig. 5

Light level immunolocalization of Hax-1 in untransfected CHO cells (a) and cells expressing wild type (b) or G592R Kv3.3 (c). Left panels show low power views and right panels show Hax-1 immunostaining at higher magnification. Scale bars under c apply to all pairs of panels in (a–c). In cells expressing G592R Kv3.3, the peripheral staining for Hax-1 resembles that of cells expressing the wild-type channel (green asterisks in right panel of e). With the mutant channel, however, a high proportion of cells develop dense Hax-1 inclusions (multivesicular bodies), associated with extracellular deposits (red arrows in right panel of e). Images are representative of 20 independent fields of cells. Electron immunolocalization of Hax-1 in untransfected CHO cells (d) and those expressing wild type (e) or G592R Kv3.3 (f). Hax-1 staining is found uniformly under the plasma membrane in cells expressing wild type Kv3.3. In G592R Kv3.3 cells, Hax-1 is localized not only to the plasma membrane but in large late endosomal/lysosomal structures. Panels are representative of 23, 20, and 87 images of untransfected CHO, wild-type Kv3.3 and G592R Kv3.3 cells, respectively. g Quantification of overall intensity of Hax-1 immunostaining in plates of untransfected CHO cells and cells expressing wild type or G592R Kv3.3 (n = 20 fields. Data are presented as mean ± SEM, one way ANOVA, Tukey’s multiple comparison test). h Quantification of the percentages of cells with dense Hax-1-containing inclusions in the three cell types (n = 20 fields of cells in each condition. Data are presented as mean ± SEM. One-way ANOVA, Tukey’s multiple comparisons test). i Immunoblots with quantification of the increase in levels of multivesicular body marker CD63 in cells expressing mutant Kv3.3. This increase was reversed by pretreatment of the cells with the TBK1 inhibitor 10 µM MRT67307 (n = 4 independent experiments; data are presented as mean ± SEM. Two-tailed paired t test). Source data and uncropped Western blots are provided as a Source Data file.

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