Fig. 4: TBK1 activity regulates Kv3.3 channel inactivation. | Nature Communications

Fig. 4: TBK1 activity regulates Kv3.3 channel inactivation.

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

Fig. 4

a Western blots (top) demonstrating that co-immunoprecipitation of Hax-1 with either wild type or G592R Kv3.3 is prevented by a TBK1 inhibitor (MRT67307, 10 μM); Bottom panels show quantification of the relative levels of Hax-1 co-immunoprecipitated with Kv3.3 with and without inhibitor (values are mean ± SEM; n = 3 independent experiments, two-tailed unpaired t test). b Representative voltage clamp traces showing that TBK1 inhibition produces rapid inactivation of wild type and G592R Kv3.3 currents. A dashed line indicates 100 msec after the onset of depolarization. c Concentration-response plots for effects of TBK1 inhibitor on inactivation. Left plot quantifies inactivation at +70 mV, 100 ms after onset of depolarization (I(100 ms)/I(peak)), while right panel shows amounts of inactivation at the end of the 600 ms depolarizing pulses (I(SS)/I(peak)). Also shown are data for the ΔN1-78 Kv3.3 mutant (n = 4 independent experiments, data are mean ± SEM). d, e Representative voltage-clamp traces showing that overexpression of TBK1 slows inactivation of wild type and G592R Kv3.3 currents. Traces at +70 mV are shown in red and superimposed at right for comparison. f Quantification of effect of TBK1 overexpression on inactivation measured 600 ms after depolarization to +70 mV for wild type and mutant channels (values are mean ± SEM; TBK1 overexpression in wild type, n = 4; vector alone in wild type, n = 8; TBK1 overexpression in G592RKv3.3 cells, n = 5; vector alone in G592R Kv3.3 cells, n = 5, two-tailed paired t test). g and h, Potential interactions between Kv3.3 and TBK1 activity. Basal TBK1 activity is required for Hax-1 binding to the Kv3.3 C-terminus, which leads to formation of a channel-associated actin cytoskeleton (g left). Inhibition of TBK1 (g right) causes dissociation of Hax-1, allowing the Kv3.3 cytoplasmic N-terminus to occlude the pore (inactivation). G592R Kv3.3 channels bind more total TBK1 and pTBK1 than wild-type channels (h left) and have slower inactivation. As with wild-type channels, inhibition of TBK1 (h right) causes dissociation of Hax-1 and rapid inactivation. Source data and uncropped Western blots are provided as a Source Data file.

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