KV1.5–KVβ1.3 Recycling Is PKC-Dependent

Figures

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  Figure 1. PKC inhibition and RACK1 block results in a smaller magnitude of the KV1.5–KVβ1.3 current. Representative currents and I–V relationships in control conditions (a) along the activation curves, in a dashed line (right), after PKC inhibition with hispidin (b) and in the presence of anti-RACK1 antibody into the internal solution (d). Graphs shown in (c) show a summary of the magnitude KV1.5–KVβ1.3 current measured at the end of +60 mV depolarizing pulses under control conditions and after inhibition of PKC (with different inhibitors) and after RACK1 block with anti-RACK1 present in the internal solution. Note that data for calphostin C and bisindolylmaleimide II are taken from data previously reported in Macias et al., 2014 [10].

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  Figure 2. PKC inhibition decreases the KV1.5–KVβ1.3 levels in the plasma membrane. HEK293 cells expressing KV1.5–KVβ1.3 channels treated or not with calphostin C (3 μM for 2 h). Cells were treated with buffer (lanes 1 and 2) or with proteinase K externally (lanes 3 and 4). Left panel shows a representative experiment (a). The two arrows indicate the molecular weight at which digested KV1.5 (surface channel) and undigested KV1.5 (intracellular form) appear. β-actin was used as a loading control. Densitometric analysis of 5 experiments as those shown in panel a. Black bars represent, normalized to control, total protein levels; grey bars, normalized to control, proteinase-K-digested KV1.5–KVβ1.3 channel as a representation of plasma membrane levels (b). Each value represents the mean ± SEM of five experiments. * p < 0.05.

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  Figure 3. PKC inhibition modifies the KV1.5 recycling. Recycling was analyzed with a biotinylation test (see Materials and Methods). Western blot of internalized channels (biotinylated) at different times (30, 60 and 120 min) under control and after PKC inhibition with calphostin C (a). Densitometric analysis of all the experiments performed (b). Each value represents the mean ± SEM of three experiments. * p < 0.05.

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  Figure 4. The inhibition of PKC enhances the internalization of KV1.5–KVβ1.3 channels. Representative confocal images of HEK293 cells transfected with KV1.5–KVβ1.3 under control conditions and after treatment with calphostin C. Fluorescence profiles and fluorescence correlation of internalized channels and plasma membrane ones (bottom) in control and after calphostin-C treatment. Note that control cells show mostly a high red signal (plasma membrane channels), whereas those treated with calphostin C present a weaker red signal, but a much greater green signal (internalized channels, which did not recycle to the cell membrane).

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  Figure 5. Influence of PKC inhibition on KV1.5–HA–KVβ1.3 mobility in HEK293. (a) KV1.5–HA labelled by fluorescence staining of live cell under control conditions and after PKC inhibition by calphostin C treatment and visualized at different times. Bar colors show the time from the calphostin-induced PKC inhibition as the beginning of the experiment. (b) Frames extracted at 0, 30, 60 and 120 min from Supplemental Video 1A for control and Supplemental Video 2B for calphostin C. Scale bar represents 10 μm in all images.