Trafficking-deficient G572R-hERG and E637K-hERG activate stress and clearance pathways in endoplasmic reticulum - PubMed (original) (raw)

Trafficking-deficient G572R-hERG and E637K-hERG activate stress and clearance pathways in endoplasmic reticulum

Ying Wang et al. PLoS One. 2012.

Abstract

Background: Long QT syndrome type 2 (LQT2) is the second most common type of all long QT syndromes. It is well-known that trafficking deficient mutant human ether-a-go-go-related gene (hERG) proteins are often involved in LQT2. Cells respond to misfolded and trafficking-deficient proteins by eliciting the unfolded protein response (UPR) and Activating Transcription Factor (ATF6) has been identified as a key regulator of the mammalian UPR. In this study, we investigated the role of ER chaperone proteins (Calnexin and Calreticulin) in the processing of G572R-hERG and E637K-hERG mutant proteins.

Methods: pcDNA3-WT-hERG, pcDNA3-G572R-hERG and pcDNA3-E637K-hERG plasmids were transfected into U2OS and HEK293 cells. Confocal microscopy and western blotting were used to analyze subcellular localization and protein expression. Interaction between WT or mutant hERGs and Calnexin/Calreticulin was tested by coimmunoprecipitation. To assess the role of the ubiquitin proteasome pathway in the degradation of mutant hERG proteins, transfected HEK293 cells were treated with proteasome inhibitors and their effects on the steady state protein levels of WT and mutant hERGs were examined.

Conclusion: Our results showed that levels of core-glycosylated immature forms of G572R-hERG and E637K-hERG in association with Calnexin and Calreticulin were higher than that in WT-hERG. Both mutant hERG proteins could activate the UPR by upregulating levels of active ATF6. Furthermore, proteasome inhibition increased the levels of core-glycosylated immature forms of WT and mutant hERGs. In addition, interaction between mutant hERGs and Calnexin/Calreticulin was stronger after proteasome inhibition, compared to WT-hERG. These results suggest that trafficking-deficient G572R-hERG and E637K-hERG mutant proteins can activate ER stress pathways and are targeted to the proteasome for degradation. Calnexin and Calreticulin play important roles in these processes.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Confocal imaging of WT-hERG and G572R-hERG/E637K-hERG channels in U2OS cells.

U2OS cells were transfected with WT-hERG, G572R-hERG and E637K-hERG plasmids and co-stained with anti-hERG (green) and anti-Calnexin or anti-Calreticulin (red) antibodies, as indicated. Calnexin and Calreticulin localize to the ER (top row) while WT-hERG (second row) localizes to both plasma membrane (indicated by white arrows) and cytoplasm. G572R-hERG (third row) and E637K-hERG (bottom row) mutants localize exclusively to the ER, as shown by the overlap with ER markers (Calnexin and Calreticulin) (scale bar 5 µm).

Figure 2

Figure 2. Analysis of WT-hERG, G572R-hERG and E637K-hERG protein expressions in HEK293 cells.

Lysates from HEK293 cells expressing WT-hERG, G572R-hERG or E637K-hERG were immunoblotted with anti-hERG antibody. Only WT-hERG expressing cells have both the fully-glycosylated, mature form and core-glycosylated, immature form of the hERG protein. Both G572R-hERG and E637K-hERG expressing cells only have the core-gylcosylated, immature forms of the hERG protein.

Figure 3

Figure 3. Association of hERG channels with Calnexin and Calreticulin.

Lysates from HEK293 cells expressing WT-hERG, G572R-hERG and E637K-hERG were immunoprecipitated with anti-Calnexin and anti-Calreticulin antibody and immunoblotted with anti-hERG antibody. The association of Calnexin/Calreticulin with the core-glycosylated, immature forms of G572R-hERG and E637K-hERG is much stronger than that with WT-hERG.

Figure 4

Figure 4. Activation of ATF6 by G572R-hERG and E637K-hERG.

HEK293 cells were transfected with WT-hERG, G572R-hERG or E637K-hERG plasmid. Cell lysates were subjected to Western blotting with anti-ATF6 antibody. The cleaved, activated form of ATF6 at 50 kDa is only detected in G572R-hERG and E637K-hERG expressing cells but not in WT-hERG expressing cells.

Figure 5

Figure 5. Degradation of G572R-hERG and E637K-hERG mutant channels through ubiquitin-proteasome pathway.

a, HEK293 cells were transfected with WT-hERG, G572R-hERG or E637K-hERG plasmid and then treated with 20 uM lactacystin (LACT), 50 uM ALLN or 100 uM leupeptin (LEUP) for 24 hours. The cell lysates were subjected to Western blotting with anti-hERG, anti-Calnexin and anti-Calreticulin antibodies. Proteasome inhibition leads to an increase in the core-glycosylated, immature forms of hERG protein with a more significant increase seen in G572R-hERG and E637K-hERG expressing cells in comparison with WT-hERG expressing cells. b, Transfected HEK293 cells were treated with proteasome inhibitors or leupeptin as indicated. Lysates were immunoprecipitated with anti-hERG antibody and immunoblotted with anti-ubiquitin antibody. Proteasome inhibition results in the accumulation of poly-ubiquitinated G572R-hERG/E637K-hERG and immature WT-hERG (135 kDa). c, Transfected HEK293 cells were treated with cycloheximide (CHX) and harvested at indicated time points. Lysates were immunoblotted with anti-hERG antibody. Protein levels of both G572R-hERG and E637K-hERG mutants are decreased after cycloheximide treatment with half-life less than 6 hrs.

Figure 6

Figure 6. Proteasome inhibition increases association of G572R-hERG/E637K-hERG with Calnexin/Calreticulin.

HEK293 cells were transfected with WT-hERG, G572R-hERG or E637K-hERG plasmid. Cells were then treated with proteasome inhibitors and immunoprecipitated with anti-Calnexin or anti-Calreticulin antibody and immunoblotted with anti-hERG antibody. In comparison to WT-hERG, treatment with either LACT or ALLN results in an increase in binding of Calnexin/Calreticulin to G572R-hERG or E637K-hERG.

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