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

<p>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.</p

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

<p>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.</p

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

<p>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).</p

Association of hERG channels with Calnexin and Calreticulin.

<p>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.</p

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

<p>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.</p

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

<p><b>a</b>, 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>b</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). <b>c</b>, 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.</p