Hsp40 Chaperones Promote Degradation of the hERG Potassium Channel

Abstract

Loss of function mutations in the hERG (human ether-ago-go related gene or KCNH2) potassium channel underlie the proarrhythmic cardiac long QT syndrome type 2. Most often this is a consequence of defective trafficking of hERG mutants to the cell surface, with channel retention and degradation at the endoplasmic reticulum. Here, we identify the Hsp40 type 1 chaperones DJA1 (DNAJA1/Hdj2) and DJA2 (DNAJA2) as key modulators of hERG degradation. Overexpression of the DJAs reduces hERG trafficking efficiency, an effect eliminated by the proteasomal inhibitor lactacystin or with DJA mutants lacking their J domains essential for Hsc70/Hsp70 activation. Both DJA1 and DJA2 cause a decrease in the amount of hERG complexed with Hsc70, indicating a preferential degradation of the complex. Similar effects were observed with the E3 ubiquitin ligase CHIP. Both the DJAs and CHIP reduce hERG stability and act differentially on folding intermediates of hERG and the disease-related trafficking mutant G601S. We propose a novel role for the DJA proteins in regulating degradation and suggest that they act at a critical point in secretory pathway quality control.

Bag1 Co-chaperone Promotes TRC8 E3 Ligase-dependent Degradation of Misfolded Human Ether a Go-Go-related Gene (hERG) Potassium Channels

“…Over a 6-h chase in control cells, CG hERG levels decreased to around 60% of the starting amount, and the FG form increased to around 30% of initial CG hERG ( Fig. 2A), consistent with previous results (8). When Bag1 was overex-pressed, the final levels of CG and FG hERG were both markedly lower than in the control, at 40 and 20% of initial CG amounts, respectively ( Fig.…”

“…As established previously (8), hERG can be detected by Western blotting as two bands, a 135-kDa core-glycosylated (CG) immature form and a 155-kDa fully glycosylated (FG) mature form that had trafficked through the Golgi. All Bag1 isoforms were knocked down to less than 30% of the non-silencing control.…”

“…The chaperone Hsp90 is necessary for hERG trafficking (7). We found that chaperones Hsp70 (HSPA1A/B) and Hsc70 (HSPA8) are also important, as knockdown of their major co-chaperone DNAJA1/Hdj2 impairs trafficking (8). The DNAJ family cochaperones activate ATP hydrolysis and substrate binding by Hsc70/Hsp70, but individual DNAJs are biologically distinct (9).…”

“…HEK293 cells were grown on poly-L-lysine-coated plates. HeLa cells stably expressing hERG and the G601S and F805C mutants with an external HA tag after residue 443 were constructed as reported previously (8). Where indicated, HeLa cells stably expressing hERG-G601S and F805C were grown at 27°C for 1 day before lysis.…”

“…The DNAJ family cochaperones activate ATP hydrolysis and substrate binding by Hsc70/Hsp70, but individual DNAJs are biologically distinct (9). DNAJA2 promotes hERG degradation by non-productively increasing interactions with Hsc70/Hsp70 and its associated E3 ubiquitin ligase CHIP (8,10,11). Hsp70 may also be more active in hERG folding than Hsc70 (12).…”