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. 2006 Feb 17;281(7):4069-74.
doi: 10.1074/jbc.M511765200. Epub 2005 Dec 16.

Mechanisms of pharmacological rescue of trafficking-defective hERG mutant channels in human long QT syndrome

Qiuming Gong  1 , Melanie A Jones, Zhengfeng Zhou
Affiliations

Mechanisms of pharmacological rescue of trafficking-defective hERG mutant channels in human long QT syndrome

Qiuming Gong et al. J Biol Chem. .

Abstract

Long QT syndrome type 2 is caused by mutations in the human ether-a-go-go-related gene (hERG). We previously reported that the N470D mutation is retained in the endoplasmic reticulum (ER) but can be rescued to the plasma membrane by hERG channel blocker E-4031. The mechanisms of ER retention and how E-4031 rescues the N470D mutant are poorly understood. In this study, we investigated the interaction of hERG channels with the ER chaperone protein calnexin. Using coimmunoprecipitation, we showed that the immature forms of both wild type hERG and N470D associated with calnexin. The association required N-linked glycosylation of hERG channels. Pulse-chase analysis revealed that N470D had a prolonged association with calnexin compared with wild type hERG and E-4031 shortened the time course of calnexin association with N470D. To test whether the prolonged association of N470D with calnexin is due to defective folding of mutant channels, we studied hERG channel folding using the trypsin digestion method. We found that N470D and the immature form of wild type hERG were more sensitive to trypsin digestion than the mature form of wild type hERG. In the presence of E-4031, N470D became more resistant to trypsin even when its ER-to-Golgi transport was blocked by brefeldin A. These results suggest that defective folding of N470D contributes to its prolonged association with calnexin and ER retention and that E-4031 may restore proper folding of the N470D channel leading to its cell surface expression.

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Figures

Figure 1
Figure 1
Association of hERG channels with calnexin. Cell lysates of HEK293 cells stably expressing wild type hERG, N470D, N598Q or untransfected HEK293 cells (control) were immunoprecipitated with anti-calnexin antibody. The precipitates were subjected to SDS-polyacrylamide gel electrophoresis and immunoblotted with anti-hERG antibody. The lower panels are inputs probed by anti-calnexin and anti-hERG antibodies.
Figure 2
Figure 2
Time course of the association of calnexin with wild type hERG and N470D. HEK293 cells stably transfected with wild type hERG (A) and N470D, treated without (B) or with 5 μM E-4031 (C), were pulse-labeled for 30 min and chased for the indicated periods of time. The cells lysates were immunoprecipitated with polyclonal anti-calnexin antibody followed by reimmunoprecipitation with anti-hERG antibody. The immunoprecipitates were subjected to SDS-polyacrylamide gel electrophoresis and autoradiography. The amount of hERG was quantified by phosphorimaging analysis and plotted as percentage of the value at time point 0 (D). The data points are means ± standard error of the mean from three independent experiments.
Figure 3
Figure 3
Protease sensitivity of wild type hERG and the N470D mutant. Total cell membranes from cells expressing wild type hERG and the N470D mutant were treated with trypsin followed by immunoblotting with anti-hERG antibody.
Figure 4
Figure 4
Effect of brefeldin A treatment on protease sensitivity of wild type hERG and the N470D mutant. A: HEK293 cells stably transfected with wild type hERG were treated with 5 μM brefeldin A for the indicated times followed by immunoblot analysis with anti-hERG antibody. B: HEK293 cells expressing wild type hERG and the N470D mutant were treated with brefeldin A for 24 h prior to the isolation of total cell membranes. The membranes were treated with trypsin followed by immunoblotting with anti-hERG antibody.
Figure 5
Figure 5
Detergent extractability of wild type hERG and the N470D mutant. HEK293 cells stably transfected with wild type hERG or N470D mutant were solubilized in lysis buffer containing different concentrations of Triton X-100. Detergent soluble and insoluble proteins were separated by centrifugation. Equal fractions from supernatants (S) and pellets (P) were analyzed by immunoblotting with anti-hERG antibody.
Figure 6
Figure 6
Effect of E-4031 on protease sensitivity of the N470D mutant. HEK293 cells stably transfected with the N470D mutant were cultured in the presence of 5 μM E-4031 (N470D+E-4031) or 5 μM E-4031 plus 5 μM BFA (N470D+E-4031+BFA) for 24 h. Total cell membranes from the cells were treated with trypsin followed by immunoblotting with anti-hERG antibody.
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