Abstract 16522: Structural Insights Into Destabilizing HERG PAS Domain Mutations Linked to Long Qt Syndrome
Introduction: Hundreds of HERG potassium channel mutations are linked to type 2 Long QT Syndrome (LQT2). Most mutations are trafficking defective but correctable by culturing cells at reduced temperature (27 C) or with the pore blocker E4031. For a few intracellular Per-ARNT-Sim (PAS) domain mutations, trafficking phenotypes have been shown to correlate with PAS domain stability. Since HERG intracellular domains are mutational hotspots containing 1/3 (>100) of all LQT2 mutations, domain destabilization may be a major mechanism underlying LQT2. However, most mutations have not been studied and structural insights are lacking.
Results: Using a recombinant PAS domain solubility assay as a proxy for stability, we tested 57 mutations and found a strong correlation between domain solubility and trafficking phenotype (trafficking defective and uncorrectable < temperature correctable < temperature and E4031 correctable < trafficking competent). For buried residues V41 and C64, we used the protein stability prediction tool FoldX and immunoblot to show that domain stability and trafficking phenotype becomes more severe as the side chain becomes larger suggesting overpacking of the PAS domain core as a mechanism for LQT2-V41F and LQT2-C64W. This was further validated by designing second-site suppressor mutations to relieve potential side chain clashes. Immunoblot analysis showed that trafficking of LQT2-V41F is improved at 27 C with C39G or C64G, while C64A improved trafficking at 37 C. Immunoblot and current density analysis showed that trafficking of LQT2-C64W (3.8±0.8 pA/pF, n=4) is improved at 37 C with C39G (37.8±3.5 pA/pF, n=3) or Q61G (69.6±12.7 pA/pF, n=3) and LQT2-I42N (2.2±0.7 pA/pF, n=3) is improved at 37 C with Q61G (41.9±6.3 pA/pF, n=6). Additionally, site-saturation mutagenesis of residue V113 revealed loss of backbone (and not side chain) interactions as the structural basis for LQT2-delV113.
Conclusions: This study identifies HERG PAS domain destabilization as a major mechanism underlying LQT2 and demonstrates a novel bioinformatics and structure-based method for analyzing disease-associated HERG mutations. These results are also a proof of concept that strategies to improve HERG domain stability could be a viable therapeutic approach.
Author Disclosures: C.L. Anderson: None. C.E. Kuzmicki: None. R.R. Childs: None. C.J. Hintz: None. C.T. January: None. R.C. Balijepalli: None.
- © 2015 by American Heart Association, Inc.