“…1). Consistent with previous findings (Anderson et al 2006(Anderson et al , 2014Ke et al 2013Ke et al , 2014 we observed two protein bands corresponding to Kv11.1 channels: a fully glycosylated (FG) band representing membrane protein and a core glycosylated (CG) band representing intracellular protein ( Fig. 1A).…”
Key pointsr Most missense long QT syndrome type 2 (LQTS2) mutations result in Kv11.1 channels that show reduced levels of membrane expression.r Pharmacological chaperones that rescue mutant channel expression could have therapeutic potential to reduce the risk of LQTS2-associated arrhythmias and sudden cardiac death, but only if the mutant Kv11.1 channels function normally (i.e. like WT channels) after membrane expression is restored.r Fewer than half of mutant channels exhibit relatively normal function after rescue by low temperature. The remaining rescued missense mutant Kv11.1 channels have perturbed gating and/or ion selectivity characteristics.r Co-expression of WT subunits with gating defective missense mutations ameliorates but does not eliminate the functional abnormalities observed for most mutant channels.r For patients with mutations that affect gating in addition to expression, it may be necessary to use a combination therapy to restore both normal function and normal expression of the channel protein.
AbstractIn the heart, Kv11
“…1). Consistent with previous findings (Anderson et al 2006(Anderson et al , 2014Ke et al 2013Ke et al , 2014 we observed two protein bands corresponding to Kv11.1 channels: a fully glycosylated (FG) band representing membrane protein and a core glycosylated (CG) band representing intracellular protein ( Fig. 1A).…”
Key pointsr Most missense long QT syndrome type 2 (LQTS2) mutations result in Kv11.1 channels that show reduced levels of membrane expression.r Pharmacological chaperones that rescue mutant channel expression could have therapeutic potential to reduce the risk of LQTS2-associated arrhythmias and sudden cardiac death, but only if the mutant Kv11.1 channels function normally (i.e. like WT channels) after membrane expression is restored.r Fewer than half of mutant channels exhibit relatively normal function after rescue by low temperature. The remaining rescued missense mutant Kv11.1 channels have perturbed gating and/or ion selectivity characteristics.r Co-expression of WT subunits with gating defective missense mutations ameliorates but does not eliminate the functional abnormalities observed for most mutant channels.r For patients with mutations that affect gating in addition to expression, it may be necessary to use a combination therapy to restore both normal function and normal expression of the channel protein.
AbstractIn the heart, Kv11
“…Interestingly, these kinetics changes are similar to the kinetic changes on hERG when the N‐Cap (the first 23 amino acids of the protein) is deleted. Evidence from multiple groups shows that the N‐cap domain has a role in regulating WT hERG kinetics, and that the EAG domain (comprised of the N‐Cap and the PAS domain [amino acids 23–135]) interacts with various other regions of the channel to facilitate slow deactivation . Some of these important contacts have been demonstrated to be in the N‐Cap, and interaction of polar regions, as well as N‐cap flexibility, are crucial.…”
Introduction
Genetic mutations in KCNQ2 which encodes hERG, the alpha subunit of the potassium channel responsible for the IKr current, cause Long QT Syndrome, an inherited cardiac arrhythmia disorder. Electrophysiology techniques are used to correlate genotype with molecular phenotype to determine which mutations identified in patients diagnosed Long QT Syndrome are disease causing, and which are benign. These investigations are usually done using heterologous expression in cell lines, and often, epitope fusion tags are used to enable isolation and identification of the protein of interest.
Methods and Results
Here, we demonstrate through electrophysiology techniques and immunochemistry, that both N-terminal and C-terminal myc fusion tags may perturb hERG protein channel expression and kinetics of the IKr current. We also characterize the impact of two previously reported inadvertent cDNA variants on hERG channel expression and half-life.
Conclusion
Our results underscore the importance of careful characterization of the impact of epitope fusion tags and conformational sequencing prior to genotype-phenotype studies for ion channel proteins such as hERG.
“…The role of the N-terminal PAS domain in hERG folding is less clear-cut. Truncated hERG1a lacking the entire N-terminal region appears to traffic normally (Phartiyal et al 2008;Ke et al 2014); however, a significant number of LQT2-associated mutations reducing channel expression have been identified in this region (Harley et al 2012). Recent studies have investigated the impact of these mutations on both the thermal stability of the isolated domain and hERG gating kinetics (Harley et al 2012;Ke et al 2013).…”
“…; Ke et al . ); however, a significant number of LQT2‐associated mutations reducing channel expression have been identified in this region (Harley et al . ).…”
Long‐QT syndrome type‐2 (LQT2) is characterized by reduced functional expression of the human ether‐à‐go‐go related (hERG) gene product, resulting in impaired cardiac repolarization and predisposition to fatal arrhythmia. Previous studies have implicated abnormal trafficking of misfolded hERG as the primary mechanism of LQT2, with misfolding being caused by mutations in the hERG gene (inherited) or drug treatment (acquired). More generally, environmental and metabolic stresses present a constant challenge to the folding of proteins, including hERG, and must be countered by robust protein quality control (QC) systems. Disposal of partially unfolded yet functional plasma membrane (PM) proteins by protein QC contributes to the loss‐of‐function phenotype in various conformational diseases including cystic fibrosis (CF) and long‐QT syndrome type‐2 (LQT2). The prevalent view has been that the loss of PM expression of hERG is attributed to biosynthetic block by endoplasmic reticulum (ER) QC pathways. However, there is a growing appreciation for protein QC pathways acting at post‐ER cellular compartments, which may contribute to conformational disease pathogenesis. This article will provide a background on the structure and cellular trafficking of hERG as well as inherited and acquired LQT2. We will review previous work on hERG ER QC and introduce the more novel view that there is a significant peripheral QC at the PM and peripheral cellular compartments. Particular attention is drawn to the unique role of the peripheral QC system in acquired LQT2. Understanding the QC process and players may provide targets for therapeutic intervention in dealing with LQT2.
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