Abstract
Long QT Syndrome (LQTS) is an acquired or inherited disorder characterized by prolonged QT interval, exertion-triggered arrhythmias, and sudden cardiac death. One of the most prevalent hereditary LQTS subtypes, LQT2, results from loss-of-function mutations in the hERG channel, which conducts IKr, the rapid component of the delayed rectifier K+ current, critical for cardiac repolarization. The majority of LQT2 mutations result in Class 2 deficits characterized by impaired maturation and trafficking of hERG channels. Here, we have developed a high-throughput flow cytometric assay to analyze the surface and total expression of wild-type (WT) and mutant hERG channels with single-cell resolution. To test our method, we focused on 16 LQT2 mutations in the hERG Per-Arnt-Sim (PAS) domain that were previously studied via a widely used biochemical approach that compares levels of 135-kDa immature and 155-kDa fully glycosylated hERG protein to infer surface expression. We confirmed that LQT2 mutants expressed in HEK293 cells displayed a decreased surface density compared to WT hERG, and were differentially rescued by low temperature. However, we also uncovered some notable differences from the findings obtained via the biochemical approach. In particular, three mutations (N33T, R56Q, and A57P) with apparent WT-like hERG glycosylation patterns displayed up to 50% decreased surface expression. Furthermore, despite WT-like levels of complex glycosylation, these mutants have impaired forward trafficking, and exhibit varying half-lives at the cell surface. The results highlight utility of the surface labeling/flow cytometry approach to quantitatively assess trafficking deficiencies associated with LQT2 mutations, to discern underlying mechanisms, and to report on interventions that rescue deficits in hERG surface expression.
Highlights
Long QT Syndrome (LQTS) is an inherited or acquired disorder characterized by delayed cardiac action potential repolarization, which predisposes to polymorphic ventricular tachycardias, syncope, and sudden cardiac death (SCD) (Moss and Kass, 2005; Bohnen et al, 2016)
Human embryonic kidney (HEK293) cells transiently transfected with wild-type (WT) bungarotoxin binding site (BBS)-hERG-YFP displayed robust fluorescence signals for total and surface channel pools when imaged by confocal microscopy (Figure 2B)
By comparing results from the flow cytometric analyses of 16 PAS domain mutant hERG channels to published data of the same mutations assessed by the biochemical approach, we find areas of concordance that validates the assay, and some discrepancies that highlight advantages of the flow cytometry method
Summary
Long QT Syndrome (LQTS) is an inherited or acquired disorder characterized by delayed cardiac action potential repolarization, which predisposes to polymorphic ventricular tachycardias (torsade de pointes), syncope, and sudden cardiac death (SCD) (Moss and Kass, 2005; Bohnen et al, 2016). Loss-of-function mutations in several genes have been linked to LQTS (LQT1-LQT13), with around 70% occurring in genes encoding pore-forming subunits for the primary repolarizing K+ currents in ventricular cardiomyocytes – KCNQ1 (LQT1) and hERG (LQT2) (Bohnen et al, 2016). The hERG potassium channels assemble as a tetramer of four Kv11.1 α1 pore-forming subunits, and conduct IKr, the rapid component of the delayed rectifier K+ current (Trudeau et al, 1995). It has become apparent that the vast majority (∼88%) of LQT2 mutations are Class 2 type, featuring compromised channel trafficking to the plasma membrane (Anderson et al, 2006, 2014; Smith et al, 2016). Understanding the mechanisms regulating hERG trafficking and how these may be dysregulated in disease is important for molecular insights into the pathophysiology of LQT2
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