Development of a High-Throughput Flow Cytometry Assay to Monitor Defective Trafficking and Rescue of Long QT2 Mutant hERG Channels

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.

Differential Modulation of IK and ICa,L Channels in High-Fat Diet-Induced Obese Guinea Pig Atria

“…Quantification of mean QD 655 fluorescence indicated that BBS-hERG 1a-YFP alone or when combined with hERG 1b-CFP displayed similar surface density (Figure 5C, 2% increase or from surface density 1.0, n = 12 separate cultures, to 1.02 ± 0.05, 8 separate cultures), in line with published data, indicating hERG 1a traffics independently of hERG 1b to the cell surface in heterologous cells (Phartiyal et al, 2008). Negative controls expressing untagged hERG 1a and 1b displayed no QD 655 fluorescence (averaged surface density was 0.18 ± 0.006, five separate cultures), confirming that the red fluorescence signal was specific and only detected the BBS tag, consistent with our previous reports on KCNQ1 trafficking in HEK293 cells and cardiomyocytes (Aromolaran et al, 2014; Kanner et al, 2018).…”

The mutation L69P in the PAS domain of the hERG potassium channel results in LQTS by trafficking deficiency

“…It is well established that many hERG mutants with trafficking deficits can be rescued by incubating cells at low temperature; this treatment presumably supports a correct folding of the protein, which is otherwise compromised by the mutation [13,[25][26][27]. To examine whether the L69P mutation can be rescued in this manner we repeated the same experiments as in Figure 3(Ab) with cells incubated at 27°C.…”

“…For surface labeling experiments, the BBS-hERG-YFP construct was utilized as previously described [13,14], with the 13-residue bungarotoxin-binding site (BBS) introduced in the extracellular S3-S4 loop of hERG. Cell surface and total ion-channel pools were assayed by flow cytometry in live, transfected HEK293 cells [13,15]. Briefly, 48 h post-transfection, cells cultured in 12-well plates gently washed with ice cold PBS containing Ca 2+ and Mg 2+ (in mM: 0.9 CaCl 2 , 0.49 MgCl 2 , pH 7.4), and then incubated for 30 min in blocking medium (DMEM with 3% BSA) at 4ºC.…”

“…To directly assess differences in the surface expression of wt and the L69P mutant, a flow cytometric assay was performed as previously reported [13]. HEK293 cells were transfected with the BBS-hERG-YFP construct, engineered with a 13-residue high-affinity bungarotoxin binding site (BBS) extracellular tag for the efficient detection of surface channels in non-permeabilized cells with Alexa Fluor 647-conjugated bungarotoxin (BTX647).…”

Long QT Syndrome Type 2: Emerging Strategies for Correcting Class 2 KCNH2 (hERG) Mutations and Identifying New Patients

“…As Kv11.1a proteins traffic through the Golgi apparatus, the N-linked glycans on Kv11.1 α-subunit channel proteins are terminally glycosylated and their molecular mass increases to~155 kDa [34]. At the cell surface membrane, Kv11.1 channels are continually internalized and recycle back to the cell surface membrane every couple of minutes for several hours before they are targeted for degradation in lysosomes [35,36]. There are several excellent reviews that detail Kv11.1 channel trafficking, chaperones, and the biophysical function of Kv11.1 channels/I Kr [15,16,31,37] This review is focused on the newer strategies being developed to treat LQT2 and identify new LQT2 patients before they suffer a life-threatening event.…”