Ubiquitination-dependent quality control of hERG K+ channel with acquired and inherited conformational defect at the plasma membrane

Abstract

Membrane trafficking in concert with the peripheral quality control machinery plays a critical role in preserving plasma membrane (PM) protein homeostasis. Unfortunately, the peripheral quality control may also dispose of partially or transiently unfolded polypeptides and thereby contribute to the loss-of-expression phenotype of conformational diseases. Defective functional PM expression of the human ether-a-go-go-related gene (hERG) K(+) channel leads to the prolongation of the ventricular action potential that causes long QT syndrome 2 (LQT2), with increased propensity for arrhythmia and sudden cardiac arrest. LQT2 syndrome is attributed to channel biosynthetic processing defects due to mutation, drug-induced misfolding, or direct channel blockade. Here we provide evidence that a peripheral quality control mechanism can contribute to development of the LQT2 syndrome. We show that PM hERG structural and metabolic stability is compromised by the reduction of extracellular or intracellular K(+) concentration. Cardiac glycoside-induced intracellular K(+) depletion conformationally impairs the complex-glycosylated channel, which provokes chaperone- and C-terminal Hsp70-interacting protein-dependent polyubiquitination, accelerated internalization, and endosomal sorting complex required for transport-dependent lysosomal degradation. A similar mechanism contributes to the down-regulation of PM hERG harboring LQT2 missense mutations, with incomplete secretion defect. These results suggest that PM quality control plays a determining role in the loss-of-expression phenotype of hERG in certain hereditary and acquired LTQ2 syndromes.

FIGURE 1:

Intracellular potassium depletion decreases mature hERG half-life. (A) HeLa cells expressing hERG were treated for 24 h with cardiac glycosides and analyzed by immunoblotting for total hERG expression. Solid arrow, complex-glycosylated hERG; empty arrow, core-glycosylated hERG. (B) Glycosylation state of wt hERG in HeLa cell lysate after EndoH or PNGaseF digestion (3 h at 30°C) assessed with immunoblotting. (C, D) PM density and turnover of hERG determined by cell-surface (cs)-ELISA after 24-h treatment with the indicated glycoside. Data are expressed as percentage of initial hERG density. Data are means ± SEM, n ≥ 3 independent experiments, each performed in triplicate. (E) Intracellular K⁺ content of wt hERG-expressing HeLa cells after incubation with ouabain or K⁺-free (0.1 mM K⁺) media measured with flame emission spectroscopy. (F) Turnover of hERG in HeLa cells (top and middle) and H9C2_i cardiac myocytes (bottom) in the presence of 150 μg/ml cycloheximide or 300 μM ouabain as indicated. Calnexin (cal) was used as loading control. (G) Densitometry of complex-glycosylated hERG turnover based on immunoblots as shown in F. The reduction of intracellular K⁺ content is also plotted during 300 nM ouabain exposure. dig, digoxin; oua, ouabain.

FIGURE 2:

Intracellular potassium depletion destabilizes hERG channels at the plasma membrane. (A) Indirect immunostaining of PM hERG in ouabain-treated HeLa (top; bar, 10 μm) and H9C2_i cells (bottom; bar, 15 μm) by epifluorescence microscopy. Cell-surface hERG was labeled with anti-HA Ab on ice and chased for 0, 3, or 4.5 h at 37°C, fixed, and stained without permeabilization. (B) Cell-surface proteins were labeled with sulfo-NHS-SS-biotin after the indicated ouabain treatment and isolated on NeutrAvidin–agarose beads. Biotinylated hERG was detected using anti-HA Ab. Neither the core-glycosylated, ER-resident hERG nor tubulin was accessible to biotinylation. Bottom, densitometric analysis of biotinylated and total hERG pool turnover. Data are means ± SEM, n ≥ 3. (C). PM turnover of hERG in HeLa (left) and H9C2_i (right) cells determined by cs-ELISA in the presence of 300 nM glycosides. (D) Stability of CFTR, MLC1, V2R, and DRD4.4 determined by cs-ELISA after 3.5 h 300 nM ouabain treatment. (E) Cellular and PM expression of wt, F627Y, and S641A hERG measured by immunoblotting and cs-ELISA. (F) PM stability of wt, S627Y, and S641A hERG determined by cs-ELISA. Dashed line, t_1/2 of PM hERG stability in ouabain-treated HeLa cells. dig, digoxin; oua, ouabain. Data are means ± SEM, n = 3.

FIGURE 3:

Potassium depletion increases hERG protease susceptibility. (A) hERG conformational stability probed with limited proteolysis in concert with immunoblotting using isolated microsomes from HeLa cells. Microsomes were incubated with increasing concentration of chymotrypsin (top) or trypsin (bottom) for 10 min at 35ºC in either 75 mM KCl- or 75 mM NMDG-Cl–based medium. Solid arrow, complex glycosylated mature hERG (155 kDa); empty arrow, core-glycosylated hERG. Use of 10 μM valinomycin (val) and 10 μM CCCP facilitated equilibration of luminal [K⁺] with that of the medium (see insert in B). The HA-epitope tag is extracellular and located luminally in microsomes. (B) Quantitative densitometry of the remaining complex-glycosylated hERG as a function of protease concentration on A. (C) Protease resistance of wt and mutant hERG as a function of [K⁺]. Limited proteolysis in medium with the indicated K⁺ concentration (balance to 300 mOsm with NMDG) and 50 μg/ml trypsin performed as in A. Densitometric analysis of the mature hERG protease resistance was determined on immunoblots (right). (D) Protease susceptibility of hERG at low luminal [K⁺]. Limited proteolysis was performed as in A but in the absence of ionophores to preserve the low intraluminal [K⁺]. Quantification of the mature hERG remaining (bottom). (E) Trypsin (50 μg/ml) digestion was done as in A using either K⁺ or other cations that bind to the selectivity filter (SF). Na⁺ served as a negative control. val, valinomycin.

FIGURE 4:

Glycosides-induced lysosomal targeting of hERG from the cell surface. (A) Internalization of hERG in HeLa (top) and H9C2_i (bottom) cells was monitored by the Ab uptake assay at 37ºC after 1.5-h ouabain or digoxin treatment and measured by cs-ELISA as described in Materials and Methods. (B) The recycling efficiency of internalized and anti-HA–labeled hERG was determined by a cs-ELISA as described in Materials and Methods and expressed as percentage of endocytosed hERG. (C) hERG is targeted to lysosomes and colocalizes with dextran (Dx) in ouabain-treated cells. Indirect immunostaining of internalized hERG by laser confocal microscopy was visualized in HeLa cells (bar, 10 μm). Cell-surface hERG was labeled with anti-HA Ab on ice and chased at 37°C in the presence or absence of 300 nM ouabain in Ab-free medium. Texas red–conjugated dextran (50 μg/ml) was loaded overnight and chased for 4 h. Manders’ coefficient for hERG colocalization with dextran was 0.56 ± 0.08 in ouabain and 0.29 ± 0.04 in untreated cells (n = 25). (D) Immunoblot analysis of hERG degradation after treatment with cycloheximide and 300 nM ouabain in the absence or presence of lysosomal inhibitors bafilomycin A1 (Baf), NH₄Cl (NH), and/or leupeptin/pepstatin (L/P) for 3 h. Calnexin (cal) served as a loading control, and quantification of mature hERG (solid arrow) is shown in bar graph. (E, F) Histogram (E) and mean pH_v of internalized hERG-containing endocytic vesicles, determined by FRIA in HeLa cells. Anti-HA Ab and FITC-Fab were bound on ice, and FRIA was performed after 1- to 4-h chase in the presence or absence of ouabain or digoxin at 37ºC. pH are means ± SEM. The graph shows the vesicular pH at each chase point (F).

FIGURE 5:

LQT2 mutations of hERG are unstable at the PM. (A) Immunoblot analysis of wt, F805C, and G601S hERG expression at 37ºC and after 26ºC rescue for 48 h (top). PM density of hERG was determined by cs-ELISA as described in Materials and Methods (bottom). (B) Same as in A, but in H9C2_i cells. (C) Stability of rescued (r) hERG was determined at 37ºC by cs-ELISA. Rescued channels were unfolded (37°C, 2 h) before cell-surface stability measurements. (D) Recycling efficiency was determined as described in Materials and Methods and expressed as percentage of internalized hERG. The mutants were temperature rescued (r) and unfolded before recycling measurement as in C. (E–G) The luminal pH of vesicles containing rescued and internalized hERG after unfolding (37ºC, 2 h) determined in HeLa (E), H9C2_i (F), and HL-1 cardiac myocytes (G) by FRIA as described in Materials and Methods. Anti-HA Ab and FITC-Fab were internalized for 1 h at 37ºC, and FRIA was performed after chase at 37ºC. (H) Limited trypsinolysis of wt and G601S hERG analyzed by immunoblotting. G601S hERG was rescued at 26ºC and then unfolded (37ºC, 2 h) before microsome isolation. Densitometric quantification represents three independent experiments (bottom).

FIGURE 6:

Mutant hERGs are ubiquitinated at the PM and post-Golgi compartments. (A) Ubiquitination of G601S hERG was measured by denaturing immunoprecipitation and immunoblotting (IB) using the P4D1 or K63- or K48-linked chain–specific anti-Ub Abs. Cells were treated with Baf and cycloheximide for 3 h at the indicated temperature. (B) Indirect immunostaining and laser confocal microscopy shows PM accumulation of mutant hERG upon lysosomal inhibition. The PM hERG was labeled on ice with anti-HA Ab and chased for 3 h in the presence or absence of Baf. Cells were then fixed and permeabilized, and lysosomes were counterstained for Lamp1. Bar, 10 μm. (C) Ubiquitination of F805C hERG monitored as in A. ER-to-Golgi transport was inhibited with brefeldin A (4 h).

FIGURE 7:

Intracellular and extracellular potassium depletion provokes polyubiquitination of mature hERG. (A) Ubiquitination of wt and F805C hERG in ouabain-treated cells measured as in Figure 6A. Cells were incubated with 300 nM ouabain and 200 nM Baf for 4.5 h. The densitometric ubiquitin signal was normalized to the hERG in the precipitates as detected by anti-HA immunoblotting (right). Data are means ± SEM, n = 3, *p < 0.05. (B) The PM stability of wt hERG in full medium (5 mM [K⁺]_ex) or 0.1 mM [K⁺]_ex as determined by cs-ELISA. (C) Internalization rate of wt hERG in HeLa cells incubated in 300 nM ouabain, 0.1 mM [K⁺]_ex, or complete medium for the indicated time determined using cs-ELISA. (D) Effect on 0.1 mM and 5 mM [K⁺]_ex in the absence or presence of Baf on wt and F805C ubiquitination determined after 40-min incubation, as in A.

FIGURE 8:

Role of CHIP in the ubiquitin-dependent peripheral destabilization of hERG channel. (A, B) The wt, G601S, and F805C hERG PM density (A) and stability (B) determined after 4-h chase in shCHIP cells by cs-ELISA. (C) Internalization rate of hERG was monitored at 37ºC. Internalization was determined by Ab uptake in shCHIP cells for 5 min. (D) Representative results of pH_v histogram of G601S hERG-containing vesicles in shCHIP or shNT HeLa cells after 4-h chase. The mean pH_v of the individual components of multiple Gaussian distribution is indicated from a total of 433 vesicles. (E) Mean pH_v of wt, G601S, and F805C hERG in shNT and shCHIP-depleted cells measured after anti-HA Ab and FITC-Fab internalization for 1 h and chased for 2 or 4 h. hERG-expressing cells were rescued at 26ºC and then unfolded (37ºC, 2 h) before Ab labeling. (F) The wt hERG disappearance kinetics from the PM in shCHIP and shNT HeLa cells treated with ouabain or digoxin. hERG PM density was determined by cs-ELISA. (G, H) PM turnover of hERG was measured as in F, but shCHIP-expressing HeLa cells were overexpressed with wt, K30A (incapable of chaperone binding), or H260Q (catalytically inactive) myc-CHIP variant. (I) Immunoblotting of cells depicted in G and H using anti-HA and anti-CHIP Abs for detecting hERG, endogenous (gray arrow), and myc-CHIP (black arrow), respectively. Calnexin (cal) was used as loading control.

FIGURE 9:

Ubiquitin-dependent peripheral removal of hERG channel. (A, B) Effect of siCHIP on ouabain-induced ubiquitination of wt (A) and G601S hERG (B). Denaturing immunoprecipitation and Ub detection were performed as in Figure 6A. In G the densitometric analysis of ubiquitination at molecular weight >150 kDa was normalized to complex-glycosylated hERG. Data are means ± SEM, n = 3, *p < 0.05. dig; digoxin; oua; ouabain.

FIGURE 10:

ESCRT0-I is required for nonnative hERG degradation from the PM. (A) Representative pH_v histograms of internalized wt and G601S hERG containing vesicles after 4-h chase in shNT and shStam1 cells determined with FRIA. hERG was rescued at 26ºC and then unfolded (37ºC, 2 h). Anti-HA Ab and FITC-Fab were internalized for 1 h at 37ºC before the chase in Ab-free medium. (B) The mean vesicular pH of hERG-containing compartments was determined after 4-h chase in cells depleted for Tsg101, Hrs, and Stam1 (left). The lysosomal targeting of CD63/LAMP2 and dextran was not influenced by shESCRTs (right). (C) The mean pH_v of internalized wt hERG-containing compartment after ouabain or digoxin treatment for 3.5 h in HeLa cells depleted for Tsg101, Hrs, and Stam1. (D) Cell-surface stability of hERG in ouabain- or digoxin-treated shESCRT cells determined with cs-ELISA after 3.5-h chase. (F) Schematic model of hERG gating cycles and the effect of K⁺ depletion on hERG conformation. Significance was calculated against NT or treated shNT. Data are means ± SEM, n ≥ 3; *p ≤ 0.05 and **p ≤ 0.01.