Abstract
The human ether-à-go-go-related gene (hERG) encodes the channel that conducts the rapidly activating delayed rectifier potassium current (IKr) in the heart. Reduction in IKr causes long QT syndrome, which can lead to fatal arrhythmias triggered by stress. One potential link between stress and hERG function is protein kinase C (PKC) activation; however, seemingly conflicting results regarding PKC regulation of hERG have been reported. We investigated the effects of PKC activation using phorbol 12-myristate 13-acetate (PMA) on hERG channels expressed in human embryonic kidney cell line 293 (HEK293) cells and IKr in isolated neonatal rat ventricular myocytes. Acute activation of PKC by PMA (30 nM, 30 minutes) reduced both hERG current (IhERG) and IKr. Chronic activation of PKC by PMA (30 nM, 16 hours) increased IKr in cardiomyocytes and the expression level of hERG proteins; however, chronic (30 nM, 16 hours) PMA treatment decreased IhERG, which became larger than untreated control IhERG after PMA removal for 4 hours. Deletion of amino acid residues 2–354 (Δ2-354 hERG) or 1–136 of the N terminus (ΔN 136 hERG) abolished acute PMA (30 nM, 30 minutes)-mediated IhERG reduction. In contrast to wild-type hERG channels, chronic activation of PKC by PMA (30 nM, 16 hours) increased both Δ2-354 hERG and ΔN136 hERG expression levels and currents. The increase in hERG protein was associated with PKC-induced phosphorylation (inhibition) of Nedd4-2, an E3 ubiquitin ligase that mediates hERG degradation. We conclude that PKC regulates hERG in a balanced manner, increasing expression through inhibiting Nedd4-2 while decreasing current through targeting a site(s) within the N terminus.
Introduction
The human ether-à-go-go-related gene (hERG) encodes the pore-forming subunit of the channel that conducts the rapidly activating delayed rectifier potassium current (IKr) in the heart (Sanguinetti et al., 1995; Trudeau et al., 1995). IKr is crucial for cardiac repolarization and participates in determining action potential duration. Loss of function in hERG channels attributable to genetic mutations or medications results in long QT syndrome (LQTS), which predisposes affected individuals to potentially fatal cardiac arrhythmias (Curran et al., 1995; Keating and Sanguinetti, 2001). Thus, it is important to elucidate the intracellular signaling pathways that regulate hERG function. One pathway of interest in hERG regulation is the activation of protein kinase C (PKC). PKC activation may be the link between stress and induction of arrhythmias in settings that increase sympathetic drive such as chronic heart failure (Triposkiadis et al., 2009); however, PKC regulation of hERG is complex, and seemingly conflicting results have been reported. We have shown a PKC-mediated upregulation of hERG current (IhERG) and expression after chronic activation of muscarinic acetylcholine M3 receptors (Wang et al., 2014). It was also reported that chronic (hours) PKC activation enhances IKr in guinea pig ventricular myocytes owing to altered C-type inactivation (Heath and Terrar, 2000), as well as hERG expression and IhERG in hERG-expressing HEK cells (Chen et al., 2010; Krishnan et al., 2012). Several studies, however, have demonstrated that acute (minutes) activation of PKC inhibits IhERG and IKr (Cockerill et al., 2007; Wang et al., 2008, 2009; Liu et al., 2017).
In the present study, we investigated hERG expression and function after various periods of PKC activation using phorbol 12-myristate 13-acetate (PMA). Our data show that prolonged activation of PKC by PMA (10–30 nM, 4–24 hours) increased hERG protein expression and paradoxically decreased IhERG. The PKC-mediated IhERG reduction occurred within minutes after PMA application and was slowly reversible upon PMA removal. Using N terminus–deletion hERG mutants, Δ2-354 hERG and ΔN136 hERG, where amino acid residues of 2–354 and 1–136 are deleted, respectively, our results revealed that the N terminus of hERG is required for PMA-mediated IhERG reduction but not for the increase in protein expression. Chronic PKC activation increased both the current and expression of N-deletion hERG mutant channels. We further demonstrated that PKC activation delayed hERG degradation through inhibiting an E3 ubiquitin ligase Nedd4-2 (neural precursor cell expressed developmentally downregulated protein 4 subtype 2), leading to increased plasma membrane expression of hERG channels.
Materials and Methods
Molecular Biology
Wild-type (WT) hERG cDNA was provided by Dr. Gail Robertson (University of Wisconsin-Madison). The hERG mutants, including Δ2-354 hERG (N-terminal deletion of residues 2–354), ΔN136 hERG (N-terminal truncation of residues 1–136), as well as point mutants T74C and T74I, were created using the polymerase chain reaction (PCR) method and confirmed by DNA sequencing (GENEWIZ, South Plainfield, NJ). Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA) was used to transfect WT and mutant channel plasmids into HEK293 cells. The HEK cell lines stably expressing WT and mutant hERG channels were created using G418 for selection (1 mg/ml) and maintenance (0.4 mg/ml). HEK cell lines were cultured in minimum essential medium (MEM) supplemented with 10% fetal bovine serum, 1× nonessential amino acids, and 1 mM sodium pyruvate (Thermo Fisher Scientific).
Cell Treatment
PKC Activation.
PMA (Sigma-Aldrich, St. Louis, MO) at various concentrations in culture media was used to activate PKC in hERG-HEK cells for various periods at 37°C. Acute treatments were 30 minutes, and chronic treatments were 4–24 hours.
PKC Inhibition.
Cells were incubated with 10 μM bisindolylmaleimide I (BIM-1), 200 nM Gö 6983, or 200 nM sotrastaurin (STN) in culture media to inhibit PKC activation.
Analysis of Isolated Plasma Membrane Proteins.
Surface hERG proteins were isolated using the Pierce cell-surface protein isolation kit (89881; Thermo Fisher Scientific). Briefly, surface proteins of intact cells were labeled with Sulfo-NHS-SS-Biotin for 30 minutes at 4°C. The quenching solution was then added to cease the labeling reaction. Biotinylated proteins were isolated by binding to avidin agarose and eluted using SDS-PAGE sample buffer containing dithiothreitol. The isolated cell-surface proteins were analyzed via Western blot analysis.
Cleavage of Cell Surface.
Live hERG-HEK cells were treated for 20 minutes with 200 μg/ml proteinase K (P6556; Sigma-Aldrich) in culture media to cleave cell-surface hERG. The reaction was stopped by the addition of 1.8 mM EDTA and 55 μM phenylmethylsulfonyl fluoride (Sigma-Aldrich) before lysis and collection of whole-cell proteins.
Measurement of hERG Degradation Rate.
hERG-HEK cells were treated with 10 μM brefeldin A (BFA, B6542; Sigma-Aldrich) and 10 μg/ml cycloheximide (CHX, 01810; Sigma-Aldrich) in culture media to inhibit transport of proteins from the endothelial reticulum to the Golgi apparatus and prevent protein synthesis, respectively. The rate of degradation of hERG proteins was assessed by comparing amounts of mature hERG remaining after 6 hours of culture between cells treated with BFA + CHX (as control) and those treated with BFA + CHX plus PMA (30 nM).
Neonatal Rat Ventricular Myocyte Isolation and Culture.
Experiments using rats were approved by the Queen’s University animal care committee and conducted in conformity with the Canadian Council on Animal Care. Sprague-Dawley neonatal rats of either sex at 1 day of age were sacrificed by decapitation, followed by heart removal. Ventricular myocytes were isolated by enzymatic dissociation, as described in detail (Tschirhart et al., 2019). Myocytes were cultured in 10% fetal bovine serum-containing Dulbecco’s modified Eagle’s medium/Ham’s F-12 medium (Invitrogen, Burlington, ON) on coverslips overnight. Various treatments were then performed, and Cs+-mediated IKr was recorded (Zhang, 2006; Guo et al., 2007).
Patch-Clamp Recording
IhERG was recorded using the whole-cell voltage-clamp method. The pipette solution consisted of (in mM) the following: 135 KCl, 5 EGTA, 5 MgATP, and 10 HEPES (pH 7.2 with KOH). The bath solution consisted of (in millimolars) of the following: 135 NaCl, 5 KCl, 10 HEPES, 10 glucose, 1 MgCl2, and 2 CaCl2 (pH 7.4 with NaOH). To elicit hERG current, cells were depolarized to voltages between −70 and 70 mV in 10-mV increments for 4 seconds and then repolarized to −50 mV for 5 seconds before returning to the holding potential of −80 mV. IhERG amplitude for WT and N-deletion/truncation hERG were analyzed using the peak tail current following the 50 mV depolarizing step. Activation-voltage relationships were constructed by normalizing peak tail current at each depolarizing step to that at 50 mV depolarization. Voltage of half-activation (V1/2) was determined by fitting the activation-voltage relationships to the Boltzmann function (Gang and Zhang, 2006). For recordings of native IKr in cultured neonatal rat ventricular myocytes, the pipette solution contained (in mM) 135 CsCl, 5 MgATP, 10 EGTA, and 10 HEPES (pH 7.2 with CsOH). The bath solution contained (in mM) 135 CsCl, 1 MgCl2, 10 glucose, 10 HEPES, and 10 μM nifedipine (pH 7.4 with CsOH). From a holding potential of −80 mV, the Cs+-mediated IKr was evoked by depolarizing steps to voltages between −70 and 70 mV in 10-mV increments for 1 second. Tail currents upon repolarization to the holding potential of −80 mV were plotted against depolarizing steps to construct tail current-voltage relationships in control and treated groups of ventricular myocytes (Zhang, 2006; Guo et al., 2007). Patch-clamp experiments were conducted at room temperature (22 ± 1°C).
Western Blot Analysis
After treatment, proteins were isolated from whole-cell lysate. Briefly, the cells were washed twice with ice-cold phosphate buffer saline (PBS) solution and collected into a 1.5- ml Eppendorf tube with 1 ml of ice-cold PBS. After centrifugation at 100g for 4 minutes at 4°C, the pellets were resuspended in radioimmunoprecipitation assay lysis buffer with Complete protease inhibitor cocktail (Sigma-Aldrich). For detection of phosphorylated neural precursor cell expressed developmentally downregulated protein 4 subtype 2 (Nedd4-2), PhosSTOP phosphatase inhibitor (Roche Applied Science, Indianapolis, IN) was also added to the lysis buffer. After sonication, the lysates were centrifuged at 10,000g for 10 minutes at 4°C. The supernatants were collected, and protein concentration was determined using the DC protein assay kit (Bio-Rad, Hercules, CA). To avoid protein degradation, every step was performed on ice before the addition of SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) sample buffer. For each sample, 15 μg of whole-cell protein was diluted in 50 μl SDS-PAGE sample buffer containing 5% β-mercaptoethanol, boiled for 5 minutes, and loaded into an 8% polyacrylamide gel, separated by electrophoresis, and transferred overnight onto a polyvinylidene fluoride membrane. Membranes were blocked for 1 hour in 0.1% Tween 20 in Tris-buffered saline containing 5% nonfat milk. The membranes were then immunoblotted with appropriate primary antibodies for 2 hours at room temperature. Horseradish peroxidase–conjugated secondary antibodies and ECL detection kit (GE Healthcare, Little Chalfont, UK) were used to detect protein signals on X-ray film (Fuji, Minato, Toyko, Japan). The BLUeye prestained protein ladder (GeneDireX, Taiwan) was used to determine the molecular weights of proteins. For quantification of Western blot data, images on X-ray films were scanned with a high-resolution scanner (Epson Perfection V800 photo color scanner; Epson, Nagano, Japan). Band intensities from each gel, measured using Image Laboratory software (Bio-Rad Laboratories), were first normalized to actin intensities in the same lanes; the band intensities of treatment group(s) were then normalized to their respective control(s) in the same gel and expressed as relative values.
Quantitative Real-Time PCR
Total RNA was extracted from cells using a Total RNA mini kit (Geneaid Biotech Ltd., Taiwan). RNA concentration was measured using a Nanodrop ND-1000 spectrophotometer (Nano Drop, Wilmington, DE). After treatment with DNase I (M0303S; New England Biolabs), the Omniscript reverse transcription kit (Qiagen, Hilden, Germany) was used for reverse transcription of total RNA (1 μg) to cDNA. Quantitative real-time PCR was performed with TaqMan Gene Expression Master Mix (Thermo Fisher Scientific) and a Model 7500 thermal cycler (Applied Biosystems, Foster City, CA). For internal control, a housekeeping gene, GAPDH, was used. Oligonucleotide primers (hERG: Assay ID Hs04234270_g1; GAPDH: Assay ID Hs03929097_g1) were obtained from Thermo Fisher Scientific. The real-time PCR protocol was 2 minutes at 50°C, then 10 minutes at 95°C, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Data were calculated using the 2−ΔΔCT method (Livak and Schmittgen, 2001) and expressed as fold changes of hERG normalized to GAPDH.
Reagents and Antibodies
Bisindolylmaleimide I (BIM-1, ab144264) was purchased from Abcam (Cambridge, MA). Gö 6983 (CAS no. 133053-19-7) and Sotrastaurin (STN, also known as AEB071, CAS no. 425637-18-9) were purchased from Cayman Chemical Company (Ann Arbor, MI). Goat anti-hERG (C20, sc-15968, C-terminal), mouse anti-hERG (F-12, sc-377388, N-terminal), and goat anti-integrin β1 (sc-6622) primary antibodies and mouse anti-goat IgG-HRP secondary antibody (sc-2354) were purchased from Santa Cruz Biotechnology (Dallas, TX). Mouse anti-hERG (DT-331, S5-Pore linker) antibody was purchased from D.I.V.A.L. (Toscana S.R.L., Florence, Italy). Mouse anti-actin primary antibody (A4700) was purchased from Sigma-Aldrich. Rabbit antiphosphorylated Nedd4-2 (p-Nedd4-2, Ser-448, 8063S) primary antibody, horse anti-mouse (7076S), and goat anti-rabbit (7074S) secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA). The PKC sampler kit containing isoenzyme specific antibodies (611421) was obtained from BD Biosciences (Franklin Lakes, NJ). Electrolytes and chemicals for patch-clamp recordings were purchased from Sigma-Aldrich.
Statistical Analysis
All data are expressed as the mean ± S.E.M. For experiments where multiple groups were being compared with control, a one-way analysis of variance (ANOVA) with Dunnett’s post-hoc test was used. For experiments with multiple groups, a one-way ANOVA with Tukey’s post-hoc test was used. For experiments between two groups, a two-tailed unpaired Student’s t test was used. A P value ≤0.05 was considered statistically significant.
Results
Chronic PMA Activation Increases hERG Expression but Decreases Current.
The phorbol ester PMA was used to study the effects of PKC activation on hERG-HEK cells. To observe changes in hERG expression, cells were treated with PMA, and whole-cell proteins were analyzed. On Western blots, hERG protein displays two bands with molecular masses of 135 and 155 kDa. The 135-kDa band represents the intracellular, immature core-glycosylated form of the channel proteins; the 155-kDa band is the mature fully glycosylated form in the plasma membrane (Zhou et al., 1998). First, we determined the time-dependent effects of PMA on hERG expression. hERG-HEK cells from the same passage were separated into culture plates and treated with 30 nM PMA. At different time points, cells were collected, and hERG expression was examined using Western blot analysis. Goat anti-hERG antibody (C-20, C-terminal) was used as the main primary antibody. PMA (30 nM) increased hERG expression in a time-dependent manner; the increase started at 0.5 hour after treatment, became significant at 4 hours, and reached a maximal effect at 6 hours. Treatment at 12 and 24 hours caused further increases in two of five experiments; but, overall, a maximal effect was achieved 6 hours after treatment (Fig. 1A).
PMA is a PKC activator (Thomas et al., 2003; Cockerill et al., 2007). To confirm that the effects of PMA on hERG are indeed mediated by PKC activation, we used a PKC inhibitor, BIM-1, to inhibit PKC activation. The addition of BIM-1 (10 μM) to PMA (30 nM) for 6-hour treatment abolished the PMA-mediated increase in hERG expression (Fig. 1B), indicating that PMA increases hERG expression through PKC activation. BIM-1 alone had no effect on hERG expression (Fig. 1B).
The concentration-response relationship of PMA-induced increase in hERG expression was examined by treating hERG-HEK cells with PMA for 6 hours at various concentrations. Whereas 10 nM PMA significantly increased hERG expression and 30 nM PMA caused a further increase in hERG expression, 100 and 1000 nM PMA did not cause any further increase (Fig. 2A).
Increased hERG protein expression is usually accompanied by increased IhERG. We recorded IhERG using a whole-cell voltage-clamp method. In contrast to the increased hERG expression, treatments with PMA decreased IhERG. Whereas 6-hour treatment with 10 nM PMA significantly decreased IhERG and 30 nM PMA caused a further decrease in IhERG, 100, 300, and 1000 nM PMA did not cause a further decrease (Fig. 2B).
hERG Channels Are Membrane-Bound and Functional after Chronic PMA Treatment.
One possible explanation for the discrepancy between increased 155-kDa hERG expression and decreased IhERG after PMA treatment is that the 155-kDa hERG protein is not at the plasma membrane. We performed two experiments to determine whether the 155-kDa hERG after PMA treatment (30 nM, 6 hours) is localized at the cell surface. First, we isolated cell-surface protein using a biotinylation method and examined hERG expression using Western blot analysis. PMA treatment increased the membrane-bound 155-kDa hERG proteins (Fig. 3A). Second, we cleaved cell-surface hERG by treating hERG-HEK cells with proteinase K (PK, 200 µg/ml, 20 minutes). We previously demonstrated that PK selectively cleaves plasma-membrane-bound 155-kDa hERG proteins without affecting the intracellularly localized immature 135-kDa hERG proteins (Lamothe et al., 2016). The 155-kDa hERG bands, but not 135-kDa hERG bands, in both control and PMA-treated (30 nM, 6 hours) cells were completely cleaved by PK (Fig. 3B). Thus, the PMA-increased 155-kDa hERG is in the plasma membrane.
Mature hERG expression can be increased by two mechanisms: accelerated protein synthesis and/or decreased hERG degradation. We examined the mRNA levels in control and PMA- (30 nM, 2 and 6 hours) treated hERG-HEK cells. PMA did not increase hERG mRNA level (Fig. 3C). To determine whether PMA treatment delays degradation of mature hERG channels, we first treated hERG-HEK cells with 10 μg/ml cycloheximide (CHX, a protein synthesis inhibitor) and 10 µM brefeldin A (BFA, an ER-to-Golgi transit inhibitor). This treatment prevents protein synthesis and forward trafficking and therefore allows for observation of the rate of degradation of mature hERG channels. Western blot analysis of whole-cell protein revealed that, compared with cells treated with BFA and CHX, cells with additional PMA treatment had significantly more mature (155-kDa) hERG remaining after 6 hours of treatment (Fig. 3D). Thus, PMA (30 nM) delayed hERG degradation, leading to an increased membrane expression of hERG channels. We previously demonstrated that hERG degradation is mediated by the ubiquitin ligase Nedd4-2 (Guo et al., 2012). Nedd4-2 activity is controlled by phosphorylation, which leads to inhibition of its catalytic activity (Debonneville et al., 2001). To determine whether Nedd4-2 plays a role in PMA-mediated hERG increase, we examined the effects of PMA treatment (30 nM, 6 hours) on the expression levels of total and phosphorylated Nedd4-2 using Western blot analysis. PMA increased the amount of phosphorylated Nedd4-2 without affecting the amount of total Nedd4-2 protein (Fig. 3E), providing an explanation for PMA-mediated increase in hERG expression through delayed degradation.
Acute PMA Treatment Reduces IhERG.
We reasoned that PKC activation may exert two independent effects on hERG channels: inhibiting channel activity and increasing protein expression. To isolate hERG inhibition from increased channel expression, we examined an acute (30-minute) treatment of hERG-HEK cells with 30 nM PMA, which does not lead to increased hERG protein expression (Fig. 1A). Acute PMA treatment decreased IhERG (Fig. 4A) and shifted the voltage of half-maximal activation (V1/2) by 10.7 mV to the positive-voltage direction (Fig. 4B). PMA treatment did not change the slope factor of the activation curve. In an attempt to prevent the PMA-mediated effects on IhERG, we used BIM-1 (10 μM); however, since BIM-1 (10 μM) alone inhibits IhERG (Thomas et al., 2004a), its effects on PMA-mediated IhERG reduction could not be addressed (Fig. 4A). On the other hand, although BIM-1 (10 μM) treatment alone did not shift V1/2 (data not shown), it prevented the PMA-mediated shift of the hERG activation curve (Fig. 4B), indicating that the PMA-mediated effects on IhERG were through PKC activation.
Our data so far indicate that PMA-mediated IhERG reduction occurs in tens of minutes, but PMA-mediated increase in hERG expression occurs within hours after PMA treatment. We reasoned that, upon PMA removal, if the PMA-induced reduction in IhERG recovers faster than the PMA-induced increase in hERG expression, we should be able to observe a PMA-mediated increase in IhERG associated with increased channel density. Our results showed that this is indeed the case. We treated hERG-HEK cells with PMA (30 nM) for 16 hours. We then washed the cells with fresh media three times to remove PMA and continued to culture the cells in fresh media. Over the course of the next 6 hours, IhERG gradually increased to a magnitude greater than that of untreated controls (Fig. 4C). After 6-hour recovery, V1/2 of IhERG was also restored to that of untreated controls (Fig. 4D). This result is consistent with an increased abundance of functional hERG channels after PMA treatment. We propose that PKC directly inhibits hERG channels through a reversible modification. The inhibition is relieved upon PMA removal, whereas the enhanced protein expression by PMA is still maintained because of the relatively slow turnover rate of hERG channels (Ficker et al., 2003).
Acute PMA Treatment Reduces, but Chronic PMA Treatment Increases, IKr in Neonatal Rat Ventricular Myocytes.
To determine whether the differential acute and chronic effects of PMA treatments on expressed hERG channels also apply to native cardiomyocytes, we examined the effects of PMA on IKr in neonatal rat ventricular myocytes. Ventricular myocytes were treated with PMA (30 nM) for either 30 minutes or 16 hours in culture conditions. After treatment, ventricular myocytes were transferred to the recording chamber. Families of Cs+-mediated IKr (IKr-Cs) were recorded using whole-cell voltage-clamp method. As shown in Fig. 5, whereas a 30-minute treatment with PMA decreased IKr (Fig. 5A), a 16-hour treatment increased IKr (Fig. 5B). These results are generally consistent with our results obtained from hERG-HEK cells. Thus, while a 30-minute treatment with PMA (30 nM) decreased both IKr and IhERG, a 16-hour treatment with PMA (30 nM) increased IKr and hERG expression; however, a difference exists; whereas chronic (16 hours) PMA-induced increase in IhERG recorded from HEK cells is only seen after PMA removal for 4 hours (Fig. 4C), chronic (16 hours) PMA-induced increase in IKr recorded from cardiomyocytes is observed immediately after treatment (Fig. 5B).
PMA-Induced Effect on IhERG Is Mediated by PKC Activation.
PMA activates not only PKC isoforms but also other proteins that are regulated by diglycerides such as Ras guanyl nucleotide-releasing protein (Ebinu et al., 2000). We showed that the PKC inhibitor BIM-1 prevented PMA-induced effects on hERG expression and activation curve of IhERG (Fig. 1B; Fig. 4B). Whereas BIM-1 is considered a “selective” PKC inhibitor, it can also inhibit other important signaling kinases such as p70S6K and p90RSK (Alessi, 1997; Roberts et al., 2005). Furthermore, our data demonstrated that BIM-1 also directly inhibits IhERG (Fig. 4A). To further confirm that PKC activation is responsible for the PMA-induced effects on hERG, we examined the effects of two additional PKC inhibitors, Gö 6983 and STN, on hERG channels. Neither Gö 6983 (200 nM) nor STN (200 nM) acutely affected IhERG (data not shown), but both completely prevented acute PMA treatment (30 minutes, 30 nM) induced IhERG reduction (Fig. 6A), and chronic PMA treatment (16 hours, 30 nM) induced an IhERG increase after 6-hour PMA removal (Fig. 6B). Western blot analysis showed that they also prevented chronic PMA treatment (16 hours, 30 nM)-induced increase in hERG protein expression (Fig. 6C). These results further indicate that PMA-mediated changes in hERG are through PKC activation.
PMA-Mediated Effects on hERG Expressed in HEK Cells May Not Be due to the PKC Depletion.
There are various PKC isoforms. To investigate the effects of PMA treatment on the expression of PKC isoforms in our hERG-HEK cells, we used a PKC sampler kit containing isoenzyme-specific antibodies (611421; BD Biosciences). As can been seen in Fig. 7, with the isoform-specific antibodies of the kit, all PKC isoforms were detected. Treatment of hERG-HEK cells with 30 nM PMA for up to 24 hours caused a time-dependent depletion of the expression level of PKCα and PKCθ and did not affect the expressions of PKCβ, PKCε, PKCσ, PKCι, and PKCλ up to 18 hours (Fig. 7); however, 30 nM PMA treatment of 6 hours did not cause depletion of any PKC isoenzymes (Fig. 7).
The N Terminus of hERG Is Required for PKC-Mediated Inhibition of Current but not Channel Expression.
We investigated the molecular mechanisms underlying PMA-mediated IhERG reduction. Previous studies have suggested that the N terminus of hERG is required for PKC-mediated IhERG reduction (Cockerill et al., 2007; Liu et al., 2017). We obtained novel evidence that the N terminus is involved in PMA-mediated IhERG reduction. As shown in Fig. 8A, after a 6-hour PMA (30 nM) treatment, an increase in 155-kDa hERG expression was observed using either an anti-C-terminus or anti-S5-pore hERG antibody; however, a decrease in 155-kDa hERG was observed when an anti-N-terminus hERG antibody was used in the same samples (Fig. 8A). We believe activation of PKC modifies the N terminus of hERG, and such modification interferes with binding of the N-terminal targeting antibody to the protein, resulting in decreased hERG detection despite the increase in overall hERG expression. We then investigated the correlation between PMA-mediated reduction in IhERG and a decrease in 155-kDa hERG detection with the anti-N-terminal antibody. As shown in Fig. 8B, Western blot analysis using the anti-C-terminus antibody did not reveal a significant change in 155-kDa hERG expression after PMA (30 nM) treatment up to 60 minutes; however, PMA (30 nM)-mediated reduction in 155-kDa detection with the anti-N-terminus antibody started upon 20-minute treatment and was more obvious upon 30- and 60-minute treatments. This time course is consistent with PMA-mediated reduction in IhERG, indicating that modification of the N terminus is associated with PKC-mediated IhERG reduction. This notion is further supported by our data shown in Fig. 8C. After a 16-hour treatment of hERG-HEK cells with PMA (30 nM), Western blot analysis using the anti-C-terminus antibody revealed an increased hERG expression. In contrast, Western blot analysis of the same protein samples using the anti-N-terminus antibody revealed a decreased hERG (155-kDa) expression, indicating that PMA-induced PKC activation modified the N terminus of the channel. Upon removal of PMA, the 155-kDa hERG expression detected with the anti-C-terminus antibody remained at an increased level up to 6 hours. Intriguingly, the decreased 155-kDa hERG detection with the anti-N-terminus antibody recovered significantly at 4 and 6 hours after PMA removal (Fig. 8C), a time course similar to the recovery of IhERG upon PMA removal (Fig. 4C). These observations support the notion that modification of the N terminus by PMA is involved in hERG channel inhibition, and recovery from this inhibition leads to an increase in IhERG (Fig. 4C) associated with increased protein expression.
To investigate more completely the role of the N terminus in PMA-mediated IhERG inhibition, we deleted amino acid residues 2–354 of the N terminus of hERG to construct the Δ2-354 hERG mutant. This mutant was developed based on previous studies showing that the Δ2-354 hERG mutant produces functional channels (Wang et al., 1998). In contrast to the effects on WT hERG, acute treatment of Δ2-354 hERG cells with PMA (30 nM, 30 minutes) had no effect on either the current amplitude (Fig. 9A) or the V1/2 of Δ2-354 hERG channels (Fig. 9B). On the other hand, the N terminus is not required for PKC upregulation of hERG expression; chronic PMA treatment (30 nM, 16 hours) increased expression of Δ2-354 hERG channels (Fig. 9C). Furthermore, the increased protein expression is associated with an increase in current (IΔ2-354 hERG) (Fig. 9D) without shifting the activation curve (Fig. 9E). The PMA-mediated increases in Δ2-354 hERG expression (Fig. 9C) and IΔ2-354 hERG (Fig. 9D) were prevented by the presence of BIM-1 (10 μM), indicating that the effects are mediated through PKC activation.
Our results so far indicate that PKC-mediated IhERG inhibition involves N-terminus modification of hERG channels. This notion is in line with previous studies, which propose that hERG inhibition by PKC occurs through phosphorylation of sites, particularly T74, in the N-terminal region (Thomas et al., 2003; Cockerill et al., 2007). In these previous studies using ΔPKC-hERG, a channel in which 17 of the 18 putative phosphorylation sites have been mutated to Ala, it was found that acute PKC activation still shifted the V1/2 or inhibited the current of mutant channels (Thomas et al., 2003; Cockerill et al., 2007); however, mutations of T74 (i.e., T74A, T74V, T74D, and T74E) resulted in nonfunctional channels, so the role of this phosphorylation site could not be examined (Thomas et al., 2003; Cockerill et al., 2007). We created T74C and T74I mutant hERG constructs. Unfortunately, neither the T74C nor T74I mutant produced a functional channel. Eight putative PKC phosphorylation sites (S354, S278, S250, S179, T174, T162, T74, and S26) were removed by the Δ2-354 hERG mutant. To narrow down the site potentially responsible for PKC-mediated hERG inhibition, we created a 1–136 truncation mutant, ΔN136 hERG, which removed only T74 and S26 putative phosphorylation sites. The ΔN136 hERG mutant generated robust currents (Fig. 10). In contrast to the effects on WT hERG (Fig. 4A), acute PMA treatment (30 nM, 30 minutes) did not decrease the ΔN136 hERG current (IΔN136 hERG) (Fig. 10A). On the other hand, chronic PMA treatment (16 hours, 30 nM) increased both IΔN136 hERG (Fig. 10B) and ΔN136 hERG protein expression (Fig. 10C). The increase in both IΔN136 hERG and ΔN136 hERG protein expression upon chronic PMA treatment (16 hours, 30 nM) was prevented by the presence of STN (200 nM) (Fig. 10, B and C).
Discussion
The hERG channel plays a crucial role in cardiac repolarization, and a loss of its function is a major cause of LQTS, which can lead to dangerous arrhythmias and sudden death (Curran et al., 1995; Keating and Sanguinetti, 2001). It has been recognized that sympathetic activation related to physical or emotional stress can exacerbate cardiac events in patients with LQTS (Wilde et al., 1999; Schwartz et al., 2001; Winter et al., 2018). To that end, PKC is under investigation as a potential link between stress conditions and reduced hERG function; however, controversial effects of PKC on hERG function have been reported (Thomas et al., 2003; Cockerill et al., 2007; Wang et al., 2008, 2009, 2014; Chen et al., 2010; Krishnan et al., 2012; Liu et al., 2017). By investigating the effects of PKC activation on both hERG current and expression on a time scale ranging from minutes to hours, our data, for the first time, uncovered that PKC differentially regulates hERG protein expression and function; PKC activation chronically enhances channel expression (hours) through delaying channel degradation while inhibiting channel function with a faster time course (tens of minutes) through a mechanism that involves the N terminus of the channel.
Our results showed that PKC activation inhibited IhERG and positively shifted the V1/2 of channel activation by approximately 10 mV after a 30-minute PMA (30 nM) application (Fig. 4, A and B). This observation is consistent with previous literature investigating acute PKC activation (Thomas et al., 2003; Cockerill et al., 2007; Wang et al., 2008, 2009; Liu et al., 2017). Our results further demonstrated that PKC-mediated IhERG inhibition is slowly reversible. Although a 16-hour treatment with PMA (30 nM) led to inhibition of IhERG and a positive shift in V1/2, replacement of PMA media with fresh media resulted in a gradual increase of IhERG, which became larger than IhERG of control cells after 4 hours of recovery, and V1/2 of activation returned to control (Fig. 4, C and D). We propose that replacement of the media resulted in the gradual removal of a reversible modification from the channel, allowing for recovery of IhERG from inhibition. The fact that IhERG in PMA-treated cells after recovery is greater than that in control cells demonstrates that the increased channel proteins after PKC activation are not only membrane-bound, but also functional. hERG protein turnover time is around 12 hours (Ficker et al., 2003). We demonstrated that, upon PMA removal, the recovery of inhibited channel function is faster than hERG protein turnover, resulting in greater IhERG in PMA-treated cells owing to increased hERG protein (Fig. 4C; Fig. 8C).
Our Western blot analysis revealed novel evidence of PMA-mediated modification of the N terminus of hERG channels. After chronic PMA treatment, an increase in mature hERG expression was evident using a C-terminal antibody (Fig. 1; Fig. 2A), as well as with a S5P-binding antibody (Fig. 8A); however, detection of the same samples using an N-terminal-targeting antibody showed decreased expression of the mature hERG protein (Fig. 8A). We propose that PKC-mediated modulation of the N-terminal region of hERG interferes with the antibody-binding epitope and is involved in the inhibition of hERG. With 5- to 60-minute treatments of hERG-HEK cells with PMA, hERG expression was not changed when detected using the C-terminal antibody. However, detection using the N-terminal antibody demonstrated a time-dependent decrease in mature hERG expression (Fig. 8B) that was associated with decreased IhERG (Fig. 4A). Furthermore, a 16-hour treatment with PMA (30 nM) led to both reduction in IhERG (Fig. 4C) and mature hERG detection with the anti-N-terminal antibody (Fig. 8C). Removal of PMA resulted in recovery of IhERG (Fig. 4C) and recovery of mature hERG detection with the anti-N-terminal antibody (Fig. 8C). These results indicate that PKC-mediated IhERG inhibition involves N-terminus modification of channels. This conclusion is in line with previous studies that have proposed that hERG inhibition by PKC occurs through phosphorylation of sites, particularly T74, in the N-terminal region (Cockerill et al., 2007; Liu et al., 2017); however, mutations of T74 resulted in nonfunctional channels, so the role of T74 in PKC- mediated IhERG inhibition could not be directly examined (Thomas et al., 2003; Cockerill et al., 2007). In the present study, we demonstrated that PMA treatment of 30 minutes did not decrease the current of Δ2-354 or ΔN136 hERG mutant channels. Moreover, PMA treatment of 16 hours increased both expression and current of Δ2-354 and ΔN136 hERG mutant channels. Therefore, the N terminus is required for PKC-mediated inhibition of hERG channel function but not for PKC-mediated increase in expression. Consistent with previous studies (Thomas et al., 2003; Cockerill et al., 2007), our results demonstrated that disrupting the putative T74 phosphorylation site by new mutations, T74C and T74I, led to nonfunctional channels; however, our results showed that deletion of the two putative phosphorylation sites S26 and T74 (i.e., ΔN136) abolished the acute PMA treatment (30 minutes)-mediated inhibition of hERG currents (Fig. 10A). Since removal of the putative phosphorylation sites, including S26, did not affect PMA-mediated effects on hERG current (Thomas et al., 2003; Cockerill et al., 2007), these results strongly support the notion that PMA targets residue T74 to mediate the inhibition of IhERG.
The PKC-mediated hERG increase may not be due to enhanced transcription since hERG mRNA level was not affected by 2 and 6 hours of PMA treatment (Fig. 3C). On the other hand, our results revealed that the degradation rate of mature hERG was reduced after PMA treatment. In hERG-HEK cells treated with cycloheximide (a protein synthesis inhibitor) and brefeldin A (an ER to Golgi transit inhibitor), PMA treatment slowed the degradation of 155-kDa hERG proteins (Fig. 3D). PMA treatment also led to an increase in phosphorylated Nedd4-2 without affecting total Nedd4-2 level (Fig. 3E), indicating that PMA treatment inhibits Nedd4-2 activity. Since Nedd4-2 mediates hERG degradation (Guo et al., 2012), its inhibition by PMA treatment would result in reduced ubiquitination and degradation of hERG, leading to accumulation of mature channels in the plasma membrane. Previous studies demonstrated that PKC mediated inhibition of Nedd4-2 through the serum- and glucocorticoid-inducible kinase, as well as muscarinic acetylcholine M3 receptor delays hERG degradation, leading to increased hERG expression (Lamothe and Zhang, 2013; Wang et al., 2014). Nonetheless, other mechanisms should also be considered. For example, previous studies have proposed that chronic PKC activation enhances hERG expression through accelerated post-transcriptional channel synthesis (Chen et al., 2010; Krishnan et al., 2012).
It is important to note that the regulation of hERG by PKC may be shaped by the unique profile of PKC isoenzymes activated by specific signaling pathways. Our data showed that treatment of hERG-HEK cells with 30 nM PMA caused a time-dependent depletion of the expression level of PKCα and PKCθ, but it did not affect the expression of PKCβ, PKCε, PKCσ, PKCι, and PKCλ up to 18 hours (Fig. 7). Moreover, the depletion became obvious only after 6-hour treatment (Fig. 7). PMA-induced IhERG inhibition occurred at 30 minutes (Fig. 4A), and PMA-induced increase in hERG expression occurred at 4 hours (Fig. 1A). Thus, PKC depletion may not play an essential role in PMA-mediated effects on hERG channels in hERG-HEK cells. PMA activates various PKC isozymes (McFerran et al., 1995), whereas specific PKC isoform signaling can be achieved by the activation of distinct G protein–coupled receptor pathways (Liu et al., 2017). Nonetheless, there are some similarities in hERG/IKr responses to PKC activation induced by PMA and α1-AR or M3 receptor stimulation. For example, acute agonism of α1-AR or M3 receptors inhibits hERG current and positively shifts V1/2 in a manner that involves the N terminus of hERG (Thomas et al., 2004b; Cockerill et al., 2007; Liu et al., 2017), whereas chronic activation of α1-AR or M3 receptors increases hERG expression and current (Chen et al., 2010; Wang et al., 2014; Mahati et al., 2016). On the other hand, angiotensin II–mediated PKC activation inhibits IhERG but does not shift V1/2, and the N terminus of hERG is not involved in the inhibition (Liu et al., 2017). It was proposed that PKCα mediates IhERG inhibition after α1-AR activation, whereas PKCε mediates inhibition after AT1 receptor activation (Liu et al., 2017). Future investigations are required to uncover specific isoenzymes and downstream pathways involved in hERG regulation with aspects of functional regulation versus channel expression. In addition, although the effects of both acute (30 minutes) and chronic (16 hours) PMA treatment were similar for IhERG in HEK cells and IKr in cardiomyocytes, chronic PMA-induced increase in IhERG recorded from HEK cells was seen only after 4 hours of PMA removal (Fig. 4C), but chronic PMA-induced increase in IKr recorded from ventricular myocytes was observed immediately after treatment (Fig. 5B). The molecular mechanisms for such a difference are complex and warrant future investigation. Differences in endogenous PKC isoenzymes and phosphatases, as well as their responses to prolonged PMA treatment could be responsible. Moreover, there is a possibility that PKC activation results in the activation of another kinase that then phosphorylates the hERG channel. Future studies in a more purified system with a purified kinase will be required to demonstrate a direct phosphorylation by specific enzymes/kinases.
In summary, we have demonstrated that PMA-mediated PKC activation regulates hERG channels through two opposing mechanisms: increasing hERG expression and decreasing hERG current. Whereas PMA-mediated PKC activation is not physiologic, our data nonetheless raise the possibility that a buffer system may exist regarding PKC-mediated hERG regulation whereby PKC-mediated hERG current inhibition is countered by an increase in hERG channel abundance.
Authorship Contributions
Participated in research design: Sutherland-Deveen, Wang, Lamothe, Zhang.
Conducted experiments: Sutherland-Deveen, Wang, Lamothe, Guo, Li, Yang, Du.
Performed data analysis: Sutherland-Deveen, Wang, Tschirhart, Guo, Li, Yang.
Wrote or contributed to the writing of the manuscript: Sutherland-Deveen, Wang, Zhang.
Footnotes
- Received November 8, 2018.
- Accepted April 17, 2019.
This work was supported by the Canadian Institutes of Health Research [Grant MOP 72911] to S.Z.
Abbreviations
- ANOVA
- analysis of variance
- BFA
- brefeldin A
- BIM-1
- bisindolylmaleimide I
- CHX
- cycloheximide
- HEK
- human embryonic kidney
- hERG
- human ether-à-go-go-related gene
- IhERG
- hERG current
- IKr
- rapidly activating delayed rectifier potassium current
- LQTS
- long QT syndrome
- MEM
- minimum essential medium
- Nedd4-2
- neural precursor cell expressed developmentally downregulated protein 4 subtype 2
- PCR
- polymerase chain reaction
- PK
- proteinase K
- PKC
- protein kinase C
- PMA
- phorbol 12-myristate 13-acetate
- STN
- sotrastaurin
- WT
- wild-type
- Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics