Progesterone Impairs Human Ether-a-go-go-related Gene (HERG) Trafficking by Disruption of Intracellular Cholesterol Homeostasis*

The prolongation of QT intervals in both mothers and fetuses during the later period of pregnancy implies that higher levels of progesterone may regulate the function of the human ether-a-go-go-related gene (HERG) potassium channel, a key ion channel responsible for controlling the length of QT intervals. Here, we studied the effect of progesterone on the expression, trafficking, and function of HERG channels and the underlying mechanism. Treatment with progesterone for 24 h decreased the abundance of the fully glycosylated form of the HERG channel in rat neonatal cardiac myocytes and HERG-HEK293 cells, a cell line stably expressing HERG channels. Progesterone also concentration-dependently decreased HERG current density, but had no effect on voltage-gated L-type Ca2+ and K+ channels. Immunofluorescence microscopy and Western blot analysis show that progesterone preferentially decreased HERG channel protein abundance in the plasma membrane, induced protein accumulation in the dilated endoplasmic reticulum (ER), and increased the protein expression of C/EBP homologous protein, a hallmark of ER stress. Application of 2-hydroxypropyl-β-cyclodextrin (a sterol-binding agent) or overexpression of Rab9 rescued the progesterone-induced HERG trafficking defect and ER stress. Disruption of intracellular cholesterol homeostasis with simvastatin, imipramine, or exogenous application of cholesterol mimicked the effect of progesterone on HERG channel trafficking. Progesterone may impair HERG channel folding in the ER and/or block its trafficking to the Golgi complex by disrupting intracellular cholesterol homeostasis. Our findings may reveal a novel molecular mechanism to explain the QT prolongation and high risk of developing arrhythmias during late pregnancy.

Progesterone (P4) 2 is an important steroid hormone involved in the female menstrual cycle, pregnancy, and embryogenesis. In the normal menstrual cycle, P4 levels increase from 0. 6 -4.5 nmol/liter during the preovulatory phase to 10.5-80 nmol/liter during the luteal phase (1). It was reported that P4 at Ͻ100 nmol/liter, which is comparable with that occurring during the luteal phase, had no significant effect on the QT interval (2)(3)(4). However, recent studies show that P4 may shorten the action potential duration or QT interval (5). This is probably because P4 at this level enhances the rapid component of the delayed rectifier K ϩ current and inhibits L-type Ca 2ϩ currents (6). If pregnancy occurs, P4 levels are initially maintained at the luteal level, but may increase to 1 mol/liter at term (7). Elevated levels of P4 are important for the implantation of the embryo and the maintenance of a conducive environment for the embryo. At such a high level, the corrected QT interval is significantly prolonged (8,9). This explains why pregnant women are more susceptible to ventricular arrhythmias during pregnancy, labor, and delivery (10 -12). However, it is interesting to note that in the patients with inherited long QT syndrome (LQTS) the risk for cardiac events is not higher during pregnancy than during other periods in life (10,13). These findings suggest that the mechanisms for pregnancy-induced cardiac events in normal healthy women are different from those in the patients with inherited LQTS.
In the fetus, the P4 level was reported to be very high (ϳ4.5 mol/liter) in umbilical cord vein during late stages of pregnancy (7). At this stage, longer QT and corrected QT intervals were reported in fetal magnetocardiography and noninvasive fetal electrocardiography (14,15). More importantly, the rate of sudden death is higher in the uterus than at most other times in the human life cycle. Although the major reasons remain unknown, several studies of newborns suggest that QT prolongation and the increased risk of ventricular arrhythmias may account for the significant mortality (16,17). This may be the same for stillbirth as several case reports indicate that LQTS is one cause for otherwise unexplained fetal demise (18).
The human ether-a-go-go-related gene (HERG) K ϩ channel, which encodes the ␣-subunit of the rapid component of the delayed rectifier K ϩ current (I Kr ), is largely responsible for the repolarization of action potentials in cardiac myocytes. Inherited mutations or drug-induced blockade of the HERG channel prolongs QT intervals and increases the risk of lethal arrhythmia. Trafficking to the plasma membrane is very important for HERG channel, and defects in trafficking, caused either by mutation of HERG gene or by drugs, can affect HERG function significantly.
The function of the HERG channel is important for embryo development because impaired HERG channel function not only induces LQTS in embryo, but also affects the structure of the heart (19,20) and even causes embryonic lethality (20). However, the effect of P4 on HERG K ϩ channel function remains unclear. Because the P4 level during late pregnancy is very high, we hypothesized that P4 may contribute to prolonged QT intervals and the sudden cardiac death in the fetus and perhaps in pregnant women as well. This study was therefore designed to examine the effect of P4 on protein expression, trafficking, and function of HERG K ϩ channels in heterological expression systems and rat neonatal cardiac myocytes.

EXPERIMENTAL PROCEDURES
Reagents-PD98059 and LY294002 were from Merck. Simvastatin was from Eurodrug Laboratories (Belgium). Sulfo-NHS-LC-Biotin and Pierce Streptavidin UltraLink Resin were from Thermo Scientific. All other reagents were from Sigma. P4, cholesterol, and RU486 were dissolved in ethanol as stock solutions. Cycloheximide, PD98059, and LY294002 were dissolved in dimethyl sulfoxide as stock solutions. The final ethanol and dimethyl sulfoxide concentrations were Ͻ0.2%. The GFP-Rab9 plasmid was a gift from Dr Suzanne Pfeffer, Stanford University.
Isolation of Rat Neonatal Cardiac Myocytes-Rat neonatal cardiac myocytes were isolated from 1-2-day-old Sprague-Dawley rats via serial pancreatin (Sigma)/collagenase II (Worthington Biochemical) digestion. Briefly, rat hearts were minced in ice-cold dissociation buffer (116 mM NaCl, 20 mM Hepes, 0.8 mM Na 2 HPO 4 , 5.6 mM glucose, 5.4 mM KCl, 0.8 mM MgSO 4 , pH 7.35) with pancreatin and collagenase II and then transferred into a sterilized tissue dissociation bottle. The digestion was carried out in a 37°C water bath with occasional shaking. The suspension from the first digestion was discarded. The suspensions from the second to the sixth digestions were collected and resuspended in fetal bovine serum. After all the digestions, cells were centrifuged and resuspended in culture medium (DMEM supplemented with 10% fetal bovine serum, 5% horse serum, and penicillin-streptomycin) and placed in the incubator for ϳ1 h. The cardiac myocyte-rich suspension was centrifuged and resuspended in culture medium. To prevent the proliferation of other type of cells, 100 M BrdUrd was added in the first 24 h. The medium for rat neonatal cardiac myocytes were changed every other day.
Isolation of Plasma Membrane Proteins by Biotinylation-Briefly, HERG-HEK293 cells were washed with ice-cold PBS three times and then incubated with 1 mg/ml Sulfo-NHS-LC-Biotin for 30 min at 4°C, followed by washing with PBS ϩ 100 mM glycine for three times to quench and remove excess biotin reagent and byproducts. Cells were lysed as described previously (21). Whole cell lysates were incubated with Pierce Streptavidin UltraLink Resin for 1 h at room temperature. The resin was washed with binding buffer (0.1 M phosphate, 0.15 M NaCl, pH 7.2) four times. The collected samples were subjected for Western blotting analysis.
Western Blot Analysis-Western blot analysis was performed using whole cell lysates as described previously (21). Anti-myc mouse monoclonal 9E10 and anti-tubulin rabbit monoclonal antibodies were from Sigma. Other antibodies were from Santa Cruz Biotechnology. All gels illustrated in the figures are representative examples from four to eight independent experiments. ␤-Tubulin and hypoxanthine phosphoribosyltransferase were applied as internal controls to normalize protein loading. The intensity of bands was quantified using LabWorks TM Image Analysis software (UVP).
Confocal Microscopy-HERG-HEK293 cells grown on glass coverslips were subject to formaldehyde fixation before indirect immunofluorescent staining. The method was described in our previous publication (21).To study the plasma membrane HERG K ϩ channel distribution, cells were incubated with anti-K v 11.1 (HERG extracellular epitope, residues 430 -445, between S1 and S2 loop) antibody (Alomone Labs, Jerusalem, Israel) without permeabilization, and the primary antibody was detected with Alexa Fluor 488-conjugated anti-rabbit Ig (Invitrogen).
Filipin Staining-HERG-HEK293 cells were fixed with formaldehyde, and glycine was used to quench extra formaldehyde. Cells were then stained with filipin (Sigma-Aldrich) and viewed by fluorescence microscopy at magnification of ϫ20 or ϫ40 times using a UV filter set.
Cellular Cholesterol Measurement-An AmplexRed Cholesterol Assay kit (Molecular Probes) was used to measure the cellular cholesterol level following the manufacturer's instructions.
Statistics-Values presented are as means Ϯ S.E. ANOVA and Tukey's post hoc tests were employed to assess statistical significance, and p values of Ͻ 0.05 were considered to be significant.

RESULTS
P4 Impairs the Maturation of HERG K ϩ Channels-There are two forms of the HERG protein in HERG-HEK293 cells. The 135-kDa form represents the core-glycosylated protein located mainly in the ER, and the 155-kDa form represents the fully glycosylated protein located mainly in the Golgi complex and plasma membrane. P4 at 0.5 mol/liter or higher significantly decreased the amount of the mature (fully glycosylated) form, but had no significant effect on the immature (core-glycosylated) form of the HERG K ϩ channel (Fig. 1A). HERG coassembles with its ␤-subunit Mirp1 to conduct I Kr . Similar results were also found in the HERG-HEK293 cells transfected with Mirp1, the ␤-subunit of HERG/I Kr (supplemental Fig. 1). These data suggest that P4 may impair HERG trafficking in the presence or absence of its ␤-subunit.
In a subsequent time course study, the significant inhibitory effect on channel maturation was observed after treatment with P4 for 8 h (Fig. 1B). The effect of P4 was reversible as removal of P4 for 6 h successfully rescued the mature form of HERG (Fig.  1C). The above data suggest that the effect of P4 on HERG maturation is both concentration-and time-dependent.
P4 Significantly Decreases HERG Current Intensity-We also tested whether P4 can affect HERG function by recording K ϩ currents by whole cell patch clamping. Current protocols are stated in figure legends. As shown in Fig. 2, A and B, treatment with P4 (0.5-5 mol/liter, 24 h) concentration-dependently decreased current density at the peak of tail current in HERG-HEK293 cells. Acute P4 treatment (0.05-5 mol/liter) for 15 min failed to affect HERG K ϩ current density (data not shown), which is in agreement with the previous study on I Kr (6). These data suggest that the long term effect of P4 was not secondary to its direct interaction with HERG channels. Similar results were observed in the CHO cells overexpressing HERG K ϩ channels (supplemental Fig. 2). P4 also significantly reduced HERG current density in the HEK293 cells expressing both HERG and Mirp1 (Fig. 2C). These data suggest that P4 may also inhibit the function of HERG/I Kr .
P4 Preferentially Decreases HERG Channel Protein in the Plasma Membrane-We further investigated the effect of P4 on HERG K ϩ channel subcellular compartmentalization. To detect the surface expression, cells were incubated with anti-K v 11.1 raised against the extracellular loop between S1 and S2 domains. Confocal microscopic examination showed the fluorescent HERG signals at the cell surface (Fig. 3A). The signals were quantified with the software ImageJ as shown in the right panel. It was found that P4 significantly reduced HERG expression on the plasma membrane (Control: 50.5 Ϯ 7.7 versus P4: 10.4 Ϯ 5.0, p Ͻ 0.05). To confirm further the preferential effect of P4, cells were permeabilized and double-stained with antimyc and anti-calnexin (ER marker) or anti-GM130 (Golgi complex marker) antibodies. P4 caused ER dilation (dotted green  signal, anti-calnexin), and HERG K ϩ channels (red signal) accumulated in the dilated ER (Fig. 3B). In contrast, there is no co-localization between the accumulated HERG K ϩ channels (red signal) and the Golgi complex (green signal, anti-GM130) (Fig. 3C). This result suggests that P4 treatment may cause ER stress and affect HERG K ϩ channel folding and trafficking.
To confirm our findings, we labeled plasma membrane protein with biotinylation. Fig. 4A shows that the fully glycosylated form of HERG protein is the predominant form present in the plasma membrane. P4 treatment significantly reduced biotinylation-labeled plasma membrane HERG protein. These data suggest that P4 may mainly reduce the plasma membrane protein.
To study P4-induced ER stress that may impair HERG trafficking from the ER, we further examined the protein expression of C/EBP homologous protein (CHOP), a hallmark of ER stress. We found that P4 increased CHOP protein expression starting from 4 h of treatment in a time-dependent manner (Fig. 4B, left panels). This is mimicked by thapsigargin, which induces ER stress via blocking ER Ca 2ϩ -ATPase and disturbing ER Ca 2ϩ homeostasis (Fig. 4B, right panels). These data clearly suggest that P4 may induce a HERG trafficking defect via promoting ER stress.

Effect of P4 Is Neither P4 Receptor-mediated nor via de Novo
Protein Synthesis-RU486, a P4 receptor antagonist, decreased the mature form of HERG K ϩ channel slightly, but failed to block the inhibitory effect of P4 on HERG maturation (Fig. 5A). These data suggest that the effect of P4 is not mediated by the nuclear P4 receptor. This is also supported by our functional data that P4 decreased HERG K ϩ current density in CHO cells (supplemental Fig. 2), which do not express P4 receptors (23). Cycloheximide, a protein synthesis inhibitor significantly decreased the total amount of HERG protein but failed to prevent/abolish the effect of P4 on HERG K ϩ channel maturation (Fig. 5B). These results indicate that the effect of P4 on HERG channel trafficking is neither via stimulation of nuclear P4 receptor nor via de novo protein synthesis.
The role of protein kinases was also investigated. We found that inhibitors of MAPK, PI3K/Akt, cAMP, or PKA failed to abolish the effect of P4 (supplemental Figs. 3 and 4).
Effect of P4 Is Reversed by a HERG Channel Blocker and Low Culture Temperature-Both E-4031, a HERG channel blocker, and low temperature (27°C) improve the proper folding of HERG channel in ER (24). As shown in Fig. 5, C and D, both maneuvers rescued the HERG channel trafficking-defect caused by P4. These data imply that P4 may affect HERG channel folding in ER and/or block its trafficking to Golgi complex.
Effect of P4 on Cholesterol Level and Distribution-Protein folding/trafficking can be directly or indirectly affected by cholesterol (25)(26)(27)(28). We examined the role of cholesterol in the regulatory effect of P4 on HERG K ϩ channel maturation. The free cholesterol in the cells was stained with filipin. As shown in Fig. 6A, P4 caused free cholesterol accumulation in the cytosol in a concentration-dependent manner (1-5 M). 2-Hydroxypropyl-␤-cyclodextrin (HPCD), a sterol-binding agent, redis-tributed cholesterol in the compartments of cells (Fig. 6B), which is consistent with previous reports (29).
The effect of P4 on cholesterol content was also examined. As shown in Fig. 6C, P4 had no significant effect, whereas simvastatin (10 M, an inhibitor of de novo cholesterol synthesis) significantly decreased free and total cholesterol levels in HERG-HEK293 cells. These data suggest that P4 can only affect the distribution of cholesterol, but had no effect on the amount of total and free forms of cholesterol.
P4 Blocks HERG Channel Trafficking via Disturbing Intracellular Cholesterol Homeostasis-Western blot data showed that HPCD reversed the effect of P4 on HERG channel maturation, but had no effect on that of V630A, a HERG trafficking mutant (Fig. 7A). These data suggest that cholesterol is important for the manifestation of the P4-induced trafficking defect, but does not mediate that caused by HERG trafficking mutants.
Rab9 is important for cholesterol trafficking, and overexpression of Rab9 prevents cholesterol accumulation (30). We found in the present study that overexpression of Rab9, which itself had no effect on HERG trafficking, partially reversed the HERG trafficking defect caused by P4 (Fig. 7B). These data confirm that cholesterol accumulation is responsible for the P4-induced HERG trafficking defect.
To confirm these findings, we decreased the cholesterol level with simvastatin, accumulated free cholesterol in the late endosome/lysosome with imipramine, or delivered a large amount of exogenous cholesterol into the cells. We found that all of these treatments mimicked the effect of P4. In addition, simvastatin failed to reverse the effect of cholesterol (Fig. 7C). Similar effects were also observed in HERG currents. As shown in supplemental Fig. 5, both cholesterol and simvastatin decreased HERG currents. Application of cholesterol together with simvastatin failed to reverse the effect of simvastatin. These data suggest that not only the cholesterol level, but also the distribution of free cholesterol, is important for HERG maturation.
We further tested the involvement of cholesterol in P4-induced ER stress. As shown in Fig. 7D, HPCD reversed P4-induced up-regulation of CHOP expression, but failed to affect thapsigargin-induced CHOP expression. These data further indicate that P4 may impair HERG maturation by disturbing intracellular cholesterol homeostasis and the subsequent ER stress.
Specificity of P4 on HERG K ϩ Channel Trafficking-In this series of experiments, we first studied whether P4 has a similar effect on L-type Ca v 1.2 calcium channels. The human cardiac Ca v 1.2CM (22), and its accessory subunits, ␤ 2a and ␣ 2 ␦, were transfected into HEK293 cells. Two protocols, IV protocol (Fig.  8A) and tail protocol (Fig. 8B), were employed to record I Ba that flowed through the Ca v 1.2CM channels. As shown in the right panels of Fig. 8, A and B, the current densities recorded with both protocols were not affected by P4 (5 M, 24 h). The effect of P4 on endogenous voltage-gated K ϩ current was also examined. As shown in Fig. 8C, P4 had no significant effect on current density of endogenous voltage-gated K ϩ current. K v 1.5 channel undergoes similar glycosylation and trafficking to HERG. The fully glycosylated form (75 kDa) mainly represents the plasma membrane channel, whereas the core-glycosylated form (68KDa) mainly represents the immature form in ER (31). As shown in Fig. 8D, treatment with P4 (5 M) for 24 h only impaired the trafficking of HERG, but had no effect on that of K v 1.5 K ϩ channels. Taken together, the above data clearly demonstrated that P4 specifically induced a HERG K ϩ channel trafficking defect.
P4 Impaired Maturation of ERG K ϩ Channels in Rat Neonatal Cardiac Myocytes-There are also two forms of ERG protein (160 and 120 kDa) in rat neonatal cardiac myocytes, which are consistent with the mature and immature forms of rat ERG1a as previously reported (32). We found that treatment with 5 M P4 for 24 h significantly decreased the mature form of the ERG K ϩ channel (Fig. 9A) and the current density of ERG K ϩ current (Fig.  9B), suggesting that P4 may also impair the maturation and function of ERG channels in the native cardiac tissue.

DISCUSSION
HERG trafficking defects are one of the main causes of LQTS. Trafficking can be impaired by mutations of the channel (33) or induced by drugs such as probucol, cardiac glycosides, fluoxetine, norfluoxetine, pentamidine, arsenic trioxide, and celas-trol (31,34). We report here that a HERG trafficking defect can also be induced by excessive P4 hormone. The effects of P4 on I Kr current density and the channel protein trafficking were further confirmed in HERG-HEK293 cells transfected with Mirp1 and in neonatal cardiac myocytes. The impaired ERG/I Kr may, in turn, induce imbalance of heart electrical stability and therefore development of ventricular arrhythmias. This may suggest a mechanism to explain why the corrected QT intervals are longer in pregnancy and why women at late pregnancy are more susceptible to ventricular arrhythmias. However, for the LQTS patients, the trafficking of the mutated HERG is already blocked. Thus, P4 may not be able to further impair the blocked trafficking of the mutated HERG channels in the inherited LQTS patients. For this reason, our results may also explain why the incidence of arrhythmias is not higher in pregnancy than in other periods in the life of the LQTS patients (3,13).
During the late phase of pregnancy, the P4 level in fetal circulation (ϳ4.5 mol/liter) is much higher than that in maternal blood (1 mol/liter). This may imply that P4 has a greater impact on the fetal heart and could explain the higher rate of Apart from the Western blots and patch clamp data, the impaired HERG protein trafficking by P4 was further confirmed with the confocal microscopy. P4 preferentially decreased Golgi and plasma membrane protein expression. More importantly, P4 treatment induced ER stress and dilation. Immature HERG protein may be stuck in the dilated ER. This may prevent the maturation and trafficking of HERG into Golgi and plasma membrane.
To investigate the mechanism, we first examined whether the effect of P4 is mediated by nuclear P4 receptors. We found that RU486, a P4 receptor blocker, failed to reverse the effect of P4 on HERG K ϩ channel trafficking. In addition, the inhibitory effect of P4 on HERG currents in CHO cells, a P4 receptornegative cell line (23), further supports the hypothesis that the effect of P4 is nuclear receptor-independent. In addition, P4 did not decrease the total amount of HERG protein. Cycloheximide, a protein synthesis inhibitor, decreased total protein expression but failed to block the P4 effect. These results indicate that the effect of P4 is not via altering protein synthesis.
The role of protein kinases was also investigated. We found that the effect of P4 is not secondary to activation of MAPK, PI3K/ Akt, cAMP, or PKA.
Cholesterol homeostasis is very important for protein folding and trafficking. Cholesterol may affect membrane protein folding either directly or indirectly. For example, nicotinic acetylcholine receptor contains internal binding sites for cholesterol, and cholesterol binding stabilizes nicotinic acetylcholine receptor protein structure. Overloading of free cholesterol may also directly cause ER stress and ER dilation (35) and therefore impairs protein folding. We therefore examined whether the P4-induced HERG trafficking defect involves cholesterol. We found in the present study that P4 induced intracellular free cholesterol accumulation, which further induces ER stress and ER dilation. Because P4 can insert into lipid bilayers and perturb membrane function and lipid mobility (36), direct interaction between P4 and lipids may be responsible for the action of P4 on intracellular cholesterol homeostasis. HPCD is a sterolbinding agent, which redistributes cholesterol in the cellular compartments. HPCD also can disrupt membrane lipid rafts and affect functions of membrane receptors, ion channels, transporters, and protein kinases located in/related to lipid rafts. We found that HPCD abolished the effect of P4 on cholesterol homeostasis, ER stress, and HERG trafficking. Rab9 is important for cholesterol trafficking. Overexpression of Rab9 prevents cholesterol accumulation (30). We found in this study that Rab9 also reversed HERG trafficking defects. These data confirm that the P4-induced HERG trafficking defect is secondary to the accumulation of cholesterol. In addition, disrupting intracellular cholesterol homeostasis with simvastatin, imipramine, or exogenous delivery of cholesterol mimicked the effect of P4 on HERG K ϩ channel trafficking. Thus, impaired cholesterol  processing (high levels, low levels, or disrupted distribution) may contribute to HERG K ϩ channel defects caused by P4.
The mechanisms underlying cholesterol-induced ER stress are still not clear. It has been reported that free cholesterol accumulation in the ER membrane activates the unfolded protein response (37). The unfolded protein response may induce ER-phagy, which selectively sequesters and tightly packs ER membranes into autophagosomes (38). In ER, cholesterol is also an important factor for ER-Golgi membrane transport. Sterol depletion in the ER inhibits the ER-to-Golgi transport of secretory membrane proteins (25). In addition, inhibition of the early stage of de novo cholesterol synthesis can also affect isoprenoid intermediates, which are very important for the membrane anchoring and activation of small G proteins such as Ras, Rho, and Rab. The dysfunction of these G proteins can affect protein trafficking (27,28).
Interestingly, we found in the present study that the trafficking of K v 1.5 channels was not affected by P4. These data suggest that the mechanism for the effect of P4 may not be relevant to K v 1.5 trafficking. More studies are warranted to examine how exactly cholesterol regulates ER function/stress and its specificity on protein trafficking.
In summary, P4 may disturb intracellular cholesterol homeostasis and block HERG channel trafficking, which may prolong the QT intervals of both mother and fetus and increase the risk of developing ventricular arrhythmias. Our study may reveal a new mechanism for pregnancy-associated LQTS and provide new approaches to prevent ventricular arrhythmias and sudden death. Cells were activated from the holding potential (Ϫ100 mV) via a series of depolarizing pulses (Ϫ60 to 40 mV) for 900 ms. n ϭ 9 -12. B, I-V curve plotted from the peak tail currents. Cells were activated via a series of depolarizing pulses (Ϫ60 to 100 mV) for 20 ms, and the tail currents were recorded at the voltage of Ϫ50 mV. n ϭ 7-12. C, effect of P4 on endogenous voltage-gated K ϩ currents of HEK293 cells. I-V relation curve was plotted from the currents measured at the end of depolarizing test pulses. Cells were activated from the holding potential (Ϫ70 mV) via a series of depolarizing pulses (Ϫ60 to 120 mV) for 5 s. n ϭ 9. Current and time scales are shown in the insets of different representative tracings (A-C). D, effect of P4 on the trafficking of K v 1.5 and HERG. Both myc-K v 1.5 and myc-HERG were transiently transfected into HEK293 cells. Cells were treated with P4 or vehicle for 24 h. Anti-myc antibody was used to detect both myc-K v 1.5 and myc-HERG. For these two potassium channels, the upper bands (arrows) indicate the mature or fully glycosylated form, and the lower bands (arrowheads) indicate the immature or core-glycosylated form. Mean Ϯ S.E. (error bars) are shown. n ϭ 5. *, p Ͻ 0.05.