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Abstract

Channels responsible for slowly activating delayed-rectifier potassium current (I _Ks) are composed of KCNQ1 and KCNE1 subunits, and these channels play a role in the repolarization of cardiac action potentials. Recently, we showed that the antihyperlipidemic drug probucol, which induces QT prolongation, decreases the I _Ks after 24-h treatment. In the present study, we investigated the effects of three cholesterol-lowering agents (probucol, an enhancer of cholesterol efflux; simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor; and triparanol, a 3β-hydroxysterol-▵24-reductase inhibitor) on cholesterol synthesis, the KCNQ1 current (I _KCNQ1), and the I _Ks to clarify the differences in the modes of action of these agents on the I _Ks. Probucol did not inhibit cholesterol synthesis and had no effect on I _KCNQ1, while I _Ks decreased after 24-h treatment. Simvastatin inhibited cholesterol synthesis and decreased I _KCNQ1 and I _Ks. Additionally, the activation kinetics of I _Ks became faster, compared with that of control I _Ks. Triparanol inhibited cholesterol synthesis but did not reduce I _KCNQ1 and I _Ks. However, the activation kinetics of I _Ks became faster. Our data indicated that the mechanism by which probucol inhibits I _Ks was not mediated by the inhibition of cholesterol synthesis but depended on an interaction with the KCNQ1/KCNE1 complex. Meanwhile, the reduction in cholesterol induced by simvastatin and triparanol is one of the mechanisms that affects the kinetics of I _ks.

Keywords

KCNQ1 KCNE1 probucol simvastatin triparanol QT prolongation

Introduction

Slowly activating delayed-rectifier potassium current (I _Ks) plays an important role in the repolarization of action potentials in human atrial and ventricular muscles.¹ The I _Ks channel is composed of four α-subunits and two accessory β-subunits.² KCNQ1 encodes the pore-forming α-subunit and produces fast activating (time constant: 103 ms) and deactivating (time constant: 222 ms) currents (I _KCNQ1).³ The β-subunit encoded by KCNE1 does not yield functional channels by itself; however, the coexpression of KCNQ1/KCNE1 slows the current activation (time constant: 1.3 s)³ and stabilizes the open conformation of KCNQ1 subunits.⁴ The dysfunction of I _Ks channels as a result of the genetic mutation of KCNQ1 or KCNE1 causes congenital long QT syndrome (LQT1 or LQT5).⁵

The KCNQ1 and KCNE1 subunits are transported to the plasma membrane and form I _Ks channel complexes in lipid rafts on the plasma membrane.^6,7 Lipid rafts are specialized microdomains of the cellular membrane composed of glycosphingolipids, sphingomyelin, cholesterol, and phospholipid.⁸ The depletion of cholesterol by 5-methl-β-cyclodextrin induces lipid raft disruption, indicating that cholesterol is an important component of lipid rafts.⁹

Probucol is an antihyperlipidemic drug that is used to reduce blood cholesterol levels in patients with primary hypercholesterolemia. When used clinically, the prolongation of QT intervals and the induction of torsades de pointes (TdP) have been reported.¹⁰ Several mechanisms have been postulated to explain probucol-induced QT prolongation, such as the inhibition of hERG channel trafficking¹¹ and hERG channel degradation.¹² In a previous article, we demonstrated that the I _Ks decreased after 24-h treatment with probucol in Chinese hamster ovary (CHO)-K1 cells with a stable expression of KCNQ1/KCNE1 channels, suggesting that a reduction in the I _Ks might be responsible for QT prolongation.¹³ Because hERG and I _Ks channels are reportedly localized to lipid rafts, cholesterol might be a key factor in the effects of probucol.

In the present study, we focused on the cholesterol-lowering effect of probucol as a possible cause of I _Ks reduction. The cholesterol-lowering mechanism of probucol is postulated to increase cholesterol efflux and to enhance reverse cholesterol transport through the activation of cholesterol ester transfer protein or scavenger receptor class B type I.¹⁴ However, the exact mechanism responsible for the cholesterol-lowering effect remains uncertain, and the effects of probucol on cellular cholesterol metabolism are unclear. On the other hand, the cholesterol-lowering mechanisms of simvastatin and triparanol are well known. Simvastatin inhibits 3-hydroxy-3-methylglutaryl coenzyme A reductase (a key rate-limiting enzyme in cholesterol biosynthesis),¹⁵ while triparanol inhibits 3β-hydroxysterol-▵24-reductase (DHCR24),¹⁶ as DHCR24 catalyzes the last step in cholesterol biosynthesis. These different sites of action of cholesterol biosynthesis inhibitors affect the cholesterol content of cells in different manners.^17,18 Therefore, evaluating the effects of different cholesterol-lowering compounds on I _Ks is of interest. Here, we examined I _Ks inhibition mechanisms of probucol and two types of cholesterol biosynthesis inhibitors using CHO-K1 cells expressing both KCNQ1 and KCNE1 or KCNQ1 subunits alone.

Materials and methods

Cell lines

CHO-K1 cells expressing KCNQ1 (KCNQ1-CHO-K1) or KCNQ1/KCNE1 (KCNQ1/KCNE1-CHO-K1) channels were prepared by retroviral-mediated transduction with the KCNQ1 or KCNQ1/KCNE1 genes, respectively. The methods used to prepare these cells were described previously.¹³ CHO-K1 cells were used as the host cells for the following reasons. First, CHO-K1 cells are suitable for electrophysiological experiments involving heterologously expressed ion channels because they are adherent cells and the background ionic currents are low.¹⁹ Second, the expression of ion channels in CHO-K1 cells is relatively easy, and these cells can be maintained for a long time with a stable expression level.¹⁹ Third, CHO-K1 cells are reported to have caveolin, which is an important component of caveolae, where some ion channels are presumed to exist.²⁰

Reagents

The inhibitors of cholesterol synthesis, simvastatin (Wako Pure Chemical Industries, Ltd, Osaka, Japan) and triparanol (Sigma-Aldrich, St Louis, Missouri, USA), were dissolved in 10 mM dimethyl sulfoxide (DMSO; Sigma-Aldrich) to prepare stock solutions. Probucol (Wako) was dissolved in 30 mM ethanol (EtOH; Wako) to prepare a stock solution. The microtubule-dependent vesicle transport inhibitor brefeldin A (Wako) was dissolved in 10 mM DMSO to prepare a stock solution. As vehicles, 0.1% DMSO or 0.1% EtOH was used.

The concentrations used for probucol (0.03–30 μM), simvastatin (0.01–3 μM), and triparanol (0.01–10 μM) were selected for the following reasons. First, the maximum plasma concentration (C _max) of patients who received probucol ranged from 35.2 to 75.9 μM, and these values were sufficiently higher than the concentration required for I _ks inhibition (1 μM).²¹ And second, the half maximal inhibitory concentration (IC₅₀) values of simvastatin and triparanol for cholesterol synthesis inhibition were reported to be 0.018 and 0.8 μM in rat liver tissue, respectively.^22,23 In this study, the IC₅₀ values of simvastatin and triparanol were 0.15 and 4.6 μM in the CHO-K1 cells, respectively. The concentrations used in the electrophysiological studies covered these IC₅₀ values.

Dulbecco’s modified Eagle’s medium (DMEM), phosphate-buffered saline (PBS (−1)), antibiotic/antimycotic solution, hygromycin B, geneticin, trypsin/ethylenediaminetetraacetic acid (EDTA), and amphotericin B were purchased from Invitrogen (Carlsbad, California, USA). Fetal bovine serum (FBS) was obtained from Equitech-Bio (Kerrville, Texas, USA). [¹⁴C] Acetic acid sodium salt was purchased from GE Health Care UK Ltd (Little Chalford, Buckinghamshire, UK).

Cell cultures

KCNQ1-CHO-K1 or KCNQ1/KCNE1-CHO-K1 cells were maintained in DMEM supplemented with 10% FBS, 1% antibiotic/antimycotic solution, and 0.5 mg/mL of geneticin with or without 0.5 mg/mL of hygromycin B at 37°C in a 5% CO₂ incubator.

Pretreatment for 24 h

When cells cultured in F225 flasks reached about 80% confluence, they were divided into F75 flasks and incubated for 24 h in normal culture medium. Then, the culture medium in the flasks was exchanged with (−) FBS and (−) antibiotic/antimycotic medium containing the vehicle alone or various concentrations of the test compounds. The cells were then further incubated for 24 h. After 24-h treatment, the cells were washed with PBS (−) twice and dispersed using trypsin/EDTA.

Planar patch-clamp electrophysiology

Electrophysiological recordings were performed using the IonWorks Quattro systems (Molecular devices, Sunnyvale, California, USA) in the population patch-clamp mode. The operation protocol for this system was described previously.¹³ Briefly, for I _KCNQ1 or I _Ks recordings, the compositions of the external and internal solutions were as follows: the external solution contained 120 sodium–gluconate, 17 sodium chloride, 4 potassium chloride (KCl), 1 magnesium chloride (MgCl₂), 2 calcium chloride, 10 glucose, and 10 hydroxyethyl piperazineethanesulfonic acid (HEPES; in mM; pH 7.4 with 1 M potassium hydroxide (KOH)), while the internal solution contained 140 KCl, 1 MgCl₂, 1 ethylene glycol tetraacetic acid, and 20 HEPES (in mM; pH 7.25 with 1 M KOH). To establish the perforate patch configuration, 100 μg/mL of amphotericin B was added to the internal solution. Wells with a series resistance of 20 MΩ or less were not included in the analysis. The current signal was sampled at 2.5 kHz.

Voltage protocol for I _KCNQ1and I _Ks experiments

To evaluate the effects of acute and 24-h treatment with the cholesterol-lowering compounds (probucol, simvastatin, and triparanol) and brefeldin A, the same voltage clamp pulses were used. To characterize the voltage dependency of I _KCNQ1 or I _Ks activation, I _KCNQ1 or I _Ks were evoked using 3-s depolarizing step pulses to +20 mV from a holding potential of −80 mV, followed by a repolarization pulse to −50 mV for 1s. A step pulse to −90 mV with 80-ms duration was applied before the test pulses, and the small current elicited by this step pulse was used to subtract the current leakage. The amplitude of I _KCNQ1 or I _Ks was measured as the difference in the current levels between the holding potential and the depolarizing test pulses. To evaluate the effects of 24-h treatment, only the precompound current was recorded, since the compounds were washed out before the current recording. The residual current ratio for the 24-h treatment was calculated by dividing the mean value of the wells treated with the compound by that of the wells treated with the vehicle alone.

Measurement of cholesterol synthesis

A total of 2 × 10⁵ KCNQ1/KCNE1-CHO-K1 cells were treated with the vehicle or a cholesterol-lowering compound (probucol, simvastatin, or triparanol) for 24 h. Then, 2 μCi of [¹⁴C] acetic acid sodium salt was added and the cells were incubated for an additional 2 h. The cells were washed twice with saline, and the lipids were extracted with 800 μL of hexane-2-propanol (3:2, v/v) for 30 min. The solvents were evaporated under a nitrogen gas stream at 40°C. The samples were resuspended in 50 μL of petroleum ether, applied to thin layer chromatography glass plate coated with silver nitrate, and developed using chloroform–acetone (85:15, v/v). The radioactivity in the cholesterol and the total radioisotope count were analyzed using the BAS2500 imaging plate system (GE Health Care, Little Chalford, Buckinghamshire, UK).

Measurement of cholesterol in cells

A total of 2 × 10⁶ KCNQ1/KCNE1-CHOK1 cells were cultured on 10-cm plastic dishes. After incubation with the vehicle or a cholesterol-lowering compound (probucol, simvastatin, or triparanol) for 24 h, the cells were washed twice with saline. The cellular lipids were extracted with 10 mL of hexane-2-propanol (3:2, v/v) containing 20 µg of d7-cholesterol as the internal standard. The solvents were evaporated under a nitrogen gas stream at 40°C, and the residue was dissolved in 1 mL of EtOH. Extract solutions of 1 μL was chromatographed on an Atlantis dC18 (4.6mm × 150 mm, 3 µm; Waters Corporation, Milford, Massachusetts, USA) at 40°C using a mobile phase consisting of distilled water/methanol (initial) and 2-propanol (final) at a flow rate of 0.6 mL/min. The cholesterol contents along with the internal standard solution were measured using high-performance liquid chromatography under a gradient elution with quarto-pole/time-of-flight mass spectrometry (Waters Corporation) operated in the positive atmospheric pressure ionization mode and detected using selected ion monitoring.

Data analysis

Nonlinear curve fittings were performed to calculate the activation time constants of I _Ks using Origin 5.0 software (OriginLab, Northampton, Massachusetts, USA). The data were represented as the mean ± standard error of the mean. Statistical significance was evaluated using the Dunnett’s multiple comparison test.

Results

Effects of cholesterol-lowering compounds on I _KCNQ1

It was reported that treatment with probucol for 24 h decreased I _Ks of KCNQ1/KCNE1-CHO-K1 cells.¹³ We examined the direct effect of probucol on the I _KCNQ1 using KCNQ1-CHO-K1 cells. Probucol treatment did not have any effect on I _KCNQ1 (Figure 1(a)). Nevertheless, the cholesterol synthesis inhibitor, simvastatin, decreased I _KCNQ1 by approximately 30% at 0.1 μM and above (Figure 1(b)). On the other hand, another cholesterol synthesis inhibitor, triparanol, did not decrease the I _KCNQ1 at 0.3 and 1 μM (Figure 1(b)). After treatment with 3 and 10 μM for 24 h, the formation of a high-resistance seal (gigaseal) was not observed. The exact reason for this finding is not clear, but the cell membrane likely become relatively fragile because of the decrease in cholesterol and the trypsin/EDTA treatment required for cell dispersion. A microtubule-dependent vesicle transport inhibitor, brefeldin A, completely abolished I _KCNQ1 at 3 μM in the same experiment (Figure 1(b)).

Figure 1.

Effects of cholesterol-lowering compounds on I _KCNQ1 in KCNQ1-CHO-K1 cells after 24-h treatment. (a) The changes in the I _KCNQ1 after 24-h treatment with the vehicle control (0.1% ethanol (EtOH); n = 83); or 1 and 3 μM of probucol (n = 77 and 86, respectively), (b) the vehicle control (0.1% dimethyl sulfoxide (DMSO); n = 48); 0.03, 0.1, and 0.3 μM of simvastatin (n = 47, 38, and 22, respectively); 0.3 and 1 μM of triparanol (n = 31 and 19, respectively); or 3 μM of brefeldin A (n = 24) are shown. The current amplitude was measured at the end of a steady-state current at +20 mV. The current values after treatment with the compounds were normalized according to the value for the vehicle control. *p < 0.05, **p < 0.01, ***p < 0.001, compared with the vehicle control. CHO: Chinese hamster ovary.

Effects of cholesterol-lowering reagents on cholesterol synthesis and cholesterol content in KCNQ1/KCNE1-CHO-K1 cells after 24-h treatment

The effects of cholesterol-lowering compounds on cholesterol synthesis and the cholesterol content in KCNQ1/KCNE1-CHO-K1 cells are shown in Figure 2. Neither the inhibition of cholesterol synthesis nor a reduction in the cholesterol content was observed after treatment with probucol (Figure 2(a) and (b)). Treatment with simvastatin or triparanol for 24 h inhibited cholesterol synthesis in the cells (Figure 2(a)) and reduced the cholesterol content (Figure 2(c)). The IC₅₀ values of simvastatin and triparanol for cholesterol synthesis inhibition were 0.15 and 4.6 μM, respectively (Figure 2(a)). The cholesterol content of the cells was reduced to approximately 15% after treatment with simvastatin (0.1 μM; p < 0.01; Figure 2(c)). Triparanol significantly decreased the cholesterol content in a concentration-dependent manner, and a reduction of approximately 20% was observed after treatment with 10 μM (p < 0.001; Figure 2(c)).

Figure 2.

Effects of cholesterol-lowering compounds on the inhibition of cholesterol synthesis and the cholesterol content in KCNQ1/KCNE1-CHO-K1 cells after 24-h treatment. (a) Cells were treated for 24 h with the vehicle control (0.1% EtOH or 0.1% DMSO); 0.03, 0.1, 0.3, 1, 3, 10, or 30 μM of probucol; 0.01, 0.03, 0.1, 0.3, 1, or 3 μM of simvastatin; or 0.01, 0.03, 0.1, 0.3, 1, 3 or 10 μM of triparanol. The synthesis of cholesterol from [C¹⁴] acetic acid was measured and normalized according to the value for the vehicle control (n = 3 each; *p < 0.05, ***p < 0.001, compared with the control). (b and c) KCNQ1/KCNE1-CHO-K1 cells were treated for 24 h with the vehicle control; 0.1, 0.3, or 1 μM of probucol; 0.03 or 0.1 μM of simvastatin; or 1, 3, or 10 μM of triparanol. The cholesterol contents of the treated cells were measured and normalized according to the value for the vehicle control (n = 3, respectively, *p < 0.05, **p < 0.01, ***p < 0.001, compared with the control). CHO: Chinese hamster ovary; DMSO: dimethyl sulfoxide; EtOH: ethanol.

Effects of cholesterol-lowering compounds on I _Ks

An acute effect of probucol on I _ks was not observed at concentrations up to 10 μM in a previously reported article.¹³ Treatment with simvastatin or triparanol did not induce any acute effects on I _Ks in KCNQ1/KCNE1-CHO-K1 (Figure 3(a)). After 24-h treatment, probucol significantly decreased the I _Ks in a concentration-dependent manner from 0.03 μM and almost abolished the current at 1 μM (Figure 3(b)). The effects of 24-h treatment with simvastatin or triparanol on I _Ks are shown in Figure 3(c). Treatment with simvastatin at 0.03, 0.1, or 0.3 μM significantly decreased the I _Ks by approximately 20% (p < 0.001). In contrast, triparanol did not reduce the I _Ks until 3 μM. Brefeldin A completely abolished the I _Ks at 3 μM (Figure 3(c)).

Figure 3.

Acute and 24-h treatment effects of cholesterol-lowering compounds on I _Ks in KCNQ1/KCNE1-CHO-K1 cells. (a) Changes in I _Ks after acute treatment with the vehicle control (n = 16); 0.03, 0.1, and 0.3 μM of simvastatin (n = 5, 5, and 6, respectively); or 0.3, 1, and 3 μM of triparanol (n = 6, 6, and 6, respectively). The current amplitude was measured at the end of a steady-state current at +20 mV. The current values after treatment with the compounds were normalized according to the value for the vehicle control (0.1% DMSO). (b) Change in I _Ks after 24-h treatment with the vehicle control (0.1% EtOH; n = 64); or 0.03, 0.1, 0.3, and 1 μM of probucol (n = 80 each). (c) Changes in I _Ks after 24-h treatment with the vehicle control (0.1% DMSO; n = 40); 0.03, 0.1, and 0.3 μM of simvastatin (n = 36, 30, and 40, respectively); 0.3, 1, and 3 μM of triparanol (n = 40, 35, and 43, respectively); or 3 μM of brefeldin A (n = 13). ***p < 0.001, compared with the vehicle control. CHO: Chinese hamster ovary; DMSO: dimethyl sulfoxide; EtOH: ethanol.

Effects of cholesterol-lowering compounds on the activation kinetics of I _Ks

Figure 4(a) shows representative traces of I _Ks and I _KCNQ1 evoked by depolarizing pulses at +20 mV. In the absence of KCNE1, I _KCNQ1 were activated with rapid kinetics with depolarizing pulses. The coexpression of KCNQ1 and KCNE1 slowed the activation kinetics of I _KCNQ1. Figure 4(b) shows the typical current traces produced by the treatment with probucol, simvastatin, or triparanol. After treatment with probucol at 1 μM for 24 h, the I _Ks amplitude decreased greatly and the activation kinetics became faster, compared with the control I _Ks. Simvastatin decreased the I _Ks amplitude and accelerated the activation kinetics. Triparanol did not decrease the I _Ks amplitude, but the activation kinetics became faster (Figure 4(b)). Figure 4(c) summarizes the activation time constants for I _Ks. The activation time constants at +20 mV were significantly decreased by treatment with probucol, simvastatin, or triparanol.

Figure 4.

Effects of cholesterol-lowering compounds on the kinetics of I _Ks and KCNQ1 after 24-h treatment. (a) Representative I _Ks and I _KCNQ1 traces elicited by a step pulse to +20 mV from a holding potential of −80 mV in cells treated with the vehicle control (0.1% DMSO) using KCNQ1/KCNE1-CHO-K1 and KCNQ1-CHO-K1 cells. (b) Representative current traces elicited by a step pulse to +20 mV from a holding potential of −80 mV in cells treated with the vehicle control (0.1% DMSO), 1 μM of probucol, 0.3 μM of simvastatin, or 3 μM of triparanol using KCNQ1/KCNE1-CHO-K1 cells. (c) Time constants for I _Ks activation in the vehicle control (0.1% DMSO), 1 μM of probucol, 0.3 μM of simvastatin, or 3 μM of triparanol (n = 8 each; ***p < 0.001, compared with the vehicle control) using KCNQ1/KCNE1-CHO-K1 cells. CHO: Chinese hamster ovary; DMSO: dimethyl sulfoxide.

Discussion

Probucol has been used as a cholesterol-lowering reagent for the treatment of hyperlipidemia, but clinical reports have indicated that it can cause QT prolongation and ventricular arrhythmias, including TdP.^10,24 In our previous article, we postulated that a reduction in I _Ks channels on the plasma membrane might be responsible for the prolongation of the QT interval.¹³ One possible mechanism involves the cholesterol-lowering effect of probucol, since cholesterol in the plasma membrane has an important role in the formation of lipid rafts and the distribution of ion channels to these lipid rafts.^25,26 I _Ks channels are composed of KCNQ1 and KCNE1 subunits and are transported to and function in caveolin-rich lipid rafts (caveolae)^6,7; the depletion of cholesterol leads to the breakdown of the caveolae. This mechanism is thought to function in CHO cells, since caveolin exists endogenously in CHO cells²⁰ and I _Ks channels are presumed to exist in the caveolae. However, probucol did not affect either cholesterol synthesis or cholesterol content, suggesting that cholesterol metabolism is not involved in the probucol-induced inhibition of I _Ks. Furthermore, a novel lipid-lowering agent AGI-1067, which is a metabolically stable derivative of probucol and has a similar cholesterol-lowering mechanism to probucol,²⁷ did not prolong QT intervals.²⁸ This fact supports the hypothesis that probucol reduces the I _Ks by the mechanisms unrelated to cholesterol-lowering effects.

Our previous data showed that probucol decreases the number of channel complexes functioning as I _Ks channels,¹³ but whether probucol preferentially targets KCNQ1, KCNE1, or KCNQ1/KCNE1 complex remained unclear. One possible mechanism explaining the chronic effects of probucol on I _Ks is the inhibition of KCNQ1 translocation from the endoplasmic reticulum to the Golgi apparatus. Brefeldin A, which inhibits microtubule-dependent vesicle transport,²⁹ abolished I _Ks and I _KCNQ1. Meanwhile, probucol inhibited I _Ks but had no effect on I _KCNQ1, indicating that probucol is unlikely to inhibit KCNQ1 trafficking.

Probucol did not affect I _KCNQ1 when only KCNQ1 was expressed, so that the target of probucol was thought to be KCNE1 or the KCNQ1/KCNE1 complex. If probucol binds exclusively to the KCNE1 protein and interferes with the ability of KCNE1 to associate with KCNQ1, I _KCNQ1 might be observed after treatment with probucol. However, probucol almost completely abolished I _Ks, suggesting that probucol might exert its effect by interacting with the KCNQ1/KCNE1 complex. Although the exact mechanism responsible for probucol’s effect remains unclear, the most probable mechanism for the reduction of I _ks by probucol is the degradation of the KCNQ1/KCNE1 complex through the ubiquitin–proteasome system.³⁰

On the other hand, simvastatin and triparanol are thought to affect I _Ks by inhibiting cholesterol synthesis. Ion channels have been shown to exist in caveolae or lipid rafts³¹ and a decrease in the cholesterol level led to the degradation of caveolae and lipid rafts in various cells,^6,18 suggesting that distribution of ion channels is greatly disturbed by cholesterol synthesis inhibitors. Ion channels, such as l-type calcium channels, that are regulated by phosphorylation and dephosphorylation through hormone receptors are localized in caveolae or lipid rafts and form large channel complexes.³¹ I _Ks channels are similarly regulated and exist with caveolin-3 in heart cells; thus, I _Ks channels are thought to be located in caveolae/lipid rafts. The existence of caveolin protein in the lipid rafts was dramatically decreased by various cholesterol inhibitors,¹⁸ suggesting that a small decrease in cholesterol in CHO cells might have significant effects on caveolae formation and the integration of the I _Ks channel complex.

The KCNE1 subunit slows the activation kinetics of the I _KCNQ1 when associated with KCNQ1 channels. If the association between KCNE1 and KCNQ1 subunits is weakened after a reduction in cholesterol levels, the channel might show the property resembling KCNQ1 channel alone and activation kinetics of the residual I _Ks might become faster. The faster activation kinetics of residual I _Ks after treatment with simvastatin and triparanol can likely be explained by this effect. Triparanol is known to increase the production of desmosterol,¹⁶ a precursor of cholesterol. However, the increase in desmosterol might not compensate for the role of cholesterol for the formation of caveolae, and the integration of KCNQ1 and KCNE1 complex might be disturbed. This was supported by the finding that treatment of triparanol inhibited insulin receptor, which was located in lipid rafts and reduced insulin-stimulated glucose uptake.¹⁸ On the other hand, desmosterol sufficiently sustained the proliferation of J744-D cells in the absence of cholesterol.³² This action of desmosterol may be related to the fact that triparanol did not reduce the amplitude of I _Ks.

The present findings are very interesting from the standpoint that probucol and cholesterol synthesis inhibitors, which decrease cholesterol in clinical applications, regulate I _Ks channel function through different mechanisms. These differences are thought to be important when we consider the risk of QT interval prolongation.

Footnotes

Acknowledgments

We wish to thank Ms Misato Nakagawa, Ms Risa Hashimoto, and Mr Takuho Ishii for their technical support in measuring the cholesterol levels, and Ms Hiromi Ohashi for collecting the IonWorks Quattro data.

Conflict of interest

The authors declared no conflicts of interest.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

References

Nerbonne

. Molecular basis of functional voltage-gated K⁺ channel diversity in the mammalian myocardium. J Physiol 2000; 525(Pt 2): 285–298.

Chen

Kim

Rajan

Charybdotoxin binding in the I(Ks) pore demonstrates two MinK subunits in each channel complex. Neuron 2003; 40: 15–23.

Seebohm

Lerche

Pusch

A kinetic study on the stereospecific inhibition of KCNQ1 and I(Ks) by the chromanol 293B. Br J Pharmacol 2001; 134: 1647–1654.

Jespersen

Grunnet

Olesen

. The KCNQ1 potassium channel: from gene to physiological function. Physiology (Bethesda). 2005; 20: 408–416.

Chiang

Roden

. The long QT syndromes: genetic basis and clinical implications. J Am Coll Cardiol 2000; 36: 1–12.

Balijepalli

Delisle

Balijepalli

Kv11.1 (ERG1) K⁺ channels localize in cholesterol and sphingolipid enriched membranes and are modulated by membrane cholesterol. Channels (Austin) 2007; 1: 263–272.

Nakamura

Kurokawa

Bai

Progesterone regulates cardiac repolarization through a nongenomic pathway: an in vitro patch-clamp and computational modeling study. Circulation 2007; 116: 2913–2922.

O'Connell

Martens

Tamkun

. Localization of ion channels to lipid Raft domains within the cardiovascular system. Trend Cardiovasc Med 2004; 14: 37–42.

Zidovetzki

Levitan

. Use of cyclodextrins to manipulate plasma membrane cholesterol content: evidence, misconceptions and control strategies. Biochim Biophys Acta 2007; 1768: 1311–1324.

10.

Matsuhashi

Onodera

Kawamura

Probucol-induced QT prolongation and torsades de pointes. Jpn J Med 1989; 28: 612–615.

11.

Guo

Massaeli

Identification of IKr and its trafficking disruption induced by probucol in cultured neonatal rat cardiomyocytes. J Pharmacol Exp Ther 2007; 321: 911–920.

12.

Guo

Shallow

Involvement of caveolin in probucol-induced reduction in hERG plasma-membrane expression. Mol Pharmacol 2011; 79: 806–813.

13.

Taniguchi

Uesugi

Arai

Chronic probucol treatment decreases the slow component of the delayed-rectifier potassium current in cho cells transfected with KCNQ1 and KCNE1: a novel mechanism of QT prolongation. J Cardiovasc Pharmacol 2012; 59: 377–386.

14.

Yamashita

Matsuzawa

. Where are we with probucol: a new life for an old drug? Atherosclerosis 2009; 207: 16–23.

15.

Goldstein

Brown

. Regulation of the mevalonate pathway. Nature 1990; 343: 425–430.

16.

Avigan

Steinberg

Vroman

Studies of cholesterol biosynthesis. I. The identification of desmosterol in serum and tissues of animals and man treated with MER-29. J Biol Chem 1960; 235: 3123–3126.

17.

Mullen

Luscher

Scharnagl

Effect of simvastatin on cholesterol metabolism in C2C12 myotubes and HepG2 cells, and consequences for statin-induced myopathy. Biochem Pharmacol 2010; 79: 1200–1209.

18.

Sanchez-Wandelmer

Davalos

Herrera

Inhibition of cholesterol biosynthesis disrupts lipid raft/caveolae and affects insulin receptor activation in 3T3-L1 preadipocytes. Biochim Biophys Acta 2009; 1788: 1731–1739.

19.

Sawada

Yoshinaga

Automated patch clamping. Patch Clamp Technol 2012, pp. 323–332. Japan: Springer Japan.

20.

Schilling

Opitz

Wiesenthal

Translocation of endothelial nitric-oxide synthase involves a ternary complex with caveolin-1 and NOSTRIN. Mol Biol Cell 2006; 17: 3870–3880.

21.

Heeg

Tachizawa

. Plasma levels of probucol in man after single and repeated oral doses (author's transl). Nouv Presse Med 1980; 9: 2990–2994.

22.

Mosley

Kalinowski

Schafer

Tissue-selective acute effects of inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase on cholesterol biosynthesis in lens. J Lipid Res 1989; 30: 1411–1420.

23.

Bae

Paik

. Cholesterol biosynthesis from lanosterol: development of a novel assay method and characterization of rat liver microsomal lanosterol delta 24-reductase. Biochem J 1997; 326(Pt 2): 609–616.

24.

Tamura

Ueki

Ohtsuka

Probucol-induced QT prolongation and syncope. Jpn Circ J 1994; 58: 374–377.

25.

Brown

London

. Functions of lipid rafts in biological membranes. Annu Rev Cell Dev Biol 1998; 14: 111–136.

26.

Simons

Toomre

. Lipid rafts and signal transduction. Nat Rev Mol Cell Biol 2000; 1: 31–39.

27.

Sundell

Somers

Meng

AGI-1067: a multifunctional phenolic antioxidant, lipid modulator, anti-inflammatory and antiatherosclerotic agent. J Pharmacol Exp Ther 2003; 305: 1116–1123.

28.

Tardif

Gregoire

Schwartz

Effects of AGI-1067 and probucol after percutaneous coronary interventions. Circulation 2003; 107: 552–528.

29.

Sciaky

Presley

Smith

Golgi tubule traffic and the effects of brefeldin A visualized in living cells. J Cell Biol 1997; 139: 1137–1155.

30.

Mearini

Schlossarek

Willis

The ubiquitin-proteasome system in cardiac dysfunction. Biochim Biophys Acta 2008; 1782: 749–763.

31.

Dart

. Lipid microdomains and the regulation of ion channel function. J Physiol 2010; 588: 3169–3178.

32.

Rodriguez-Acebes

de la Cueva

Fernandez-Hernando

Desmosterol can replace cholesterol in sustaining cell proliferation and regulating the SREBP pathway in a sterol-delta24-reductase-deficient cell line. Biochem J 2009; 420: 305–315.