Effects of rosiglitazone, an antidiabetic drug, on Kv3.1 channels.

Rosiglitazone is a thiazolidinedione-class antidiabetic drug that reduces blood glucose and glycated hemoglobin levels. We here investigated the interaction of rosiglitazone with Kv3.1 expressed in Chinese hamster ovary cells using the whole-cell patch-clamp technique. Rosiglitazone rapidly and reversibly inhibited Kv3.1 currents in a concentration-dependent manner (IC50 = 29.8 µM) and accelerated the decay of Kv3.1 currents without modifying the activation kinetics. The rosiglitazone-mediated inhibition of Kv3.1 channels increased steeply in a sigmoidal pattern over the voltage range of -20 to +30 mV, whereas it was voltage-independent in the voltage range above +30 mV, where the channels were fully activated. The deactivation of Kv3.1 current, measured along with tail currents, was also slowed by the drug. In addition, the steady-state inactivation curve of Kv3.1 by rosiglitazone shifts to a negative potential without significant change in the slope value. All the results with the use dependence of the rosiglitazone-mediated blockade suggest that rosiglitazone acts on Kv3.1 channels as an open channel blocker.

The antidiabetic drug rosiglitazone blocks Kv1.5 potassium channels in an open state.

An antidiabetic drug, rosiglitazone is a member of the drug class of thiazolidinedione. Although restrictions on use due to the possibility of heart toxicity have been removed, it is still a drug that is concerned about side effects on the heart. We here examined, using Chinese hamster ovary cells, the action of rosiglitazone on Kv1.5 channels, which is a major determinant of the duration of cardiac action potential. Rosiglitazone rapidly and reversibly inhibited Kv1.5 currents in a concentration-dependent manner (IC50 = 18.9 µM) and accelerated the decay of Kv1.5 currents without modifying the activation kinetics. In addition, the deactivation of Kv1.5 current, assayed with tail current, was slowed by the drug. All of the results as well as the use-dependence of the rosiglitazone-mediated blockade indicate that rosiglitazone acts on Kv1.5 channels as an open channel blocker. This study suggests that the cardiac side effects of rosiglitazone might be mediated in part by suppression of Kv1.5 channels, and therefore, raises a concern of using the drug for diabetic therapeutics.

Effects of iloperidone on hERG 1A/3.1 heterotetrameric channels.

OBJECTIVES: Iloperidone is an atypical antipsychotic drug that is widely used for the treatment of schizophrenia. hERG 3.1, alternatively spliced form of hERG 1A, is considered a potential target for an antipsychotic drug. The present study was designed to study the effects of iloperidone on hERG 1A/3.1 heterotetrameric channels. METHODS: The interactions of iloperidone with hERG 1A/3.1 heterotetrameric channels stably expressed in HEK cells were investigated using the whole-cell patch-clamp technique and western blot analysis. RESULTS: Iloperidone inhibited the hERG 1A/3.1 tail currents at -50 mV in a concentration-dependent manner with an IC50 value of 0.44 µM. The block of hERG 1A/3.1 currents by iloperidone was voltage-dependent and increased over a range of voltage for channel activation. However, the block by iloperidone was voltage-independent at more depolarized potentials where the channels were fully activated. A fast application of iloperidone inhibited the hERG 1A/3.1 current elicited by a 5-s depolarizing pulse to +60 mV to fully inactivate the hERG 1A/3.1 currents. Iloperidone also induced a rapid and reversible inhibition of hERG 1A/3.1 tail currents during repolarization. However, iloperidone had no effect on either hERG 1A or hERG 1A/3.1 channel trafficking to the cell membrane. CONCLUSIONS: Our results indicated that iloperidone concentration-dependently inhibited hERG 1A/3.1 currents by preferentially interacting with the open states of channels, but not by the disruption of membrane trafficking or surface membrane expression of hERG 1A and hERG 1A/3.1 channel proteins.

Norquetiapine blocks the human cardiac sodium channel Nav1.5 in a state-dependent manner.

Quetiapine, an atypical antipsychotic drug, is used for the treatment of schizophrenia and acute mania. Although a previous report showed that quetiapine blocked hERG potassium current, quetiapine has been considered relatively safe in terms of cardiovascular side effects. In the present study, we used the whole-cell patch-clamp technique to investigate the effect that quetiapine and its major metabolite norquetiapine can exert on human cardiac sodium channels (hNav1.5). The half-maximal inhibitory concentrations of quetiapine and norquetiapine at a holding potential of -90 mV near the resting potential of cardiomyocytes were 30 and 6 µM, respectively. Norquetiapine as well as quetiapine was preferentially bound in the inactivated state of the hNav1.5 channel. Norquetiapine slowed the recovery from inactivation of hNav1.5 and consequently induced strong use-dependent inhibition. Our results indicate that norquetiapine blocks hNav1.5 current in concentration-, state- and use-dependent manners, suggesting that the blockade of hNav1.5 current by norquetiapine may shorten the cardiac action potential duration and reduce the risk of QT interval prolongation induced by the inhibition of hERG potassium currents.

Characteristics of Metal-Metal Nano-Area Contact Resistance for Less Than 5 nm Technology Node.

Recently, better understanding of nano-area is required for 5 nm or less technology node. In particular, the high contact resistance generated in a nano-area significantly degrades the device performance. In this study, we propose a direct contact resistance measurement method without a test structure by separate processes to improve the nano-area contact resistance. The nano-area contact resistance of Ti-Ti and Cu-Cu decreased from 6.46 MΩ to 1.08 MΩ and from 3.78 MΩ to 1.48 MΩ, respectively, when the metal line and native layer formed on the surface were removed. In addition, it is confirmed that the contact resistance decreased with an increase in bonding strength in the case of nano-area homo-metal contact. However, the contact resistance is affected by the tunneling effect and bond energy according to the distance between the first layers of atoms in the case of nano-area hetero-metal contact.

Effects of cariprazine on hERG 1A and hERG 1A/3.1 potassium channels.

Cariprazine is a novel atypical antipsychotic drug that is widely used for the treatment of schizophrenia and bipolar mania/mixed disorder. We used the whole-cell patch-clamp technique to investigate the effects of cariprazine on hERG channels that are stably expressed in HEK cells. Cariprazine inhibited the hERG 1A and hERG 1A/3.1 tail currents at -50 mV in a concentration-dependent manner with IC50 values of 4.1 and 12.2 µM, respectively. The block of hERG 1A currents by cariprazine was voltage-dependent, and increased over a range of voltage for channel activation. Cariprazine shifted the steady-state inactivation curve of the hERG 1A currents in a hyperpolarizing direction and produced a use-dependent block. A fast application of cariprazine inhibited the hERG 1A currents elicited by a 5 s depolarizing pulse to +60 mV to fully inactivate the hERG 1A currents. During a repolarizing pulse wherein the hERG 1A current was deactivated slowly, cariprazine rapidly and reversibly blocked the open state of the hERG 1A current. However, cariprazine did not affect hERG 1A and hERG 1A/3.1 channel trafficking to the cell membrane. Our results indicated that cariprazine concentration-dependently inhibited hERG 1A and hERG 1A/3.1 currents by preferentially interacting with the open states of the hERG 1A channel, but not by the disruption of hERG 1A and hERG 1A/3.1 channel protein trafficking. Our study examined cariprazine's mechanism of action provides a biophysical profile that is necessary to assess the potential therapeutic effects of this drug.

Development of a New Electrochemical Atomic Force Microscopy Tool for Obtaining Surface Morphology Through Electrochemical Reaction.

Electrochemical atomic force microscopy is a complex electrochemical analysis method that has been applied in many fields. Electrochemical atomic force microscopy consists of an electrochemical cell, electrochemical analysis system, and atomic force microscopy. To simultaneously analyze the electrochemical system and the atomic force microscopy, an electrochemical cell with a suitable structure is needed. We developed the electrochemical atomic force microscopy analysis system using a self-developed electrochemical cell. To confirm the in-situ analytical ability of the developed electrochemical atomic force microscopy analysis system, we observed the electrochemical corrosion process of copper with respect to changes in pH of a sulfuric acid solution. The operation of the electrochemical atomic force microscopy tool was verified by experiments on the electrochemical corrosion of copper, and the factors affecting the corrosion process were examined. It was confirmed that electrochemical atomic force microscopy can perform electrochemical analysis and atomic force microscopy image analysis at the same time.

Antidepressant drug paroxetine blocks the open pore of Kv3.1 potassium channel.

In patients with epilepsy, depression is a common comorbidity but difficult to be treated because many antidepressants cause pro-convulsive effects. Thus, it is important to identify the risk of seizures associated with antidepressants. To determine whether paroxetine, a very potent selective serotonin reuptake inhibitor (SSRI), interacts with ion channels that modulate neuronal excitability, we examined the effects of paroxetine on Kv3.1 potassium channels, which contribute to highfrequency firing of interneurons, using the whole-cell patch-clamp technique. Kv3.1 channels were cloned from rat neurons and expressed in Chinese hamster ovary cells. Paroxetine reversibly reduced the amplitude of Kv3.1 current, with an IC50 value of 9.43 µM and a Hill coefficient of 1.43, and also accelerated the decay of Kv3.1 current. The paroxetine-induced inhibition of Kv3.1 channels was voltage-dependent even when the channels were fully open. The binding (k+1) and unbinding (k-1) rate constants for the paroxetine effect were 4.5 µM-1s-1 and 35.8 s-1, respectively, yielding a calculated KD value of 7.9 µM. The analyses of Kv3.1 tail current indicated that paroxetine did not affect ion selectivity and slowed its deactivation time course, resulting in a tail crossover phenomenon. Paroxetine inhibited Kv3.1 channels in a usedependent manner. Taken together, these results suggest that paroxetine blocks the open state of Kv3.1 channels. Given the role of Kv3.1 in fast spiking of interneurons, our data imply that the blockade of Kv3.1 by paroxetine might elevate epileptic activity of neural networks by interfering with repetitive firing of inhibitory neurons.

Effects of norquetiapine, the active metabolite of quetiapine, on cloned hERG potassium channels.

Quetiapine is an atypical antipsychotic drug that is widely used for the treatment of schizophrenia. It is mainly metabolized by a cytochrome P450 system in the liver. Norquetiapine is a major active metabolite in humans with a pharmacological profile that differs distinctly from that of quetiapine. We used the whole-cell patch-clamp technique to investigate the effects of norquetiapine on hERG channels that are stably expressed in HEK cells. Quetiapine and norquetiapine inhibited the hERG tail currents at -50mV in a concentration-dependent manner with IC50 values of 8.3 and 10.8µM, respectively, which suggested equal potency. The block of hERG currents by norquetiapine was voltage-dependent with a steep increase over a range of voltages for channel activation. However, at more depolarized potentials where the channels were fully activated, the block by norquetiapine was voltage-independent. The steady-state inactivation curve of the hERG currents was shifted to the hyperpolarizing direction in the presence of norquetiapine. Norquetiapine did not produce a use-dependent block. A fast application of norquetiapine inhibited the hERG current elicited by a 5s depolarizing pulse to +60mV, which fully inactivated the hERG currents, suggesting an inactivated-state block. During a repolarizing pulse wherein the hERG current was slowly deactivated, albeit remaining in an open state, a fast application of norquetiapine rapidly and reversibly inhibited the open state of the hERG current. Our results indicated that quetiapine and norquetiapine had equal potency in inhibiting hERG tail currents. Norquetiapine inhibited the hERG current by preferentially interacting with the open and/or inactivated states of the channels.

Dapoxetine induces neuroprotective effects against glutamate-induced neuronal cell death by inhibiting calcium signaling and mitochondrial depolarization in cultured rat hippocampal neurons.

Selective serotonin reuptake inhibitors (SSRIs) have an inhibitory effect on various ion channels including Ca2+ channels. We used fluorescent dye-based digital imaging, whole-cell patch clamping and cytotoxicity assays to examine whether dapoxetine, a novel rapid-acting SSRI, affect glutamate-induced calcium signaling, mitochondrial depolarization and neuronal cell death in cultured rat hippocampal neurons. Pretreatment with dapoxetine for 10min inhibited glutamate-induced intracellular free Ca2+ concentration ([Ca2+]i) increases in a concentration-dependent manner (Half maximal inhibitory concentration=4.79µM). Dapoxetine (5µM) markedly inhibited glutamate-induced [Ca2+]i increases, whereas other SSRIs such as fluoxetine and citalopram only slightly inhibited them. Dapoxetine significantly inhibited the glutamate-induced [Ca2+]i responses following depletion of intracellular Ca2+ stores by treatment with thapsigargin. Dapoxetine markedly inhibited the metabotropic glutamate receptor agonist, (S)-3,5-dihydroxyphenylglycine-induced [Ca2+]i increases. Dapoxetine significantly inhibited the glutamate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-induced [Ca2+]i responses in either the presence or absence of nimodipine. Dapoxetine also significantly inhibited AMPA-evoked currents. However, dapoxetine slightly inhibited N-methyl-D-aspartate (NMDA)-induced [Ca2+]i increases. Dapoxetine markedly inhibited 50mMK+-induced [Ca2+]i increases. Dapoxetine significantly inhibited glutamate-induced mitochondrial depolarization. In addition, dapoxetine significantly inhibited glutamate-induced neuronal cell death and its neuroprotective effect was significantly greater than fluoxetine. These data suggest that dapoxetine reduces glutamate-induced [Ca2+]i increases by inhibiting multiple pathways mainly through AMPA receptors, voltage-gated L-type Ca2+ channels and metabotropic glutamate receptors, which are involved in neuroprotection against glutamate-induced cell death through mitochondrial depolarization.

Inhibition of cloned hERG potassium channels by risperidone and paliperidone.

Risperidone and one of its active metabolites, paliperidone, are widely used for the treatment of schizophrenia. We used a patch-clamp study to investigate the effects of paliperidone on hERG potassium channels expressed in HEK cells. Western blot analyses were used to study the effects of risperidone and paliperidone on hERG and hERG 3.1 isoform channel trafficking. Risperidone and paliperidone inhibited the hERG tail currents in a concentration-dependent manner with IC50 values of 0.16 and 0.57 µM, respectively. The block of hERG currents by paliperidone was voltage-dependent, increasing over a range of voltages for channel activation. A fast application of paliperidone inhibited the hERG current elicited by a 5-s depolarizing pulse to +60 mV to fully inactivate the hERG currents, suggesting an inactivated state block. A fast application of paliperidone during repolarization reversibly inhibited the hERG tail currents in a concentration-dependent manner with a IC50 value of 1.26 µM. Kinetic analysis of paliperidone interaction with the open state of the hERG channels showed that the rate constants of association (k +1) and dissociation (k -1) for paliperidone were 0.45 µM-1 s-1 and 1.07 s-1, respectively. Paliperidone shifted the steady-state inactivation curve of the hERG currents in a hyperpolarizing direction and also produced a use-dependent block. Risperidone and paliperidone had no effect on hERG and hERG 3.1 channel trafficking to the cell membrane. Our results indicated that paliperidone inhibited the hERG current by preferentially interacting with the open and inactivated states of the channel, but not by disruption of hERG channel protein trafficking.

Mechanism of inhibition by chlorpromazine of the human pain threshold sodium channel, Nav1.7.

Chlorpromazine is a phenothiazine derivative which is primarily used for schizophrenia and occasionally for migraine. Because Nav1.7 plays an important role in pain sensation, we investigated whether chlorpromazine blocks the human Nav1.7 (hNav1.7) sodium current in HEK293 cells stably expressing hNav1.7 using the whole-cell patch-clamp technique. The peak current of hNav1.7 was reduced by chlorpromazine in a concentration-dependent manner with a half-maximal inhibitory concentration of 25.9±0.6µM and a Hill coefficient of 2.3±0.1. The calmodulin inhibitory peptide did not abolish the blockade of hNav1.7 currents by chlorpromazine. The blockade of hNav1.7 currents by chlorpromazine was completely and repeatedly reversible after washout. The half-maximal voltage of activation of hNav1.7 was not changed by chlorpromazine. However, chlorpromazine caused hyperpolarized the steady-state inactivation of hNav1.7. The recovery from inactivation in the presence of chlorpromazine was slower than in the absence of chlorpromazine. Chlorpromazine also showed strong use-dependent inhibition of the hNav1.7 current. Our results demonstrate that chlorpromazine blocks the hNav1.7 current in concentration-, state- and use-dependent manners and suggest that it merits further study for potential use in pain management.

Trifluoperazine blocks the human cardiac sodium channel, Nav1.5, independent of calmodulin.

Trifluoperazine is a phenothiazine derivative which is mainly used in the management of schizophrenia and also acts as a calmodulin inhibitor. We used the whole-cell patch-clamp technique to study the effects of trifluoperazine on human Nav1.5 (hNav1.5) currents expressed in HEK293 cells. The 50% inhibitory concentration of trifluoperazine was 15.5 ± 0.3 µM and the Hill coefficient was 2.7 ± 0.1. The effects of trifluoperazine on hNav1.5 were completely and repeatedly reversible after washout. Trifluoperazine caused depolarizing shifts in the activation and hyperpolarizing shifts in the steady-state inactivation of hNav1.5. Trifluoperazine also showed strong use-dependent inhibition of hNav1.5. The blockade of hNav1.5 currents by trifluoperazine was not affected by the whole cell dialysis of the calmodulin inhibitory peptide. Our results indicated that trifluoperazine blocks hNav1.5 current in concentration-, state- and use-dependent manners rather than via calmodulin inhibition.

Blockade of Kv1.5 channels by the antidepressant drug sertraline.

Sertraline, a selective serotonin reuptake inhibitor (SSRI), has been reported to lead to cardiac toxicity even at therapeutic doses including sudden cardiac death and ventricular arrhythmia. And in a SSRI-independent manner, sertraline has been known to inhibit various voltage-dependent channels, which play an important role in regulation of cardiovascular system. In the present study, we investigated the action of sertraline on Kv1.5, which is one of cardiac ion channels. The eff ect of sertraline on the cloned neuronal rat Kv1.5 channels stably expressed in Chinese hamster ovary cells was investigated using the whole-cell patch-clamp technique. Sertraline reduced Kv1.5 whole-cell currents in a reversible concentration-dependent manner, with an IC 50 value and a Hill coefficient of 0.71 µM and 1.29, respectively. Sertraline accelerated the decay rate of inactivation of Kv1.5 currents without modifying the kinetics of current activation. The inhibition increased steeply between -20 and 0 mV, which corresponded with the voltage range for channel opening. In the voltage range positive to +10 mV, inhibition displayed a weak voltage dependence, consistent with an electrical distance δ of 0.16. Sertraline slowed the deactivation time course, resulting in a tail crossover phenomenon when the tail currents, recorded in the presence and absence of sertraline, were superimposed. Inhibition of Kv1.5 by sertraline was use-dependent. The present results suggest that sertraline acts on Kv1.5 currents as an open-channel blocker.

Blockade of Kv1.5 by paroxetine, an antidepressant drug.

Paroxetine, a selective serotonin reuptake inhibitor (SSRI), has been reported to have an effect on several ion channels including human ether-a-go-go-related gene in a SSRI-independent manner. These results suggest that paroxetine may cause side effects on cardiac system. In this study, we investigated the effect of paroxetine on Kv1.5, which is one of cardiac ion channels. The action of paroxetine on the cloned neuronal rat Kv1.5 channels stably expressed in Chinese hamster ovary cells was investigated using the whole-cell patch-clamp technique. Paroxetine reduced Kv1.5 whole-cell currents in a reversible concentration-dependent manner, with an IC 50 value and a Hill coefficient of 4.11 µM and 0.98, respectively. Paroxetine accelerated the decay rate of inactivation of Kv1.5 currents without modifying the kinetics of current activation. The inhibition increased steeply between -30 and 0 mV, which corresponded with the voltage range for channel opening. In the voltage range positive to 0 mV, inhibition displayed a weak voltage dependence, consistent with an electrical distance δ of 0.32. The binding (k+1) and unbinding (k-1) rate constants for paroxetine-induced block of Kv1.5 were 4.9 µM(-1)s(-1) and 16.1 s(-1), respectively. The theoretical K D value derived by k-1/k+1 yielded 3.3 µM. Paroxetine slowed the deactivation time course, resulting in a tail crossover phenomenon when the tail currents, recorded in the presence and absence of paroxetine, were superimposed. Inhibition of Kv1.5 by paroxetine was use-dependent. The present results suggest that paroxetine acts on Kv1.5 currents as an open-channel blocker.

Mechanism of inhibition by olanzapine of cloned hERG potassium channels.

Olanzapine is widely used in the treatment of schizophrenia and related psychoses. We investigated the effects of olanzapine on human ether-a-go-go related gene (hERG) channels stably expressed in human embryonic kidney (HEK) cells using the whole-cell patch-clamp technique. Olanzapine inhibited hERG tail currents at -50mV in a concentration-dependent manner with an IC50 value of 8.0µM and a Hill coefficient of 0.9. The voltage-dependent inhibition of the hERG currents by olanzapine increased steeply in the voltage range of channel activation. Olanzapine also shifted the steady-state activation curve of the hERG currents in a hyperpolarizing direction. At more depolarized potentials where the channels were fully activated (between 0 and +50mV), the olanzapine inhibition was voltage-independent. The steady-state inactivation curve of the hERG currents was shifted in the hyperpolarizing direction in the presence of olanzapine. A fast application of olanzapine induced a reversible inhibition of the hERG tail currents during repolarization in a concentration-dependent manner with an IC50 value of 11.9µM, suggesting an open-channel block. Olanzapine also decreased the hERG current elicited by a 5s depolarizing pulse to +60mV to inactivate the hERG currents, suggesting an inhibition of the activated (open and/or inactivated) states of the channels. These results indicated that olanzapine inhibited the hERG current by preferentially interacting with the activated states of the channel.

Endoxifen, the active metabolite of tamoxifen, inhibits cloned hERG potassium channels.

The effects of tamoxifen, and its active metabolite endoxifen (4-hydroxy-N-desmethyl-tamoxifen), on hERG currents stably expressed in HEK cells were investigated using the whole-cell patch-clamp technique and an immunoblot assay. Tamoxifen and endoxifen inhibited hERG tail currents at -50mV in a concentration-dependent manner with IC50 values of 1.2 and 1.6µM, respectively. The steady-state activation curve of the hERG currents was shifted to the hyperpolarizing direction in the presence of endoxifen. The voltage-dependent inhibition of hERG currents by endoxifen increased steeply in the voltage range of channel activation. The inhibition by endoxifen displayed a shallow voltage dependence (δ=0.18) in the full activation voltage range. A fast application of endoxifen induced a reversible block of hERG tail currents during repolarization in a concentration-dependent manner, which suggested an interaction with the open state of the channel. Endoxifen also decreased the hERG current elicited by a 5s depolarizing pulse to +60mV to inactivate the hERG currents, suggesting an interaction with the activated (open and/or inactivated) states of the channels. Tamoxifen and endoxifen inhibited the hERG channel protein trafficking to the plasma membrane in a concentration-dependent manner with endoxifen being more potent than tamoxifen. These results indicated that tamoxifen and endoxifen inhibited the hERG current by direct channel blockage and by the disruption of channel trafficking to the plasma membrane in a concentration-dependent manner. A therapeutic concentration of endoxifen inhibited the hERG current by preferentially interacting with the activated (open and/or inactivated) states of the channel.

Effects of donepezil on hERG potassium channels.

Donepezil is a potent, selective inhibitor of acetylcholinesterase, which is used for the treatment of Alzheimer's disease. Whole-cell patch-clamp technique and Western blot analyses were used to study the effects of donepezil on the human ether-a-go-go-related gene (hERG) channel. Donepezil inhibited the tail current of the hERG in a concentration-dependent manner with an IC50 of 1.3 µM. The metabolites of donepezil, 6-ODD and 5-ODD, inhibited the hERG currents in a similar concentration-dependent manner; the IC50 values were 1.0 and 1.5 µM, respectively. A fast drug perfusion system demonstrated that donepezil interacted with both the open and inactivated states of the hERG. A fast application of donepezil during the tail currents inhibited the open state of the hERG in a concentration-dependent manner with an IC50 of 2.7 µM. Kinetic analysis of donepezil in an open state of the hERG yielded blocking and unblocking rate constants of 0.54 µM(-1)s(-1) and 1.82 s(-1), respectively. The block of the hERG by donepezil was voltage-dependent with a steep increase across the voltage range of channel activation. Donepezil caused a reduction in the hERG channel protein trafficking to the plasma membrane at low concentration, but decreased the channel protein expression at higher concentrations. These results suggest that donepezil inhibited the hERG at a supratherapeutic concentration, and that it did so by preferentially binding to the activated (open and/or inactivated) states of the channels and by inhibiting the trafficking and expression of the hERG channel protein in the plasma membrane.

Raloxifene inhibits cloned Kv4.3 channels in an estrogen receptor-independent manner.

Raloxifene is widely used for the treatment and prevention of postmenopausal osteoporosis. We examined the effects of raloxifene on the Kv4.3 currents expressed in Chinese hamster ovary (CHO) cells using the whole-cell patch-clamp technique and on the long-term modulation of Kv4.3 messenger RNA (mRNA) by real-time PCR analysis. Raloxifene decreased the Kv4.3 currents with an IC50 of 2.0 µM and accelerated the inactivation and activation kinetics in a concentration-dependent manner. The inhibitory effects of raloxifene on Kv4.3 were time-dependent: the association and dissociation rate constants for raloxifene were 9.5 µM(-1) s(-1) and 23.0 s(-1), respectively. The inhibition by raloxifene was voltage-dependent (δ = 0.13). Raloxifene shifted the steady-state inactivation curves in a hyperpolarizing direction and accelerated the closed-state inactivation of Kv4.3. Raloxifene slowed the time course of recovery from inactivation, thus producing a use-dependent inhibition of Kv4.3. ß-Estradiol and tamoxifen had little effect on Kv4.3. A preincubation of ICI 182,780, an estrogen receptor antagonist, for 1 h had no effect on the inhibitory effect of raloxifene on Kv4.3. The metabolites of raloxifene, raloxifene-4'-glucuronide and raloxifene-6'-glucuronide, had little or no effect on Kv4.3. Coexpression of KChIP2 subunits did not alter the drug potency and steady-state inactivation of Kv4.3 channels. Long-term exposure to raloxifene (24 h) significantly decreased the expression level of Kv4.3 mRNA. This effect was not abolished by the coincubation with ICI 182,780. Raloxifene inhibited Kv4.3 channels by interacting with their open state during depolarization and with the closed state at subthreshold potentials. This effect was not mediated via an estrogen receptor.

Blockade of HERG human K+ channels by the antidepressant drug paroxetine.

The effects of paroxetine, a selective serotonin reuptake inhibitor, on human ether-a-go-go-related gene (HERG) channels were investigated using the whole-cell patch-clamp technique. The HERG channels were stably expressed in human embryonic kidney cells. Paroxetine inhibited the peak tail currents of the HERG channel in a concentration-dependent manner, with an IC50 value of 0.45 µM and a Hill coefficient of 0.85. These effects were reversible after wash-out of the drug. The paroxetine-induced inhibition of the HERG channels was voltage-dependent. There was a steep increase in inhibition over the voltage range of the channel opening. Also, a shallow voltage-dependent inhibition was detected over the voltage range in which the channels were fully activated. The fractional electrical distance was estimated to be 0.11. Paroxetine induced a leftward shift in the voltage-dependence of the steady-state activation of the HERG channels. Before and after application of the 1 µM paroxetine, the half-maximum activation was -14.21 mV and -27.04 mV, respectively, with no shift in the slope value. The HERG channel block was not use-dependent. The characteristics of the block were dependent on open and inactivated channel states rather than closed state. Paroxetine had no effect on activation and deactivation kinetics, steady-state inactivation. These results suggest that paroxetine blocks the HERG channels by binding to these channels in the open and inactivated states.