• The Korean Journal of Physiology and Pharmacology
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• v.20 no.5
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• pp.525-531
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• 2016

The analgesic mechanism of opioids is known to decrease the excitability of substantia gelatinosa (SG) neurons receiving the synaptic inputs from primary nociceptive afferent fiber by increasing inwardly rectifying $K^+$ current. In this study, we examined whether a ${\mu}$-opioid agonist, [D-Ala2,N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO), affects the two-pore domain $K^+$ channel (K2P) current in rat SG neurons using a slice whole-cell patch clamp technique. Also we confirmed which subtypes of K2P channels were associated with DAMGO-induced currents, measuring the expression of K2P channel in whole spinal cord and SG region. DAMGO caused a robust hyperpolarization and outward current in the SG neurons, which developed almost instantaneously and did not show any time-dependent inactivation. Half of the SG neurons exhibited a linear I~V relationship of the DAMGO-induced current, whereas rest of the neurons displayed inward rectification. In SG neurons with a linear I~V relationship of DAMGO-induced current, the reversal potential was close to the $K^+$ equilibrium potentials. The mRNA expression of TWIK (tandem of pore domains in a weak inwardly rectifying $K^+$ channel) related acid-sensitive $K^+$ channel (TASK) 1 and 3 was found in the SG region and a low pH (6.4) significantly blocked the DAMGO-induced $K^+$ current. Taken together, the DAMGO-induced hyperpolarization at resting membrane potential and subsequent decrease in excitability of SG neurons can be carried by the two-pore domain $K^+$ channel (TASK1 and 3) in addition to inwardly rectifying $K^+$ channel.

• The Korean Journal of Physiology and Pharmacology
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• v.19 no.4
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• pp.383-388
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• 2015

$K^+$ outward currents in the outer hair cells (OHCs) of circling mice (homozygous (cir/cir) mice), an animal model for human deafness (DFNB6 type), were investigated using a whole cell patch clamp technique. Littermate heterozygous (+/cir) mice of the same age (postnatal day (P) 0-P6) were used as controls. Similar slow rising $K^+$ currents were observed in both genotypes, but their biophysical and pharmacological properties were quite different. The values of Vhalf for activation were significantly different in the heterozygous (+/cir) and homozygous (cir/cir) mice ($-8.1{\pm}2.2mV$, heterozygous (+/cir) mice (n=7) and $-17.2{\pm}4.2mV$, homozygous (cir/cir) mice (n=5)). The inactivation curve was expressed by a single first order Boltzmann equation in the homozygous (cir/cir) mice, while it was expressed by a sum of two first order Boltzmann equations in the heterozygous (+/cir) mice. The $K^+$ current of homozygous (cir/cir) mice was more sensitive to TEA in the 1 to 10 mM range, while the 4-AP sensitivities were not different between the two genotypes. Removal of external $Ca^{2+}$ did not affect the $K^+$ currents in either genotype, indicating that the higher sensitivity of $K^+$ current to TEA in the homozygous (cir/cir) mice was not due to an early expression of $Ca^{2+}$ activated $K^+$ channels. Our results suggest that the $K^+$ outward current of developing homozygous (cir/cir) mice OHCs is different in both biophysical and pharmacological aspects than that of heterozygous (+/cir) mice.

• The Korean Journal of Physiology and Pharmacology
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• v.15 no.5
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• pp.291-297
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• 2011

The effect of cyclosporin A (CsA), an immunosuppressant, on human ether-a-go-go-related gene (HERG) channel as it is expressed in human embryonic kidney cells was studied using a whole-cell, patch-clamp technique. CsA inhibited the HERG channel in a concentration-dependent manner, with an $IC_{50}$ value and a Hill coefficient of $3.17{\mu}m$ and 0.89, respectively. Pretreatment with cypermethrine, a calcineurin inhibitor, had no effect on the CsA-induced inhibition of the HERG channel. The CsA-induced inhibition of HERG channels was voltage-dependent, with a steep increase over the voltage range of the channel opening. However, the inhibition exhibited voltage independence over the voltage range of fully activated channels. CsA blocked the HERG channels predominantly in the open and inactivated states rather than in the closed state. Results of the present study suggest that CsA acts directly on the HERG channel as an open-channel blocker, and it acts independently of its effect on calcineurin activity.

• The Korean Journal of Physiology and Pharmacology
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• v.12 no.1
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• pp.31-35
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• 2008

Lysophosphatidylcholine (LPC), a metabolite of membrane phospholipids by phospholipase $A_2$, has been considered responsible for the development of abnormal vascular reactivity during atherosclerosis. $Ca^{2+}$ influx was shown to be augmented in atherosclerotic artery which might be responsible for abnormal vascular reactivity. However, the mechanism underlying $Ca^{2+}$ influx change in atherosclerotic artery remains undetermined. The purpose of the present study was to examine the effects of LPC on L-type $Ca^{2+}$ current $(I_{Ca(L)})$ activity and to elucidate the mechanism of LPC-induced change of $I_{Ca(L)}$ in rabbit portal vein smooth muscle cells using whole cell patch clamp. Extracellular application of LPC increased $I_{Ca(L)}$ through whole test potentials, and this effect was readily reversed by washout. Steady state voltage dependency of activation or inactivation properties of $I_{Ca(L)}$ was not significantly changed by LPC. Staurosporine (100 nM) or chelerythrine $(3{\mu}M)$, which is a potent inhibitor of PKC, significantly decreased basal $I_{Ca(L)}$, and LPC-induced increase of $I_{Ca(L)}$ was significantly suppressed in the presence of PKC inhibitors. On the other hand, application of PMA, an activator of PKC, increased basal $I_{Ca(L)}$ significantly, and LPC-induced enhancement of $I_{Ca(L)}$ was abolished by pretreatment of the cells with PMA. These findings suggest that LPC increased $I_{Ca(L)}$ in vascular smooth muscle cells by a pathway that involves PKC, and that LPC-induced increase of $I_{Ca(L)}$ might be, at least in part, responsible for increased $Ca^{2+}$ influx in atherosclerotic artery.

• The Korean Journal of Physiology and Pharmacology
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• v.24 no.6
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• pp.545-553
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• 2020

Aripiprazole is a quinolinone derivative approved as an atypical antipsychotic drug for the treatment of schizophrenia and bipolar disorder. It acts as with partial agonist activities at the dopamine D2 receptors. Although it is known to be relatively safe for patients with cardiac ailments, less is known about the effect of aripiprazole on voltage-gated ion channels such as transient A-type K⁺ channels, which are important for the repolarization of cardiac and neuronal action potentials. Here, we investigated the effects of aripiprazole on Kv1.4 currents expressed in HEK293 cells using a whole-cell patch-clamp technique. Aripiprazole blocked Kv1.4 channels in a concentration-dependent manner with an IC₅₀ value of 4.4 μM and a Hill coefficient of 2.5. Aripiprazole also accelerated the activation (time-to-peak) and inactivation kinetics. Aripiprazole induced a voltage-dependent (δ = 0.17) inhibition, which was use-dependent with successive pulses on Kv1.4 currents without altering the time course of recovery from inactivation. Dehydroaripiprazole, an active metabolite of aripiprazole, inhibited Kv1.4 with an IC₅₀ value of 6.3 μM (p < 0.05 compared with aripiprazole) with a Hill coefficient of 2.0. Furthermore, aripiprazole inhibited Kv4.3 currents to a similar extent in a concentration-dependent manner with an IC₅₀ value of 4.9 μM and a Hill coefficient of 2.3. Thus, our results indicate that aripiprazole blocked Kv1.4 by preferentially binding to the open state of the channels.

• Molecules and Cells
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• v.37 no.3
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• pp.202-212
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• 2014

ClC-1 is a member of a large family of voltage-gated chloride channels, abundantly expressed in human skeletal muscle. Mutations in ClC-1 are associated with myotonia congenita (MC) and result in loss of regulation of membrane excitability in skeletal muscle. We studied the electrophysiological characteristics of six mutants found among Korean MC patients, using patch clamp methods in HEK293 cells. Here, we found that the autosomal dominant mutants S189C and P480S displayed reduced chloride conductances compared to WT. Autosomal recessive mutant M128I did not show a typical rapid deactivation of Cl- currents. While sporadic mutant G523D displayed sustained activation of $Cl^-$ currents in the whole cell traces, the other sporadic mutants, M373L and M609K, demonstrated rapid deactivations. $V_{1/2}$ of these mutants was shifted to more depolarizing potentials. In order to identify potential effects on gating processes, slow and fast gating was analyzed for each mutant. We show that slow gating of the mutants tends to be shifted toward more positive potentials in comparison to WT. Collectively, these six mutants found among Korean patients demonstrated modifications of channel gating behaviors and reduced chloride conductances that likely contribute to the physiologic changes of MC.

• The Korean Journal of Physiology and Pharmacology
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• v.23 no.6
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• pp.483-491
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• 2019

Cordycepin exerts neuroprotective effects against excitotoxic neuronal death. However, its direct electrophysiological evidence in Alzheimer's disease (AD) remains unclear. This study aimed to explore the electrophysiological mechanisms underlying the protective effect of cordycepin against the excitotoxic neuronal insult in AD using whole-cell patch clamp techniques. ${\beta}$-Amyloid ($A{\beta}$) and ibotenic acid (IBO)-induced injury model in cultured hippocampal neurons was used for the purpose. The results revealed that cordycepin significantly delayed $A{\beta}$ + IBO-induced excessive neuronal membrane depolarization. It increased the onset time/latency, extended the duration, and reduced the slope in both slow and rapid depolarization. Additionally, cordycepin reversed the neuronal hyperactivity in $A{\beta}$ + IBO-induced evoked action potential (AP) firing, including increase in repetitive firing frequency, shortening of evoked AP latency, decrease in the amplitude of fast afterhyperpolarization, and increase in membrane depolarization. Further, the suppressive effect of cordycepin against $A{\beta}$ + IBO-induced excessive neuronal membrane depolarization and neuronal hyperactivity was blocked by DPCPX (8-cyclopentyl-1,3-dipropylxanthine, an adenosine $A_1$ receptor-specific blocker). Collectively, these results revealed the suppressive effect of cordycepin against the $A{\beta}$ + IBO-induced excitotoxic neuronal insult by attenuating excessive neuronal activity and membrane depolarization, and the mechanism through the activation of $A_1R$ is strongly recommended, thus highlighting the therapeutic potential of cordycepin in AD.

• Proceedings of the Ginseng society Conference
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• 2002.10a
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• pp.531-544
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• 2002

Ginseng is the best known and most popular herbal medicine used worldwide. Ameliorating effects of ginseng were observed on the models of scopolamine-induced, aged or hippocampal lesioned learning and memory deficits. Further beneficial effects of ginseng were observed on neuronal cell death associated with ischemia or glutamate toxicity. In spite of these beneficial effects of ginseng on the CNS, little scientific evidence shows at the cellular level. In the present study, we have employed cultures of rat hippocampal neurons and examined the direct modulation of ginseng on NMDA receptor-induced changes in $[Ca^{2+}]_i$ and -gated currents using fura-2-based digital imaging and perforated whole-cell patch-clamp techniques, respectively. We found that ginseng total saponins inhibited NMDA-induced but less effectively glutamate-induced increase in $[Ca^{2+}]_i$ Ginseng total saponins also modulated $Ca^{2+}$ transients evoked by depolarization with 50 mM KCI along with its own effects on $[Ca^{2+}]_i$. Among ginsenosides tested, ginsenoside $Rg_3$ was found to be the most potent component for ginseng actions on NMDA receptors. Furthermore, we examined the inhibitory effects ofbiotransformants of ginsenosides on NMDA receptor using purified stereoisomers of ginsenosides. 20(S)-ginsenoside $Rg_3$ and its metabolite, 20(S)-ginsenoside $Rh_3$, produced the strongest inhibition while 20(S)-ginsenoside $Rh_1$ and Compound K produced the moderate inhibition on NMDA-induced increase in $[Ca^{2+}]_i$. The data obtained suggest that the inhibition of NMDA receptors by ginseng, in particular by 20(S)-ginsenoside $Rg_3$ and its metabolite, 20(S)-ginsenoside $Rh_2$, could be one of mechanisms for ginsengmediated neuroprotective actions.

• Integrative Medicine Research
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• v.6 no.2
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• pp.149-155
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• 2017

Background: Naringenin (NRG) is a common dietary polyphenolic constituent of fruits. NRG has diverse pharmacological activities, and is used in traditional medicine to treat various diseases including gastrointestinal (GI) disorders. Interstitial cells of Cajal (ICCs) are pacemaker cells of the GI tract. In this study, the authors investigated the effects of NRG on ICCs and on GI motility in vitro and in vivo. Methods: ICCs were dissociated from mouse small intestines by enzymatic digestion. The whole-cell patch clamp configuration was used to record pacemaker potentials in cultured ICC clusters. The effects of NRG on GI motility were investigated by calculating percent intestinal transit rates (ITR) using Evans blue in normal mice. Results: NRG inhibited ICC pacemaker potentials in a dose-dependent manner. In the presence of tetraethylammonium chloride or iberiotoxin, NRG had no effect on pacemaker potentials, but it continued to block pacemaker potentials in the presence of glibenclamide. Preincubation with SQ-22536 had no effect on pacemaker potentials or on their inhibition by NRG. However, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one blocked pacemaker potential inhibition by NRG. In addition, L-NG-nitroarginine methyl ester blocked pacemaker potential inhibition by NRG. Furthermore, NRG significantly suppressed murine ITR enhancement by neostigmine in vivo. Conclusion: This study shows NRG dose-dependently inhibits ICC pacemaker potentials via a cyclic guanosine monophosphate/nitric oxide-dependent pathway and $Ca^{2+}$-activated $K^+$ channels in vitro. In addition, NRG suppressed neostigmine enhancement of ITR in vivo.

• International Neurourology Journal
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• v.22 no.4
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• pp.252-259
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• 2018

Purpose: Naftopidil ((${\pm}$)-1-[4-(2-methoxyphenyl) piperazinyl]-3-(1-naphthyloxy) propan-2-ol) is prescribed in several Asian countries for lower urinary tract symptoms suggestive of benign prostatic hyperplasia. Previous animal experiments showed that intrathecal injection of naftopidil abolished rhythmic bladder contraction in vivo. Naftopidil facilitated spontaneous inhibitory postsynaptic currents in substantia gelatinosa (SG) neurons in spinal cord slices. These results suggest that naftopidil may suppress the micturition reflex at the spinal cord level. However, the effect of naftopidil on evoked excitatory postsynaptic currents (EPSCs) in SG neurons remains to be elucidated. Methods: Male Sprague-Dawley rats at 6 to 8 weeks old were used. Whole-cell patch-clamp recordings were made using SG neurons in spinal cord slices isolated from adult rats. Evoked EPSCs were analyzed in $A{\delta}$ or C fibers. Naftopidil or prazosin, an ${\alpha}1$-adrenoceptor blocker, was perfused at $100{\mu}M$ or $10{\mu}M$, respectively. Results: Bath-applied $100{\mu}M$ naftopidil significantly decreased the peak amplitudes of $A{\delta}$ and C fiber-evoked EPSCs to $72.0%{\pm}7.1%$ (n=15) and $70.0%{\pm}5.5%$ (n=20), respectively, in a reversible and reproducible manner. Bath application of $100{\mu}M$ prazosin did not inhibit $A{\delta}$ or C fiber-evoked EPSCs. Conclusions: The present study suggests that a high concentration of naftopidil reduces the amplitude of evoked EPSCs via a mechanism that apparently does not involve ${\alpha}1$-adrenoceptors. Inhibition of evoked EPSCs may also contribute to suppression of the micturition reflex, together with nociceptive stimulation.