• The Korean Journal of Physiology and Pharmacology
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• v.1 no.5
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• pp.485-493
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• 1997

We investigated the effect of ${\alpha}-adrenergic$ and cholinergic receptor agonists on $Ca^{2+}$ current in adult rat trigeminal ganglion neurons using whole-cell patch clamp methods. The application of acetylcholine, carbachol, and oxotremorine ($50\;{\mu}M\;each$) produced a rapid and reversible reduction of the $Ca^{2+}$ current by $17{\pm}6%,\;19{\pm}3%,\;and\;18{\pm}4%$, respectively. Atropine, a muscarinic antagonist, blocked carbachol- induced $Ca^{2+}$ current inhibition to $3{\pm}1%$. Norepinephrine ($50\;{\mu}M$) reduced $Ca^{2+}$ current by $18{\pm}2%$, while clonidine ($50\;{\mu}M$), an ${\alpha}2-adrenergic$ receptor agonist, inhibited $Ca^{2+}$ current by only $4{\pm}1%$. Yohimbine, an ${\alpha}2-adrenergic$ receptor antagonist, did not block the inhibitory effect of norepinephrine on $Ca^{2+}$ current, whereas prazosin, an ${\alpha}1-adrenergic$ receptor antagonist, attenuated the inhibitory effect of norepinephrine on $Ca^{2+}$ current to $6{\pm}1%$. This pharmacology contrasts with ${\alpha}2-adrenergic$ receptor modulation of $Ca^{2+}$ channels in rat sympathetic neurons, which is sensitive to clonidine and blocked by yohimbine. Our data suggest that the modulation of voltage dependent $Ca^{2+}$ channel by norepinephrine is mediated via an α1-adrenergic receptor. Pretreatment with pertussis toxin (250 ng/ml) for 16 h greatly reduced norepinephrine- and carbachol-induced $Ca^{2+}$ current inhibition from $17{\pm}3%\;and\;18{\pm}3%\;to\;2{\pm}1%\;and\;2{\pm}1%$, respectively. These results demonstrate that norepinephrine, through an ${\alpha}1-adrenergic$ receptor, and carbachol, through a muscarinic receptor, inhibit $Ca^{2+}$ currents in adult rat trigeminal ganglion neurons via pertussis toxin sensitive GTP-binding proteins.

• The Korean Journal of Physiology and Pharmacology
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• v.7 no.2
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• pp.59-63
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• 2003

The medial vestibular nucleus (MVN) neurons are controlled by excitatory synaptic transmission from the vestibular afferent and commissural projections, and by inhibitory transmission from interneurons. Spontaneous synaptic currents of MVN neurons were studied using whole cell patch clamp recording in slices prepared from 13- to 17-day-old rats. The spontaneous inhibitory postsynaptic currents (sIPSCs) were significantly reduced by the $GABA_A$ antagonist bicuculline ($20{\mu}M$), but were not affected by the glycine antagonist strychnine ($1{\mu}M$). The frequency, amplitude, and decay time constant of sIPSCs were $4.3{\pm}0.9$ Hz, $18.1{\pm}2.0$ pA, and $8.9{\pm}0.4$ ms, respectively. Spontaneous excitatory postsynaptic currents (sEPSCs) were mediated by non-NMDA and NMDA receptors. The specific AMPA receptor antagonist GYKI-52466 ($50{\mu}M$) completely blocked the non-NMDA mediated sEPSCs, indicating that they are mediated by an AMPA-preferring receptor. The AMPA mediated sEPSCs were characterized by low frequency ($1.5{\pm}0.4$ Hz), small amplitude ($13.9{\pm}1.9$ pA), and rapid decay kinetics ($2.8{\pm}0.2$ ms). The majority (15/21) displayed linear I-V relationships, suggesting the presence of GluR2-containing AMPA receptors. Only 35% of recorded MVN neurons showed NMDA mediated currents, which were characterized by small amplitude and low frequency. These results suggest that the MVN neurons receive excitatory inputs mediated by AMPA, but not kainate, and NMDA receptors, and inhibitory transmission mediated by $GABA_A$ receptors in neonatal rats.

• The Korean Journal of Physiology and Pharmacology
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• v.18 no.6
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• pp.503-508
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• 2014

Spontaneous myometrial contraction (SMC) in pregnant uterus is greatly related with gestational age and growing in frequency and amplitude toward the end of gestation to initiate labor. But, an accurate mechanism has not been elucidated. In human and rat uterus, all TRPCs except TRPC2 are expressed in pregnant myometrium and among them, TRPC4 are predominant throughout gestation, suggesting a possible role in regulation of SMC. Therefore, we investigated whether the TRP channel may be involved SMC evoked by mechanical stretch in pregnant myometrial strips of rat using isometric tension measurement and patch-clamp technique. In the present results, hypoosmotic cell swelling activated a potent outward rectifying current in G protein-dependent manner in rat pregnant myocyte. The current was significantly potentiated by $1{\mu}M$ lanthanides (a potent TRPC4/5 stimulator) and suppressed by $10{\mu}M$ 2-APB (TRPC4-7 inhibitor). In addition, in isometric tension experiment, SMC which was evoked by passive stretch was greatly potentiated by lanthanide ($1{\mu}M$) and suppressed by 2-APB ($10{\mu}M$), suggesting a possible involvement of TRPC4/5 channel in regulation of SMC in pregnant myometrium. These results provide a possible cellular mechanism for regulation of SMC during pregnancy and provide basic information for developing a new agent for treatment of premature labor.

• The Korean Journal of Physiology and Pharmacology
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• v.22 no.1
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• pp.71-80
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• 2018

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 high-frequency 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 $IC_{50}$ value of $9.43{\mu}M$ 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 $4.5{\mu}M^{-1}s^{-1}$ and $35.8s^{-1}$, respectively, yielding a calculated $K_D$ value of $7.9{\mu}M$. 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 use-dependent 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.

• Journal of Physiology & Pathology in Korean Medicine
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• v.28 no.2
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• pp.162-168
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• 2014

Fruits of Schisandra chinensis (SC) Baill are considered a traditional herbal medicine for the treatment and alleviation of various diseases. The purpose of this study was to investigate the anti-cancer effects of SC extract in human breast adenocarcinoma cells (MCF-7). We used human breast adenocarcinoma cell line, MCF-7 cells. We examined cell death by MTT assay and caspase 3 and 9 assay with SC extract. To examine the inhibitory effects of SC extract, cell cycle (sub G1) analysis and mitochondrial membrane depolarization was done the MCF-7 cells after one day with SC extract. In addition, to investigate the transient receptor potential melastatin 7 (TRPM7) currents, we used the whole cell patch clamp techniques. Furthermore, TRPM7 channels were overexpressed in human embryonic kidney (HEK) 293 cells to identify the role of TRPM7 channels in MCF-7 cell growth and survival. SC extract inhibited the growth of MCF-7 cells in a dose-dependent fashion. Also we showed that SC extract induced apoptosis in MCF-7 cells by MTT assay, caspase 3 and 9 assay, sub-G1 analysis and mitochondrial membrane depolarization. SC extract inhibited the TRPM7 currents in MCF-7 cells and in TRPM7 overexpressed HEK 293 cells. Furthermore, TRPM7 channel overexpression in HEK 293 cells exacerbated SC extract-induced cell death. Our findings provide insight into unraveling the effects of SC extract in human breast adenocarcinoma cells and developing therapeutic agents against breast cancer.

• Proceedings of the Korean Society of Applied Pharmacology
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• 2003.11a
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• pp.112-112
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• 2003

Voltage-gated $K^{+}$ (Kv) channels represent a structurally and functionally diverse group of membrane proteins. These channels play an important role in determining the length of the cardiac action potential and are the targets for antiarrhythmic drugs. Many $K^{+}$ channel genes have been cloned from human myocardium and functionally contribute to its electrical activity. One of these channels, Kv1.5, is one of the more cardiovascular-specific $K^{+}$ channel isoforms identified to date and forms the molecular basis for an ultra-rapid delayed rectifier $K^{＋}$ current found in human atrium. Thus, the blocker of hKv1.5 is expected to be an ideal antiarrhythmic drug for atrial fibrillation. Chelidonine was isolated from Chelidonium majus L. We examined the effect of chelidonine on the hKv1.5 current expressed in Ltk-cells using whole cell mode of patch clamp techniques. Chelidonine selectively inhibited the hKv1.5 current expressed in Ltk-cells in a concentration-dependent manner, whereas did not affect the HERG current expressed in HEK-293 cells. Additionally, chelidonine reduced the tail current amplitude recorded at -50 mV after 250 ms depolarizing pulses to +60 mV, and slowed the deactivation time course resulting in a 'crossover' phenomenon when the tail currents recorded under control conditions and in the presence of chelidonine were superimposed. We found that chelidonine also inhibited the $K^{+}$ current in isolated human atrial myocytes where hKv1.5 channels were predominantly expressed. Furthermore, we examined the effects of chelidonine on the action potentials in rabbit hearts using conventional microelectrode technique. Chelidonine prolonged the action potential durations (APD) of atrial, ventricular myocytes and Purkinje fibers in a dose-dependent manner. However, the effect of chelidonine on atrial APD was frequency-dependent whereas the effect of chelidonine on the APDs of ventricular myocytes and Purkinje fibers was not frequency- dependent. Also, the selective action of chelidonine on heart was more potent than dofetilide, $K^{+}$ channel blocker.

• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.110-110
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• 2017

Stomata are the main gateways for water and air transport between leaves and the environment. Inward-rectifying potassium channels regulate photo-induced stomatal opening. Rice contains three inward rectifying shaker-like potassium channel proteins, OsKAT1, OsKAT2 and OsKAT3. Among these, only OsKAT2 is specifically expressed in guard cells. Here, we investigated the functions of OsKAT2 in stomatal regulation using three dominant negative mutant proteins, OsKAT2(T235R), OsKAT2(T285A) and OsKAT2(T285D), which are altered in amino acids in the channel pore and at a phosphorylation site. Yeast complementation and patch clamp assays showed that all three mutant proteins lost channel activity. However, among plants overexpressing these mutant proteins, only plants overexpressing OsKAT2(T235R) showed significantly less water loss than the control. Moreover, overexpression of this mutant protein led to delayed photo-induced stomatal opening and increased drought tolerance. Our results indicate that OsKAT2 is an inward-rectifying shaker-like potassium channel that mainly functions in stomatal opening. Interestingly, overexpression of OsKAT2(T235R) did not cause serious defects in growth or yield in rice, suggesting that OsKAT2 is a potential target for engineering plants with improved drought tolerance without yield penalty.

• International Journal of Oral Biology
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• v.34 no.3
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• pp.151-155
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• 2009

The role of $Cl^-$ channels in regulatory volume decrease (RVD) in human salivary gland acinar cells was examined using a whole-cell patch clamp technique. Human tissues were obtained from healthy volunteers or from patients with oromaxillofacial tumors. During the measurements, $K^+$-free solutions were employed to eliminate contamination of whole-cell conductance by $K^+$ currents. When the cells were exposed to a 70% hypotonic solution, outward-rectifying currents, which were not observed in the resting state, were found to have significantly increased both in human labial and parotid gland acinar cells. The amplitudes of the currents were reduced in a low $Cl^-$ bath solution. Furthermore, the addition of $100{\mu}M$ 5-Nitro-2- (3-phenyl propylamino) benzoic acid (NPPB) or $100{\mu}M$ 4,4'-diisothio cyanatostilbene-2,2'-disulphonic acid (DIDS), known to partially block $Cl^-$ channels, significantly inhibited these currents. Its outward-rectifying current profile, shift in reversal potential in a low $Cl^-$ bath solution and pharmacological properties suggest that this is a $Ca^{2+}$-independent, volume activated $Cl^-$ current. We conclude therefore that volume activated $Cl^-$ channels play a putative role in RVD in human salivary gland acinar cells.

• International Journal of Oral Biology
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• v.33 no.4
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• pp.217-221
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• 2008

Bicuculline is one of the most commonly used $GABA_A$ receptor antagonists in electrophysiological research. Because of its poor water solubility, bicuculline quaternary ammonium salts such as bicuculline methiodide (BMI) and bicuculline methbromide are preferred. However, a number of studies have shown that BMI has non-$GABA_A$ receptor-mediated effects. The substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) is implicated in the processing of nociceptive signaling. In this study, we investigated whether BMI has non-GABA receptor-mediated activity in Vc SG neurons using a whole cell patch clamp technique. SG neurons were depolarized by application of BMI ($20{\mu}M$) using a high $Cl^-$ pipette solution. GABA ($30-100{\mu}M$) also induced membrane depolarization of SG neuron. Although BMI is known to be a $GABA_A$ receptor antagonist, GABA-induced membrane depolarization was enhanced by co-application with BMI. However, free base bicuculline (fBIC) and picrotoxin (PTX), a $GABA_A$ and $GABA_C$ receptor antagonist, blocked the GABA-induced response. Furthermore, BMI-induced membrane depolarization persisted in the presence of PTX or an antagonist cocktail consisting of tetrodotoxin ($Na^+$ channel blocker), AP-5 (NMDA receptor antagonist), CNQX (non-NMDA receptor antagonist), and strychnine (glycine receptor antagonist). Thus BMI induces membrane depolarization by directly acting on postsynaptic Vc SG neurons in a manner which is independent of $GABA_A$ receptors. These results suggest that other unknown mechanisms may be involved in BMI-induced membrane depolarization.

• Progress in Medical Physics
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• v.12 no.1
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• pp.59-70
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• 2001

Adrenal chromaffin cells secrete catecholamine in response to acetylcholine. The secretory response has absolute requirement for extracellular calcium, indicating that $Ca^{2+}$ influx through voltage operated $Ca^{2+}$ channels is the primary trigger of the secretion cascade. Although the existence of various types of $Ca^{2+}$ channels has been explored using patch clamp technique in adrenal chromaffin cells, there is still disagreement with the types of $Ca^{2+}$ channels existed in different species. Therefore, we have tried to identify several distinct types of $Ca^{2+}$ channels in rat chromaffin cells. By using nicardipine(L type channel blocker), $\omega$-CgTx GVIA(N type channel blocker), and $\omega$-AgaTx VIA(P type channel blocker), it was identified that L, N, and P type $Ca^{2+}$ channel exist in rat adrenal chromaffin cells and the order of contribution of each channel type to whole cell $Ca^{2+}$ current was L type> N type> P type. type> P type.