• Progress in Medical Physics
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• v.14 no.1
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• pp.54-67
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• 2003

The voltage-dependence of N-type calcium current inactivation is U-shaped with the degree of inactivation roughly mirroring inward current. This voltage-dependence has been reported to result from a purely voltage-dependent mechanism. However, $Ca^{2＋}$-dependent inactivation of N-channels has also been reported. We have investigated the role of $Ca^{2＋}$ in N-channel inactivation by comparing the effects of $Ba^{2＋}$and $Ca^{2＋}$ on whole-cell N-current in rat superior cervical ganglion neurons. For individual cells in-activation was always larger in $Ca^{2＋}$ than in $Ba^{2＋}$ even when internal EGTA (11 mM) was replaced with BAPTA (20 mM). The inactivation vs. voltage relationship was U-shaped in both divalent cations. The enhancement of inactivation by $Ca^{2＋}$ was inversely related with the magnitude of inactivation in $Ba^{2＋}$ as if the mechanisms of inactivation were the same in both $Ba^{2＋}$ and $Ca^{2＋}$. In support of this idea we could separate fast ( ${\gamma}$ ～150 ms) and slow ( ${\gamma}$ ～ 2500 ms) components of inactivation in both $Ba^{2＋}$and $Ca^{2＋}$ using 5 sec voltage steps. Differential effects were observed on each component with $Ca^{2＋}$ enhancing the magnitude of the fast component and the speed of the slow component. The larger amplitude of fast component indicates that the more channels inactivate via this pathway with $Ca^{2＋}$ than with $Ba^{2＋}$, but the stable time constants support the idea the fast inactivation mechanism is identical in $Ba^{2＋}$and $Ca^{2＋}$. The results do not support a $Ca^{2＋}$-dependent mechanism for fast inactivation. However, the $Ca^{2＋}$-induced acceleration of the slowly inactivating component could result from a $Ca^{2＋}$-dependent process.

• The Korean Journal of Physiology and Pharmacology
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• v.12 no.2
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• pp.51-58
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• 2008

Cardiac fibroblasts constitute one of the largest cell populations in the heart, and contribute to structural, biochemical, mechanical and electrical properties of the myocardium. Nonetheless, their cardiac functions, especially electrophysiological properties, have often been disregarded in studies. $Ca^{2+}$-activated $K^+\;(K_{Ca})$ channels can control $Ca^{2+}$ influx as well as a number of $Ca^{2+}$-dependent physiological processes. We, therefore, attempted to identify and characterize $K_{Ca}$ channels in rat Cardiac fibroblasts. First, we showed that the cells cultured from the rat ventricle were cardiac fibroblasts by immunostaining for discoidin domain receptor 2 (DDR-2), a specific fibroblast marker. Secondly, we detected the expression of various $K_{Ca}$ channels by reverse transcription polymerase chain reaction (RT-PCR), and found all three family members of $K_{Ca}$ channels, including large conductance $K_{Ca}$ (BK-${\alpha}1-\;and\;-{\beta}1{\sim}4$subunits), intermediate conductance $K_{Ca}$ (IK), and small conductance $K_{Ca}$ (SK$1{\sim}4$ subunits) channels. Thirdly, we recorded BK, IK, and SK channels by whole cell mode patch clamp technique using their specific blockers. Finally, we performed cell proliferation assay to evaluate the effects of the channels on cell proliferation, and found that the inhibition of IK channel increased the cell proliferation. These results showed the existence of BK, IK, and SK channels in rat ventricular fibroblasts and involvement of IK channel in cell proliferation.

• Molecules and Cells
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• v.37 no.9
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• pp.650-655
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• 2014

Mesenchymal stem cells (MSCs) can differentiate into neural cells to treat nervous system diseases. Magnetic resonance is an ideal means for cell tracking through labeling cells with superparamagnetic iron oxide (SPIO). However, no studies have described the neural differentiation ability of SPIO-labeled MSCs, which is the foundation for cell therapy and cell tracking in vivo. Our results showed that bone marrow-derived mesenchymal stem cells (BM-MSCs) labeled in vitro with SPIO can be induced into neural-like cells without affecting the viability and labeling efficiency. The cellular uptake of SPIO was maintained after labeled BM-MSCs differentiated into neural-like cells, which were the basis for transplanted cells that can be dynamically and non-invasively tracked in vivo by MRI. Moreover, the SPIO-labeled induced neural-like cells showed neural cell morphology and expressed related markers such as NSE, MAP-2. Furthermore, whole-cell patch clamp recording demonstrated that these neural-like cells exhibited electrophysiological properties of neurons. More importantly, there was no significant difference in the cellular viability and $[Ca^{2+}]_i$ between the induced labeled and unlabeled neural-like cells. In this study, we show for the first time that SPIO-labeled MSCs retained their differentiation capacity and could differentiate into neural-like cells with high cell viability and a good cellular state in vitro.

• The Korean Journal of Physiology and Pharmacology
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• v.20 no.2
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• pp.193-200
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• 2016

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 effect 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 $0.71{\mu}M$ 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 ${\delta}$ 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.

• 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.

• The Korean Journal of Physiology and Pharmacology
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• v.20 no.1
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• pp.75-82
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• 2016

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 $4.11{\mu}M$ 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 ${\delta}$ of 0.32. The binding ($k_{+1}$) and unbinding ($k_{-1}$) rate constants for paroxetine-induced block of Kv1.5 were $4.9{\mu}M^{-1}s^{-1}$ and $16.1s^{-1}$, respectively. The theoretical $K_D$ value derived by $k_{-1}/k_{+1}$ yielded $3.3{\mu}M$. 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.

• The Korean Journal of Pharmacology
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• v.32 no.2
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• pp.283-292
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• 1996

The spinal dorsal horn is the area where primary afferent fibers terminate and cutaneous sensory information is processed. A number of putative neurotransmitter substances, including excitatory and inhibitory amino acids and peptides, are present in this region. In this study, single neurons of the spinal dorsal horn were acutely isolated and the properties of whole cell current and responses to excitatory and inhibitory neurotransmitters were studied by patch clamp technique. Transverse slice ($(300{\mu}m$) of lumbar spinal cords from young rats$(7{\sim}14\;days)$ were sequentially treated with two pretenses(pronase 0.75 mg/ml and thermolysin 0.75 mg/ml), then single neurons were mechanically dissociated. These neurons showed near-intact morphology such as multipolar, ellipsoidal and bipolar, and pyramidal cells and we recorded the typical whole cell currents of $K^+$, $Ca^{2+}$ and ligand-operated channels from these neurons. Glutamate $(30{\mu}M)$ and N-methyl-D-aspartate(NMDA, $30{\mu}M)$ induced inward currents of $117{\pm}12.4$ pA(n=5) and $49{\pm}6.9$ pA(n=3), respectively. Glycine $(1{\mu}M)$ potentiated glutamate-induced currents $4{\sim}5$ times and NMDA-induced currents $8{\sim}10$ times. In addition, glycine $(30{\mu}M)$ induced Inward current ($31{\pm}6.1$ nA, n=2), which was rapidly desensitized after the peak to a new steady-state level. However, the inward currents induced by ${\gamma}-amino$ butyric acid(GABA, $1{\mu}M$) decreased continuously after the peak($226{\pm}41.6$ pA, n=3) under the similar experimental condition. The ionic currents and pharmacological responses of isolated neurons in this work were similar to those observed in vivo or in vitro spinal cord slice, indicating that acutely isolated neurons could be effectively used for further pharmacological studies.

• The Korean Journal of Physiology and Pharmacology
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• v.10 no.2
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• pp.71-77
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• 2006

The goal of this study was to analyze the effects of genistein, a widely used tyrosine kinase inhibitor, on cloned Shaw-type $K^+$ currents, Kv3.1 which were stably expressed in Chinese hamster ovary (CHO) cells, using the whole-cell configuration of patch-clamp techniques. In whole-cell recordings, genistein at external concentrations from 10 to $100{\mu}M$ accelerated the rate of inactivation of Kv3.1 currents, thereby concentration-dependently reducing the current at the end of depolarizing pulse with an $IC_{50}$ value of $15.71{\pm}0.67{\mu}M$ and a Hill coefficient of $3.28{\pm}0.35$ (n=5). The time constant of activation at a 300 ms depolarizing test pulses from -80 mV to +40 mV was $1.01{\pm}0.04$ ms and $0.90{\pm}0.05$ ms (n=9) under control conditions and in the presence of $20{\mu}M$ genistein, respectively, indicating that the activation kinetics was not significantly modified by genistein. Genistein $(20{\mu}M)$ slowed the deactivation of the tail current elicited upon repolarization to -40 mV, thus inducing a crossover phenomenon. These results suggest that drug unbinding is required before Kv3.1 channels can close. Genistein-induced block was voltage-dependent, increasing in the voltage range $(-20\'mV{\sim}0\'mV)$ for channel opening, suggesting an open channel interaction. Genistein $(20{\mu}M)$ produced use-dependent block of Kv3.1 at a stimulation frequency of 1 Hz. The voltage dependence of steady-state inactivation of Kv3.1 was not changed by $20{\mu}M$ genistein. Our results indicate that genistein blocks directly Kv3.1 currents in concentration-, voltage-, time-dependent manners and the action of genistein on Kv3.1 is independent of tyrosine kinase inhibition.

• The Korean Journal of Physiology and Pharmacology
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• v.7 no.2
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• pp.73-77
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• 2003

The effects of nitric oxide on the vestibular function recovery following unilateral labyrinthectomy (UL) were studied. Sprague-Dawley male rats, treated with nitric oxide liberating agent sodium nitroprusside (SNP) and NOS inhibitor $N^G$-nitro-L-arginine methyl ester (L-NAME), were subjected to destruction of the unilateral vestibular apparatus, and then spontaneous nystagmus was observed in the rat. To explore the effects of nitric oxide on the neuronal excitability, whole cell patch clamp technique was applied on isolated medial vestibular nuclear neurons. The frequency of spontaneous nystagmus in SNP treated rats was lesser than that of spontaneous nystagmus in control animals. In contrast, pre-UL treatment with L-NAME resulted in a significant increase in spontaneous nystagmus frequency. In addition, SNP increased the frequency of spontaneous action potential in isolated medial vestibular nuclear neurons. Potassium currents of the vestibular nuclear neurons were inhibited by SNP. After blockade of calcium dependent potassium currents by high EGTA (11 mM) in a pipette solution, SNP did not inhibit outward potassium currents. 1H-[1,2,4] oxadiazolo [4,3-a] quinozalin-1-one (ODQ), a specific inhibitor of soluble guanylyl cyclase, inhibited the effects of SNP on the spontaneous firing and the potassium current. These results suggest that nitric oxide after unilateral labyrinthectomy would help to facilitate vestibular compensation by inhibiting calcium-dependent potassium currents through increasing intracellular cGMP, and consequently would increase excitability in ipsilateral vestibular nuclear neurons.