• Restorative Dentistry and Endodontics
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• 제25권3호
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• pp.407-420
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• 2000

삼차신경절의 뉴런이 구강악안면영역에서의 촉각, 입각, 온도각 및 통각 등 다양한 감각을 중추신경계로 전달하는 역할을 하는 것은 주지의 사실이다. 이러한 신경전달에 있어서 이온통로는 감각정보를 전달하는데 핵심적인 역할을 수행하며 특히 소디움 통로는 활동전위의 발생에 중요하다. 소디움 통로는 tetrodotoxin-sensitive(TTX-s) 및 tetrodotoxin-resistant(TTX-r) 통로로 나누어지는 데 이 중 TTX-r 통로에 발생되는 tetrodotoxin-resistant sodium current(TTX-r $I_{Na}$)는 capsaicin에 민감한 일차구심신경세포에서 유해자극에 의해 통각신호를 발생시키고 전달하는데 중요하다. 또한 칼슘 통로는 시냅스 전도에 있어서 필수적인 역할을 수행하고 있다 한편 치과영역에서 치수의 진정 목적으로 eugenol이 흔히 사용되고 있다. 그러나 eugenol의 그 작용 기전에 대해서 현재까지 이온 통로에 대한 상세한 결과가 없는 실정이며 최근의 보고에 의하면 eugenol이 capsaicin 수용기를 통하여 감각신경에 대한 억제작용을 나타낸다고 한다. 따라서 본 실험은 eugenol과 capsaicin이 흰쥐의 삼차신경절의 TTX-r $I_{Na}$와 칼슘통로에 어떠한 영향을 미치는지를 알아보고 eugenol이 capsaicin 수용기를 통하여 작용하는지를 검증하고자 시행되었다. 삼차신경절 뉴런은 100~150g의 흰쥐의 삼차신경절로부터 외과적으로 절제하여 통법의 화학적 및 기계적 처리를 통해 단일세포로 분리하였고 이를 whole-cell patch clamp 방법을 이용하여 시행한 바 다음과 같은 결론을 얻었다. 1. 1mM의 dugenol은 흰쥐 삼차신경절 뉴런의 TTX-r $I_{Na}$와 HVA $I_{Ca}$를 억제하였다. 2. $1{\mu}m$의 capsaicin은 흰쥐 삼차신경절 뉴런의 TTX-r $I_{Na}$와 HVA $I_{Ca}$를 억제하였다. 3. Capsazepine은 capsaicin의 HVA $I_{Ca}$에 대한 억제작용을 차단하였다. 4. Capsazepine은 capsaicin의 HVA $I_{Ca}$에 대한 억제작용을 차단하지 못하였다. 결론적으로 eugenol과 capsaicin은 tetrodotoxin-resistant sodium current(TTX-r $I_{Na}$)와 high voltage-activated calcium current(HVA $I_{Ca}$)를 모두 억제하는 것으로 나타났으며, 이러한 작용이 통각의 발생과 시냅스 전달과정을 차단하여 치수 진정 목적으로 많이 사용하는 eugenol의 작용기전으로 판단된다. 한편 capsaicin의 길항제인 capsazepine을 전처치하였을 때에도 eugenol의 HVA $I_{Ca}$에 대한 억제효과는 변화가 없었다. 이와같은 결과로 보아 HVA $I_{Ca}$에 관한 한 eugenol은 capsaicin 수용기를 통하여 나타나지 않는 것으로 사료된다.

• Journal of Ginseng Research
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• 제39권2호
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• pp.169-177
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• 2015

Background: Ginsenoside Rd (GSRd), one of the most abundant ingredients of Panax ginseng, protects the heart via multiple mechanisms including the inhibition of $Ca^{2+}$ influx.We intended to explore the effects of GSRd on L-type $Ca^{2+}$ current ($I_{Ca,L}$) and define the mechanism of the suppression of $I_{Ca,L}$ by GSRd. Methods: Perforated-patch recording and whole-cell voltage clamp techniques were applied in isolated rat ventricular myocytes. Results: (1) GSRd reduced $I_{Ca,L}$ peak amplitude in a concentration-dependent manner [half-maximal inhibitory concentration $(IC_{50})=32.4{\pm}7.1{\mu}mol/L$] and up-shifted the current-voltage (I-V) curve. (2) GSRd ($30{\mu}mol/L$) significantly changed the steady-state activation curve of $I_{Ca,L}$ ($V_{0.5}:-19.12{\pm}0.68$ vs. $-6.26{\pm}0.38mV$; n = 5, p < 0.05) and slowed down the recovery of $I_{Ca,L}$ from inactivation [the time content (${\zeta}$) from 91 ms to 136 ms, n = 5, p < 0.01]. (3) A more significant inhibitive effect of GSRd ($100{\mu}mol/L$) was identified in perforated-patch recording when compared with whole-cell recording [$65.7{\pm}3.2%$ (n = 10) vs. $31.4{\pm}5.2%$ (n = 5), p < 0.01]. (4) Pertussis toxin ($G_i$ protein inhibitor) completely abolished the $I_{Ca,L}$ inhibition induced by GSRd. There was a significant difference in inhibition potency between the two cyclic adenosine monophosphate elevating agents (isoprenaline and forskolin) prestimulation [$55{\pm}7.8%$ (n = 5) vs. $17.2{\pm}3.5%$ (n = 5), p < 0.01]. (5) 1H-[1,2,4]Oxadiazolo[4,3-a]-quinoxalin-1-one (a guanylate cyclase inhibitor) and N-acetyl-$\small{L}$-cysteine (a nitric oxide scavenger) partly recovered the $I_{Ca,L}$ inhibition induced by GSRd. (6) Phorbol-12-myristate-13-acetate (a protein kinase C activator) and GF109203X (a protein kinase C inhibitor) did not contribute to the inhibition of GSRd. Conclusion: These findings suggest that GSRd could inhibit $I_{Ca,L}$ through pertussis toxin-sensitive G protein ($G_i$) and a nitric oxide-cyclic guanosine monophosphate-dependent mechanism.

• 한국발생생물학회지:발생과생식
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• 제15권1호
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• pp.31-39
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• 2011

Change in intracellular $Ca^{2+}$-concentration ($[Ca^{2+}]_i$) is an essential event for egg activation and further development. $Ca^{2+}$ ion is originated from intracellular $Ca^{2+}$-store via inositol 1,4,5-triphosphate receptor and/or $Ca^{2+}$ influx via $Ca^{2+}$ channel. This study was performed to investigate whether changes in $Ca^{2+}$/calmodulin dependent protein kinase II (CaM KII) activity affect $Ca^{2+}$ influx during artificial egg activation with ethanol using $Ca^{2+}$ monitoring system and whole-cell patch clamp technique. Under $Ca^{2+}$ ion-omitted condition, $Ca^{2+}$-oscillation was stopped within 30 min post microinjection of porcine sperm factor, and ethanol-induced $Ca^{2+}$ increase was reduced. To investigate the role of CaM KII known as an integrator of $Ca^{2+}$- oscillation during mammalian egg fertilization, CaM KII activity was tested with a specific inhibitor KN-93. In the eggs treated with KN-93, ethanol failed to induce egg activation. In addition, KN-93 inhibited inward $Ca^{2+}$ current ($I_{Ca}$) in a time-dependent manner in whole-cell configuration. Immunostaining data showed that the voltage-dependent $Ca^{2+}$ channels were distributed along the plasma membrane of mouse egg and 2-cell embryo. From these results, we suggest that $Ca^{2+}$ influx during fertilization might be controlled by CaM KII activity.

• The Korean Journal of Physiology
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• 제27권1호
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• pp.93-105
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• 1993

The present study was performed to investigate the properties of ionic currents elicited by voltage pulses in the unfertilized eggs of mouse and hamster by using the whole cell voltage clamp techniques and to find out if there are any differences in properties between eggs of the two rodents. In addition, the modulatory effect of G proteins and protein kinase C (PKC) on the ionic channels were observed. The inward current in hamster eggs was shown to be due to $Ca^{2+}\;current\;(i_{ca})$). The current voltage relations of these currents in hamster egg were analogous to those in mouse eggs. The amplitude of $i_{ca}$ in the hamster egg was larger than that in the mouse egg ($-3.12{\pm}1.07\;nA\;vs.\;-1.71{\pm}0.71\;nA,\;mean{\pm}\;SD$). These results suggest that the $Ca^{2+}$ channels in both kinds of eggs have similar channel properties but their density, and/or conduct ance per unit area is higher in hamster eggs than in mouse eggs. Outward currents in eggs of both mouse and hamster were carried by $K^+$. In hamster eggs, they appeared to comprise at least two components; a transient outward component ($i_{to}$) and a steady state component ($i_{\infty}.$ The $i_{to}$ was found to be dependent on intracellular $Ca^{2+}$ concentration; whereas on the other hand $i_{\infty}\;was\;Ca^{2+}$-independent. $Ca^{2+}$ currents were increased in eggs treated with GTP (or $GTP{\gamma}S$) or fluoroaluminate ($AIF_4^-$). In the hamster egg these increments were antagonized by GDP (or $GDP{\beta}S$) application. In contrast to the enhancement of $i_{ca},\;i_k$ was reduced following GTP (or $GTP{\gamma}S$) perfusion in mouse eggs. The transient component ($i_{to}$) in hamster eggs was increased by adding GTP but decreased by phorbol ester, TPA or dioctanoyl glycerol (DOG). Simultaneous application of $GTP{\gamma}S$ and DOG suppressed $i_{to}$ more effectively than a single application or DOG or TPA. From the above results, we have shown that ionic currents elicited by voltage pulses existed in the unfertilized eggs of mouse and hamster. There are at least two types of currents, $i_{ca}\;and\;i_k$ in mouse eggs, while three types, $i_{ca},\;Ca^{2+}$-dependent $i_k$ and $Ca^{2+}$-independent $i_k$ exist in hamster eggs. ionic channels in these eggs may be regulated either directly by GTP and PKC or indirectly by the substances linked with GTP and PKC.

• The Korean Journal of Physiology and Pharmacology
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• 제8권2호
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• pp.69-76
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• 2004

A variety of G protein coupled receptors (GPCRs) are expressed in the presynaptic terminals of central and peripheral synapses and play regulatory roles in transmitter release. The patch-clamp whole-cell recording technique, applied to the calyx of Held presynaptic terminal in brainstem slices of rodents, has made it possible to directly examine intracellular mechanisms underlying the GPCR-mediated presynaptic inhibition. At the calyx of Held, bath-application of agonists for GPCRs such as $GABA_B$ receptors, group III metabotropic glutamate receptors (mGluRs), adenosine $A_1$ receptors, or adrenaline ${\alpha}2$ receptors, attenuate evoked transmitter release via inhibiting voltage-activated $Ca^{2+}$ currents without affecting voltage-activated $K^+$ currents or inwardly rectifying $K^+$ currents. Furthermore, inhibition of voltage-activated $Ca^{2+}$ currents fully explains the magnitude of GPCR-mediated presynaptic inhibition, indicating no essential involvement of exocytotic mechanisms in the downstream of $Ca^{2+}$ influx. Direct loadings of G protein ${\beta}{\gamma}$ subunit $(G{\beta}{\gamma})$ into the calyceal terminal mimic and occlude the inhibitory effect of a GPCR agonist on presynaptic $Ca^{2+}$ currents $(Ip_{Ca})$, suggesting that $G{\beta}{\gamma}$ mediates presynaptic inhibition by GPCRs. Among presynaptic GPCRs glutamate and adenosine autoreceptors play regulatory roles in transmitter release during early postnatal period when the release probability (p) is high, but these functions are lost concomitantly with a decrease in p during postnatal development.

• The Korean Journal of Physiology and Pharmacology
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• 제9권6호
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• pp.353-361
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• 2005

The interaction of cyclosporine A (CsA), an immunosuppressant, with rat brain Kv1.5 (Kv1.5) channels, which were stably expressed in Chinese hamster ovary cells, was investigated using the whole-cell patch-clamp technique. CsA reversibly blocked Kv1.5 currents at +50 mV in a reversible concentrationdependent manner with an apparent $IC_{50}$ of 1.0μM. Other calcineurin inhibitors (cypermethrin, autoinhibitory peptide) had no effect on Kv1.5 and did not prevent the inhibitory effect of CsA. Fast application of CsA led to a rapid and reversible block of Kv1.5, and the onset time constants of the CsA-induced block were decreased in a concentration-dependent manner. The CsA-induced block of Kv1.5 channels was voltage-dependent, with a steep increase over the voltage range of channel opening. However, the block exhibited voltage independence over the voltage range in which channels were fully activated. The rate constants for association and dissociation of CsA were $7.0{\mu}M{-1}s^{-1}$ and $8.1s^{-1}$, respectively. CsA slowed the deactivation time course, resulting in a tail crossover phenomenon. Block of Kv1.5 by CsA was use-dependent. CsA also blocked Kv1.3 currents at +50 mV in a reversible concentration-dependent manner with an apparent $IC_{50}$ of $1.1{\mu}M$. The same effects of CsA on Kv1.3 were also observed in excised inside-out patches when applied to the internal surface of the membrane. The present results suggest that CsA acts directly on Kv1.5 currents as an open-channel blocker, independently of the effects of CsA on calcineurin activity.

• The Korean Journal of Physiology and Pharmacology
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• 제15권5호
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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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• 제26권5호
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• pp.397-404
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• 2022

Fesoterodine, an antimuscarinic drug, is widely used to treat overactive bladder syndrome. However, there is little information about its effects on vascular K⁺ channels. In this study, voltage-dependent K⁺ (Kv) channel inhibition by fesoterodine was investigated using the patch-clamp technique in rabbit coronary artery. In whole-cell patches, the addition of fesoterodine to the bath inhibited the Kv currents in a concentration-dependent manner, with an IC₅₀ value of 3.19 ± 0.91 μM and a Hill coefficient of 0.56 ± 0.03. Although the drug did not alter the voltage-dependence of steady-state activation, it shifted the steady-state inactivation curve to a more negative potential, suggesting that fesoterodine affects the voltage-sensor of the Kv channel. Inhibition by fesoterodine was significantly enhanced by repetitive train pulses (1 or 2 Hz). Furthermore, it significantly increased the recovery time constant from inactivation, suggesting that the Kv channel inhibition by fesoterodine is use (state)-dependent. Its inhibitory effect disappeared by pretreatment with a Kv 1.5 inhibitor. However, pretreatment with Kv2.1 or Kv7 inhibitors did not affect the inhibitory effects on Kv channels. Based on these results, we conclude that fesoterodine inhibits vascular Kv channels (mainly the Kv1.5 subtype) in a concentration- and use (state)-dependent manner, independent of muscarinic receptor antagonism.

• 대한수의학회지
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• 제38권2호
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• pp.280-289
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• 1998

Voltage-sensitive ion channels contribute to establishment of the cell excitablity and the generation of the cellular function. At hamster oocytes in the primitive stage during developing process, an inward current elicited by voltage pulses was found to be carried mainly by $Ca^{2+}$. Even at present, $Ca^{2+}$ channels serve as the most probable route to pass this inward current but there is no evidence of the presence of this channels in eggs. To date, both the characteristic properties and the physiological role in the early stage of development remain unclear. Here we examined the characteristic properties of the inward current and changes in this currents at unfertilized oocytes, fertilized zygotes and two-cell embryos using whole-cell voltage clamp technique. The inward current carried reportedly by $Ca^{2+}$ was remained following removing external $Ca^{2+}$ but completely abolished by further replacement of impermeants such as tetramethylammonium ion ($TMA^+$) or $choline^+$ instead of $[Na^+]_0$. Tetrodotoxin did not affect on this inward current remained at $[Ca^{2+}]_0$-free condition. Removal of $Na^+$ ion out of the experimental solution clearly decreased the current. After adding 2mM $Ca^{2+}$ to the $Na^+$-free media, the inward current was restored. Interestingly, this current carried by either $Ca^{2+}$ or $Na^+$ was decreased by the reduction of intracellular $Cl^-$ concentration, or by $Cl^-$ channel blockers such as niflumic acid, DIDS and SITS. When $Cl^-$ concentration was lowered without changes in other ionic components, this inward current was reduced. At fertilized oocytes and two-cell embryos, the inward current carried by $Ca^{2+}$ and $Na^+$ was severely reduced. Also $Cl^-$ component could not be observed. From these results, the inward current is composed of $Ca^{2+}$, $Na^+$ and $Cl^-$ component, suggesting that the channel carrying this inward current is not selective specifically to $Ca^{2+}$. During early stage of development, the voltage-sensitive ion current seems not to contribute essentially to the cell cleavage and differentiation. The loss of $Cl^-$ component after fertilization suggests that $Cl^-$ may play a role in maintaining the viability of unfertilized ova.

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 2003년도 정기총회 및 학술발표회
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• pp.39-39
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• 2003

In our previous study, WEHI-231, an immature B cell line, showed intractable increase in [C $a^{2+}$]$_{c}$ after the B-cell receptor (BCR) ligation and treatment with 2-aminoethoxydiphenylborate (2-APB), which was never observed in Bal-17, a mature B cell line (Nam et al., 2003, FEBS Lett). In this study, a whole cell voltage clamp study revealed a specific expression of a novel type of $K^{+}$ current, namely voltage-independent background-type $K^{+}$ channels (IK-bg), in WEHI-231 cells. IK-bg was dramatically increase by the application of 2-APB (50 $\square$M), which induced severe hyperpolarization of WEHI-231 from -45 ㎷ to -90 ㎷, When dialyzed with $Mg^{2+}$ and ATP-free pipette solution, a spontaneous development of IK-bg and membrane hyperpolarization were observed. IK-bg was insensitive to classical $K^{+}$ channel blockers (TEA, glibenclamide, $Ba^{2+}$(1 mM)), whereas blocked by quinine and quinidine in a voltage-dependent manner ($IC_{50}$/=6~9 $\square$M at +60㎷). Phorbol myrstate, a PKC activator, decreased the amplitude of IK-bg. Extracellular acidification (pH 6.5) slightly inhibited IK-bg. Arachidonic acid, riluzole, or hyposmotic stress could not affect the IK-bg after the full development by the intracellular dialysis with Mg-ATP-free solution. In a cell-attached mode of single channel recording from WEHI231, we found two types of voltage-independent $K^{+}$ channels with unitary conductance of 300 pS and 120 pS, respectively. Both channels showed very short mean open times and their open probabilities were increase by the application of 2-APB. In Bal-17 cells, no such $K^{+}$ current was observed in 50 cells tested. In summary, WEHI-231 immature B cells express background $K^{+}$ channels. The pharmacological properties and the large unitary conductance suggest that novel types of two-pore domain $K^{+}$ channels (2-P-K channels) might be expressed in WEHI-231, which may provide an intriguing targets of signal transduction in the immature B lymphocytes.e B lymphocytes.