• 한국발생생물학회지:발생과생식
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• 제24권4호
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• pp.297-306
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• 2020

Repetitive changes in the intracellular calcium concentration ([Ca²⁺]i) triggers egg activation, including cortical granule exocytosis, resumption of second meiosis, block to polyspermy, and initiating embryonic development. [Ca²⁺]i oscillations that continue for several hours, are required for the early events of egg activation and possibly connected to further development to the blastocyst stage. The sources of Ca²⁺ ion elevation during [Ca²⁺]i oscillations are Ca²⁺ release from endoplasmic reticulum through inositol 1,4,5 tri-phosphate receptor and Ca²⁺ ion influx through Ca²⁺ channel on the plasma membrane. Ca²⁺ channels have been characterized into voltage-dependent Ca²⁺ channels (VDCCs), ligand-gated Ca²⁺ channel, and leak-channel. VDCCs expressed on muscle cell or neuron is specified into L, T, N, P, Q, and R type VDCs by their activation threshold or their sensitivity to peptide toxins isolated from cone snails and spiders. The present study was aimed to investigate the localization pattern of N and P/Q type voltage-dependent calcium channels in mouse eggs and the role in fertilization. [Ca²⁺]i oscillation was observed in a Ca²⁺ contained medium with sperm factor or adenophostin A injection but disappeared in Ca²⁺ free medium. Ca²⁺ influx was decreased by Lat A. N-VDCC specific inhibitor, ω-Conotoxin CVIIA induced abnormal [Ca²⁺]i oscillation profiles in SrCl₂ treatment. N or P/Q type VDC were distributed on the plasma membrane in cortical cluster form, not in the cytoplasm. Ca²⁺ influx is essential for [Ca²⁺]i oscillation during mammalian fertilization. This Ca²⁺ influx might be controlled through the N or P/Q type VDCCs. Abnormal VDCCs expression of eggs could be tested in fertilization failure or low fertilization eggs in subfertility women.

• Journal of Microbiology and Biotechnology
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• 제25권8호
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• pp.1371-1379
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• 2015

The Ca_v1.2 Ca²⁺ channel is essential for cardiac and smooth muscle contractility and many physiological functions. We mutated single, double, and quadruple sites of the four potential Asn (N)-glycosylation sites in the rabbit Ca_v1.2 into Gln (Q) to explore the effects of Nglycosylation. When a single mutant (N124Q, N299Q, N1359Q, or N1410Q) or Ca_v1.2/WT was expressed in Xenopus oocytes, the biophysical properties of single mutants were not significantly different from Ca_v1.2/WT. In comparison, the double mutant N124,299Q showed a positive shift in voltage-dependent gating. Furthermore, the quadruple mutant (QM; N124,299,1359,1410Q) showed a positive shift in voltage-dependent gating as well as a reduction of current. We tagged EGFP to the QM, double mutants, and Ca_v1.2/WT to chase the mechanisms underlying the reduced currents of QM. The surface fluorescence intensity of QM was weaker than that of Ca_v1.2/WT, suggesting that the reduced current of QM arises from its lower surface expression than Ca_v1.2/WT. Tunicamycin treatment of oocytes expressing Ca_v1.2/WT mimicked the effects of the quadruple mutations. These findings suggest that Nglycosylation contributes to the surface expression and voltage-dependent gating of Ca_v1.2.

• The Korean Journal of Physiology
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• 제25권2호
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• pp.125-131
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• 1991

Inactivation properties of Ca current in the unfertilized eggs of mouse were studied by using the whole cell voltage clamp technique and single microelectrode voltage clamp technique. Membrane potential was held at -80 mV and step depolarization was applied from -50 mV to 50 mV for $200{\sim}500\;ms$. Peak of inward Ca currents was $-2{\sim}-4\;nA$ at a membrane Potentials from -20 mV to 0 mV and outward currents were not observed within the membrane voltage range studied $(-50{\sim}50\;mV)$. Inward currents were fully inactivated within 200 ms after the onset of step depolarization. As the membrane became depolarized, time constant of inactivation (${\tau}$) was decreased but remained around $20{\sim}30\;ms$ beyond 10 mV. When $Ca^{2+}$ was used as a charge earlier, inactivation of inward $Ca^{2+}$ current also occured and time course of inactivation was similar to that of $Ca^{2+}$ currents as charge carrier. In the bathing solution containing high potassium $(131\;mM\;K^+)$, process of inactivation was not changed except a parallel decrease of value for the entire range of membrane potential. Steady-state inactivation of the $current(h_{\infty})$ obtained from the double pulse experiment showed the voltage-dependent change. These results suggested that inactivation of Ca currents in the unfertilized eggs of mouse was voltage-dependent.

• The Korean Journal of Physiology
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• 제27권2호
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• pp.139-150
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• 1993

We have reported that dopamine potentiates spontaneous contractions dose-dependently in guinea-pig antral circular muscle strips (Hwang et al, 1991). To clarify the underlying excitatory mechanism of dopamine on the gastric smooth muscle, the effects of dopamine on voltage-dependent $Ca^{2+}\;currents\;and\;Ca^{2+}\;-dependent\;K^+\;currents$ were observed in enzymatically dispersed guinea-pig gastric myocytes using the whole-cell voltage-clamp technique. Experiments were also done using isometric tension recording and conventional intracellular microelectrode techniques. 1) The effect of dopamine on the spontaneous contraction of antral circular muscle strips of the guinea-pig was excitatory in a dose-dependent manner, and was blocked by phentolamine, an ${\alpha}-adrenoceptor$ blocker. 2) The slow waves were not changed by dopamine. 3) The voltage-operated inward $Ca^{2+}$ current was not influenced by dopamine. 4) The $Ca^{2+}\;-dependent\;K^+$ outward current, which might reflect the changes of intracellular calcium concentration, was enhanced by dopamine. This effect was abolished by phentolamine. 5) The enhancing effect of dopamine on the $Ca^{2+}\;-dependent\;K^+$ current disappeared with heparin which is known to block the action of $InsP_3$. From these results, it is suggested that dopamine acts via $InsP_3-mediated\;Ca^{2+}$ mobilization from intracellular stores and such action potentiates the spontaneous contraction of guinea-pig gastric smooth muscle.

• The Korean Journal of Physiology
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• 제27권2호
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• pp.115-122
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• 1993

There is evidence that noradrenaline enhances spontaneous contractions dose-dependently in guinea-pig antral circular muscle. To investigate the mechanism of this excitatory action, slow waves and membrane currents were recorded using conventional microelectrode techniques in muscle strips and the whole cell patch clamp technique in isolated gastric myocytes. On recording slow waves, noradrenaline $(10^{-5}\;M)$ induced the hyperpolarization of the membrane potential, although the shape of the slow waves became tall and steep. Also, spike potentiaIs occurred at the peaks of slow waves. These changes were completely reversed by administration of phentolamine $(10^{-5}\;M),\;an\;{\alpha}-adrenoceptor$ blocker. Noradrenaline-induced hyperpolarization was blocked by apamin $(10^{-7}\;M)$, a blocker of a class of $Ca^{2+}\;-dependent\;K^+$ channels. To investigate the mechanisms for these effects, we performed whole cell patch clamp experiments. Norndrenaline increased voltage-dependent $Ca^{2+}$ currents in the whole range of test potentials. Noradrenaline also increased $Ca^{2+}\;-dependent\;K^+$\;currents, and this effects was abolished by apamin. These results suggest that the increase in amplitude and the generation of spike potentials on slow waves was caused by the activation of voltage-dependent $Ca^{2+}$ channel via adrenoceptors, and hyperpolarization of the membrane potential was mediated by activation of apamin-sensitive $Ca^{2+}\;-dependent\;K^+\;channels$.

• The Korean Journal of Physiology
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• 제28권1호
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• pp.51-59
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• 1994

This study was carried out to elucidate the excitatory mechanisms of Substance P in the antral circular muscle, using isometric contraction recording, conventional microelectrode method and whole-cell patch clamp technique. Substance P produced tonic and phasic contractions in a dose-dependent manner and depolarized membrane potential with increased amplitude of slow waves in muscle strips. Voltage-dependent $Ca^{2+}$ currents were increased by the application of Substance P from a holding potential of -60mV to 50mV in 10mV steps and this effect was blocked by the addition of an antagonist. Also Substance P increased transient and spontaneous oscillatory $K^+$ outward currents. The enhanced outward currents were abolished by apamin in dispersed single cells. These results suggest that the depolarization of membrane potential by Substance P activates voltage-dependent $Ca^{2+}$ channels, which represents an excitatory response in the antral circular muscle and led to an increase in $Ca^{2+}\;activated\;K^+\;currents$.

• The Korean Journal of Physiology and Pharmacology
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• 제12권6호
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• pp.323-330
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• 2008

The properties of voltage dependent $Ca^{2+}$ current (VDCC) were investigated in interstitial cells of Cajal (ICC) distributed in the myenteric layer (ICC-MY) of guinea-pig antrum. In tissue, ICC-MY showed c-Kit positive reactions and produced driving potentials with the amplitude and frequency of about 62 mV and 2 times $min^{-1}$ respectively, in the presence of $1{\mu}M$ nifedipine. Single ICC-MY isolated by enzyme treatment also showed c-Kit immunohistochemical reactivity. These cells were also identified by generation of spontaneous inward current under $K^+$ -rich pipette solution. The voltage clamp experiments revealed the amplitude of - 329 pA inward current at irregular frequency. With $Cs^+$-rich pipette solution at $V_h=-80\;mV$, ICC-MY produced voltage-dependent inward currents (VDIC), and nifedipine ($1{\mu}M$) blocked VDIC. Therefore, we successfully isolated c-Kit positive single ICC from guinea-pig stomach, and found that ICC-MY potently produced dihydropiridine sensitive L-type voltage-dependent $Ca^{2+}$ currents ($VDCC_L$).

• Journal of Ginseng Research
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• 제24권1호
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• pp.18-22
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• 2000

앞의 연구에서 우리는 진세노사이드가 신경세포에 존재하는 high-threshold voltage-dependent $Ca^{2+}$ channel을 억제한다는 것을 발표하였다. 그러나, 이러한 연구는 진세노사이드가 여러 칼슘 채널subtypes중 어느 특정 칼슘 채널만을 선택적으로 조절한다는 것을 보여주지는 않았다. 따라서 이 연구에서 우리는 여러 칼슘 채널subtypes에 선택적으로 작용하는 약물 혹은 toxins을 이용하여 진세노사이드가 어느 종류의 칼슘 채널 subtypes를 억제하는가를 bovine chromaffin cell을 이용하여 연구하였다. 사용한 물질은nimodipine(L-type 칼슘 채널 길항제), $\omega$-conotoxin GVIA (N-type $Ca^{2+}$ channel 길항제), $\omega$-agatoxin IVA(P-type 칼슘 채널 길항제)이었다. 연구 결과 진세노사이드는 bovine chromaffin 세포에 존재하는 high-threshold 칼슘 current을 투여 농도별로 억제하였다. $IC_{50}$/은 약 120 $\mu$g/ml인 것으로 나타났다. nimodipine은 진세노사이드에 의한 칼슘 currents억제 작용에 영향을 미치지 않은 것으로 나타났다. 그러나, $\omega$-conotoxin GVIA, $\omega$-agatoxin IVA 및 nimodipine+$\omega$-conotoxin GVIA+$\omega$-agatoxin IVA을 처리한 세포에서는 진세노사이드에 의한 칼슘 currents억제 작용이 현저하게 줄어 들었다. 이러한 연구 결과들은 진세노사이드가 L-type 칼슘 채널은 억제하지 않고, 주로 N-, p-, 및 Q-type칼슘 채널을 억제한다는 것을 보여주고 있다

• The Korean Journal of Physiology and Pharmacology
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• 제21권2호
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• pp.215-223
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• 2017

The effects of acidic pH on several voltage-dependent ion channels, such as voltage-dependent $K^+$ and $Ca^{2+}$ channels, and hyperpolarization-gated and cyclic nucleotide-activated cation (HCN) channels, were examined using a whole-cell patch clamp technique on mechanically isolated rat mesencephalic trigeminal nucleus neurons. The application of a pH 6.5 solution had no effect on the peak amplitude of voltage-dependent $K^+$currents. A pH 6.0 solution slightly, but significantly inhibited the peak amplitude of voltage-dependent $K^+$ currents. The pH 6.0 also shifted both the current-voltage and conductance-voltage relationships to the depolarization range. The application of a pH 6.5 solution scarcely affected the peak amplitude of membrane currents mediated by HCN channels, which were profoundly inhibited by the general HCN channel blocker $Cs^+$ (1 mM). However, the pH 6.0 solution slightly, but significantly inhibited the peak amplitude of HCN-mediated currents. Although the pH 6.0 solution showed complex modulation of the current-voltage and conductance-voltage relationships, the midpoint voltages for the activation of HCN channels were not changed by acidic pH. On the other hand, voltage-dependent $Ca^{2+}$ channels were significantly inhibited by an acidic pH. The application of an acidic pH solution significantly shifted the current-voltage and conductance-voltage relationships to the depolarization range. The modulation of several voltage-dependent ion channels by an acidic pH might affect the excitability of mesencephalic trigeminal nucleus neurons, and thus physiological functions mediated by the mesencephalic trigeminal nucleus could be affected in acidic pH conditions.

• Biomolecules & Therapeutics
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• 제1권2호
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• pp.171-176
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• 1993

Purpose of the present study was to clarify the role of noradrenergic neural activities in hypothalamus for either triggering or maintaining hypertension in deoxycorticosterone (DOCA)-salt hypertensive rats. Two groups of animals were prepared: 1) normotensive Wistar rats and 2) DOCA-salt induced hypertensive rats. Voltage dependent $^{45}Ca^{++}$ uptake, endogenous norepinephrine release, and the catecholamine content in the hypothalamus of DOCA-salt hypertensive and normotensive Wistar rats were compared. Animals at 4, 6 and 16 week-old of two groups were sacrificed by decapitation and hypothalamus was dissected out. Voltage dependent calcium uptake and norepinephrine release were determined from hypothalamic synaptosomes either in low potassium or high potassium stimulatory condition by using $^{45}Ca^{++}$ isotope and HPLC-ECD technique. Degrees of voltage dependent $^{45}Ca^{++}$ uptake and norepinephrine release in hypothalamic synaptosomes of 16-week-old DOCA-salt hypertensive rats were significantly greater than those of age matched normotensive control rats. The norepinephrine and dopamine contents of hypothalamus were about the same in two groups of animals. These results suggest that the alteration of evoked norepinephrine release related to calcium uptake in hypothalamus may play a role in the maintenance of hypertension in DOCA-salt hypertensive rats.