• Animal cells and systems
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• 제12권3호
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• pp.137-141
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• 2008

Molecular cloning revealed the three isoforms($Ca_v3.1,\;Ca_v3.2,\;and\;Ca_v3.3$) of the T-type calcium channel subfamily. Expression studies exhibited their distinctive electrophysiological and pharmacological properties, accounting for diverse properties of T-type calcium channel currents previously characterized from isolated cells. However, electrophysiological properties of ion channels have shown to be more diversified by their splice variants. We here searched splice variants of rat $Ca_v3.1$ T-type channel by reverse-transcription-polymerase chain reaction(RT-PCR) to further explore diversity of $Ca_v3.1$. Interestingly, analyses of cloned RT-PCR products displayed that there were at least four splicing variants of rat $Ca_v3.1$ in the loop connecting repeats III and IV. Southern blot analyses indicated that the predominantly detected variant in brain was $Ca_v3.1a$(492 bp), which were rarely detected in most of peripheral tissues. Other two variants($Ca_v3.1c$, 546 bp; $Ca_v3.1d$, 525 bp) were detected in most of the tissues examined. The smallest isoform($Ca_v3.1b$, 471 bp) was rarely detected all the tissues. Electrophysiological characterization of the splicing variants indicated that the splice variants differ in inactivation kinetics and the voltage dependence of activation and inactivation as well.

• The Korean Journal of Physiology and Pharmacology
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• 제21권2호
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• pp.241-249
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• 2017

Plasma membrane hyperpolarization associated with activation of $Ca^{2+}$-activated $K^+$ channels plays an important role in sperm capacitation during fertilization. Although Slo3 (slowpoke homologue 3), together with the auxiliary ${\gamma}^2$-subunit, LRRC52 (leucine-rich-repeat-containing 52), is known to mediate the pH-sensitive, sperm-specific $K^+$ current KSper in mice, the molecular identity of this channel in human sperm remains controversial. In this study, we tested the classical $BK_{Ca}$ activators, NS1619 and LDD175, on human Slo3, heterologously expressed in HEK293 cells together with its functional interacting ${\gamma}^2$ subunit, hLRRC52. As previously reported, Slo3 $K^+$ current was unaffected by iberiotoxin or 4-aminopyridine, but was inhibited by ~50% by 20 mM TEA. Extracellular alkalinization potentiated hSlo3 $K^+$ current, and internal alkalinization and $Ca^{2+}$ elevation induced a leftward shift its activation voltage. NS1619, which acts intracellularly to modulate hSlo1 gating, attenuated hSlo3 $K^+$ currents, whereas LDD175 increased this current and induced membrane potential hyperpolarization. LDD175-induced potentiation was not associated with a change in the half-activation voltage at different intracellular pHs (pH 7.3 and pH 8.0) in the absence of intracellular $Ca^{2+}$. In contrast, elevation of intracellular $Ca^{2+}$ dramatically enhanced the LDD175-induced leftward shift in the half-activation potential of hSlo3. Therefore, the mechanism of action does not involve pH-dependent modulation of hSlo3 gating; instead, LDD175 may modulate $Ca^{2+}$-dependent activation of hSlo3. Thus, LDD175 potentially activates native KSper and may induce membrane hyperpolarization-associated hyperactivation in human sperm.

• Journal of Photoscience
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• 제3권3호
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• pp.133-136
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• 1996

Calcium modulates the activity of guanylate cyclase and plays a key role in dark and light adaptation in the visual system. We have measured the Ca$^{2+}$, K$^+$ and Na$^+$ concentration in dark and light adapted bullfrog's (Rana catesbeiana) vitreous humor by using the atomic absorption spectrophotometer. The calcium concentration of the light adapted bullfrog's vitreous humor was higher than that of the dark adapted bullfrog's vitreous humor. This means that ion activity between the photoreceptor and vitreous humor side is light dependent and we have found that a Ca$^{2+}$ channel and Na$^+$ - Ca$^{2+}$ exchanger exist in the vitreous humor.

• Journal of Ginseng Research
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• 제27권3호
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• pp.120-128
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• 2003

Alternative medicines such as herbal products are increasingly being used for preventive and therapeutic purposes. Ginseng is the best known and most popular herbal medicine used worldwide. In spite of some beneficial effects of ginseng on the nervous system, little scientific evidence shows at the cellular level. In the present study, I have examined the direct modulation of ginseng total saponins and individual ginsenosides on the activation of $Ca^{2+}$ channels and NMDA-gated channels in cultured rat dorsal root ganglion (DRG) and hippocampal neurons, respectively. In DRG neurons, application of ginseng total saponins suppressed high-voltage-activated $Ca^{2+}$ channel currents and ginsenoside Rg$_3$, among the 11 ginsenosides tested, produced the strongest inhibition on $Ca^{2+}$ channel currents. Occlusion experiments using selective $Ca^{2+}$ channel blockers revealed that ginsenoside Rg$_3$ could modulate L-, N-, and P/Q-type currents. In addition, ginsenoside Rg$_3$ also proved to be an active component of ginseng actions on NMDA receptors in cultured hippocampal neurons. Application of ginsenoside Rg$_3$ suppressed NMDA-induced [Ca$^{2+}$]$_{i}$ increase and -gated channels using fura-2-based digital imaging and patch-clamp techniques, respectively. These results suggest that the modulation of $Ca^{2+}$ channels and NMDA receptors by ginsenoside Rg$_3$ could be part of the pharmacological basis of ginseng actions in the peripheral and central nervous systems.ous systems.

• The Korean Journal of Physiology and Pharmacology
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• 제4권6호
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• pp.463-469
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• 2000

It has been well known that 4-aminopyridine (4-AP) has an excitatory effect on vascular smooth muscle due to causing membrane depolarization by blocking $K^+-channel$. However, we observed that 4-AP had an inhibitory effect on the mesenteric artery of rat. Therefore, we investigated the mechanism of 4-AP-induced vasorelaxation. The mesenteric arcuate artery and its branches were isolated and cut into ring. The ring segment was immersed in HEPES-buffered solution and its isometric tension was measured. 4-AP $(0.1{\sim}10\;mM)$ induced a concentration-dependent relaxation, which was unaffected by NO synthase inhibitor, $N^G-nitro-L-arginine$ methylester $(100\;{\mu}M)$ or soluble guanylate cyclase inhibitor, methylene blue $(100\;{\mu}M).$ Glibenclamide $(100\;{\mu}M)$, ATP-sensitive $K^+$ channel blocker, did not exert any effect on the 4-AP-induced vasorelaxation. 4-AP relaxed the sustained contraction induced by 100 mM $K^+$ or $Ca^{2+}$ ionophore, A23187 $(100\;{\mu}M)$ in a dose-dependent manner. In addition, 4-AP significantly decreased the phasic contractile response to norepinephrine in the absence of extracellular $Ca^{2+}$. However, 4-AP did not block the $^{45}Ca$ influx of rat aorta. From the above results, we suggest that 4-AP may not block the $Ca^{2+}$ influx through $Ca^{2+}-channel,$ but act as a nonspecific vasorelaxant in arterial smooth muscle.

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 2001년도 학술 발표회 진행표 및 논문초록
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• pp.36-36
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• 2001

Small conductance $Ca^{2＋}$-activated $K^{+}$ channels (or S $K_{Ca}$ channels) are a group of $K^{＋}$-selective ion channels activated by sub-micromolar concentrations of intracellular $Ca^{2＋}$ independent of membrane voltage. We expressed a cloned S $K_{Ca}$ channel, rSK2, in Xenopus oocytes and investigated the monovalent cation selectivity of the channels. We have used site-directed mutagenesis and macro-channel recordings to identify amino acid residues influencing the ion selectivity.(omitted)d)

• Journal of Ginseng Research
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• 제23권4호
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• pp.230-234
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• 1999

혈관의 평활근 세포막에 존재하는 포타슘채널은 근세포의 막전압을 조절하여 근수축 및 이완을 조절한다. 네가지 유형의 포타슘채널이 근세포막에 존재하며 이중 전도도가 큰 칼슘의존성-포타슘채널$(BK_{Ca})$은 평활근 막전압 조절에 중요한 기능을 담당하는 채널로 알려져 있다. 현재 홍삼 복합사포닌이 혈관 평활근의 이완을 증진시켜 혈압강하를 촉진시킨다고는 알려져 있으나 어떤 분자적 기전이나 전기생리학 기전으로 작용하는지 정확히 알려져 있지 않다. 본 연구는 홍삼 복합사포닌 및 사포닌 $Rg_3$ 성분이 토끼 관상동맥 평활근 세포의 $BK_{Ca}$채널의 활성을 증진시켜 막전압을 과분극시키고 곧 평활근 이완을 촉진한다는 가설을 테스트하였다. 관상동맥 평활근세포의 $BK_{Ca}$채널은 막전압 의존성, 외향정류(outward rectification) 특성을 보였고 단일채널의 전도도는 200pS으로 측정되었으며 charybdotoxin 및 tetraethylammonium에 억제되는 약리학적 특성을 보였다 Whole-cell $BK_{Ca}$활성은 홍삼 복합사포닌에 의해서 농도 의존적으로 증가되었으나 막전압 의존성은 변화되지 않았으며, 단일채널이 열리는 시간은 증가되었다. 홍삼 사포닌 $Rg_3$성분도 막전압 의존성에는 영향을 주지 않으면서$BK_{Ca}$의 활성을 증가시켰으며 단일채널이 열리는 시간도 증가시켰다. 따라서 홍삼 복합사포닌 및 사포닌 $Rg_3$성분은 $BK_{Ca}$의 활성을 증가시켜 막전압을 과분극시켜 평활관의 이완을 촉진한다고 여겨진다.

• The Korean Journal of Physiology and Pharmacology
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• 제9권2호
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• pp.87-94
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• 2005

It is not clear whether $Ca^{2+}-induced$ $Ca^{2+}$ release from the sarcoplasmic reticulum (SR) is involved in the regulation of atrial natriuretic peptide (ANP) release. Previously, we have shown that nifedipine increased ANP release, indicating that $Ca^{2+}$ entry via voltage-gated L-type $Ca^{2+}$ channel activation decreases ANP release. The purpose of the present study was two-fold: to define the role of SR $Ca^{2+}$ release in the regulation of ANP release and whether $Ca^{2+}$ entry via L-type $Ca^{2+}$ channel is prerequisite for the SR-related effect on ANP release. Experiments were performed in perfused beating rabbit atria. Ryanodine, an inhibitor of SR $Ca^{2+}$ release, increased atrial myocytic ANP release ($8.69{\pm}3.05$, $19.55{\pm}1.09$, $27.31{\pm}3.51$, and $18.91{\pm}4.76$% for 1, 2, 3, and $6{\mu}M$ ryanodine, respectively; all P<0.01) with concomitant decrease in atrial stroke volume and pulse pressure in a dose-dependent manner. In the presence of thapsigargin, an inhibitor of SR $Ca^{2+}$ pump, ryanodine-induced increase in ANP release was not observed. Thapsigargin attenuated ryanodine-induced decrease in atrial dynamic changes. Blockade of L-type $Ca^{2+}$ channel with nifedipine abolished ryanodine-induced increase in ANP release ($0.69{\pm}5.58$% vs. $27.31{\pm}3.51$%; P＜0.001). In the presence of thapsigargin and ryanodine, nifedipine increased ANP release and decreased atrial dynamics. These data suggest that $Ca^{2+}$-induced $Ca^{2+}$ release from the SR is inversely involved in the regulation of atrial myocytic ANP release.

• The Korean Journal of Physiology and Pharmacology
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• 제9권1호
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• pp.29-35
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• 2005

To prove the buffering contribution of mitochondria to the increase of intracellular $Ca^{2+}$ level ($[Ca^{2+}]_i$) via background nonselective cation channel (background NSCC), we examined whether inhibition of mitochondria by protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) affects endothelial $Ca^{2+}$ entry and $Ca^{2+}$ buffering in freshly isolated rabbit aortic endothelial cells (RAECs). The ratio of fluorescence by fura-2 AM ($R_{340/380}$) was measured in RAECs. Biological state was checked by application of acetylcholine (ACh) and ACh ($10{\mu}M$) increased $R_{340/380}$ by $1.1{\pm}0.15$ ($mean{\pm}S.E.$, n=6). When the external $Na^+$ was totally replaced by $NMDG^+$, $R_{340/380}$ was increased by $1.19{\pm}0.17$ in a reversible manner (n=27). $NMDG^+$-induced $[Ca^{2+}]_i$ increase was followed by oscillatory decay after $[Ca^{2+}]_i$ reached the peak level. The increase of $[Ca^{2+}]_i$ by $NMDG^+$ was completely suppressed by replacement with $Cs^+$. When $1{\mu}M$ CCCP was applied to bath solution, the ratio of $[Ca^{2+}]_i$ was increased by $0.4{\pm}0.06$ (n=31). When $1{\mu}M$ CCCP was used for pretreatment before application of $NMDG^+$, oscillatory decay of $[Ca^{2+}]_i$ by $NMDG^+$ was significantly inhibited compared to the control (p＜0.05). In addition, $NMDG^+-induced$ increase of $[Ca^{2+}]_i$ was highly enhanced by pretreatment with $2{\mu}M$ CCCP by $320{\pm}93.7$%, compared to the control ($mean{\pm}S.E.$, n=12). From these results, it is concluded that mitochondria might have buffering contribution to the $[Ca^{2+}]_i$ increase through regulation of the background NSCC in RAECs.

• Asian-Australasian Journal of Animal Sciences
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• 제15권7호
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• pp.949-956
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• 2002

There are several physiological and pharmacological evidences indicating that opening of voltage dependent $Ca^{2+}$ channels play a critical role in induction of acrosome reaction in mammalian sperm. We determined the intracellular free $Ca^{2+}$ concentration in ejaculated goat sperm using a fluorescent, $Ca^{2+}$-specific probe, Fura2/AM, after the suspension of sperm in KRB medium, capable of sustaining capacitation and the acrosome reaction. We used nifedipine, D-600 and diltiazem, the $Ca^{2+}$ channel antagonists belonging to the classes of dihydropyridines, phenylalkylamines and benzothiazepines, to investigate the possibility that L-type voltage gated $Ca^{2+}$ channels play a role in the progesterone-stimulated exocytotic response. Progesterone promoted a rise in intracellular $Ca^{2+}$ in goat sperm and addition of nifedipine (100 nM) just prior to progesterone induction, significantly inhibited both intracellular $Ca^{2+}$ rise and exocytosis suggesting that $Ca^{2+}$ channels are involved in the process. However, the intracellular $Ca^{2+}$ increase during the process of capacitation was not affected with the addition of nifedipine suggesting a role of focal channel for $Ca^{2+}$ during capacitation. Studies using monensin and nigericin, two monovalent cation ionophores showed that an influx of $Na^+$ also may play a role in the opening of $Ca^{2+}$ channels. These results strongly suggests that the entry of $Ca^{2+}$ channels with characteristics similar to those of L-type, voltage-sensitive $Ca^{2+}$ channels found in cardiac and skeletal muscle, is a crucial step in the sequence of events leading to progesterone induced acrosome reaction in goat sperm.