• Clinical and Experimental Pediatrics
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• 제57권10호
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• pp.445-450
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• 2014

Purpose: Familial hypokalemic periodic paralysis (HOKPP) is an autosomal dominant channelopathy characterized by episodic attacks of muscle weakness and hypokalemia. Mutations in the calcium channel gene, CACNA1S, or the sodium channel gene, SCN4A, have been found to be responsible for HOKPP; however, the mechanism that causes hypokalemia remains to be determined. The aim of this study was to improve the understanding of this mechanism by investigating the expression of calcium-activated potassium ($K_{Ca}$) channel genes in HOKPP patients. Methods: We measured the intracellular calcium concentration with fura-2-acetoxymethyl ester in skeletal muscle cells of HOKPP patients and healthy individuals. We examined the mRNA and protein expression of KCa channel genes (KCNMA1, KCNN1, KCNN2, KCNN3, and KCNN4) in both cell types. Results: Patient cells exhibited higher cytosolic calcium levels than normal cells. Quantitative reverse transcription polymerase chain reaction analysis showed that the mRNA levels of the $K_{Ca}$ channel genes did not significantly differ between patient and normal cells. However, western blot analysis showed that protein levels of the KCNMA1 gene, which encodes $K_{Ca}$1.1 channels (also called big potassium channels), were significantly lower in the membrane fraction and higher in the cytosolic fraction of patient cells than normal cells. When patient cells were exposed to 50 mM potassium buffer, which was used to induce depolarization, the altered subcellular distribution of BK channels remained unchanged. Conclusion: These findings suggest a novel mechanism for the development of hypokalemia and paralysis in HOKPP and demonstrate a connection between disease-associated mutations in calcium/sodium channels and pathogenic changes in nonmutant potassium channels.

• ALGAE
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• 제36권4호
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• pp.315-326
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• 2021

Ion channels are membrane protein complexes mediating passive ion flux across the cell membranes. Every organism has a certain set of ion channels that define its physiology. Dinoflagellates are ecologically important microorganisms characterized by effective physiological adaptability, which backs up their massive proliferations that often result in harmful blooms (red tides). In this study, we used a bioinformatics approach to identify homologs of known ion channels that belong to 36 ion channel families. We demonstrated that the versatility of the dinoflagellate physiology is underpinned by a high diversity of ion channels including homologs of animal and plant proteins, as well as channels unique to protists. The analysis of 27 transcriptomes allowed reconstructing a consensus ion channel repertoire (channelome) of dinoflagellates including the members of 31 ion channel families: inwardly-rectifying potassium channels, two-pore domain potassium channels, voltage-gated potassium channels (K_v), tandem K_v, cyclic nucleotide-binding domain-containing channels (CNBD), tandem CNBD, eukaryotic ionotropic glutamate receptors, large-conductance calcium-activated potassium channels, intermediate/small-conductance calcium-activated potassium channels, eukaryotic single-domain voltage-gated cation channels, transient receptor potential channels, two-pore domain calcium channels, four-domain voltage-gated cation channels, cation and anion Cys-loop receptors, small-conductivity mechanosensitive channels, large-conductivity mechanosensitive channels, voltage-gated proton channels, inositole-1,4,5-trisphosphate receptors, slow anion channels, aluminum-activated malate transporters and quick anion channels, mitochondrial calcium uniporters, voltage-dependent anion channels, vesicular chloride channels, ionotropic purinergic receptors, animal volage-insensitive cation channels, channelrhodopsins, bestrophins, voltage-gated chloride channels H⁺/Cl^- exchangers, plant calcium-permeable mechanosensitive channels, and trimeric intracellular cation channels. Overall, dinoflagellates represent cells able to respond to physical and chemical stimuli utilizing a wide range of G-protein coupled receptors- and Ca²⁺-dependent signaling pathways. The applied approach not only shed light on the ion channel set in dinoflagellates, but also provided the information on possible molecular mechanisms underlying vital cellular processes dependent on the ion transport.

• International Journal of Oral Biology
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• 제40권4호
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• pp.211-216
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• 2015

Nitric Oxide (NO) is an important signaling molecule in the nociceptive process. Our previous study suggested that high concentrations of sodium nitroprusside (SNP), a NO donor, induce a membrane hyperpolarization and outward current through large conductances calcium-activated potassium ($BK_{ca}$) channels in substantia gelatinosa (SG) neurons. In this study, patch clamp recording in spinal slices was used to investigate the sources of $Ca^{2+}$ that induces $Ca^{2+}$-activated potassium currents. Application of SNP induced a membrane hyperpolarization, which was significantly inhibited by hemoglobin and 2-(4-carboxyphenyl) -4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (c-PTIO), NO scavengers. SNP-induced hyperpolarization was decreased in the presence of charybdotoxin, a selective $BK_{Ca}$ channel blocker. In addition, SNP-induced response was significantly blocked by pretreatment of thapsigargin which can remove $Ca^{2+}$ in endoplasmic reticulum, and decreased by pretreatment of dentrolene, a ryanodine receptors (RyR) blocker. These data suggested that NO induces a membrane hyperpolarization through $BK_{ca}$ channels, which are activated by intracellular $Ca^{2+}$ increase via activation of RyR of $Ca^{2+}$ stores.

• The Korean Journal of Physiology and Pharmacology
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• 제25권3호
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• pp.227-237
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• 2021

Carbon monoxide (CO) is a cardioprotectant and potential cardiovascular therapeutic agent. Human cardiac fibroblasts (HCFs) are important determinants of myocardial structure and function. Large-conductance Ca2+-activated K+ (BK) channel is a potential therapeutic target for cardiovascular disease. We investigated whether CO modulates BK channels and the signaling pathways in HCFs using whole-cell mode patch-clamp recordings. CO-releasing molecules (CORMs; CORM-2 and CORM-3) significantly increased the amplitudes of BK currents (IBK). The CO-induced stimulating effects on IBK were blocked by pre-treatment with specific nitric oxide synthase (NOS) blockers (L-NG-monomethyl arginine citrate and L-NG-nitroarginine methyl ester). 8-bromo-cyclic GMP increased IBK. KT5823 (inhibits PKG) or ODQ (inhibits soluble guanylate cyclase) blocked the CO-stimulating effect on IBK. Moreover, 8-bromo-cyclic AMP also increased IBK, and pre-treatment with KT5720 (inhibits PKA) or SQ22536 (inhibits adenylate cyclase) blocked the CO effect. Pre-treatment with N-ethylmaleimide (a thiol-alkylating reagent) also blocked the CO effect on IBK, and DL-dithiothreitol (a reducing agent) reversed the CO effect. These data suggest that CO activates IBK through NO via the NOS and through the PKG, PKA, and S-nitrosylation pathways.

• The Korean Journal of Physiology and Pharmacology
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• 제11권5호
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• pp.215-219
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• 2007

Small and large conductance $Ca^{2+}$-activated $K^+(SK_{Ca}\;and\;BK_{Ca})$ channels are implicated in the modulation of neuronal excitability. We investigated how changes in peripheral $K_{Ca}$ channel activity affect mechanical sensitivity as well as the afferent fiber type responsible for $K_{Ca}$ channel-induced mechanical sensitivity. Blockade of $SK_{Ca}$ and $BK_{Ca}$ channels induced a sustained decrease of mechanical threshold which was significantly attenuated by topical application of capsaicin onto afferent fiber and intraplantar injection of 1-ethyl-2-benzimidazolinone. NS1619 selectively attenuated the decrease of mechanical threshold induced by charybdotoxin, but not by apamin. Spontaneous flinching and paw thickness were not significantly different after $K_{Ca}$ channel blockade. These results suggest that mechanical sensitivity can be modulated by $K_{Ca}$ channels on capsaicin-sensitive afferent fibers.

• The Korean Journal of Physiology
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• 제25권1호
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• pp.17-26
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• 1991

Single smooth muscle cells of the rabbit pulmonary artery were isolated by treatment with collagenase and elastase. Using the patch clamp technique, potassium channel activity was recorded from the inside-out membrane patch. The channel had a sin히e channel conductance of about 360 pS in symmetrical concentration of K on both sides of the patch, 150 mM, and had a linear current-voltage relationship. During the application of 10 mM tetraethylammonium (TEA) to the intracellular membrane surface, the amplitude of single channel current was reduced and very rapid flickering appeared. The open probability $(P_0)$ of this channel was increased by increasing positivity of the potential across the patch membrane, with e-fold increase by 20 mV depolarization, and by increasing the internal $Ca^{2+}$ concentration. These findings are consistent with those of large conductance Ca-activated K channels reported in other tissues. But the shortening of the mean open time by increasing $[Ca^{2+}]_i$, was an unexpected result and one additional closed state which might be arisen from a block of the open channel by Ca binding was suggested. The $P_0-membrane$ potential relationship was modulated by internal pH. Decreasing pH reduced $P_0$. Increasing pH not only increased $P_0$ but also weakened the voltage dependency of the channel opening. The modulation of Ca-activated K channel by pH was thought to be related to the mechanism of regulation of vascular tone by the pH change.

• 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 Yeungnam Medical Science
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• 제11권2호
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• pp.363-374
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• 1994

흰쥐의 적출 배뇨근에 대한 수종의 potassium 통로개방제의 작용을 관찰하고, 배뇨근에 존재하는 potassium 통로의 특성을 알아보기 위하여 체중 250~350g의 흰쥐 (Sprague-Dawley)를 단두하여 희생시킨 후 방광을 적출하였다. 적출된 방광으로 부터 $1.5mm{\times}1.5cm$의 배뇨근 수평절편을 만들어 1ml의 Tyrode 영양액을 포함하는 적출근편실험조에 현수하고 등척성장력을 측정하여 polygraph에 묘기하였다. 배뇨근절편은 potassium 통로 개방제인 pinacidil, BRL 38227 및 RP 52891의 누적 농도 첨가에 의하여 그 기본장력이 농도의존적으로 감소하였는데 그 작용강도는 RP 52891, pinacidil 그리고 BRL 38227의 순이었다. 전위 의존성 potassium 통로 봉쇄제인 procaine은 배뇨근 절편의 기본장력에 영향을 미치지 못했으며, pinacidil, BRL 38227 및 RP 52891에 의한 기본장력감소작용에 대해서도 영향을 미치지 못하였다. 칼슘 의존성 potassium 통로봉쇄제인 apamin은 배뇨근의 기본장력에 유의한 변화를 가져오지 못하였고, potassium 통로 개방제들에 대하여는 상경적 길항작용을 나타내지는 않았으나 BRL 38227과 RP 52891의 최고효능을 유의하게 감소시켰다. ATP 의존성 potassium 통로봉쇄제인 glibenclamide는 배뇨근 절편의 기본장력을 증가시키고, pinacidil을 상경적으로 길항하였으며, BRL 38227과 RP 52891을 상경적으로 길항하는 동시에 그 최대효능을 감소시켰다. 췌장의 ${\beta}$-세포에서 ATP 의존성 potassium 통로를 개방시켜 인슐린의 분비를 억제하는 galanin은 흰쥐의 배뇨근을 수축시켰다. 이상의 결과를 종합하면, 흰쥐의 배뇨근에서는 새로운 potassium 통로 개방제인 RP 52891의 배뇨근 이완작용이 pinacidil보다 강한 것으로 관찰되었다. 또 흰쥐 배뇨근에서는 ATP 의존성이며, glibenclamide 반응성인 potassium 통로가 존재 한다고 생각되는데, 이는 췌장의 ${\beta}$-세포에 있는 ATP 의존성 potassium 통로와는 다른 특성을 가진 것으로 추측된다.

• The Korean Journal of Physiology and Pharmacology
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• 제2권1호
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• pp.95-100
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• 1998

The conductance change evoked by step depolarization was studied in primarily cultured rat adrenal chromaffin cells using patch-clamp and capacitance measurement techniques. When we applied a depolarizing pulse to a chromaffin cell, the inward calcium current was followed by an outward current and depolarization-induced exocytosis was accompanied by an increase in conductance trace. The slow inward tail current which has the same time course as the conductance change was observed in current recording. The activation of slow tail current was calcium-dependent. Reversal potentials agreed with Nernst equation assuming relative permeability of $Cs^+\;to\;K^+$ is 0.095. The outward current and tail current were blocked by apamin (200 nM) and d-tubocurarine (2 mM). The conductance change was blocked by apamin and did not affect membrane capacitance recording. We confirmed that conductance change after depolarization comes from the activation of the SK channel and can be blocked by application of the SK channel blockers. Consequently, it is necessary to consider blocking of the SK channel during membrane capacitance recording.

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 2003년도 정기총회 및 학술발표회
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• pp.41-41
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• 2003

Small conductance calcium-activated potassium channels (or SKCa channels) are potassium selective, voltage-independent, and activated by intracellular calcium concentration. These channels play important roles in excitable cells such as neuron in the central nervous system (Vergara et al., 1998). The activity of SKCa channels underlies the slow afterhyperpolarization that inhibits neuronal cell firing (Hille, 1991; Vergara et al.,1998). Until now, N-terminal region of rSK2 isn't characterized. To study the role of N-terminus, we constructed the N-terminal deletion mutant and characterized by electrophysiological means. Interestingly, N-terminal deletion mutant be trafficked to membrane couldn't evoke any ionic currents. Thus, N-terminal region has a role in functional rSK2 channel formation. To elucidate the function of N-terminal region, (His)6-conjugated protein was purified and filtrated by affinity column chromatography. Surprisingly, N-terminal region was shown in tetramer size that was supported by cross-linking result. Thus, we predicted that N-terminal region might be involved in the tetramerization of rSK2.