• 대한의생명과학회지
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• 제18권2호
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• pp.96-103
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• 2012

Kv 4.2 ion channel protein has an ability to open at subthreshold membrane potentials and to recover quickly from inactivation. That is very important for neuronal signal transmission in vertebrate brain. In order to purify Kv 4.2 protein, the novel purification methods were experimented. The purification procedure utilized chromatography on DE-52 ion exchange column and affinity chromatography on a WGA-Sepharose 4B, and Kv 4.2 affinity column chromatography. It was found that 0.5% (wt./vol.) Triton X-100 detergent in lysis buffer worked well for Kv 4.2 protein solubilization from rat brain membrane. Protein quantitative determination was conducted by BCA method at 562 nm for each purification step to avoid determination interference of protein at 280 nm by detergent. The confirmation of Kv 4.2 existence and amount is performed using by SDS-PAGE/immunoblotting or 96-well dot blotting. The Kv 4.2 without interacting protein that contains carbohydrate, was purified from novel biochemical 3-steps purification method for further research.

• 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.

• 생명과학회지
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• 제19권10호
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• pp.1484-1488
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• 2009

가족성 저칼륨성 주기성 마비란 상염색체 우성 유전 질환으로 저칼륨혈증을 동반한 간헐적인 가역적 이완성 근육 마비를 특징으로 한다. 세포내 저류된 칼륨으로 인해 저칼륨혈증이 지속되고 근세포 활성이상으로 인해 마비가 발생하는 것으로 알려져 있다. 이러한 증상발현의 분자생물학적 기전을 확인하기 위해 세포 내 칼륨이온을 세포 밖으로 이동시키는 지연성 정류형 채널 단백질의 일종인 KCNQ3와 KCNQ5를 대상으로, 정상인과 환자에서 채취한 골격근 세포를 생리적 세포외 정상 칼륨농도인 4 mM과 탈분극 유도를 위한 고칼륨농도인 50mM에 노출시켜 단백질의 양적 변화 유무를 확인하였다. 유전자 발현양상을 확인하기 위해 mRNA의 양적 변화를 확인한 결과 모든 조건에서 유의한 변화가 관찰되지 않아 정상 칼륨조건과 고칼륨조건이 두 유전자발현의 변화를 야기하지 않음을 확인하였다. 그러나 단백질 양을 관찰한 결과 환자의 골격근 세포가 50 mM의 칼륨농도에 노출되는 경우 KCNQ3 단백질은 세포질 내에서 증가하고 세포막 내에서 감소하였다. 이는 환자의 골격근 세포가 고농도의 세포외 칼륨에 의해 탈분극 되는 경우 재분극에 중요한 기능을 담당하는 KCNQ3 채널 단백질이 세포질 내로 이동하여 재분극 형성의 장애를 초래하고 이로 인해 근세포 활성이 일어나지 않게 되어 마비를 유발할 수 있음을 시사하는 결과로 본 질환의 새로운 발병 기전을 설명할 수 있는 근거로 생각된다.

• 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.

• Journal of Plant Biotechnology
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• 제37권4호
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• pp.539-548
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• 2010

Potassium ($K^+$) is one of the most abundant cations in higher plant. It comprises about 10% of plant dry weight and it plays roles in numerous functions such as osmo- and turgor regulation, charge balance of plasma membrane and control of stomata and organ movement. Several potassium transporters and potassium channels regulate $K^+$ homeostasis in response to $K^+$ uptake systems. In this review, we describe the biological, biochemical and physiological characteristics of shaker like potassium channels in higher plant. Especially, we searched the rice genome databases and analysized expressed genes, genome structures and protein domain characteristics of shaker like potassium channels.

• Clinical and Experimental Pediatrics
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• 제61권9호
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• pp.271-278
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• 2018

Purpose: Abnormal potassium channels expression affects vessel function, including vascular tone and proliferation rate. Diverse potassium channels, including voltage-gated potassium (Kv) channels, are involved in pathological changes of pulmonary arterial hypertension (PAH). Since the role of the Kv1.7 channel in PAH has not been previously studied, we investigated whether Kv1.7 channel expression changes in the lung tissue of a monocrotaline (MCT)-induced PAH rat model and whether this change is influenced by the endothelin (ET)-1 and reactive oxygen species (ROS) pathways. Methods: Rats were separated into 2 groups: the control (C) group and the MCT (M) group (60 mg/kg MCT). A hemodynamic study was performed by catheterization into the external jugular vein to estimate the right ventricular pressure (RVP), and pathological changes in the lung tissue were investigated. Changes in protein and mRNA levels were confirmed by western blot and polymerase chain reaction analysis, respectively. Results: MCT caused increased RVP, medial wall thickening of the pulmonary arterioles, and increased expression level of ET-1, ET receptor A, and NADPH oxidase (NOX) 4 proteins. Decreased Kv1.7 channel expression was detected in the lung tissue. Inward-rectifier channel 6.1 expression in the lung tissue also increased. We confirmed that ET-1 increased NOX4 level and decreased glutathione peroxidase-1 level in pulmonary artery smooth muscle cells (PASMCs). ET-1 increased ROS level in PASMCs. Conclusion: Decreased Kv1.7 channel expression might be caused by the ET-1 and ROS pathways and contributes to MCT-induced PAH.

• The Korean Journal of Physiology and Pharmacology
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• 제4권6호
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• pp.445-453
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• 2000

The present investigation tested the hypothesis that the activation of protein kinase G (PKG) leads to a phosphorylation of $Ca^{2+}-activated$ potassium channel $(K_{Ca}\;channel)$ and is involved in the activation of $K_{Ca}$ channel activity in cerebral arterial smooth muscle cells of the rabbit. Single-channel currents were recorded in cell-attached and inside-out patch configurations of patch-clamp techniques. Both molsidomine derivative 3-morpholinosydnonimine-N-ethylcarbamide $(SIN-1,\;50\;{\mu}M)$ and 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate $(8-pCPT-cGMP,\;100\;{\mu}M),$ a membrane-permeable analogue of cGMP, increased the $K_{Ca}$ channel activity in the cell-attached patch configuration, and the effect was removed upon washout of the drugs. In inside-out patches, single-channel current amplitude was not changed by SIN-1 and 8-pCPT-cGMP. Application of ATP $(100\;{\mu}M),$ cGMP $(100\;{\mu}M),$ ATP+cGMP $(100\;{\mu}M\;each),$ PKG $(5\;U/{\mu}l),$ ATP $(100\;{\mu}M)+PKG\;(5\;U/{\mu}l),$ or cGMP $(100\;{\mu}M)+PKG\;(5\;U/{\mu}l)$ did not increase the channel activity. ATP $(100\;{\mu}M)+cGMP\;(100\;{\mu}M)+PKG\;(5\;U/{\mu}l)$ added directly to the intracellular phase of inside-out patches increased the channel activity with no changes in the conductance. The heat-inactivated PKG had no effect on the channel activity, and the effect of PKG was inhibited by 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate, Rp-isomer $(Rp-pCPT-cGMP,\;100\;{\mu}M),$ a potent inhibitor of PKG or protein phosphatase 2A (PP2A, 1 U/ml). In the presence of okadaic acid (OA, 5 nM), PP2A had no effect on the channel activity. The $K_{Ca}$ channel activity spontaneously decayed to the control level upon washout of ATP, cGMP and PKG, and this was prevented by OA (5 nM) in the medium. These results suggest that the PKG-mediated phosphorylations of $K_{Ca}$ channels, or some associated proteins in the membrane patch increase the activity of the $K_{Ca}$ channel, and the activation may be associated with the vasodilating action.

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 2003년도 정기총회 및 학술발표회
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• pp.45-45
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• 2003

We isolated a novel ankyrin-repeat containing protein, rSIAP (rSlo Interacting Ankyrin-repeat Protein), as an interacting protein to the cytosolic domain of the alpha-subunit of rat large-conductance Ca$\^$2+/-activated K$\^$+/ channel (rSlo) by yeast two-hybrid screening. Affinity pull-down assay showed the direct and specific interaction between rSIAP and rSlo domain. The channel-binding proteins can be classified into several categories according to their functional effects on the channel proteins, i.e. signaling adaptors, scaffolding net, molecular tuners, molecular chaperones, etc. To obtain initial clues on its functional roles, we investigated the cellular localization of rSIAP using immunofluorescent staining. The results showed the possible co-localization of rSlo and rSIAP protein near the plasma membrane, when co-expressed in CHO cells. We then investigated the functional effects of rSIAP on the rSlo channel using electrophysiological means. The co-expression of rSIAP accelerated the activation of rSlo channel. These effects were initiated at the micromolar [Ca$\^$2+/]$\_$i/ and gradually increased as [Ca$\^$2+/]$\_$i/ raised. Interestingly, rSIAP decreased the inactivation kinetics of rSlo channel at micromolar [Ca$\^$2+/]$\_$i/, while the rate was accelerated at sub-micromolar [Ca$\^$2+/]$\_$i/. These results suggest that rSIAP may modulate the activity of native BK$\_$Ca/ channel by altering its gating kinetics depending on [Ca$\^$2+/]$\_$i/. To localize critical regions involved in protein-protein interaction between rSlo and rSIAP, a series of sub-domain constructs were generated. We are currently investigating sub-domain interaction using both of yeast two-hybrid method and in vitro binding assay.

• 한국수정란이식학회지
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• 제30권4호
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• pp.277-282
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• 2015

Cryopreservation affects osmotic tolerance and intracellular ion concentration through changes in expression levels of water and ion channels. Control of these changes is important for cell survival after cryopreservation. Relatively little is known about changes in $K^+$ channel expression compared to water channel expression. This study was performed to investigate changes in TASK-2 channel (KCNK5: potassium channel, subfamily K, member 5), a member of two-pore domain $K^+$ channel family, in cryopreserved mouse ovaries. Cryopreservation increased TASK-2 mRNA expression in mouse ovaries. In addition, TASK-2 protein expression was upregulated in vitrified and slowly frozen ovaries. TASK-2 protein was expressed in all area of granulosa cells that surround the oocyte within the follicle, except nucleus. Viability of cells overexpressed with TASK-2 was higher than that of vector-transfected cells. Our results found that TASK-2 expression was increased by cryopreservation and overexpression of TASK-2 decreased cryopreservation-induced cell death. These results suggest that TASK-2 upregulation might reduce cryodamage.

• 한국작물학회:학술대회논문집
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• 한국작물학회 2017년도 9th Asian Crop Science Association conference
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• pp.110-110
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• 2017

Stomata are the main gateways for water and air transport between leaves and the environment. Inward-rectifying potassium channels regulate photo-induced stomatal opening. Rice contains three inward rectifying shaker-like potassium channel proteins, OsKAT1, OsKAT2 and OsKAT3. Among these, only OsKAT2 is specifically expressed in guard cells. Here, we investigated the functions of OsKAT2 in stomatal regulation using three dominant negative mutant proteins, OsKAT2(T235R), OsKAT2(T285A) and OsKAT2(T285D), which are altered in amino acids in the channel pore and at a phosphorylation site. Yeast complementation and patch clamp assays showed that all three mutant proteins lost channel activity. However, among plants overexpressing these mutant proteins, only plants overexpressing OsKAT2(T235R) showed significantly less water loss than the control. Moreover, overexpression of this mutant protein led to delayed photo-induced stomatal opening and increased drought tolerance. Our results indicate that OsKAT2 is an inward-rectifying shaker-like potassium channel that mainly functions in stomatal opening. Interestingly, overexpression of OsKAT2(T235R) did not cause serious defects in growth or yield in rice, suggesting that OsKAT2 is a potential target for engineering plants with improved drought tolerance without yield penalty.