• Journal of Microbiology and Biotechnology
• /
• v.25 no.8
• /
• pp.1371-1379
• /
• 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 and Pharmacology
• /
• v.15 no.1
• /
• pp.17-22
• /
• 2011

Quercetin mainly exists in the skin of colored fruits and vegetables as one of flavonoids. Recent studies show that quercetin, like other flavonoids, has diverse pharmacological actions. However, relatively little is known about quercetin effects in the regulations of ligand-gated ion channels. In the previous reports, we have shown that quercetin regulates subsets of homomeric ligand-gated ion channels such as glycine, 5-$HT_{3A}$ and ${\alpha}7$ nicotinic acetylcholine receptors. In the present study, we examined quercetin effects on heteromeric neuronal ${\alpha}3{\beta}4$ nicotinic acetylcholine receptor channel activity expressed in Xenopus oocytes after injection of cRNA encoding bovine neuronal ${\alpha}3$ and ${\beta}4$ subunits. Treatment with acetylcholine elicited an inward peak current ($I_{ACh}$) in oocytes expressing ${\alpha}3{\beta}4$ nicotinic acetylcholine receptor. Co-treatment with quercetin and acetylcholine inhibited $I_{ACh}$ in oocytes expressing ${\alpha}3{\beta}4$ nicotinic acetylcholine receptors. The inhibition of $I_{ACh}$ by quercetin was reversible and concentration-dependent. The half-inhibitory concentration ($IC_{50}$) of quercetin was $14.9{\pm}0.8\;{\mu}M$ in oocytes expressing ${\alpha}3{\beta}4$ nicotinic acetylcholine receptor. The inhibition of $I_{ACh}$ by quercetin was voltage-independent and non-competitive. These results indicate that quercetin might regulate ${\alpha}3{\beta}4$ nicotinic acetylcholine receptor and this regulation might be one of the pharmacological actions of quercetin in nervous systems.

• The Korean Journal of Physiology and Pharmacology
• /
• v.15 no.4
• /
• pp.195-201
• /
• 2011

The flavonoid quercetin is a low molecular weight compound generally found in apple, gingko, tomato, onion and other red-colored fruits and vegetables. Like other flavonoids, quercetin has diverse pharmacological actions. However, relatively little is known about the influence of quercetin effects in the regulation of ligand-gated ion channels. Previously, we reported that quercetin regulates subsets of nicotinic acetylcholine receptors such as ${\alpha}3{\beta}4$, ${\alpha}7$ and ${\alpha}9{\alpha}10$. Presently, we investigated the effects of quercetin on muscle-type of nicotinic acetylcholine receptor channel activity expressed in Xenopus oocytes after injection of cRNA encoding human fetal or adult muscle-type of nicotinic acetylcholine receptor subunits. Acetylcholine treatment elicited an inward peak current ($I_{ACh}$) in oocytes expressing both muscle-type of nicotinic acetylcholine receptors and co-treatment of quercetin with acetylcholine inhibited $I_{ACh}$. Pre-treatment of quercetin further inhibited $I_{ACh}$ in oocytes expressing adult and fetal muscle-type nicotinic acetylcholine receptors. The inhibition of $I_{ACh}$ by quercetin was reversible and concentration-dependent. The $IC_{50}$ of quercetin was $18.9{\pm}1.2{\mu}M$ in oocytes expressing adult muscle-type nicotinic acetylcholine receptor. The inhibition of $I_{ACh}$ by quercetin was voltage-independent and non-competitive. These results indicate that quercetin might regulate human muscle-type nicotinic acetylcholine receptor channel activity and that quercetin-mediated regulation of muscle-type nicotinic acetylcholine receptor might be coupled to regulation of neuromuscular junction activity.

• The Korean Journal of Physiology and Pharmacology
• /
• v.17 no.2
• /
• pp.175-180
• /
• 2013

Resveratrol is a phytoalexin found in grapes, red wine, and berries. Resveratrol has been known to have many beneficial health effects, such as anti-cancer, neuroprotective, anti-inflammatory, and life-prolonging effects. However, relatively little is known about the effects of resveratrol on the regulation of ligand-gated ion channels. We have previously reported that resveratrol regulates subsets of homomeric ligand-gated ion channels such as those of 5-$HT_{3A}$ receptors. The ${\gamma}$-aminobutyric $acid_C$($GABA_C$) receptor is mainly expressed in retinal bipolar cells and plays an important role in visual processing. In the present study, we examined the effects of resveratrol on the channel activity of homomeric $GABA_C$ receptor expressed in Xenopus oocytes injected with cRNA encoding human $GABA_C$ ${\rho}$ subunits. Our data show that the application of GABA elicits an inward peak current ($I_{GABA}$) in oocytes that express the $GABA_C$ receptor. Resveratrol treatment had no effect on oocytes injected with $H_2O$ or with $GABA_C$ receptor cRNA. Co-treatment with resveratrol and GABA inhibited $I_{GABA}$ in oocytes with $GABA_C$ receptors. The inhibition of $I_{GABA}$ by resveratrol was in a reversible and concentration-dependent manner. The $IC_{50}$ of resveratrol was $28.9{\pm}2.8{\mu}M$ in oocytes expressing $GABA_C$ receptor. The inhibition of $I_{GABA}$ by resveratrol was in voltage-independent and non-competitive manner. These results indicate that resveratrol might regulate $GABA_C$ receptor expression and that this regulation might be one of the pharmacological actions of resveratrol on the nervous system.

• Proceedings of the Korean Society of Crop Science Conference
• /
• 2017.06a
• /
• pp.194-194
• /
• 2017

Camelina (Camelina sativa L.) is a potential bio-energy crop that has short life cycle about 90 days and contains high amount of unsaturated fatty acid which is adequate to bio-diesel production. Enhancing environmental stress tolerance is a main issue to increase not only crop productivity but also big mass production. CsRCI2s (Rare Cold Inducible 2) are cold and salt stress related protein that localized at plasma membrane (PM) and assume to be membrane potential regulation factor. These proteins can be divide into C-terminal tail (CsRCI2D/E/F/G) or no-tail group (CsRCI2A/B/C/H). However, function of CsRCI2s are less understood. In this study, physiological responses and functional characterization of CsRCI2s of Camelina under salt stress were analyzed. Full-length CsRCI2s (A/B/E/F) and CsPIP2;1 sequences were confirmed from Camelina genome browser. Physiological investigations were carried out using one- or four-week-old Camelina under NaCl stress with dose and time dependent manner. Transcriptional changes of CsRCI2A/B/E/F and CsPIP2;1 were determined using qRT-PCR in one-week-old Camelina seedlings treated with NaCl. Translational changes of CsRCI2E and CsPIP2;1 were confirmed with western-blot using the antibodies. Water transport activity and membrane potential measurement were observed by cRNA injected Xenopus laevis oocyte. As results, root growth rate and physiological parameters such as stomatal conductance, chlorophyll fluorescence, and electrolyte leakage showed significant inhibition in 100 and 150 mM NaCl. Transcriptional level of CsPIP2;1 did not changed but CsRCI2s were significantly increased by NaCl concentration, however, no-tail type CsRCI2A and CsRCI2B increased earlier than tail type CsRCI2E and CsRCI2F. Translational changes of CsPIP2;1 was constitutively maintained under NaCl stress. But, accumulation of CsRCI2E significantly increased by NaCl stress. CsPIP2;1 and CsRCI2A/B/E/F co-expressed Xenopus laevis oocyte showed decreased water transport activity as 61.84, 60.30, 62.91 and 76.51 % at CsRCI2A, CsRCI2B, CsRCI2E and CsRCI2F co-expression when compare with single expression of CsPIP2;1, respectively. Moreover, oocyte membrane potential was significantly hyperpolarized by co-expression of CsRCI2s. However, higher hyperpolarized level was observed in tail-type CsRCI2E and CsRCI2F than others, especially, CsRCI2E showed highest level. It means transport of $Na^+$ ion into cell is negatively regulated by expression of CsRCI2s, and, function of C-terminal tail is might be related with $Na^+$ ion influx. In conclusion, accumulation of NaCl-induced CsRCI2 proteins are related with $Na^+$ ion exclusion and prevent water loss by CsPIP2;1 under NaCl stress.

• Korean Journal of Environmental Agriculture
• /
• v.19 no.2
• /
• pp.147-153
• /
• 2000

Cigarette smoking deals a harmful effect directly to smokers and even to non-smokers through environmental tobacco smoke. The major damaging component in cigarette smoke is nicotine which converts to various carcinogens. Among the carcinogenic metabolites, nitrosamine-4-(methylnitrosamino)-1- (3-pyridyl)-1- butanone (NNK) is responsible for many types of lung cancers. Recent studies report that activation of NNK is markedly inhibited in the presence of cotinine, a safer metabolite from nicotine. It is well known that tea extract have potentials to prevent cancers. This study aims to correlate green tea's potential for cancer prevention with an accelerated formation of cotinine. In the presence of tea extract, a nicotine to cotinine conversion was studied in established cell lines and xenopus oocytes. Among three lines of cell used, PLC/PRF5 and 293 cells showed a fast turnover from nicotine to cotinine while HepG2 cell line showed a marginal difference between groups treated and non-treated with tea extract. A microinjection procedure using Xenopus oocyte was utilized to probe for the effect of tea extract in accelerating nicotine conversion to cotinine. According to this procedure, tea extract's unusual potential for converting nicotine to cotinine is also substantiated. Overall, this present study indicated that tea extract have an unusual effect on conversion of nicotine to cotinine in cells.

• Journal of Life Science
• /
• v.18 no.1
• /
• pp.114-119
• /
• 2008

The molecular machinery controlling cell cycle is centered around the regulation of the activity of maturation-promoting factor (MPF), a complex composed of a catalytic Cdc2 and the cyclinB regulatory subunit. Cdc2 kinase is inactivated by phosphorylation of inhibitory kinase, Wee1. It has been known that there are three different Wee1 kinases in the mammalian cell, Wee1A, Wee1B and Myt1. To investigate the regulatory mechanism of Wee1 kinases, the phosphorylation and degradation of Wee1A and Wee1B were checked in the Xenopus oocyte cell cycle. When Wee1 kinases were injected into frog oocyte, Wee1B was more stable than Wee1A. Wee1A and Wee1B kinase were phosphorylated by many kinases such as PKA and Akt. The roles of amino or carboxyl terminal in mouse Wee1A or Wee1B kinase were investigated using chimeric constructs. The degree of protein phosphorylation, degradation and cell cycle progression were different between chimeric constructs. The amino domain of Wee1A was implicated in the protein phosphorylation and degradation while amino domain of Wee1B and carboxyl domain of Wee1A were involved in the activity regulation. These results suggested that the domains of Wee1 kinase have different and significant roles in regulating the Wee1 kinases in the cell cycle progression.

• Journal of Physiology & Pathology in Korean Medicine
• /
• v.17 no.4
• /
• pp.1037-1049
• /
• 2003

Numerous lines of evidence indicate that some of the neurotoxicity associated with Alzheimer's disease (AD) is due to proteolytic fragments of the amyloid precursor protein (APP). Most research has focused on the amyloid 6 (M). However, the possible role of other cleaved products of APP is less clear. Lately It has been reported that a recombinant carboxy-terminal 105 amino acid fragment (CT105) of APP induced strong nonselective inward currents in Xenopus oocyte. In a brain with Alzheimer's disease (AD), to investigate the roles of carboxyl-terminal fragment (CT105) of amyloid precursor protein (APP) in apoptosis processes possibly linked to neurodegeneration associated with AD, we examined the effects of the CT of APP with 105 amino acid residues (CT105) on the alteration of apoptosis triggers in neubroblastoma cells. We have investigated whether Radix Polygalae and Rhizoma Acori Graminei mixture extract (RP+RAG) inhibits CT105-induced apoptosis of neuroblastoma cells. We found that RP+RAG inhibits CT105-induced apoptosis in SK-N-SH cells. Treatment of the cells with RP+RAG inhibited CT105-induced DNA fragmentation and Tunel assay of nuclear chromatin and inhibited the caspase-3 expression in SK-N-SH cells. As the result of this study, In RP+RAG group, the apoptosis in the nervous system is inhibited, the repair against the degerneration of neuroblastoma cells by CT105 expression is promoted. These results indicate that RP+RAG possess strong inhibitory effect of apoptosis in the nervous system and repair effect against the degeneration of neuroblastoma cells by CT105 expression

• Journal of Embryo Transfer
• /
• v.19 no.2
• /
• pp.89-99
• /
• 2004

Stretch-activated channels (SACs) responds to membrane stress with changes in open probability (Po). They play essential roles in regulation of cell volume and differentiation, vascular tone, and in hormonal secretion. SACs highly present in Xenopus oocytes and Ascidian oocytes are suggested to be involved in the regulation of pH and fluid transport to balance the osmotic pressure, but remain unclear in mammanlian oocytes. This study was investigated to find the presence of SACs in hamster oocytes and to examine their electrophysiological properties. To infer a role of SAC in relation to the development of early stage, we followed up to the stage of two-cell zygote with patch clamp techniques. Single channels were elicited by negative pressure (lower than ­15 cm$H_2O$). Interestingly, SACs were dependent on permeable cations such as $Na^+$ or $K^+$. As permeable cation removed from both sides across the membrane, SAC activity completely disappeared. When permeable cations present only in intracellular compartment, outward currents appeared at positive potentials. In contrast to this, inward currents occurred only at the negative voltage when permeable cation absent in cell interior. These result suggests that SAC carry cations through the nonselective cation channel (NSC channel). Taken together, we found that stretch activated channels present in hamster oocyte and the channel may carry cations through NSC channels. This stretch activated-NSC channels may play physiological role(s) in oocyte growth, maturation, fertilization and embryogenesis in fertilized oocytes to two-cell zygotes of hamster.

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.34 no.9
• /
• pp.1367-1374
• /
• 2005

Amino acid transporters play an important role in supplying nutrition to normal and cancer cells for cell proliferation. Amino acid transport system L is a major nutrient transport system responsible for the $Na^+$-independent transport of neutral amino acids including several essential amino acids. The system L is divided into two major subgroups, the L-tyre amino acid transporter 1 (LAT1) and the L-type amino acid transporter 2 (LAT2). In the present study, we have examined the expression and functional characterization of system L amino acid transporters in FOB human osteoblast cells. RT-PCR and western blot analysis have revealed that the FOB cells expressed LAT1, LAT2 together with their associating protein 4F2hc. The uptakes of $[^{14}C]_L$-leucine by FOB cells are $Na^+$-independent and almost completely inhibited by system L amino acid transporter selective inhibitor, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH). These results suggest that the transport of neutral amino acids including several essential amino acids for cellular nutrition into the FOB human osteoblast cells is mediated by system L amino acid transporters.