• Animal cells and systems
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• 제2권4호
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• pp.471-476
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• 1998

The G protein-activated inwardly rectifying $K^＋$ channel (GIRK1) was coex-pressed in Xenopus oocytes along with the $5-HT_{1A}$ receptor, a 7-helix receptor known to be coupled to $K^＋$ channels in many neural tissues. Thus, the activation of the $5-HT_{1A}$ receptor by its agonist leads to the opening of GIRK1. The GIRK1 current was measured using the two electrode voltage clamp technique with bath application of 5-HT in the presence of various external potassium concentrations $[K^＋]_0$. GIRK1 showed a strong inward rectification since only hyperpolarizing voltages evoked inward currents. $K^{+}$ was the major ion carrier as evidenced by about 44㎷ voltage shift corresponding to a 10-fold external 〔$K^＋$〕 change. 5-HT induced a concentration-dependent inward $K^＋$ current ($EC_{50}{\equation omitted}10.7nM$) which was blocked by $Ba^{2＋}$. Pertussis toxin (PTX) pre-treatment reduced the $K^＋$ current by as much as about 70%, suggesting that PTX-sensitive G protein ($G_i or G_o$ type) are involved in the $5-HT_{1A}$ receptor-GIRK1 coupling in Xenopus oocytes.

• The Korean Journal of Physiology
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• 제19권2호
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• pp.101-111
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• 1985

Transient inward current (TI) was studied by the two micro-electrode voltage clamp technique in the sinoatrial node of the rabbit. The author confirmed that in $10^{-6}$ M ouabain TI was found in the SA node and investigated the effects of ions, $(Na^+,\;K^+,\;Ca^{2+})$, $\beta-agonist$ (isoprenaline), local anesthetics (quinidine, lidocaine) and Ca-blockers ($Co^{2+}$, verapamil, diltiazem) on the TI recorded during depolarizing voltage clamp pulses to -40 and -20 mV. The results obtained were as follows ; 1) $10^{-6}M$ ouabain increased the frequency of sinus action potential and decreased the amplitude, especially overshoot of action potential. TI was induced by the depolarizing voltage clamp Pulses and the magnitude of the slow inward current (isi) decreased and the time course was slowed by the same depolarizing pulses. 2) 30% $Na^{+}$ and 24mM $K^+$ decreased by $10^{-6}M$ ouabain and 6 mM $Ca^{2+}$ and $10^{-7}M$ isoprenaline increased TI, $i_{si}$ and current oscillations. 3) Quinidine $(5\times10^{-7}M)$ reduced TI and $i_{si}$ but lidocaine $(10^6\;-10^5M)$ didn't reduced or increase TI. Current oscillations increased and isi decreased by lidocaine. 4) Ca-blockers decreased the amplitude and the frequency of sinus action potential. TI and $i_{si}$ decreased significantly but were not abolished completely at the concentrations used in this experiment. Verapamil and diltiazem had inhibitory action on TI in $2\times10^{-7}M$ concentration and showed very slow recovery after wasting out with normal Tyrode solution.

• 대한약학회:학술대회논문집
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• 대한약학회 2003년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2-2
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• pp.178.3-178.3
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• 2003

P2X receptors are ligand gated cation channels activated by the binding of extracellular adenosine 5'-triphosphate (ATP) and classified into 7 subtype families. $P2X_1$ receptors are abundantly expressed in smooth muscle mediates blood vessel and mediate constriction upon binding of neuronal ATP. The activation of $P2X_3$ receptor by ATP has been known to initiate the pain signaling in the peripheral nervous system, which is involved in chronic inflammatory nociception and neuropathic pain by nerve injury. (omitted)

• Journal of Microbiology and Biotechnology
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• 제33권2호
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• pp.203-210
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• 2023

Taste is classified into five types, each of which has evolved to play its respective role in mammalian survival. Sour taste is one of the important ways to judge whether food has gone bad, and the sour taste receptor (PKD2L1) is the gene behind it. Here, we investigated whether ʟ-pyroglutamic acid interacts with sour taste receptors through electrophysiology and mutation experiments using Xenopus oocytes. R299 of hPKD2L1 was revealed to be involved in ʟ-pyroglutamic acid binding in a concentration-dependent manner. As a result, it is possible to objectify the change in signal intensity according to the concentration of ʟ-pyroglutamic acid, an active ingredient involved in the taste of kimchi, at the molecular level. Since the taste of other ingredients can also be measured with the method used in this experiment, it is expected that an objective database of taste can be created.

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 2003년도 정기총회 및 학술발표회
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• pp.55-55
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• 2003

P2X$_3$ receptor, a member of P2 purine receptors, is a ligand-gated ion channel activated by extracellular ATP as an endogenous ligand, and highly localized in peripheral and central sensory neurons. The activation of P2X3 receptor by ATP as the pronociceptive effect has been known to initiate the pain signaling involved in chronic inflammatory nociception and neuropathic pain by nerve injury, implicating the possibility of new drug development to control pains. In this study, we have developed a two electrode voltage clamp (TEVC) assay system to evaluate the inhibitory activity of several newly synthesized PPADS and a novel non-ionic antagonist against ATP activation of human P2X3 receptor. PPADS derivatives include several pyridoxine and pyridoxic acid analogs to study the effects of phosphate and aldehyde functional groups in PPADS. All new PPADS analogs were less potent than PPADS at human P2X$_3$ receptors, however, LDD130, a non-ionic analog showed potent antagonistic property with $IC_{50}$/ of 8.34 pM. In order to uncover the structure activity relationships of LDD130, and design new structural analogs, we synthesized and investigated a few structural variants of LDD130, and the results will be discussed in this presentation.

• Archives of Pharmacal Research
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• 제26권1호
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• pp.28-33
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• 2003

Ginsenosides, major active ingredients of Panax ginseng, are known to regulate excitatory ligand-gated ion channel activity such as nicotinic acetylcholine and NMDA receptor channel activity. However, it is not known whether ginsenosides affect inhibitory ligand-gated ion channel activity. We investigated the effect of ginsenosides on human recombinant $GABA_A$ receptor (${\alpha}_1{\beta}_1{\gamma}_{2s}$) channel activity expressed in Xenopus oocytes using a two-electrode voltage-clamp technique. Among the eight individual ginsenosides examined, namely, $Rb_1$, $Rb_2$, Rc, Rd, Re, Rf, $Rg_1$ and $Rg_2$, we found that Rc most potently enhanced the GABA-induced inward peak current ($I_{GABA}$). Ginsenoside Rc alone induced an inward membrane current in certain batches of oocytes expressing the $GABA_A$ receptor. The effect of ginsenoside Rc on $I_{GABA}$ was both dose-dependent and reversible. The half-stimulatory concentration ($EC_{50}$) of ginsenoside Rc was 53.2$\pm$12.3 $\mu$M. Both bicuculline, a $GABA_A$ receptor antagonist, and picrotoxin, a $GABA_A$ channel blocker, blocked the stimulatory effect of ginsenoside Rc on $I_{GABA}$. Niflumic acid (NFA) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), both $CI^{-1}$ channel blockers, attenuated the effect of ginsenoside Rc on I$I_{GABA}$. This study suggests that ginsenosides regulated $GABA_A$ receptor expressed in Xenopus oocytes and implies that this regulation might be one of the pharmacological actions of Panax ginseng.

• Molecules and Cells
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• 제20권1호
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• pp.69-73
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• 2005

The flavonoid, quercetin, is a low molecular weight substance found in apple, tomato and other fruit. Besides its antioxidative effect, quercetin, like other flavonoids, has a wide range of neuropharmacological actions including analgesia, and motility, sleep, anticonvulsant, sedative and anxiolytic effects. In the present study, we investigated its effect on mouse 5-hydroxytryptamine type 3 ($5-HT_{3A}$) receptor channel activity, which is involved in pain transmission, analgesia, vomiting, and mood disorders. The $5-HT_{3A}$ receptor was expressed in Xenopus oocytes, and the current was measured with the two-electrode voltage clamp technique. In oocytes injected with $5-HT_{3A}$ receptor cRNA, quercetin inhibited the 5-HT-induced inward peak current ($I_{5-HT}$) with an $IC_{50}$ of $64.7{\pm}2.2{\mu}M$. Inhibition was competitive and voltage-independent. Point mutations of pre-transmembrane domain 1 (pre-TM1) such as R222T and R222A, but not R222D, R222E and R222K, abolished inhibition, indicating that quercetin interacts with the pre-TM1 of the $5-HT_{3A}$ receptor.

• Biomolecules & Therapeutics
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• 제24권4호
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• pp.410-417
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• 2016

Quercetin is a flavonoid usually found in fruits and vegetables. Aside from its antioxidative effects, quercetin, like other flavonoids, has a various neuropharmacological actions. Quercetin-3-O-rhamnoside (Rham1), quercetin-3-O-rutinoside (Rutin), and quercetin-3-(2(G)-rhamnosylrutinoside (Rham2) are mono-, di-, and tri-glycosylated forms of quercetin, respectively. In a previous study, we showed that quercetin can enhance ${\alpha}7$ nicotinic acetylcholine receptor (${\alpha}7$ nAChR)-mediated ion currents. However, the role of the carbohydrates attached to quercetin in the regulation of ${\alpha}7$ nAChR channel activity has not been determined. In the present study, we investigated the effects of quercetin glycosides on the acetylcholine induced peak inward current ($I_{ACh}$) in Xenopus oocytes expressing the ${\alpha}7$ nAChR. $I_{ACh}$ was measured with a two-electrode voltage clamp technique. In oocytes injected with ${\alpha}7$ nAChR copy RNA, quercetin enhanced $I_{ACh}$, whereas quercetin glycosides inhibited $I_{ACh}$. Quercetin glycosides mediated an inhibition of $I_{ACh}$, which increased when they were pre-applied and the inhibitory effects were concentration dependent. The order of $I_{ACh}$ inhibition by quercetin glycosides was Rutin${\geq}$Rham1>Rham2. Quercetin glycosides-mediated $I_{ACh}$ enhancement was not affected by ACh concentration and appeared voltage-independent. Furthermore, quercetin-mediated $I_{ACh}$ inhibition can be attenuated when quercetin is co-applied with Rham1 and Rutin, indicating that quercetin glycosides could interfere with quercetin-mediated ${\alpha}7$ nAChR regulation and that the number of carbohydrates in the quercetin glycoside plays a key role in the interruption of quercetin action. These results show that quercetin and quercetin glycosides regulate the ${\alpha}7$ nAChR in a differential manner.

• Molecules and Cells
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• 제19권1호
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• pp.137-142
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• 2005

We showed recently that ginsenosides inhibit the activity of various types of ion channel. Here we have investigated the role of the carbohydrate component of ginsenoside $Rg_3$ in the inhibition of $Na^+$ channels. The channels were expressed in Xenopus oocytes by injecting cRNAs encoding rat brain Nav1.2 ${\alpha}$ and ${\beta}1$ subunits, and analyzed by the two-electrode voltage clamp technique. Treatment with $Rg_3$ reversibly inhibited the inward $Na^+$ peak current ($I_{Na}$) with an $IC_{50}$ of $32.2{\pm}4.5{\mu}M$, and the inhibition was voltage-dependent. To examine the role of the sugar moiety, we prepared a straight chain form of the second glucose and a conjugate of this glucose with 3-(4-hydroxyphenyl) propionic acid hydrazide (HPPH). Neither derivative inhibited $I_{Na}$. Treatment with the carbohydrate portion of ginsenoside $Rg_3$, sophorose [${\beta}-D-glucopyranosyl$ ($1{\rightarrow}2$)-${\beta}-glucopyranoside$], or the aglycone (protopanaxadiol), on their own or in combination had no effect on $I_{Na}$. These observations indicate that the carbohydrate portion of ginsenoside $Rg_3$ plays an important role in its effect on the $Na^+$ channel.

• Journal of Ginseng Research
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• 제24권4호
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• pp.168-175
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• 2000

Xenopus oocytes를 이용하여 인삼의 유효 성분으로 알려진 Ginseng total saponin(GTS)의 신호 전달 기작을 two electrode voltage clamp 방법을 이용하여 연구하였다. GTS는 세포 바깥에 처리했을 때 -2OmV보다 더 positive한 voltage에서 커다란 outward current를 유도하였다. 그러나, 세포 안쪽에 GTS를 injection할 경우 아무런 효과가 없는 것으로 나타났다. GTS처리에 의한 outward current유발 효과는 GTS 투여 용량에 의존적인 것으로 나타났다(EC$_{50}$ : 4.4 $\mu\textrm{g}$/ml). GTS의 작용은 $Ca^{2+}$-activated Cl- channel blocker인 niflumic acid에 의하여 차단되었다. 칼슘 chelator인 BAPIA와 IP$_3$ 수용체 길항제인 heparin을 세포내 injection에 의하여 차단되었다. 또한 active phospholipase C inhibitor(PLC)인U-73122를 세포 바깥에 전처리할 경우에도 GTS의 작용이 부분적으로 억제되는 것으로 나타났다. 백일해 독소를 전처리할 경우GTS의 작용은 억제되지 않은 것으로 나타났으나, GTP analog인 GTP${\gamma}$S를 세포내 injection할 경우 GTS의 작용은 억제되는 것으로 나타났다. 이러한 연구 결과는 GTS가 oocytes세포막 성분과 상호 작용에 의하여 $Ca^{2+}$-activated Cl- channel이 열리도록 하고, 이 과정에 PLC활성 및 백일해 독소에 민감하지 않은 G단백질활성 및 IP3에 민감한 세포내 $Ca^{2+}$-activated로부터 칼슘 방출을 유도하는 것으로 나타났다났다