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

• ALGAE
• /
• 제36권4호
• /
• pp.315-326
• /
• 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.

• The Korean Journal of Physiology and Pharmacology
• /
• 제10권4호
• /
• pp.199-205
• /
• 2006

The functional expression of potassium $(K^+)$ channels has electrophysiologically been studied in bone cells from several species, however, their identity and regulation of gene expressions in bone cells are not well known. In the present study, to investigate how $K^+$ channel expressions are regulated by estrogen, we measured changes of transcript levels of various $Ca^{2+}$-activated ($K_{Ca}$) and ATP-sensitive $K^+$ channels in rat osteoblastic ROS 17/2.8 cells after treatment with estrogen. Application of 17${\beta}$-estradiol $(E_2)$ for 24 h and 48 h increased mRNA and protein expressions of inwardly rectifying $K^+$ channel (Kir) 6.2 and type 2 small conductance $K_{Ca}$ channel (SK2), respectively. Combined treatment of cells with 17${\beta}-E_2$ and ICI 182,780, a pure antiestrogen, suppressed 17${\beta}-E_2$-induced alterations of SK2 and Kir6.2 mRNA levels. In addition, treatment of cells with U0126, a specific inhibitor of extracellular receptor kinases (ERK)1/2, and SP600125, a specific inhibitor of c-jun N-terminal kinase (JNK) blocked the enhancing effects of 17${\beta}-E_2$ on SK2 and Kir6.2 protein expressions. On the other hand, blocking of p38 mitogen-activated protein kinase had no effect. Taken together, these results indicate that 17${\beta}-E_2$ modulates SK2 and Kir6.2 expressions through the estrogen receptor, involving ERK1/2 and JNK activations.

• The Korean Journal of Physiology
• /
• 제30권2호
• /
• pp.209-218
• /
• 1996

Acetylcholine (ACh) activates the inwardly rectifying muscarinic $K^{+}$ channel in rat atrial cells via pertussis toxin (PTX)-sensitive G-protein ($G_k$) coupled with the muscarinic receptor (mAChR). Although this $K^{+}\;(K_{ACh})$ channel function has reported to be modulated by the phosphorylation process, a kinase and phosphatase involved in these processes are still unclear. Since either PKA or PKC was not effective on this ATP-modulation, the present study examined the possible involvement of the protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP) in the function of the $K_{ACh}$ Channel. In the inside-out (I/O) patch preparation excised from the adult rat atrial cell, when activated by 10 ${\mu}M$ ACh in the pipette and 100 ${\mu}M$ GTP in the bath, the mean open time (${\tau}_{o}$) and the channel activity ($K_{ACh}$) was 1.13 ms (n=5) and 0.19 (n=6), respectively. Following the application of 1 mM ATP into the bath, ${\tau}_{o}$ increased by 34% (1.54 ms, n=5) and $K_{ACh}$ by 66% (0.28, n=6). Channel function elevated by ATP was lasted after washout of ATP. However, this ATP-induced increase in the $K_{ACh}$ channel function did not occur in pretreated cells with genistein ($50{\sim}100 {\mu}M$), a selective PTK inhibitor, but occurred in pretreated cells with equimolar daidzein, a negative control of the genistein. On the contrary, PTP which acts on tyrosine residue conversely reversed both ATP-induced increased ${\tau}_{o}$ by 32% (1.20 ms, n=3) and $K_{ACh}$ by 41% (0.15, n=3), respectively. Taken together, these results suggest that $K_{ACh}$ channel may, at least partly, be regulated by the tyrosyl phosphorylation, although it is unclear where this process exerts on the muscarinic signal transduction pathway comprising the mAChR-$G_{k}$-the $K_{ACh}$ channel.

• The Korean Journal of Physiology and Pharmacology
• /
• 제4권2호
• /
• pp.81-90
• /
• 2000

The types of $K^+$ channel which determine the pattern of spontaneous action potential (SAP) were investigated using whole-cell variation of patch clamp techniques under current- and voltage-clamp recording conditions in rat clonal pituitary $GH_3$ cells. Heterogeneous pattern of SAP activities was changed into more regular mode with elongation of activity duration and afterhyperpolarization by treatment of TEA (10 mM). Under this condition, exposure of the class III antiarrhythmic agent E-4031 $(5\;{\mu}M)$ to $GH_3$ cells hardly affected SAP activities. On the other hand, the main $GH_3$ stimulator thyrotropin-releasing hormone (TRH) still produced its dual effects (transient hyperpolarization and later increase in SAP frequency) in the presence of TEA. However, addition of $BaCl_2$ (2 mM) in the presence of TEA completely blocked SAP repolarization process and produced membrane depolarization in all tested cells. This effect was observed even in TEA-untreated cells and was not mimicked by higher concentration of TEA (30 mM). Also this barium-induced membrane depolarization effect was still observed after L-type $Ca^{2+}$ channel was blocked by nicardipine $(10\;{\mu}M).$ These results suggest that barium-sensitive current is important in SAP repolarization process and barium itself may have some depolarizing effect in $GH_3$ cells.

• The Korean Journal of Physiology and Pharmacology
• /
• 제14권3호
• /
• pp.145-150
• /
• 2010

Adenosine (Ado) is an important mediator of the endogenous defense against ischemia-induced injury in the heart. The action of Ado is mediated by activation of G protein-gated inwardly rectifying $K^+$ (GIRK) channels. In turn, GIRK channels are inhibited by reducing phosphatidylinositol 4,5-bisphosphate ($PIP_2$) through Gq protein-coupled receptors (GqPCRs). We previously found that GIRK channels activated by acetylcholine, a muscarinic M2 acetylcholine receptor agonist, are inhibited by GqPCRs in a receptor-specific manner. However, it is not known whether GIRK channels activated by Ado signaling are also regulated by GqPCRs. Presently, this was investigated in mouse atrial myocytes using the patch clamp technique. GIRK channels were activated by $100\;{\mu}M$ Ado. When Ado was repetitively applied at intervals of 5~6 min, the amplitude of second Ado-activated GIRK currents ($I_{K(Ado)}$) was $88.3{\pm}3.7%$ of the first $I_{K(Ado)}$ in the control. Pretreatment of atrial myocytes with phenylephrine, endothelin-1, or bradykinin prior to a second application of Ado reduced the amplitude of the second $I_{K(Ado)}$ to $25.5{\pm}11.6%$, $30.5{\pm}5.6%$, and $96.0{\pm}2.7%$, respectively. The potency of $I_{K(Ado)}$ inhibition by GqPCRs was different with that observed in acetylcholine-activated GIRK currents ($I_{K(ACh)}$) (endothelin-1>phenylephrine>bradykinin). $I_{K(Ado)}$ was almost completely inhibited by $500\;{\mu}M$ of the $PIP_2$ scavenger neomycin, suggesting low $PIP_2$ affinity of $I_{K(Ado)}$. Taken together, these results suggest that the crosstalk between GqPCRs and the Ado-induced signaling pathway is receptor-specific. The differential change in $PIP_2$ affinity of GIRK channels activated by Ado and ACh may underlie, at least in part, their differential responses to GqPCR agonists.

• The Korean Journal of Physiology and Pharmacology
• /
• 제8권2호
• /
• pp.69-76
• /
• 2004

A variety of G protein coupled receptors (GPCRs) are expressed in the presynaptic terminals of central and peripheral synapses and play regulatory roles in transmitter release. The patch-clamp whole-cell recording technique, applied to the calyx of Held presynaptic terminal in brainstem slices of rodents, has made it possible to directly examine intracellular mechanisms underlying the GPCR-mediated presynaptic inhibition. At the calyx of Held, bath-application of agonists for GPCRs such as $GABA_B$ receptors, group III metabotropic glutamate receptors (mGluRs), adenosine $A_1$ receptors, or adrenaline ${\alpha}2$ receptors, attenuate evoked transmitter release via inhibiting voltage-activated $Ca^{2+}$ currents without affecting voltage-activated $K^+$ currents or inwardly rectifying $K^+$ currents. Furthermore, inhibition of voltage-activated $Ca^{2+}$ currents fully explains the magnitude of GPCR-mediated presynaptic inhibition, indicating no essential involvement of exocytotic mechanisms in the downstream of $Ca^{2+}$ influx. Direct loadings of G protein ${\beta}{\gamma}$ subunit $(G{\beta}{\gamma})$ into the calyceal terminal mimic and occlude the inhibitory effect of a GPCR agonist on presynaptic $Ca^{2+}$ currents $(Ip_{Ca})$, suggesting that $G{\beta}{\gamma}$ mediates presynaptic inhibition by GPCRs. Among presynaptic GPCRs glutamate and adenosine autoreceptors play regulatory roles in transmitter release during early postnatal period when the release probability (p) is high, but these functions are lost concomitantly with a decrease in p during postnatal development.

• Asian-Australasian Journal of Animal Sciences
• /
• 제29권12호
• /
• pp.1675-1681
• /
• 2016

The aim of this study was to detect positional candidate genes located within the support interval (SI) regions based on the results of red blood cell, mean corpuscular volume (MCV), and mean corpuscular hemoglobin quantitative trait locus (QTL) in Sus scrofa chromosome 13, and to verify the correlation between specific single-nucleotide polymorphisms (SNPs) located in the exonic region of the positional candidate gene and the three genetic traits. The flanking markers of the three QTL SI regions are SW38 and S0215. Within the QTL SI regions, 44 genes were located, and runt-related transcription factor 1, dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A), and potassium inwardly-rectifying channel, subfamily J, member 15 KCNJ15-which are reported to be related to the hematological traits and clinical features of Down syndrome-were selected as positional candidate genes. The ten SNPs located in the exonic region of the three genes were detected by next generation sequencing. A total of 1,232 pigs of an $F_2$ resource population between Landrace and Korean native pigs were genotyped. To investigate the effects of the three genes on each genotype, a mixed-effect model which is the considering family structure model was used to evaluate the associations between the SNPs and three genetic traits in the $F_2$ intercross population. Among them, the MCV level was highly significant (nominal $p=9.8{\times}10^{-9}$) in association with the DYRK1A-SNP1 (c.2989 G$F_2$ intercross, our approach has limited power to distinguish one particular positional candidate gene from a QTL region.

• 고려인삼학회:학술대회논문집
• /
• 고려인삼학회 2002년도 학술대회지
• /
• pp.66-83
• /
• 2002

Recently, we have provided evidence that ginsenosides, the active components of Panax ginseng, utilize pertussis toxin (PTX)-insensitive $G{\alpha}_{q/11}-phospholipase\;C-{\beta}3(PLC-{\beta}3)$ signal transduction pathway for the enhancement of $Ca^{2+}-activated\;Cl^{-}$ current in the Xenopus oocyte (British J. Pharmacol. 132, 641-647, 2001; JBC 276, 48797-48802, 2001). Other investigators have shown that stimulation of receptors linked to $G{\alpha}-PLC$ pathway inhibits the activity of G proteincoupled inwardly rectifying $K^+$ (GIRK) channel. In the present study, we sought to determine whether ginsenosides influenced the activity of GIRK 1 and GIRK 4 (GIRK 1/4) channels expressed in the Xenopus oocyte, and if so, the underlying signal transduction mechanism. In oocyte injected with GIRK 1/4 channel cRNAs, bath-applied ginsenosides inhibited high potassium (HK) solution-elicited GIRK current $(EC_{50}:4.9{\pm}4.3\;{\mu}g/ml).$ Pretreatment of the oocyte with PTX reduced the HK solution-elicited GIRK current by $49\%,$ but it did not alter the inhibitory ginsenoside effect on GIRK current. Prior intraoocyte injection of cRNA(s) coding $G{\alpha}_q,\;G{\alpha}_{11}\;or\;G{\alpha}_q/G{\alpha}_{11},\;but\;not\;G{\alpha}_{i2}\;or\;G{\alpha}_{oA}$ attenuated the inhibitory ginsenoside effect. Injection of cRNAs coding $G{\beta}_{1{\gamma}2}$ also attenuated the ginsenoside effect. Similarly, injection of the cRNAs coding regulators of G protein signaling 1, 2 and 4 (RGS1, RGS2 and RGS4), which interact with $G{\alpha}_i\;and/or\;G{\alpha}_{q/11}$ and stimulates the hydrolysis of GTP to GDP in active GTP-bound $G{\alpha}$ subunit, resulted in a significant reduction of ginsenoside effect on GIRK current. Preincubation of GIRK channel-expressing oocyte in PLC inhibitor (U73122) or protein kinase C (PKC) inhibitor (staurosporine or chelerythrine) blocked the inhibitory ginsenoside effect on GIRK current. On the other hand, intraoocyte injection of BAPTA, a free $Ca^{2+}$ chelator, had no significant effect on the ginsenoside action. Taken together, these results suggest that ginsenosides inhibit the activity of GIRK 1/4 channel expressed in the Xenopus oocyte through a PTX-insensitive and $G{\alpha}_{q/11}$-,PLC-and PKC-mediated signal transduction pathway.

• Journal of Ginseng Research
• /
• 제23권4호
• /
• pp.235-238
• /
• 1999

[ $K_{ATP}$ ]채널은 세포내 ATP에 의해서 억제되는 포타슘 채널로서 혈관평활근, 골격근 및 체장의 ${\beta}$세포 막에 존재하여, 근세포의 막전압 조절을 통하여 근수축 및 이완을 조절할 뿐만 아니라 췌장의 ${\beta}$세포로부터 인슐린분비를 조절한다. 홍삼 복합사포닌 및 사포닌 $Rg_3$ 성분은 토끼 관상동맥 평활근세포의 칼슘의존성-포타슘채널$(BK_{Ca})$의 활성을 증가시켜 막전압의 과분극을 유발하여 혈관평활근을 이완시킨다. 사포닌 $Rg_3$성분은 홍삼의 복합사포닌 성분보다 $BK_{Ca}$에 더 높은 활성을 보이기 때문에 본 연구는 사포닌 $Rg_3$성분이 토끼 관상동맥 단일 평활근세포의 ${\beta}$채널의 활성도를 조절하는지를 팻치클램프 방법으로 기록하였다. 막전압 의존성과 함께 내향전류(inward rectification)특성을 보이는 ${\beta}$채널의 활성을 토끼 관상동맥 평활근 세포로부터 기록하였다. 이 ${\beta}$채널은 ATP와 giyburide에 의해서 억제되었으며 minoxidil에 의해서 활성이 증가되었다. 홍삼 사포닌 $Rg_3$성분은 $K_{ATP}$채널의 전류극대치에는 영향을 주지 않고 전류의 inactivation을 억제시켜 결과적으로 $K_{ATP}$채널의 활성을 증가시켰으며, 단일 KhTr채널이 열리는 시간도 증가시켰다. 따라서 본 실험 결과는 사포닌 $Rg_3$성분이 KATP채널의 활성을 증가시켜 막전압을 조절하여 관상동맥 평활근의 이완을 촉진한다고 여겨진다.