• Archives of Pharmacal Research
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• v.27 no.3
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• pp.305-313
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• 2004

The effect of diazoxide, a $K^{+}$channel opener, on apoptotic cell death was investigated in HepG2 human hepatoblastoma cells. Diazoxide induced apoptosis in a dose-dependent manner and this was evaluated by flow cytometric assays of annexin-V binding and hypodiploid nuclei stained with propidium iodide. Diazoxide did not alter intracellular $K^{+}$concentration, and various inhibitors of $K^{+}$channels had no influence on the diazoxide-induced apoptosis; this implies that $K^{+}$channels activated by diazoxide may be absent in the HepG2 cells. However, diazoxide induced a rapid and sustained increase in intracellular $Ca^{2+}$ concentration, and this was completely inhibited by the extracellular $Ca^{2+}$ chelation with EGTA, but not by blockers of intracellular $Ca^{2+}$ release (dantrolene and TMB-8). This result indicated that the diazoxide-induced increase of intracellular $Ca^{2+}$ might be due to the activation of a Ca2+ influx pathway. Diazoxide-induced $Ca^{2+}$ influx was not significantly inhibited by either voltage-operative $Ca^{2+}$ channel blockers (nifedipinen or verapamil), or by inhibitors of $Na^{+}$, $Ca^{2+}$-exchanger (bepridil and benzamil), but it was inhibited by flufenamic acid (FA), a $Ca^{2+}$-permeable nonselective cation channel blocker. A quantitative analysis of apoptosis by flow cytometry revealed that a treatment with either FA or BAPTA, an intracellular $Ca^{2+}$ chelator, significantly inhibited the diazoxide-induced apoptosis. Taken together, these results suggest that the observed diazoxide-induced apoptosis in the HepG2 cells may result from a $Ca^{2+}$ influx through the activation of $Ca^{2+}$-permeable non-selective cation channels. These results are very significant, and they lead us to further suggest that diazoxide may be valuable for the therapeutic intervention of human hepatomas.

• The Korean Journal of Physiology and Pharmacology
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• v.11 no.5
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• pp.227-231
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• 2007

The classic type of transient receptor potential channel(TRPC) is a molecular candidate for $Ca^{2+}$-permeable cation channel in mammalian cells. TRPC5 is rapidly desensitized after activation by G protein-coupled receptor. Herein we report the effect of dimethyl sulfoxide(DMSO) on the desensitization of TRPC5. TRPC5 was initially activated by muscarinic stimulation with $50{\mu}M$ carbachol(CCh) and then decayed rapidly even in the presence of CCh(desensitization). DMSO in the pipette solution slowed the rate of this desensitization. Under the control conditions, TRPC5 current spontaneously declined to $6{\pm}1%$ of the initial peak amplitude 60 sec after CCh application and to $1{\pm}0.5%$ after 120 sec. But, in the presence of 0.01%, 0.1% and 1% DMSO, TRPC5 current spontaneously declined to $55{\pm}2%,\;68{\pm}1%\;and\;100{\pm}0.2%$ of the initial peak amplitude 60 sec after CCh application and to $38{\pm}2%,\;61{\pm}1%\;and\;100{\pm}1%$ after 120 see, respectively. The results suggest that DMSO can internally attenuate the desensitization of TRPC5 current through unknown mechanisms that remain to be elucidated.

• The Korean Journal of Physiology and Pharmacology
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• v.23 no.3
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• pp.219-227
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• 2019

Polycystic kidney disease 2-like-1 (PKD2L1), polycystin-L or transient receptor potential polycystin 3 (TRPP3) is a TRP superfamily member. It is a calcium-permeable non-selective cation channel that regulates intracellular calcium concentration and thereby calcium signaling. Although the calmodulin (CaM) inhibitor, calmidazolium, is an activator of the PKD2L1 channel, the activating mechanism remains unclear. The purpose of this study is to clarify whether CaM takes part in the regulation of the PKD2L1 channel, and if so, how. With patch clamp techniques, we observed the current amplitudes of PKD2L1 significantly reduced when co-expressed with CaM and $CaM{\triangle}N$. This result suggests that the N-lobe of CaM carries a more crucial role in regulating PKD2L1 and guides us into our next question on the different functions of two lobes of CaM. We also identified the predicted CaM binding site, and generated deletion and truncation mutants. The mutants showed significant reduction in currents losing PKD2L1 current-voltage curve, suggesting that the C-terminal region from 590 to 600 is crucial for maintaining the functionality of the PKD2L1 channel. With PKD2L1608Stop mutant showing increased current amplitudes, we further examined the functional importance of EF-hand domain. Along with co-expression of CaM, ${\triangle}EF$-hand mutant also showed significant changes in current amplitudes and potentiation time. Our findings suggest that there is a constitutive inhibition of EF-hand and binding of CaM C-lobe on the channel in low calcium concentration. At higher calcium concentration, calcium ions occupy the N-lobe as well as the EF-hand domain, allowing the two to compete to bind to the channel.

• International Journal of Oral Biology
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• v.43 no.1
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• pp.23-27
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• 2018

Increased intracellular levels of $Ca^{2+}$ are generally thought to negatively regulate lipolysis in mature adipocytes, whereas store-operated $Ca^{2+}$ entry was recently reported to facilitate lipolysis and attenuate lipotoxicity by inducing lipophagy. Transient receptor potential mucolipin1 (TRPML1), a $Ca^{2+}$-permeable non-selective cation channel, is mainly expressed on the lysosomal membrane and plays key roles in lysosomal homeostasis and membrane trafficking. However, the roles of TRPML1 in lipolysis remains unclear. In this study, we examined whether the channel function of TRPML1 induces lipolysis in mature adipocytes. We found that treatment of mature adipocytes with ML-SA1, a specific agonist of TRPML1, solely upregulated extracellular glycerol release, but not to the same extent as isoproterenol. In addition, knockdown of TRPML1 in mature adipocytes significantly reduced autophagic flux, regardless of ML-SA1 treatment. Our findings demonstrate that the channel function of TRPML1 partially contributes to lipid metabolism and autophagic membrane trafficking, suggesting that TRPML1, particularly the channel function of TRPML1, is as therapeutic target molecule for treating obesity.

• ALGAE
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• v.36 no.4
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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.

• The Korean Journal of Physiology and Pharmacology
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• v.27 no.2
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• pp.187-196
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• 2023

Transient receptor potential canonical (TRPC) channels are non-selective calcium-permeable cation channels. It is suggested that TRPC4β is regulated by phospholipase C (PLC) signaling and is especially maintained by phosphatidylinositol 4,5-bisphosphate (PIP₂). In this study, we present the regulation mechanism of the TRPC4 channel with PIP₂ hydrolysis which is mediated by a channel-bound PLCδ1 but not by the G_qPCR signaling pathway. Our electrophysiological recordings demonstrate that the Ca²⁺ via an open TRPC4 channel activates PLCδ1 in the physiological range, and it causes the decrease of current amplitude. The existence of PLCδ1 accelerated PIP₂ depletion when the channel was activated by an agonist. Interestingly, PLCδ1 mutants which have lost the ability to regulate PIP₂ level failed to reduce the TRPC4 current amplitude. Our results demonstrate that TRPC4 self-regulates its activity by allowing Ca²⁺ ions into the cell and promoting the PIP₂ hydrolyzing activity of PLCδ1.

• The Korean Journal of Physiology and Pharmacology
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• v.13 no.1
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• pp.27-32
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• 2009

The effects of oxidized low-density lipoprotein(OxLDL) and its major lipid constituent lysophosphatidylcholine(LPC) on $Ca^{2+}$ entry were investigated in cultured human umbilical endothelial cells(HUVECs) using fura-2 fluorescence and patch-clamp methods. OxLDL or LPC increased intracellular $Ca^{2+}$ concentration($[Ca^{2+}]_i$), and the increase of $[Ca^{2+}]_i$ by OxLDL or by LPC was inhibited by $La^{3+}$ or heparin. LPC failed to increase $[Ca^{2+}]_i$ in the presence of an antioxidant tempol. In addition, store-operated $Ca^{2+}$ entry(SOC), which was evoked by intracellular $Ca^{2+}$ store depletion in $Ca^{2+}$-free solution using the sarcoplasmic reticulum $Ca^{2+}$ pump blocker, 2, 5-di-t-butyl-l,4-benzohydroquinone(BHQ), was further enhanced by OxLDL or by LPC. Increased SOC by OxLDL or by LPC was inhibited by U73122. In voltage-clamped cells, OxLDL or LPC increased $[Ca^{2+}]_i$ and simultaneously activated non-selective cation(NSC) currents. LPC-induced NSC currents were inhibited by 2-APB, $La^{3+}$ or U73122, and NSC currents were not activated by LPC in the presence of tempol. Furthermore, in voltage-clamped HUVECs, OxLDL enhanced SOC and evoked outward currents simultaneously. Clamping intracellular $Ca^{2+}$ to 1 ${\mu}M$ activated large-conductance $Ca^{2+}$-activated $K^+(BK_{ca})$ current spontaneously, and this activated $BK_{ca}$ current was further enhanced by OxLDL or by LPC. From these results, we concluded that OxLDL or its main component LPC activates $Ca^{2+}$-permeable $Ca^{2+}$-activated NSC current and $BK_{ca}$ current simultaneously, thereby increasing SOC.

• The Korean Journal of Physiology and Pharmacology
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• v.24 no.4
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• pp.287-298
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• 2020

Ca²⁺ signaling of endothelial cells plays a critical role in controlling blood flow and pressure in small arteries and arterioles. As the impairment of endothelial function is closely associated with cardiovascular diseases (e.g., atherosclerosis, stroke, and hypertension), endothelial Ca²⁺ signaling mechanisms have received substantial attention. Increases in endothelial intracellular Ca²⁺ concentrations promote the synthesis and release of endothelial-derived hyperpolarizing factors (EDHFs, e.g., nitric oxide, prostacyclin, or K⁺ efflux) or directly result in endothelial-dependent hyperpolarization (EDH). These physiological alterations modulate vascular contractility and cause marked vasodilation in resistance arteries. Transient receptor potential (TRP) channels are nonselective cation channels that are present in the endothelium, vascular smooth muscle cells, or perivascular/sensory nerves. TRP channels are activated by diverse stimuli and are considered key biological apparatuses for the Ca²⁺ influx-dependent regulation of vasomotor reactivity in resistance arteries. Ca²⁺-permeable TRP channels, which are primarily found at spatially restricted microdomains in endothelial cells (e.g., myoendothelial projections), have a large unitary or binary conductance and contribute to EDHFs or EDH-induced vasodilation in concert with the activation of intermediate/small conductance Ca²⁺-sensitive K⁺ channels. It is likely that endothelial TRP channel dysfunction is related to the dysregulation of endothelial Ca²⁺ signaling and in turn gives rise to vascular-related diseases such as hypertension. Thus, investigations on the role of Ca²⁺ dynamics via TRP channels in endothelial cells are required to further comprehend how vascular tone or perfusion pressure are regulated in normal and pathophysiological conditions.

• Molecules and Cells
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• v.27 no.2
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• pp.167-173
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• 2009

The classical type of transient receptor potential (TRPC) channel is a molecular candidate for $Ca^{2+}$-permeable cation channels in mammalian cells. Because TRPC4 and TRPC5 belong to the same subfamily of TRPC, they have been assumed to have the same physiological properties. However, we found that TRPC4 had its own functional characteristics different from those of TRPC5. TRPC4 channels had no constitutive activity and were activated by muscarinic stimulation only when a muscarinic receptor was co-expressed with TRPC4 in human embryonic kidney (HEK) cells. Endogenous muscarinic receptor appeared not to interact with TRPC4. TPRC4 activation by $GTP{\gamma}S$ was not desensitized. TPRC4 activation by $GTP{\gamma}S$ was not inhibited by either Rho kinase inhibitor or MLCK inhibitor. TRPC4 was sensitive to external pH with $pK_a$ of 7.3. Finally, TPRC4 activation by $GTP{\gamma}S$ was inhibited by the calmodulin inhibitor W-7. We conclude that TRPC4 and TRPC5 have different properties and their own physiological roles.

• The Korean Journal of Physiology and Pharmacology
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• v.14 no.6
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• pp.419-425
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• 2010

Mast cells are activated by specific allergens and also by various nonspecific stimuli, which might induce physical urticaria. This study investigated the functional expression of temperature sensitive transient receptor potential vanilloid (TRPV) subfamily in the human mast cell line (HMC-1) using whole-cell patch clamp techniques. The temperature of perfusate was raised from room temperature (RT, $23{\sim}25^{\circ}C$) to a moderately high temperature (MHT, $37{\sim}39^{\circ}C$) to activate TRPV3/4, a high temperature (HT, $44{\sim}46^{\circ}C$) to activate TRPV1, or a very high temperature (VHT, $53{\sim}55^{\circ}C$) to activate TRPV2. The membrane conductance of HMC-1 was increased by MHT and HT in about 50% (21 of 40) of the tested cells, and the I/V curves showed weak outward rectification. VHT-induced current was 10-fold larger than those induced by MHT and HT. The application of the TRPV 4 activator $3{\alpha}$-phorbol 12,13-didecanoate ($4{\alpha}$ PDD, $1\;{\mu}M$) induced weakly outward rectifying currents similar to those induced by MHT. However, the TRPV3 agonist camphor or TRPV1 agonist capsaicin had no effect. RT-PCR analysis of HMC-1 demonstrated the expression of TRPV4 as well as potent expression of TRPV2. The $[Ca^{2+}]_c$ of HMC-1 cells was also increased by MHT or by $4{\alpha}$ PDD. In summary, our present study indicates that HMC-1 cells express $Ca^{2+}$-permeable TRPV4 channels in addition to the previously reported expression of TRPV2 with a higher threshold of activating temperature.