• 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.

• International Journal of Oral Biology
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• v.40 no.4
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• pp.211-216
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• 2015

Nitric Oxide (NO) is an important signaling molecule in the nociceptive process. Our previous study suggested that high concentrations of sodium nitroprusside (SNP), a NO donor, induce a membrane hyperpolarization and outward current through large conductances calcium-activated potassium ($BK_{ca}$) channels in substantia gelatinosa (SG) neurons. In this study, patch clamp recording in spinal slices was used to investigate the sources of $Ca^{2+}$ that induces $Ca^{2+}$-activated potassium currents. Application of SNP induced a membrane hyperpolarization, which was significantly inhibited by hemoglobin and 2-(4-carboxyphenyl) -4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (c-PTIO), NO scavengers. SNP-induced hyperpolarization was decreased in the presence of charybdotoxin, a selective $BK_{Ca}$ channel blocker. In addition, SNP-induced response was significantly blocked by pretreatment of thapsigargin which can remove $Ca^{2+}$ in endoplasmic reticulum, and decreased by pretreatment of dentrolene, a ryanodine receptors (RyR) blocker. These data suggested that NO induces a membrane hyperpolarization through $BK_{ca}$ channels, which are activated by intracellular $Ca^{2+}$ increase via activation of RyR of $Ca^{2+}$ stores.

• The Korean Journal of Physiology and Pharmacology
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• v.5 no.6
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• pp.511-519
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• 2001

Nimopidine, one of dihydropyridine derivatives, has been widely used to pharmacologically identify L-type Ca currents. In this study, it was tested if nimodipine is a selective blocker for L-type Ca currents in sensory neurons and heterologous system. In mouse dorsal root ganglion neurons (DRG), low concentrations of nimodipine $(<10\;{\mu}M),$ mainly targeting L-type Ca currents, blocked high-voltage-activated calcium channel currents by ${\sim}38%.$ Interestingly, high concentrations of nimodipine $(>10\;{\mu}M)$ further reduced the 'residual' currents in DRG neurons from ${\alpha}_{1E}$ knock-out mice, after blocking L-, N- and P/Q-type Ca currents with $10\;{\mu}M$ nimodipine, $1\;{\mu}M\;{\omega}-conotoxin$ GVIA and 200 nM ${\omega-agatoxin$ IVA, indicating inhibitory effects of nimodipine on R-type Ca currents. Nimodipine $(>10\;{\mu}M)$ also produced the inhibition of both low-voltage-activated calcium channel currents in DRG neurons and ${\alpha}_{1B}\;and\;{\alpha}_{1E}$ subunit based Ca channel currents in heterologous system. These results suggest that higher nimodipine $(>10\;{\mu}M)$ is not necessarily selective for L-type Ca currents. While care should be taken in using nimodipine for pharmacologically defining L-type Ca currents from native macroscopic Ca currents, nimodipine $(>10\;{\mu}M)$ could be a useful pharmacological tool for characterizing R-type Ca currents when combined with toxins blocking other types of Ca channels.

• Journal of Korean Neurosurgical Society
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• v.39 no.3
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• pp.215-220
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• 2006

Objective : Tyrosine kinase inhibitors may be useful in the management of cerebral vasospasm. It has not yet been reported whether L-type $Ca^{2+}$ channels playa role in tyrosine kinase inhibitors-induced vascular relaxation of cerebral artery. This study was undertaken to clarify the role of L-type $Ca^{2+}$ channels in tyrosine kinase inhibitors-induced vascular relaxation, and to investigate the effect of tyrosine kinase inhibitors on L-type $Ca^{2+}$ channels currents in freshly isolated smooth muscle cells from rat basilar artery. Methods : The isolation of rat basilar smooth muscle cells was performed by special techniques. The whole cell currents were recorded by whole cell patch clamp technique in freshly isolated smooth muscle cells from rat basilar artery. Results : Patch clamp studies revealed a whole-cell current which resembles the L-type $Ca^{2+}$ current reported by others. The amplitude of this current was decreased by nimodipine and increased by Bay K 8644. Genistein[n=5], tyrphostin A-23[n=3]. A-25[n=6] $30{\mu}M$ reduced the amplitude of the L -type $Ca^{2+}$ channel current in whole cell mode. In contrast, diadzein $30{\mu}M$ [n=3]. inactive analogue of genistein, did not decrease the amplitude of the L-type $Ca^{2+}$ channels current. Conclusion : These results suggest that tyrosine kinase inhibitors such as genistein, tyrphostin A-23, A-25 may relax cerebral vessel through decreasing level of intracellular calcium, [$Ca^{2+}$]i, by inhibition of L-type $Ca^{2+}$ channel.

• Biomolecules & Therapeutics
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• v.29 no.6
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• pp.630-636
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• 2021

Eupafolin, a constituent of the aerial parts of Phyla nodiflora, has neuroprotective property. Because reducing the synaptic release of glutamate is crucial to achieving pharmacotherapeutic effects of neuroprotectants, we investigated the effect of eupafolin on glutamate release in rat cerebrocortical synaptosomes and explored the possible mechanism. We discovered that eupafolin depressed 4-aminopyridine (4-AP)-induced glutamate release, and this phenomenon was prevented in the absence of extracellular calcium. Eupafolin inhibition of glutamate release from synaptic vesicles was confirmed through measurement of the release of the fluorescent dye FM 1-43. Eupafolin decreased 4-AP-induced [Ca²⁺]_i elevation and had no effect on synaptosomal membrane potential. The inhibition of P/Q-type Ca²⁺ channels reduced the decrease in glutamate release that was caused by eupafolin, and docking data revealed that eupafolin interacted with P/Q-type Ca²⁺ channels. Additionally, the inhibition of calcium/calmodulin-dependent protein kinase II (CaMKII) prevented the effect of eupafolin on evoked glutamate release. Eupafolin also reduced the 4-AP-induced activation of CaMK II and the subsequent phosphorylation of synapsin I, which is the main presynaptic target of CaMKII. Therefore, eupafolin suppresses P/Q-type Ca²⁺ channels and thereby inhibits CaMKII/synapsin I pathways and the release of glutamate from rat cerebrocortical synaptosomes.

• The Korean Journal of Physiology and Pharmacology
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• v.23 no.5
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• pp.381-392
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• 2019

Sperm function and male fertility are closely related to pH dependent $K^+$ current (KSper) in human sperm, which is most likely composed of Slo3 and its auxiliary subunit leucine-rich repeat-containing protein 52 (LRRC52). Onion peel extract (OPE) and its major active ingredient quercetin are widely used as fertility enhancers; however, the effect of OPE and quercetin on Slo3 has not been elucidated. The purpose of this study is to investigate the effect of quercetin on human Slo3 channels. Human Slo3 and LRRC52 were co-transfected into HEK293 cells and pharmacological properties were studied with the whole cell patch clamp technique. We successfully expressed and measured pH sensitive and calcium insensitive Slo3 currents in HEK293 cells. We found that OPE and its key ingredient quercetin inhibit Slo3 currents. Inhibition by quercetin is dose dependent and this degree of inhibition decreases with elevating internal alkalization and internal free calcium concentrations. Functional moieties in the quercetin polyphenolic ring govern the degree of inhibition of Slo3 by quercetin, and the composition of such functional moieties are sensitive to the pH of the medium. These results suggest that quercetin inhibits Slo3 in a pH and calcium dependent manner. Therefore, we surmise that quercetin induced depolarization in spermatozoa may enhance the voltage gated proton channel (Hv1), and activate non-selective cation channels of sperm (CatSper) dependent calcium influx to trigger sperm capacitation and acrosome reaction.

• The Korean Journal of Physiology and Pharmacology
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• v.25 no.1
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• pp.87-94
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• 2021

Skin photoaging occurs due to chronic exposure to solar ultraviolet radiation (UV), the main factor contributing to extrinsic skin aging. Clinical signs of photoaging include the formation of deep, coarse skin wrinkles and hyperpigmentation. Although melanogenesis and skin wrinkling occur in different skin cells and have different underlying mechanisms, their initiation involves intracellular calcium signaling via calcium ion channels. The ORAI1 channel initiates melanogenesis in melanocytes, and the TRPV1 channel initiates MMP-1 production in keratinocytes in response to UV stimulation. We aimed to develop a drug that may simultaneously inhibit ORAI1 and TRPV1 activity to help prevent photoaging. We synthesized nootkatol, a chemical derivative of valencene. TRPV1 and ORAI1 activities were measured using the whole-cell patch-clamp technique. Intracellular calcium concentration [Ca2+]i was measured using calcium-sensitive fluorescent dye (Fura-2 AM). UV-induced melanin formation and MMP-1 production were quantified in B16F10 melanoma cells and HaCaT cells, respectively. Our results indicate that nootkatol (90 μM) reduced TRPV1 current by 94% ± 2% at -60 mV and ORAI1 current by 97% ± 1% at -120 mV. Intracellular calcium signaling was significantly inhibited by nootkatol in response to ORAI1 activation in human primary melanocytes (51.6% ± 0.98% at 100 μM). Additionally, UV-induced melanin synthesis was reduced by 76.38% ± 5.90% in B16F10 melanoma cells, and UV-induced MMP-1 production was reduced by 59.33% ± 1.49% in HaCaT cells. In conclusion, nootkatol inhibits both TRPV1 and ORAI1 to prevent photoaging, and targeting ion channels may be a promising strategy for preventing photoaging.

• The Korean Journal of Physiology and Pharmacology
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• v.4 no.1
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• pp.1-8
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• 2000

To investigate the contributions of intrinsic membrane properties to the spontaneous activity of medial vestibular nucleus (MVN) neurons, we assessed the effects of blocking large and small calcium-activated potassium channels by means of patch clamp recordings. Almost all the MVN neurons recorded in neonatal $(P13{\sim}P17)$ rat were shown to have either a single deep after-hyperpolarization (AHP; type A cells), or an early fast and a delayed slow AHP (type B cells). Among the recorded MVN cells, immature action potential shapes were found. Immature type A cell showed single uniform AHP and immature B cell showed a lack of the early fast AHP, and the delayed AHP was separated from the repolarization phase of the spike by a period of isopotentiality. Application of apamin and charybdotoxin (CTX), which selectively block the small and large calcium-activated potassium channels, respectively, resulted in significant changes in spontaneous firings. In both type A and type B cells, CTX (20 nM) resulted in a significant increase in spike frequency but did not induce bursting activity. By contrast, apamin (300 nM) selectively abolished the delayed slow AHP and induced bursting activity in type B cells. Apamin had no effect on the spike frequency of type A cells. These data suggest that there are differential roles of apamin and CTX sensitive potassium conductances in spontaneous firing patterns of MVN neurons, and these conductances are important in regulating the intrinsic rhythmicity and excitability.

• Journal of Biomedical Engineering Research
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• v.43 no.1
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• pp.61-70
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• 2022

Natural and synthetic forms of calcium phosphate cement (CPC) have been widely used in bone repair and augmentation. The major challenge of injectable CPC is to deliver the cells without cell death in order to regenerate new bone. The study objective was to investigate for the potential of stem cell-laden gelatin fibers containing injectable, nanocrystalline CPC to function as a delivery system. Gelatin noddle fiber method was developed to delivered cells into nCPC. Experimental groups were prepared by mixing cells with nCPC, mixing cell-laden gelatin fibers with nCPC and mixing cell-laden gelatin fibers containing BMP-2 with nCPC. Media diffusion test was conducted after dissolving the gelatin fibers. SEM examined the generated channels and delivered cell morphology. Fibers mixed with nCPC showed physical setting and hardening within 20 min after injection and showed good shape maintenances. The gelatin fibers mixed nCPC group had several vacant channels generated from the dissolved gelatin. Particularly, proliferation and attachment of the cells were observed inside of the channels. While live cells were not observed in the cell mixed nCPC group, cells delivered with the gelatin fibers into the nCPC showed good viability and increased DNA content with culture. Cell-laden gelatin fiber was a novel method for cell delivery into nCPC without cell damages. Results also indicated the osteogenic differentiation of gelatin fiber delivered cells. We suggest that the cell-laden gelatin fibers mixed with nCPC can be used as an injectable cell delivery vehicle and the addition of BMP-2 to enhances osteogenesis.

• Proceedings of the Korean Biophysical Society Conference
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• 1996.07a
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• pp.35-35
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• 1996

Nitric oxide (NO) has been reported to have many roles in vivo ranging from the neurotransmitter in brain to the relaxant in smooth muscles. Recently, using inside-out patches, Bolotina et al. (1) showed that relaxing effect of NO is aortic smooth muscle is through direct activation of Ca2+-activated K+ channels (maxi-K), resulting in hyperpolarization. (omitted)