• The Korean Journal of Physiology and Pharmacology
• /
• v.27 no.4
• /
• pp.417-426
• /
• 2023

The TRPM4 gene encodes a Ca²⁺-activated monovalent cation channel called transient receptor potential melastatin 4 (TRPM4) that is expressed in various tissues. Dysregulation or abnormal expression of TRPM4 has been linked to a range of diseases. We introduced the hemagglutinin (HA) tag into the extracellular S6 loop of TRPM4, resulting in an HA-tagged version called TRPM4-HA. This TRPM4-HA was developed to investigate the purification, localization, and function of TRPM4 in different physiological and pathological conditions. TRPM4-HA was successfully expressed in the intact cell membrane and exhibited similar electrophysiological properties, such as the current-voltage relationship, rapid desensitization, and current size, compared to the wild-type TRPM4. The presence of the TRPM4 inhibitor 9-phenanthrol did not affect these properties. Furthermore, a wound-healing assay showed that TRPM4-HA induced cell proliferation and migration, similar to the native TRPM4. Co-expression of protein tyrosine phosphatase, non-receptor type 6 (PTPN6 or SHP1) with TRPM4-HA led to the translocation of TRPM4-HA to the cytosol. To investigate the interaction between PTPN6 and tyrosine residues of TRPM4 in enhancing channel activity, we generated four mutants in which tyrosine (Y) residues were substituted with phenylalanine (F) at the N-terminus of TRPM4. The YF mutants displayed properties and functions similar to TRPM4-HA, except for the Y256F mutant, which showed resistance to 9-phenanthrol, suggesting that Y256 may be involved in the binding site for 9-phenanthrol. Overall, the creation of HA-tagged TRPM4 provides researchers with a valuable tool to study the role of TRPM4 in different conditions and its potential interactions with other proteins, such as PTPN6.

• International Journal of Oral Biology
• /
• v.42 no.4
• /
• pp.169-174
• /
• 2017

Transient receptor potential melastatin 8 (TRPM8) plays a crucial role in innocuous cool sensation, acute cold pain and cold-induced hyperalgesia during pathologic conditions. To help understand TRPM8-mediated cold perception in the dental pulp and periodontal tissues, we examined the distribution of TRPM8-immunopositive (+) axons in molar and incisor pulp and periodontal tissues using transgenic mice expressing a genetically encoded axonal tracer in TRPM8+ neurons. In the radicular pulp of the molar teeth, a small number of TRPM8+ axons were observed. TRPM8+ axons branched frequently and extensively in the core of coronal pulp, forming a network in the peripheral pulp. Some TRPM8+ axons ascended between odontoblasts and were observed in the dentinal tubule. TRPM8+ axons were linear-shaped in the radicular pulp, whereas many TRPM8+ axons showed portions shaped like beads connected with thin axonal stands at the peripheral pulp. TRPM8 was densely expressed in the bead portions. In the incisor pulp, TRPM8+ axons were occasionally observed in the core of the coronal pulp and rarely observed at the peripheral pulp. TRPM8+ axons were occasionally observed and showed a linear shape rather than a bead-like appearance in the periodontal ligament and lamina propria of the gingival tissue. These findings, showing differential distribution of TRPM8+ axons between radicular and coronal portions of the molar pulp, between incisor and molar pulp, and between dental pulp and periodontal tissues, may reflect differential cold sensitivity in these regions.

• Korean Journal of Acupuncture
• /
• v.33 no.4
• /
• pp.204-212
• /
• 2016

Objectives : The aim of the study is to investigate the effects of moxibustion on the pain behavior and expression of TRPM8 in the dorsal root ganglion(DRG) in the rat model of ambient cold(AC) exposed osteoarthritis(OA). Methods : OA was induced by the injection of $50{\mu}l$ of 2% monosodium iodoacetate(MIA) into the knee joint cavity. To examine the level of pain, weight bearing forces(WBFs) of affected limb was measured. For the AC exposure, the animals were housed in 6 h/day at $4^{\circ}C$ for 14 days after MIA injection. Moxibustion treatment was performed at EX-LE4 and EX-LE5 with 5 cons(1, 7 or 10 mg) per day for 13 days from 5 days after MIA injection. The expressions of TRPM8 in DRG were measured by western blotting analysis. Results : The WBFs of MIA-AC group were decreased significantly compared to MIA group at 2, 3, 6, 7, 8 and 9 days after arthritis induction. After the first 6 h-AC exposure, expressions of TRPM8 in MIA-AC group were increased significantly compared to those of naive group. After moxibustion treatment, only the WBFs of 7 mg treated group were restored significantly. Moreover, the over-expressions of TRPM8 were attenuated by the moxibustion treatment in AC exposed rats. Conclusions : The data suggest that AC can increase arthritic knee pain via up-regulated TRPM8 and moxibustion treatment improve the arthritic pain via modulation of TRPM8 expression in DRG in the rat model of AC exposed MIA induced arthritis.

• BMB Reports
• /
• v.48 no.1
• /
• pp.1-5
• /
• 2015

Transient Receptor Potential, Melastatin-related, member 4 (TRPM4) channels are $Ca^{2+}$-activated $Ca^{2+}$-impermeable cation channels. These channels are expressed in various types of mammalian tissues including the brain and are implicated in many diverse physiological and pathophysiological conditions. In the past several years, the trafficking processes and regulatory mechanism of these channels and their interacting proteins have been uncovered. Here in this minireview, we summarize the current understanding of the trafficking mechanism of TRPM4 channels on the plasma membrane as well as heteromeric complex formation via protein interactions. We also describe physiological implications of protein-TRPM4 interactions and suggest TRPM4 channels as therapeutic targets in many related diseases.

• Asian Pacific Journal of Cancer Prevention
• /
• v.15 no.4
• /
• pp.1551-1556
• /
• 2014

Background: Transient receptor potential melastatin 8 (TRPM8), a principle membrane receptor involved in calcium ion influx and cell signal transduction, has been found to be up-regulated in some cancer types, including melanomas. Efficiency of menthol, an agonist of TRPM8, in killing melanoma cancer cells has been reported previously, but the mechanisms remain unclear. We here determined whether in vitro cytotoxic effects of menthol on A-375 human malignant melanoma cells might be related to TRPM8 transcript expression. Materials and Methods: The $PrestoBlue^{(R)}$ cell viability assay was used to assess the in vitro cytotoxic effect of menthol after 24h of treatment. RT-PCR was used to quantify TRPM8 transcript expression levels in normal and menthol-treated cells. Cell morphology was observed under inverted phase contrast light microscopy. Results: TRPM8 transcript expression was found at low levels in A-375 cells and down-regulated in a potentially dose-dependent manner by menthol. Menthol exerted in vitro cytotoxic effects on A-375 cells with an $IC_{50}$ value of 11.8 ${\mu}M$, which was at least as effective as 5-fluorouracil ($IC_{50}=120{\mu}M$), a commonly applied chemotherapeutic drug. Menthol showed no dose-dependent cytotoxicity on HeLa cells, a TRPM8 non-expressing cell line. Conclusions: The cytotoxic effects on A-375 cells caused by menthol might be related to reduction of the TRPM8 transcript level. This suggests that menthol might activate TRPM8 to increase cytosolic $Ca^{2+}$ levels, which leads to cytosolic $Ca^{2+}$ imbalance and triggers cell death.

• Asian Pacific Journal of Cancer Prevention
• /
• v.16 no.10
• /
• pp.4469-4475
• /
• 2015

Transient receptor potential melastain 7 (TRPM7) is a bifunctional protein with dual structure of both ion channel and protein kinase, participating in a wide variety of diseases including cancer. Recent researches have reported the mechanism of TRPM7 in human cancers. However, the correlation between TRPM7 and prostate cancer (PCa) has not been well studied. The objective of this study was to investigate the potential the role of TRPM7 in the apoptosis of PC-3 cells, which is the key cell of advanced metastatic PCa. In this study, we demonstrated the influence and potential function of TRPM7 on the PC-3 cells apoptosis induced by TNF-related apoptosis inducing-ligand (TRAIL). The study also found a novel up-regulated expression of TRPM7 in PC-3 cells after treating with TRAIL. Suppression of TRPM7 by TRPM7 non-specific inhibitors ($Gd^{3+}$ or 2-aminoethoxy diphenylborate (2-APB) ) not only markedly eliminated TRPM7 expression level, but also increased the apoptosis of TRAIL-treated PC-3 cells, which may be regulated by the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling pathway accompany with up-regulated expression of cleaved Caspase-3, (TRAIL-receptor 1, death receptors 4) DR4, and (TRAIL-receptor 2, death receptors 5) DR5. Taken together, our findings strongly suggested that TRPM7 was involved in the apoptosis of PC-3 cells induced by TRAIL, indicating that TRPM7 may be applied as a therapeutic target for PCa.

• Clinical and Experimental Reproductive Medicine
• /
• v.24 no.2
• /
• pp.267-271
• /
• 1997

Apoptosis, programmed cell death, is posulated to occur in granulosa cells in ovarian follicular atresia. bcl-2 gene serves as protector from apoptosis and, thus, is associated with increased cell survival. TRPM-2 gene expression has been implicated as a trigger of apoptosis in rat prostate, uterus and mammary gland. Our objective was to determine if bcl-2 and TRPM-2 are expressed in luteinized human GC and, therefore, have regulatory functions for apoptosis in GC. Human GC were obtained via oocyte retrival from the infertile patients stimulated with exogeneous gonadotropins while undergoing IVF. GC were isolated from follicular fluid using Percoll gradient centrifugation. The GC were further purified with anti-CD45 magnetic beads to remove contaminating WBC's. RT-PCR were performed to analyze the mRNA expression of bcl-2 and TRPM-2 in the GC. The PCR primers were designed to amplify a 195 bp fragment of bcl-2 and a 174 bp fragment of TRPM-2. The PCR products were electrophoresed on 4% agarose gel. Three separate experiments indicated that both bcl-2 and TRPM-2 are concurrently expressed in human GC. We cultured granulosa cells with FSH (1 ng/ml) for 1 day to investigate the relative changes of TRPM-2 mRNA level with RNAse protection assay. When we cultured GC with serum free medium for 1 day TRPM-2 mRNA level increased with 1.3 fold, however it was decreased 0.64 fold with FSH. Therefore we conclude that bcl-2 and TRPM-2 are concurrently expressed and that the interaction of their products may be involved in GC apoptosis. And TRPM-2 may be regulated with FSH.

• Journal of Korean Medicine for Obesity Research
• /
• v.18 no.1
• /
• pp.10-18
• /
• 2018

Objectives: Metabolic syndrome is correlated with increased cardiovascular risk and characterized by several factors, including visceral obesity, hypertension, insulin resistance, and dyslipidemia. Several members of a large family of nonselective cation entry channels, e.g., transient receptor potential (TRP) melastatin 7 (TRPM7) channels have been associated with the development of cardiovascular diseases. The purpose of this study was to investigate the effects of Dangkwisoo-san, ginger and curcumin on TRPM7 channel. Methods: Human embryonic kidney (HEK) 293 cells stably transfected with the TRPM7 expression vectors were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, $5{\mu}g/mL$ blasticidin, and 0.4 mg/mL zeocin in a humidified 20% $O_2$/10% $CO_2$ atmosphere at $37^{\circ}C$. Whole-cell patch clamp recordings were obtained using an Axopatch 700B amplifier and pClamp v.10.4 software, and signals were digitalized at 5 kHz using Digidata 1422A. Results: Dangkwisoo-san extract (100, 200, 300, 400, and $500{\mu}g/mL$) inhibited the outward and inward TRPM7 whole-cell currents at dose dependent manner and the half maximal inhibitory concentration $(IC)_{50}$ of Dangkwisoo-san was $218.3{\mu}g/mL$. Also, ginger extract (100, 200, 300, 400, and $500{\mu}g/mL$) inhibited the outward and inward of TRPM7 whole-cell currents in a dose dependent manner and the $IC_{50}$ of ginger was $877.2{\mu}g/mL$. However, curcumin had no effects on TRPM7 whole-cell currents. Conclusions: These results suggest that both Dangkwisoo-san and ginger have good roles to inhibit the TRPM7 channel, suggesting that Dangkwisoo-san and ginger are considered one of the candidate agents for the treatment of metabolic syndrome such as cardiovascular disease.

• The Korean Journal of Pain
• /
• v.27 no.4
• /
• pp.326-333
• /
• 2014

Background: Nefopam is a centrally acting non-opioid analgesic agent. Its analgesic properties may be related to the inhibitions of monoamine reuptake and the N-methyl-D-aspartate (NMDA) receptor. The antinociceptive effect of nefopam has been shown in animal models of acute and chronic pain and in humans. However, the effect of nefopam on diabetic neuropathic pain is unclear. Therefore, we investigated the preventive effect of nefopam on diabetic neuropathic pain induced by streptozotocin (STZ) in rats. Methods: Pretreatment with nefopam (30 mg/kg) was performed intraperitoneally 30 min prior to an intraperitoneal injection of STZ (60 mg/kg). Mechanical and cold allodynia were tested before, and 1 to 4 weeks after drug administration. Thermal hyperalgesia was also investigated. In addition, the transient receptor potential ankyrin 1 (TRPA1) and TRP melastatin 8 (TRPM8) expression levels in the dorsal root ganglion (DRG) were evaluated. Results: Pretreatment with nefopam significantly inhibited STZ-induced mechanical and cold allodynia, but not thermal hyperalgesia. The STZ injection increased TRPM8, but not TRPA1, expression levels in DRG neurons. Pretreatment with nefopam decreased STZ-induced TRPM8 expression levels in the DRG. Conclusions: These results demonstrate that a nefopam pretreatment has strong antiallodynic effects on STZ-induced diabetic rats, which may be associated with TRPM8 located in the DRG.

• The Korean Journal of Physiology and Pharmacology
• /
• v.10 no.1
• /
• pp.7-11
• /
• 2006

Interstitial cells of Cajal (ICCs) are pacemakers in gastrointestinal tracts, regulating rhythmicity by activating nonselective cation channels (NSCCs). In the present study, we investigated the general characteristics and pH-mediated regulation of pacemaker activity in cultured interstitial cells of Cajal. Under voltage clamp mode and at the holding potential of -60 mV, the I-V relationships and difference current showed that there was no reversal potential and voltage-independent inward current. Also, when the holding potentials were changed from +20 mV to -80 mV with intervals of 20 mV, there was little difference in inward current. In pacemaker activity, the resting membrane potential (RMP) was depolarized (In pH 5.5, $23{\pm}1.5$ mV depolarized) and the amplitude was decreased by a decrease of the extracellular pH. However, in case of increase of extracellular pH, the RMP was slightly hyperpolarized and the amplitude was decreased a little. The melastatin type transient receptor potential (TRPM) channel 7 has been suggested to be required for intestinal pacemaking activity. TRPM7 produced large outward currents and small inward currents by voltage ramps, ranging from +100 to -100 mV from a holding potential of -60 mV. The inward current of TRPM7 was dramatically increased by a decrease in the extracellular pH. At pH 4.0, the average inward current amplitude measured at -100 mV was increased by about 7 fold, compared with the current amplitude at pH 7.4. Changes in the outward current (measured at +100 mV) were much smaller than those of the inward current. These results indicate that the resting membrane potential of pacemaking activity might be depolarized by external acidic pH through TRPM7 that is required for intestinal pacemaking activity.