Transient Receptor Potential Melastatin 2 Channel Research Articles

A conserved mechanism couples cytosolic domain movements to pore gating in the TRPM2 channel

Transient Receptor Potential Melastatin 2 (TRPM2) cation channels contribute to immunocyte activation, insulin secretion, and central thermoregulation. TRPM2 opens upon binding cytosolic Ca2+ and ADP ribose (ADPR). We present here the 2.5 Å cryo-electronmicroscopy structure of TRPM2 from Nematostella vectensis (nvTRPM2) in a lipid nanodisc, complexed with Ca2+ and ADPR-2'-phosphate. Comparison with nvTRPM2 without nucleotide reveals that nucleotide binding-induced movements in the protein's three "core" layers deconvolve into a set of rigid-body rotations conserved from cnidarians to man. By covalently crosslinking engineered cysteine pairs we systematically trap the cytosolic layers in specific conformations and study effects on gate opening/closure. The data show that nucleotide binding in Layer 3 disrupts inhibitory intersubunit interactions, allowing rotation of Layer 2 which in turn expands the gate located in Layer 1. Channels trapped in that "activated" state are no longer nucleotide dependent, but are opened by binding of Ca2+ alone.

Construction of HEK293T cell line stably expressing TRPM2 channel based on PiggyBac transposition system and its application in drug screening for cerebral ischemia and other diseases

To establish a cell line stably expressing the transient receptor potential melastatin 2 (TRPM2) channel for screening TRPM2 inhibitors based on PiggyBac transposition system. A plasmid PiggyBac-human TRPM2 (pPB-hTRPM2) eukaryotic expression vector was constructed using PiggyBac transposition system. The plasmid and a helper plasmid were co-transfected into HEK293T cells to express TRPM2, which was identified by fluorescence and patch-clamp assays. The high throughput screening performance was assessed with the Z ´ factor. Calcium imaging and patch clamp techniques were employed to assess the initial activity of eleven compound molecules, confirming the inhibitory effects of the primary molecules on TRPM2. The protective effect of the screened compounds on damaged cells was validated using the oxygen-glucose deprivation/reperfusion (OGD/R) injury model and CCK-8 kit. The level of cellular reactive oxygen species (ROS) was detected by flow cytometry. The neuroprotective effects of the compounds were evaluated using a transient middle cerebral artery occlusion (tMCAO) mouse model. The HEK293T cells transfected with pPB-hTRPM2-EGFP showed high TRPM2 expression. Puromycin-resistant cells, selected through screening, exhibited robust fluorescence. Whole-cell patch results revealed that induced cells displayed classical TRPM2 current characteristics comparable to the control group, showing no significant differences (P>0.05). With a Z ´ factor of 0.5416 in calcium imaging, the model demonstrated suitability for high-throughput screening of TRPM2 inhibitors. Calcium imaging and electrophysiological experiments indicated that compound 6 significantly inhibited the TRPM2 channel. Further experiments showed that 1.0 μmol/L of compound 6 enhanced cell viability (P<0.05) and reduced the level of ROS (P<0.05) of SH-SY5Y under OGD/R injury. 0.3 and 1.0 mg/kg of compound 6 reduced the cerebral infarction volume in tMCAO mice (both P<0.05). A stable TRPM2 gene expressing cell line has been successfully established using PiggyBac gene editing in this study. TRPM2 channel inhibitors were screened through calcium imaging and patch clamp techniques, and an inhibitor compound 6 was identified. This compound can alleviate cell damage after OGD/R by reducing cellular ROS levels and has a protective effect against cerebral ischemia-reperfusion injury in mice.

The effects of berberine and curcumin on cardiac, lipid profile and fibrosis markers in cyclophosphamide-induced cardiac damage: The role of the TRPM2 channel.

Cyclophosphamide (CYP) is widely used to treat various types of cancer. In addition to the therapeutic properties of this drug, unfortunately, its side effects are still not fully understood. This study investigated the protective effect of curcumin (CURC) and berberine (BER) on CYP-induced cardiac damage. Thirty-six male rats were equally divided into the control, dimethyl sulfoxide (DMSO), CYP, CYP + CURC, CYP + BER and CYP + BER + CURC groups. Troponin-I, Creatine kinase-myocardial band (CK-MB), total cholesterol, triglyceride levels in serum samples, and reactive oxygen species (ROS), poly(ADP-ribose) polymerase-1 (PARP-1), and transient receptor potential melastatin 2 (TRPM2) channel levels in heart tissue were measured using an enzyme-linked immunoassay (ELISA) kit. In addition, histopathological examination and immunohistochemical investigation of the TRPM2 channel, fibroblast specific protein-1 (FSP1), transforming growth factor-beta- 1 (TGF-β1) and α-smooth muscle actin (α-SMA) expressions were determined in heart tissue. The CYP group's troponin-I, total cholesterol, triglyceride, CK-MB, ROS, PARP-1 and TRPM2 channel levels were higher than in the other groups in the ELISA measurements (p < 0.05). In contrast, these parameters in the group treated with CURC and BER together with CYP were lower than in the CYP group (p < 0.05). Additionally, CUR and BER reduced CYP-induced pathological damage, TRPM2, FSP1, TGF-β1 and α-SMA expressions. The data showed that CYP administration can cause cardiac damage by increasing the TRPM2 channel, TGF-β1, FSP1 and α-SMA expression levels. Therefore, we concluded that CURC and BER administration following CYP application may be used as therapeutic agents to prevent CYP-induced cardiac damage.

In Silico Electrophysiological Investigation of Transient Receptor Potential Melastatin-4 Ion Channel Biophysics to Study Detrusor Overactivity.

Enhanced electrical activity in detrusor smooth muscle (DSM) cells is a key factor in detrusor overactivity which causes overactive bladder pathological disorders. Transient receptor potential melastatin-4 (TRPM4) channels, which are calcium-activated cation channels, play a role in regulating DSM electrical activities. These channels likely contribute to depolarizing the DSM cell membrane, leading to bladder overactivity. Our research focuses on understanding TRPM4 channel function in the DSM cells of mice, using computational modeling. We aimed to create a detailed computational model of the TRPM4 channel based on existing electrophysiological data. We employed a modified Hodgkin-Huxley model with an incorporated TRP-like current to simulate action potential firing in response to current and synaptic stimulus inputs. Validation against experimental data showed close agreement with our simulations. Our model is the first to analyze the TRPM4 channel's role in DSM electrical activity, potentially revealing insights into bladder overactivity. In conclusion, TRPM4 channels are pivotal in regulating human DSM function, and TRPM4 channel inhibitors could be promising targets for treating overactive bladder.

In vivo evaluation of monoclonal antibody M4M using a humanised rat model of stroke demonstrates attenuation of reperfusion injury via blocking human TRPM4 channel

Background Blocking Transient Receptor Potential Melastatin 4 (TRPM4) in rodents by our antibody M4P has shown to attenuate cerebral ischaemia-reperfusion injury. Since M4P does not interact with human TRPM4, the therapeutic potential of blocking human TRPM4 remains unclear. We developed a monoclonal antibody M4M that inhibited human TRPM4 in cultured cells. However, M4M has no effect on stroke outcome in wild-type rats. Therefore, M4M needs to be evaluated on animal models expressing human TRPM4. Methods We generated a humanised rat model using the CRISPR/Cas technique to knock-in (KI) the human TRPM4 antigen sequence. Results In primary neurons from human TRPM4 KI rats, M4M binds to hypoxic neurons, but not normoxic nor wild-type neurons. Electrophysiological studies showed that M4M blocked ATP depletion-induced activation of TRPM4 and inhibited hypoxia-associated cell volume increase. In a stroke model, administration of M4M reduced infarct volume in KI rats. Rotarod test and Neurological deficit score revealed improvement following M4M treatment. Conclusion M4M selectively binds and inhibits hypoxia-induced human TRPM4 channel activation in neurons from the humanised rat model, with no effect on healthy neurons. Use of M4M in stroke rats showed functional improvements, suggesting the potential for anti-human TRPM4 antibodies in treating acute ischaemic stroke patients.

Bidirectional regulation mechanism of TRPM2 channel: role in oxidative stress, inflammation and ischemia-reperfusion injury.

Transient receptor potential melastatin 2 (TRPM2) is a non-selective cation channel that exhibits Ca2+ permeability. The TRPM2 channel is expressed in various tissues and cells and can be activated by multiple factors, including endogenous ligands, Ca2+, reactive oxygen species (ROS) and temperature. This article reviews the multiple roles of the TRPM2 channel in physiological and pathological processes, particularly on oxidative stress, inflammation and ischemia-reperfusion (I/R) injury. In oxidative stress, the excessive influx of Ca2+ caused by the activation of the TRPM2 channel may exacerbate cellular damage. However, under specific conditions, activating the TRPM2 channel can have a protective effect on cells. In inflammation, the activation of the TRPM2 channel may not only promote inflammatory response but also inhibit inflammation by regulating ROS production and bactericidal ability of macrophages and neutrophils. In I/R, the activation of the TRPM2 channel may worsen I/R injury to various organs, including the brain, heart, kidney and liver. However, activating the TRPM2 channel may protect the myocardium from I/R injury by regulating calcium influx and phosphorylating proline-rich tyrosine kinase 2 (Pyk2). A thorough investigation of the bidirectional role and regulatory mechanism of the TRPM2 channel in these physiological and pathological processes will aid in identifying new targets and strategies for treatment of related diseases.

Impaired intracellular Ca2+ signaling contributes to age-related cerebral small vessel disease in Col4a1 mutant mice.

Humans and mice with mutations in COL4A1 and COL4A2 manifest hallmarks of cerebral small vessel disease (cSVD). Mice with a missense mutation in Col4a1 at amino acid 1344 (Col4a1+/G1344D) exhibit age-dependent intracerebral hemorrhages (ICHs) and brain lesions. Here, we report that this pathology was associated with the loss of myogenic vasoconstriction, an intrinsic vascular response essential for the autoregulation of cerebral blood flow. Electrophysiological analyses showed that the loss of myogenic constriction resulted from blunted pressure-induced smooth muscle cell (SMC) membrane depolarization. Furthermore, we found that dysregulation of membrane potential was associated with impaired Ca2+-dependent activation of large-conductance Ca2+-activated K+ (BK) and transient receptor potential melastatin 4 (TRPM4) cation channels linked to disruptions in sarcoplasmic reticulum (SR) Ca2+ signaling. Col4a1 mutations impair protein folding, which can cause SR stress. Treating Col4a1+/G1344D mice with 4-phenylbutyrate, a compound that promotes the trafficking of misfolded proteins and alleviates SR stress, restored SR Ca2+ signaling, maintained BK and TRPM4 channel activity, prevented loss of myogenic tone, and reduced ICHs. We conclude that alterations in SR Ca2+ handling that impair ion channel activity result in dysregulation of SMC membrane potential and loss of myogenic tone and contribute to age-related cSVD in Col4a1+/G1344D mice.

Vitamin D: An effective therapy against methotrexateinduced cardiotoxicity

Purpose: To determine the potential cardioprotective effect of vitamin D (VD) against methotrexate (MTX) induced cardiotoxicity.Methods: A total of thirty-five (35) rats were randomly assigned to five equal groups. Control groupreceived no treatment; MTX group received intraperitoneal (IP) injection of MTX in a single dose of 20 mg/kg on day 8; VD group received 200 IU/kg VD daily dissolved in sunflower oil orally; sunflower oil (SO) group received 1 mL/kg/day SO orally; MTX + VD group received a single dose of MTX (20 mg/kg, IP) on day 8 and VD (200 IU/kg, orally) for 21 days. Myocardial tissue samples were harvested and used for clinical chemistry, histopathological, and ultrastructural evaluation.Results: Histopathological damage in MTX group was more severe than in control group under both light and electron microscopy. Expression of transient receptor potential melastatin 2 (TRPM2) and caspase-3 markers was significantly higher in MTX group (p &lt; 0.05). Glutathione peroxidase (GSH-Px) enzyme activity in cardiac tissue was lower in MTX group, whereas malondialdehyde (MDA) levels increased significantly (p &lt; 0.05). In MTX + VD group, VD treatment alleviated histopathological damage and significantly lowered TRPM2 and caspase-3 expressions (p &lt; 0.05). Vitamin D also reduced tissue MDA levels, and increased GSH-Px activity albeit non-significantly (p &gt; 0.05),Conclusion: These findings suggest that VD exerts an ameliorative effect on MTX-induced cardiotoxicity in rats. Therefore, TRPM2 channel affords a novel therapeutic approach for treatment of diseases related to chemotherapy-induced oxidative stress.

The Role of TRPM4 in Cardiac Electrophysiology and Arrhythmogenesis.

The transient receptor potential melastatin 4 (TRPM4) channel is a non-selective cation channel that activates in response to increased intracellular Ca2+ levels but does not allow Ca2+ to pass through directly. It plays a crucial role in regulating diverse cellular functions associated with intracellular Ca2+ homeostasis/dynamics. TRPM4 is widely expressed in the heart and is involved in various physiological and pathological processes therein. Specifically, it has a significant impact on the electrical activity of cardiomyocytes by depolarizing the membrane, presumably via Na+ loading. The TRPM4 channel likely contributes to the development of cardiac arrhythmias associated with specific genetic backgrounds and cardiac remodeling. This short review aims to overview what is known so far about the TRPM4 channel in cardiac electrophysiology and arrhythmogenesis, highlighting its potential as a novel therapeutic target to effectively prevent and treat cardiac arrhythmias.

TRPM4 contribution in mouse uterine contractions.

Inappropriate uterine contractions are a matter of concern during pregnancy or menses. We identified the transient receptor potential melastatin 4 (TRPM4) ion channel as a new actor in mouse uterine contractions highlighting this protein as a potential pharmacological target for a better control of myometrial activity. Control of uterine contractions is of interest in the context of inappropriate myometrial activity during pregnancy and at time of delivery, but it is also a matter for menstrual pain. While several molecular determinants of myometrial contractions have been described, the complete distribution of roles to the various actors is far from understood. A key phenomenon is a variation in cytoplasmic Ca2+ which leads to the activation of calmodulin in smooth muscle and also in the phosphorylation of myosin allowing contraction. The Ca2+ - TRPM4 channel which is known to modulate Ca2+- fluxes in several cell types was shown to participate in vascular as well as detrusor muscle contraction. We thus designed a study to determine whether it also participates in myometrial contraction. Uterine rings were isolated from Trpm4+/+ and Trpm4-/- non-pregnant adult mice and contractions were recorded using an isometric force transducer. In basal conditions, spontaneous contractions were similar in both groups. Application of 9-phenanthrol, a pharmacological TRPM4 inhibitor, dose-dependently reduced contraction parameters in Trpm4+/+ rings with an IC50 around 2.10-6 mol/L. The effect of 9-phenanthrol was significantly reduced in Trpm4-/- rings. The effect of oxytocin was tested and was found to be stronger in Trpm4+/+ rings compared to Trpm4-/-. Under a constant stimulation by oxytocin, 9-phenanthrol still reduced contraction parameters in Trpm4+/+ rings with a smaller effect on Trpm4-/-. Altogether it indicates that TRPM4 participates in uterine contractions in mice and may thus be evaluated as a new target to control such contractions.

The Effects of N-acetylcysteine on Transient Receptor Potential Melastatin 2 Channels Activation and Expression in Testicular Tissue of Diabetic Rats.

Diabetes mellitus (DM) is a common, chronic metabolic disease that has harmful effects on many diverse tissues, including the testis. One of the ways of tissue damage is the modification of transient receptor potential melastatin 2 (TRPM2) channels by increased reactive oxygen species (ROS). In our study for the first time, it was aimed to investigate TRPM2 channel activation in testicular tissues of diabetic rats induced by streptozotosin (STZ) and to examine the efficacy of N-acetylcysteine (NAC) treatment, which is an antioxidant. In our study, 28 Wistar albino male rats aged 8-10 weeks were used, and animals were divided into four groups: control group, NAC group, DM group, and DM + NAC group. The experimental phase was designed as eight weeks. The malondialdehyde(MDA)level, which is an indicator for lipid peroxidation due to oxidative stress, was measured by the spectrophotometric method. The Tunel assay was used to determine apoptosis on testicular tissue. TRPM2 immunoreactivity was determined by the avidin-biotin-peroxidase complex method, and quantitative polymerase chain reaction (QPCR) was used to determine TRPM2 expression levels. It was seen that MDA levels were significantly increased in the DM group and decreased after NAC treatment. Similarly, it was observed that apoptosis levels, which increased significantly in diabetic rats, decreased to the levels of the control group after treatment. It was seen that TRPM2 activation and expression levels were significantly decreased in the DM group. The results of this study show that NAC regulates TRPM2 activation in the testicular tissue of patients with diabetes and has tissue-protective properties.

Curcumin attenuates hydroxychloroquine-mediated apoptosis and oxidative stress via the inhibition of TRPM2 channel signalling pathways in a retinal pigment epithelium cell line.

Hydroxychloroquine (HCQ) is used in the treatment of several diseases, such as malaria, Sjögren's disease, Covid-19, and rheumatoid arthritis. However, HCQ induces retinal pigment epithelium death via the excessive increase of cytosolic (cROS) and mitochondrial (mROS) free oxygen radical production. The transient receptor potential melastatin 2 (TRPM2) cation channel is stimulated by ADP-ribose (ADPR), cROS, and mROS, although it is inhibited by curcumin (CRC). We aimed to investigate the modulating action of CRC on HCQ-induced TRPM2 stimulation, cROS, mROS, apoptosis, and death in an adult retinal pigment epithelial 19 (ARPE19) cell line model. ARPE19 cells were divided into four groups: control (CNT), CRC (5µM for 24h), HCQ (60µM for 48h), and CRC + HCQ groups. The levels of cell death (propidium iodide positive cell numbers), apoptosis markers (caspases -3, -8, and -9), oxidative stress (cROS and mROS), mitochondria membrane depolarization, TRPM2 current density, and intracellular free Ca2+ and Zn2+ fluorescence intensity were upregulated in the HCQ group after stimulation with hydrogen peroxide and ADPR, but their levels were downregulated by treatments with CRC and TRPM2 blockers (ACA and carvacrol). The HCQ-induced decrease in retinal live cell count and cell viability was counteracted by treatment with CRC. HCQ-mediated overload Ca2+ influx and retinal oxidative toxicity were induced in an ARPE19 cell line through the stimulation of TRPM2, although they were attenuated by treatment with CRC. Hence, CRC may be a potential therapeutic antioxidant for TRPM2 activation and HCQ treatment-induced retinal oxidative injury and apoptosis.

MITOCHONDRIA: the dual function of the transient receptor potential melastatin 2 channels from cytomembrane to mitochondria.

Mitochondria are closely related to oxidative stress and play an important role in maintaining cell functional homeostasis and meeting cell energy demand. The transient receptor potential melastatin 2 (TRPM2) channel affects the occurrence and progression of diseases by regulating mitochondrial function. TRPM2 channel promotes Ca2+ influx to affect 18kDa translocator protein (TSPO), mitochondrial membrane potential (MMP), reactive oxygen species (ROS), adenosine triphosphate (ATP) production, and mitochondrial autophagy. The mechanism of Ca2+ influx into the mitochondria by TRPM2 is abundant. Interestingly, the TRPM2 channel inhibits the production of mitochondrial ROS in cancer cells and promotes the production of mitochondrial ROS in normal cells, which induces cell death in normal cells but proliferation in cancer cells. TRPM2 can be a potential target for the treatment of various diseases due to its role as a molecular link between mitochondria and Ca2+ signals.

The TRPM4 channel inhibitor 9-phenanthrol alleviates cerebral edema after traumatic brain injury in rats.

Cerebral edema (CE) exerts an important effect on brain injury after traumatic brain injury (TBI). Upregulation of transient receptor potential melastatin 4 (TRPM4) in vascular endothelial cells (ECs) results in damage to capillaries and the blood-brain barrier (BBB), which is critical for the development of CE. Many studies have shown that 9-phenanthrol (9-PH) effectively inhibits TRPM4. The current study aimed to investigate the effect of 9-PH on reducing CE after TBI. In this experiment, we observed that 9-PH markedly reduced brain water content, BBB disruption, proliferation of microglia and astrocytes, neutrophil infiltration, neuronal apoptosis and neurobehavioral deficits. At the molecular level, 9-PH significantly inhibited the protein expression of TRPM4 and MMP-9, alleviated the expression of apoptosis-related molecules and inflammatory cytokines, such as Bax, TNF-α and IL-6, near injured tissue, and diminished serum SUR1 and TRPM4 levels. Mechanistically, treatment with 9-PH inhibited activation of the PI3K/AKT/NF-kB signaling pathway, which was reported to be involved in the expression of MMP-9. Taken together, the results of this study indicate that 9-PH effectively reduces CE and alleviates secondary brain injury partly through the following possible mechanisms: ①9-PH inhibits TRPM4-mediated Na + influx and reduces cytotoxic CE; ②9-PH hinders the expression and activity of MMP-9 by inhibiting the TRPM4 channel and decreases disruption of the BBB, thereby preventing vasogenic cerebral edema. ③9-PH reduces further inflammatory and apoptotic damage to tissues.

Faulty TRPM4 channels underlie age-dependent cerebral vascular dysfunction in Gould syndrome

Gould syndrome is a rare multisystem disorder resulting from autosomal dominant mutations in the collagen-encoding genes COL4A1 and COL4A2. Human patients and Col4a1 mutant mice display brain pathology that typifies cerebral small vessel diseases (cSVDs), including white matter hyperintensities, dilated perivascular spaces, lacunar infarcts, microbleeds, and spontaneous intracerebral hemorrhage. The underlying pathogenic mechanisms are unknown. Using the Col4a1+/G394V mouse model, we found that vasoconstriction in response to internal pressure-the vascular myogenic response-is blunted in cerebral arteries from middle-aged (12 mo old) but not young adult (3 mo old) animals, revealing age-dependent cerebral vascular dysfunction. The defect in the myogenic response was associated with a significant decrease in depolarizing cation currents conducted by TRPM4 (transient receptor potential melastatin 4) channels in native cerebral artery smooth muscle cells (SMCs) isolated from mutant mice. The minor membrane phospholipid phosphatidylinositol 4,5 bisphosphate (PIP2) is necessary for TRPM4 activity. Dialyzing SMCs with PIP2 and selective blockade of phosphoinositide 3-kinase (PI3K), an enzyme that converts PIP2 to phosphatidylinositol (3, 4, 5)-trisphosphate (PIP3), restored TRPM4 currents. Acute inhibition of PI3K activity and blockade of transforming growth factor-beta (TGF-β) receptors also rescued the myogenic response, suggesting that hyperactivity of TGF-β signaling pathways stimulates PI3K to deplete PIP2 and impair TRPM4 channels. We conclude that age-related cerebral vascular dysfunction in Col4a1+/G394V mice is caused by the loss of depolarizing TRPM4 currents due to PIP2 depletion, revealing an age-dependent mechanism of cSVD.

Effect of selenium against doxorubicin-induced oxidative stress, inflammation, and apoptosis in the brain of rats: Role of TRPM2 channel

Doxorubicin (DOX) is widely used as an anticancer drug in humans' various solid and haematological tumours. Although many studies on the toxic effect of DOX are used in different organs, its impact on brain tissue has yet to be adequately studied. This study investigated the protective effect of selenium (Se) and the role of transient receptor potential melastatin‐2 (TRPM2) channel activation against brain damage caused by DOX administration. Sixty rats were randomly divided into the sham, dimethyl sulfoxide (DMSO), DOX, DOX + Se, DOX + N-(p-amylcinnamoyl) anthranilic acid (ACA), and DOX + Se + ACA groups. The reactive oxygen species (ROS), poly [ADP-ribose] polymerase 1 (PARP1), and TRPM2 channel levels in brain tissues were measured by ELISA. In addition, a histopathological examination was performed in the cerebral cortex and hippocampal areas, and the TRPM2 channel, NF-κB, and caspase-3 expression were determined immunohistochemically. The levels of ROS, PARP1 and TRPM2 channel in the DOX group were higher than in the sham and DMSO groups (P &lt; 0.05). However, these parameters were decreased in the in DOX+Se and DOX+ACA groups by the treatments of Se and ACA (P &lt; 0.05). Also, we determined that Se and ACA treatment decreased the NF-κB, caspase-3, and TRPM2 channel expression in the cerebral cortex and hippocampal areas in the DOX-induced rats. The data showed that Se and/or ACA administration together with DOX administration could be used as a protective agent against DOX-induced brain damage.

Possible role of transient receptor potential melastatin 4 channels in adrenergic contractions in mouse prostate smooth muscles.

Transient receptor potential melastatin 4 (TRPM4) cation channels are expressed in prostate glands. However, the precise role of these channels in prostate contractility remains unclear. In this study, we examined whether TRPM4 channels were involved in adrenergic contractions in the mouse prostate gland. Adrenergic contractile responses elicited by noradrenaline or electrical field stimulation of the sympathetic nerve were isometrically recorded, and the effects of 9-phenanthrol, a specific TRPM4 channel inhibitor, on those contractile responses were investigated in mouse ventral prostate preparations. 9-phenanthrol (10 or 30 μM) inhibited noradrenaline- and sympathetic nerve-evoked contractions in a concentration-dependent manner. A similar inhibitory effect was observed with another TRPM4 channel inhibitor, 4-chloro-2-(2-(naphthalene-1-yloxy) acetamido) benzoic acid (NBA; 10 μM). Inhibition by 9-phenanthrol and NBA were much greater at lower noradrenaline concentrations and lower stimulus frequencies than those of higher concentrations or frequencies. However, 9-phenanthrol did not inhibit the noradrenaline-induced contractile response when the membrane potential was decreased to approximately 0 mV in the 140 mM K+ medium. Moreover, 9-phenanthrol does not affect noradrenaline-induced increases in spontaneous contractions of cardiac atrial preparation. This agent inhibited noradrenaline-induced contractions in the posterior aorta preparation. However, the inhibitory effect was significantly weaker than that observed in the prostate gland. These results suggest that TRPM4 channels are involved in adrenergic contractions in the mouse prostate gland, possibly through membrane depolarization by their opening; therefore, they might be potential candidates for treating benign prostatic hyperplasia.

Over-activation of TRPM2 ion channel accelerates blood-spinal cord barrier destruction in diabetes combined with spinal cord injury rat.

Spinal cord injury (SCI) is a devastating neurological disorder that often results in loss of motor and sensory function. Diabetes facilitates the blood-spinal cord barrier (BSCB) destruction and aggravates SCI recovery. However, the molecular mechanism underlying it is still unclear. Our study has focused on transient receptor potential melastatin 2 (TRPM2) channel and investigated its regulatory role on integrity and function of BSCB in diabetes combined with SCI rat. We have confirmed that diabetes is obviously not conductive to SCI recovery through accelerates BSCB destruction. Endothelial cells (ECs) are the important component of BSCB. It was observed that diabetes significantly worsens mitochondrial dysfunction and triggers excessive apoptosis of ECs in spinal cord from SCI rat. Moreover, diabetes impeded neovascularization in spinal cord from SCI rat with decreases of VEGF and ANG1. TRPM2 acts as a cellular sensor of ROS. Our mechanistic studies showed that diabetes significantly induces elevated ROS level to activate TRPM2 ion channel of ECs. Then, TRPM2 channel mediated the Ca2+ influx and subsequently activated p-CaMKII/eNOS pathway, and which in turn triggered the ROS production. Consequently, over-activation of TRPM2 ion channel results in excessive apoptosis and weaker angiogenesis during SCI recovery. Inhibition of TRPM2 with 2-Aminoethyl diphenylborinate (2-APB) or TRPM2 siRNA will ameliorate the apoptosis of ECs and promote angiogenesis, subsequently enhance BSCB integrity and improve the locomotor function recovery of diabetes combined with SCI rat. In conclusion, TRPM2 channel may be a key target for the treatment of diabetes combined with SCI rat.

TRPM2 protects against cisplatin-induced acute kidney injury and mitochondrial dysfunction via modulating autophagy.

Background: Cisplatin is a widely used anti-tumor agent but its use is frequently limited by nephrotoxicity. Transient receptor potential melastatin 2 (TRPM2) is a non-selective cation channel which is generally viewed as a sensor of oxidative stress, and increasing evidence supports its link with autophagy, a critical process for organelle homeostasis. Methods: Cisplatin-induced cell injury and mitochondrial damage were both assessed in WT and Trpm2-knockout mice and primary cells. RNA sequencing, immunofluorescence staining, immunoblotting and flowcytometry were applied to interpret the mechanism of TRPM2 in cisplatin nephrotoxicity. Results: Knockout of TRPM2 exacerbates renal dysfunction, tubular injury and cell apoptosis in a model of acute kidney injury (AKI) induced by treatment with cisplatin. Cisplatin-caused tubular mitochondrial damage is aggravated in TRPM2-deficient mice and cells and, conversely, alleviated by treatment with Mito-TEMPO, a mitochondrial ROS scavenger. TRPM2 deficiency hinders cisplatin-induced autophagy via blockage of Ca2+ influx and subsequent up-regulation of AKT-mTOR signaling. Consistently, cisplatin-induced tubular mitochondrial damage, cell apoptosis and renal dysfunction in TRPM2-deficient mice are mitigated by treatment with a mTOR inhibitor. Conclusion: Our results suggest that the TRPM2 channel plays a protective role in cisplatin-induced AKI via modulating the Ca2+-AKT-mTOR signaling pathway and autophagy, providing novel insights into the pathogenesis of kidney injury.

The identification of the key residues E829 and R845 involved in transient receptor potential melastatin 2 channel gating.

Transient receptor potential melastatin 2 (TRPM2), a non-selective cation channel, is involved in many physiological and pathological processes, including temperature sensing, synaptic plasticity regulation, and neurodegenerative diseases. However, the gating mechanism of TRPM2 channel is complex, which hinders its functional research. With the discovery of the Ca2+ binding site in the S2-S3 domain of TRPM2 channel, more and more attention has been drawn to the role of the transmembrane segments in channel gating. In this study, we focused on the D820-F867 segment around the S2 domain, and identified the key residues on it. Functional assays of the deletion mutants displayed that the deletions of D820-W835 and L836-P851 destroyed channel function totally, indicating the importance of these two segments. Sequence alignments on them found three polar and charged residues with high conservation (D820, E829, and R845). D820A, E829A, and R845A which removed the charge and the side chain of the residues were tested by 500 μM adenosine diphosphate-ribose (ADPR) or 50 mM Ca2+. E829A and R845A affected the characteristic of channel currents, while D820A behaved similarly to WT, indicating the participations of E829 and R845 in channel gating. The charge reversing mutants, E829K and R845D were then constructed and the electrophysiological tests showed that E829A and E829K made the channel lose function. Interestingly, R845A and R845D exhibited an inactivation process when using 500 μM ADPR, but activated normally by 50 mM Ca2+. Our data suggested that the negative charge at E829 took a vital part in channel activation, and R845 increased the stability of the Ca2+ combination in S2-S3 domain, thus guaranteeing the opening of TRPM2 channel. In summary, our identification of the key residues E829 and R845 in the transmembrane segments of TRPM2. By exploring the gating process of TRPM2 channel, our work helps us better understand the mechanism of TRPM2 as a potential biomarker in neurodegenerative diseases, and provides a new approach for the prediction, diagnosis, and prognosis of neurodegenerative diseases.