Transient Receptor Potential Melastatin 2 Channel Research Articles

Selective inhibition of TRPM2 channel by two novel synthesized ADPR analogues.

Transient receptor potential melastatin‐2 (TRPM2) channel critical for monitoring internal body temperature is implicated in the pathological processes such as neurodegeneration. However, lacking selective and potent TRPM2 inhibitors impedes investigation and validation of the channel as a drug target. To discover novel and selective TRPM2 inhibitors, a series of adenosine 5′‐diphosphoribose analogues were synthesized, and their activities and selectivity were evaluated. Whole‐cell patch‐clamp recordings were employed for screen and evaluation of synthesized compounds. Two compounds, 7i and 8a, were identified as TRPM2 inhibitors with IC 50 of 5.7 and 5.4 μm, respectively. Both 7i and 8a inhibited TRPM2 current without affecting TRPM7, TRPM8, TRPV1 and TRPV3. These two TRPM2 inhibitors can serve as new pharmacological tools for further investigation and validation of TRPM2 channel as a drug target, and the summarized structure–activity relationship (SAR) may also provide insights into further improving existing inhibitors as potential lead compounds.

TRPM2 dependence of ROS-induced NLRP3 activation in Alzheimer's disease

Recent studies have shown that neuroinflammation plays an important role in Alzheimer's disease (AD). Microglial cells are responsible for the phagocytosis of Amyloid-β (Aβ). However, it has been demonstrated that in AD patients the efficiency of phagocytosis decreases due to proinflammatory cytokines, such as Interleukin-1β (IL-1β), which is produced through the activation of NLRP3 inflammasome. In this study, we aimed at deciphering the mechanism underlying the NLRP3 activation. The results showed that Aβ induces an increase in the level of reactive oxygen species (ROS). According to this study, ROS produced from both mitochondria and NADPH oxidase was responsible for NLRP3 activation. In addition, it was observed that this high level of ROS activated the transient receptor potential melastatin 2 (TRPM2) channel, which causes an increase in the level of intracellular calcium. The results demonstrated that in the absence of intracellular calcium, caspase-1 cannot be activated and therefore the level of IL-1β decreases. Altogether, our findings supported the role of TRPM2 channel in ROS-induced NLRP3 activation in microglial cells through the exposure to Aβ.

New insights into the interaction between ADP-ribose and human TRPM2 channel

Since adenosine 5'-diphosphoribose (ADPR) has been discovered (1) as stimulus of the human apoptosis-related ion channel transient receptor potential melastatin 2 (TRPM2), the compound has undergone a rapid ascent from being considered a mere spin-off of the cellular energy metabolism to a generally acknowledged important intracellular second messenger.

Glucagon-like peptide-1 potentiates glucose-stimulated insulin secretion via the transient receptor potential melastatin 2 channel.

The transient receptor potential melastatin 2 (TRPM2) channel, a Ca2+ permeable channel activated by cAMP, is expressed on pancreatic β-cells and is responsible for the regulation of insulin secretion. It is known that glucose-stimulated insulin secretion (GSIS) can be potentiated by glucagon like peptide-1 (GLP-1), and that the changes in the extracellular glucose concentration alter the levels of intracellular adenosine ATP and cAMP. The present study hypothesized that TRPM2 mediates the modulatory effect of GLP-1 on insulin secretion. The results demonstrated that silencing of TRPM2 eliminated GLP-1-enhanced insulin secretion, indicating the involvement of TRPM2 in this process. In addition, the results of current recordings of TRPM2 and measurement of the resulting insulin secretion in β-cells in the presence of GLP-1 and various concentrations of glucose suggest that GLP-1 regulates GSIS via the TRPM2 channel. Furthermore, inhibiting the activity or expression of TRPM2 attenuated GLP-1-induced GSIS. By using specific activators or inhibitors, the present study demonstrated that the two primary downstream effectors of the GLP-1 receptor, exchange protein directly activated by cAMP and protein kinase A, differentially influence GSIS and GLP-1-potentiated GSIS. In conclusion, the present study revealed the role of TRPM2 in GLP-1-regulated insulin secretion. The results of the present study provide a novel avenue for the prevention and treatment of diabetes and its complications.

Transient receptor potential melastatin 4 channel is required for rat dental pulp stem cell proliferation and survival.

Investigate the role of the transient receptor potential melastatin 4 (TRPM4) channel in rat dental pulp stem cell (DPSC) proliferation and survival. Immunofluorescence and FACS analysis were used to detect the stem cell marker CD90. Alizarin Red S and Oil Red O staining were used to identify osteoblast and adipocyte differentiation, respectively. To characterize TRPM4, patch-clamp recordings were obtained from single cells in the whole-cell configuration mode. The significance of TRPM4 for proliferation and survival was examined with 9-phenanthrol, a TRPM4 inhibitor during a 96-hour period of culture. Real-time Ca2+ imaging analysis with Fura-2AM was used to investigate the impact of TRPM4 on intracellular Ca2+ signals. DPSCs were CD90-positive and differentiated into osteoblasts. Patch-clamp recordings revealed currents typical of TRPM4 that were Ca2+ -activated, voltage-dependent and Na+ -conducting. Inhibition of TRPM4 resulted in a significant reduction in the cell population after a 96-hr period of culture and transformed the biphasic pattern of intracellular Ca2+ signalling into sustained oscillations. Rat DPSCs have stem cell characteristics and functional TRPM4 channels that are required for proliferation and survival. These data suggest that the shape and frequency of intracellular Ca2+ signals may mediate stem cell proliferation and survival.

Regulation of transient receptor potential melastatin 4 channel by sarcoplasmic reticulum inositol trisphosphate receptors: Role in human detrusor smooth muscle function

ABSTRACTWe recently reported key physiologic roles for Ca2+-activated transient receptor potential melastatin 4 (TRPM4) channels in detrusor smooth muscle (DSM). However, the Ca2+-signaling mechanisms governing TRPM4 channel activity in human DSM cells are unexplored. As the TRPM4 channels are activated by Ca2+, inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca2+ release from the sarcoplasmic reticulum represents a potential Ca2+ source for TRPM4 channel activation. We used clinically-characterized human DSM tissues to investigate the molecular and functional interactions of the IP3Rs and TRPM4 channels. With in situ proximity ligation assay (PLA) and perforated patch-clamp electrophysiology, we tested the hypothesis that TRPM4 channels are tightly associated with the IP3Rs and are activated by IP3R-mediated Ca2+ release in human DSM. With in situ PLA, we demonstrated co-localization of the TRPM4 channels and IP3Rs in human DSM cells. As the TRPM4 channels and IP3Rs must be located within close apposition to functionally interact, these findings support the concept of a potential Ca2+-mediated TRPM4-IP3R regulatory mechanism. To investigate IP3R regulation of TRPM4 channel activity, we sought to determine the consequences of IP3R pharmacological inhibition on TRPM4 channel-mediated transient inward cation currents (TICCs). In freshly-isolated human DSM cells, blocking the IP3Rs with the selective IP3R inhibitor xestospongin-C significantly decreased TICCs. The data suggest that IP3Rs have a key role in mediating the Ca2+-dependent activation of TRPM4 channels in human DSM. The study provides novel insight into the molecular and cellular mechanisms regulating TRPM4 channels by revealing that TRPM4 channels and IP3Rs are spatially and functionally coupled in human DSM.

Identification of the ADPR binding pocket in the NUDT9 homology domain of TRPM2.

Activation of the transient receptor potential melastatin 2 (TRPM2) channel occurs during the response to oxidative stress under physiological conditions as well as in pathological processes such as ischemia and diabetes. Accumulating evidence indicates that adenosine diphosphate ribose (ADPR) is the most important endogenous ligand of TRPM2. However, although it is known that ADPR binds to the NUDT9 homology (NUDT9-H) domain in the intracellular C-terminal region, the molecular mechanism underlying ADPR binding and activation of TRPM2 remains unknown. In this study, we generate a structural model of the NUDT9-H domain and identify the binding pocket for ADPR using induced docking and molecular dynamics simulation. We find a subset of 11 residues-H1346, T1347, T1349, L1379, G1389, S1391, E1409, D1431, R1433, L1484, and H1488-that are most likely to directly interact with ADPR. Results from mutagenesis and electrophysiology approaches support the predicted binding mechanism, indicating that ADPR binds tightly to the NUDT9-H domain, and suggest that the most significant interactions are the van der Waals forces with S1391 and L1484, polar solvation interaction with E1409, and electronic interactions (including π-π interactions) with H1346, T1347, Y1349, D1431, and H1488. These findings not only clarify the roles of a range of newly identified residues involved in ADPR binding in the TRPM2 channel, but also reveal the binding pocket for ADPR in the NUDT9-H domain, which should facilitate structure-based drug design for the TRPM2 channel.

Evaluation of the TRPM2 channel as a biomarker in breast cancer using public databases analysis

BackgroundBreast cancer is one of the most common malignancies affecting women. Recent investigations have revealed a major role of ion channels in cancer. The transient receptor potential melastatin-2 (TRPM2) is a plasma membrane and lysosomal channel with important roles in cell migration and cell death in immune cells and tumor cells. MethodsIn this study, we investigated the prognostic value of TRPM2 channel in breast cancer, analyzing public databases compiled in Oncomine™ (Thermo Fisher, Ann Arbor, MI) and online Kaplan-Meier Plotter platforms. ResultsThe results revealed that TRPM2 mRNA overexpression is significant in situ and invasive breast carcinoma compared to normal breast tissue. Furthermore, multi-gene validation using Oncomine™ showed that this channel is coexpressed with proteins related to cellular migration, transformation, and apoptosis. On the other hand, Kaplan-Meier analysis exhibited that low expression of TRPM2 could be used to predict poor outcome in ER- and HER2+ breast carcinoma patients. ConclusionsTRPM2 is a promising biomarker for aggressiveness of breast cancer, and a potential target for the development of new therapies.

Duloxetine Reduces Oxidative Stress, Apoptosis, and Ca2+ Entry Through Modulation of TRPM2 and TRPV1 Channels in the Hippocampus and Dorsal Root Ganglion of Rats.

Overload of Ca2+ entry and excessive oxidative stress in neurons are the two main causes of depression. Activation of transient receptor potential (TRP) vanilloid type 1 (TRPV1) and TRP melastatin 2 (TRPM2) during oxidative stress has been linked to neuronal survival. Duloxetine (DULOX) in neurons reduced the effects of Ca2+ entry and reactive oxygen species (ROS) through glutamate receptors, and this reduction of effects may also occur through TRPM2 and TRPV1 channels. In order to better characterize the actions of DULOX in peripheral pain and hippocampal oxidative injury through modulation of TRPM2 and TRPV1, we tested the effects of DULOX on apoptosis and oxidative stress in the hippocampal and dorsal root ganglion (DRG) neurons of rats. Freshly isolated hippocampal and DRG neurons were incubated for 24h with DULOX. In whole-cell patch-clamp and intracellular-free calcium ([Ca2+]) concentration (Fura-2) experiments, cumene hydroperoxide and ADP-ribose-induced TRPM2 currents in the neurons were inhibited by N-(p-amylcinnamoyl) anthranilic acid (ACA) and capsaicin-induced TRPV1 currents were inhibited by capsazepine (CPZ) incubations. TRPM2 and TRPV1 channel current densities, [Ca2+] concentration, apoptosis, caspase 3, caspase 9, mitochondrial depolarization, and intracellular ROS production values in the neurons were lower in the DULOX group than in controls. In addition, the above values were further decreased by DULOX + CPZ and DULOX + ACA treatments. In conclusion, TRPM2 and TRPV1 channels are involved in Ca2+ entry-induced neuronal death and modulation of the activity of these channels by DULOX treatment may account for their neuroprotective activity against apoptosis, excessive ROS production, and Ca2+ entry.

MP28-17 SPATIAL AND FUNCTIONAL INTERACTIONS OF THE TRANSIENT RECEPTOR POTENTIAL MELASTATIN-4 CHANNELS AND INOSITOL TRISPHOSPHATE RECEPTORS: NOVEL REGULATORY MECHANISM IN HUMAN DETRUSOR SMOOTH MUSCLE FUNCTION

You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology I1 Apr 2016MP28-17 SPATIAL AND FUNCTIONAL INTERACTIONS OF THE TRANSIENT RECEPTOR POTENTIAL MELASTATIN-4 CHANNELS AND INOSITOL TRISPHOSPHATE RECEPTORS: NOVEL REGULATORY MECHANISM IN HUMAN DETRUSOR SMOOTH MUSCLE FUNCTION Aaron Provence, Kiril Hristov, Eric Rovner, and Georgi V. Petkov Aaron ProvenceAaron Provence More articles by this author , Kiril HristovKiril Hristov More articles by this author , Eric RovnerEric Rovner More articles by this author , and Georgi V. PetkovGeorgi V. Petkov More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1067AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Transient receptor potential melastatin-4 (TRPM4) channels are emerging as key regulators of human detrusor smooth muscle (DSM) function. However, the cellular mechanisms regulating TRPM4 channel activity in human DSM is virtually unexplored. As a potential cellular mechanism regulating TRPM4 channel activity in human DSM, we have directed our attention to the inositol trisphosphate (IP3) receptors (IP3Rs), which facilitate the release of Ca2+ from the sarcoplasmic reticulum (SR). Since the TRPM4 channels are activated by Ca2+, IP3R-mediated Ca2+ release of the SR represents a potential Ca2+ source for TRPM4 channel activation. Using clinically-characterized human tissues, we investigated the molecular and functional interactions of the TRPM4 channels and IP3Rs in human DSM cells. METHODS In accordance with the approved IRB Pro00045232 of the Medical University of South Carolina, we examined human DSM tissues from 6 donor-patients. Using in situ proximity ligation assay (PLA) and amphotericin-B perforated patch-clamp electrophysiology, we sought to test the hypothesis that TRPM4 channels are tightly associated with the IP3Rs and are activated by IP3R-mediated Ca2+ release of the SR in human DSM. RESULTS As determined by in situ PLA, we demonstrated close co-localization of the TRPM4 channels and IP3Rs in human DSM cells. As the TRPM4 channels and IP3Rs must be located within close apposition to functionally interact, these findings support the concept of a TRPM4-IP3R regulatory mechanism in human DSM function. To investigate IP3R regulation of TRPM4 channel activity, we sought to determine the consequences of IP3R pharmacological inhibition on TRPM4 channel-mediated transient inward cation currents (TICCs). In freshly-isolated human DSM cells, the selective IP3R inhibitor xestospongin C significantly decreased TICCs. These findings suggest that the SR IP3Rs have a key role in mediating the Ca2+-dependent activation of TRPM4 channels in human DSM. CONCLUSIONS In conclusion, this study provides novel insight into the cellular mechanisms regulating TRPM4 channel activity in human DSM function. The study reveals that the TRPM4 channels and IP3Rs are spatially and functionally coupled in human DSM, information that is critical for further evaluating the potential role of the TRPM4 channels as novel therapeutic targets for overactive bladder. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e378 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Aaron Provence More articles by this author Kiril Hristov More articles by this author Eric Rovner More articles by this author Georgi V. Petkov More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Modulation of Diabetes-Induced Oxidative Stress, Apoptosis, and Ca2+ Entry Through TRPM2 and TRPV1 Channels in Dorsal Root Ganglion and Hippocampus of Diabetic Rats by Melatonin and Selenium

Neuropathic pain and hippocampal injury can arise from the overload of diabetes-induced calcium ion (Ca2+) entry and oxidative stress. The transient receptor potential (TRP) melastatin 2 (TRPM2) and TRP vanilloid type 1 (TRPV1) are expressed in sensory neurons and hippocampus. Moreover, activations of TRPM2 and TRPV1 during oxidative stress have been linked to neuronal death. Melatonin (MEL) and selenium (Se) have been considered potent antioxidants that detoxify a variety of reactive oxygen species (ROS) in neurological diseases. In order to better characterize the actions of MEL and Se in diabetes-induced peripheral pain and hippocampal injury through modulation of TRPM2 and TRPV1, we tested the effects of MEL and Se on apoptosis and oxidative stress in the hippocampal and dorsal root ganglion (DRG) neurons of streptozotocin (STZ)-induced diabetic rats. Fifty-eight rats were divided into six groups. The first group was used as control. The second group was used as the diabetic group. The third and fourth groups received Se and MEL, respectively. Intraperitoneal Se and MEL were given to diabetic rats in the fifth and sixth groups. On the 14th day, hippocampal and DRG neuron samples were freshly taken from all animals. The neurons were stimulated with a TRPV1 channel agonist (capsaicin) and a TRPM2 channel agonist (cumene hydroperoxide). We observed a modulator role of MEL and Se on intracellular free Ca2+ concentrations, current densities of TRPM2 and TRPV1 channels, apoptosis, caspase 3, caspase 9, mitochondrial depolarization, reduced glutathione, glutathione peroxidase, lipid peroxidation, and intracellular ROS production values in the neurons. In addition, procaspase 3 and 9 activities in western blot analyses of the brain cortex were also decreased by MEL and Se treatments. In conclusion, in our diabetes experimental model, TRPM2 and TRPV1 channels are involved in the Ca2+ entry-induced neuronal death and modulation of this channel activity by MEL and Se treatment may account for their neuroprotective activity against apoptosis and Ca2+ entry. Graphical Abstract Possible molecular pathways of involvement of melatonin and selenium in diabetes-induced apoptosis, oxidative stress, and calcium accumulation through TRPM2 and TRPV1 channels in the hippocampus and DRG neurons of rats. The TRPM2 channel is activated by ADP-ribose and oxidative stress although it is inhibited by ACA. The TRPV1 channel is activated by oxidative stress and capsaicin and it is blocked by capsazepine (CPZ). Diabetes can result in augmented ROS release in hippocampal and DRG neurons through polyol reactions, leading to Ca2+ uptake through TRPM2 and TRPV1 channels. Mitochondria were reported to accumulate Ca2+ provided intracellular Ca2+ rises, thereby leading to the depolarization of mitochondrial membranes and release of apoptosis-inducing factors such as caspase 3 and caspase 9. Melatonin and selenium reduce TRPM2 and TRPV1 channel activation through the modulation of polyol oxidative reactions and selenium-dependent glutathione peroxidase (GSH-Px) antioxidant pathways.

Short-Term Ketamine Treatment Decreases Oxidative Stress Without Influencing TRPM2 and TRPV1 Channel Gating in the Hippocampus and Dorsal Root Ganglion of Rats.

Calcium ions (Ca2+) are important second messengers in neurons. Ketamine (KETAM) is an anesthetic and analgesic, with psychotomimetic effects and abuse potential. KETAM modulates the entry of Ca2+ in neurons through glutamate receptors, but its effect on transient receptor potential melastatin 2 (TRPM2) and transient receptor potential vanilloid 1 (TRPV1) channels has not been clarified. This study investigated the short-term effects of KETAM on oxidative stress and TRPM2 and TRPV1 channel gating in hippocampal and dorsal root ganglion (DRG) neurons of rats. Freshly isolated hippocampal and DRG neurons were incubated for 24h with KETAM (0.3mM). The TRPM2 channel antagonist, N-(p-amylcinnamoyl)anthranilic acid (ACA), inhibited cumene hydroperoxide and ADP-ribose-induced TRPM2 currents in the neurons, and capsazepine (CPZ) inhibited capsaicin-induced TRPV1 currents. The TRPM2 and TRPV1 channel current densities and intracellular free calcium ion concentration of the neurons were lower in the neurons exposed to ACA and CPZ compared to the control neurons, respectively. However, the values were not further decreased by the KETAM+CPZ and KETAM+ACA treatments. KETAM decreased lipid peroxidation levels in the neurons but increased glutathione peroxidase activity. In conclusion, short-term KETAM treatment decreased oxidative stress levels but did not seem to influence TRPM2- and TRPV1-mediated Ca2+ entry.

Novel regulatory mechanism in human urinary bladder: central role of transient receptor potential melastatin 4 channels in detrusor smooth muscle function.

Transient receptor potential melastatin 4 (TRPM4) channels are Ca(2+)-activated nonselective cation channels that have been recently identified as regulators of detrusor smooth muscle (DSM) function in rodents. However, their expression and function in human DSM remain unexplored. We provide insights into the functional role of TRPM4 channels in human DSM under physiological conditions. We used a multidisciplinary experimental approach, including RT-PCR, Western blotting, immunohistochemistry and immunocytochemistry, patch-clamp electrophysiology, and functional studies of DSM contractility. DSM samples were obtained from patients without preoperative overactive bladder symptoms. RT-PCR detected mRNA transcripts for TRPM4 channels in human DSM whole tissue and freshly isolated single cells. Western blotting and immunohistochemistry with confocal microscopy revealed TRPM4 protein expression in human DSM. Immunocytochemistry further detected TRPM4 protein expression in DSM single cells. Patch-clamp experiments showed that 9-phenanthrol, a selective TRPM4 channel inhibitor, significantly decreased the transient inward cation currents and voltage step-induced whole cell currents in freshly isolated human DSM cells. In current-clamp mode, 9-phenanthrol hyperpolarized the human DSM cell membrane potential. Furthermore, 9-phenanthrol attenuated the spontaneous phasic, carbachol-induced and nerve-evoked contractions in human DSM isolated strips. Significant species-related differences in TRPM4 channel activity between human, rat, and guinea pig DSM were revealed, suggesting a more prominent physiological role for the TRPM4 channel in the regulation of DSM function in humans than in rodents. In conclusion, TRPM4 channels regulate human DSM excitability and contractility and are critical determinants of human urinary bladder function. Thus, TRPM4 channels could represent promising novel targets for the pharmacological or genetic control of overactive bladder.

A dual role of transient receptor potential melastatin 2 channel in cytotoxicity induced by silica nanoparticles.

Silica nanoparticles (NPs) have remarkable applications. However, accumulating evidence suggests NPs can cause cellular toxicity by inducing ROS production and increasing intracellular Ca2+ ([Ca2+]i), but the underlying molecular mechanism is largely unknown. Transient receptor potential melastatin 2 (TRPM2) channel is known to be a cellular redox potential sensor that provides an important pathway for increasing the [Ca2+]i under oxidative stress. In this study, we examined the role of TRPM2 channel in silica NPs-induced oxidative stress and cell death. By quantitation of cell viability, ROS production, [Ca2+]i, and protein identification, we showed that TRPM2 channel is required for ROS production and Ca2+ increase induced by silica NPs through regulating NADPH oxidase activity in HEK293 cells. Strikingly, HEK293 cells expressing low levels of TRPM2 were more susceptible to silica NPs than those expressing high levels of TRPM2. Macrophages from young mice showed significantly lower TRPM2 expression than those from senescent mice and had significantly lower viability after silica NPs exposure than those from senescent ones. Taken together, these findings demonstrate for the first time that TRPM2 channel acts as an oxidative stress sensor that plays a dual role in silica NPs-induced cytotoxicity by differentially regulating the NADPH oxidase activity and ROS generation.

The Transient Receptor Potential Melastatin 2 (TRPM2) Channel Contributes to β-Amyloid Oligomer-Related Neurotoxicity and Memory Impairment.

Transient receptor potential melastatin 2 (TRPM2) is an oxidative stress sensing calcium-permeable channel that is thought to contribute to calcium dysregulation associated with neurodegenerative diseases, including Alzheimer's disease. Here we show that oligomeric β-amyloid, the toxic peptide in Alzheimer's disease, facilitates TRPM2 channel activation. In mice designed to model Alzheimer's disease, genetic elimination of TRPM2 normalized deficits in synaptic markers in aged mice. Moreover, the absence of TRPM2 improved age-dependent spatial memory deficits observed in Alzheimer's mice. Our results reveal the importance of TRPM2 for neuronal toxicity and memory impairments in an Alzheimer's mouse model and suggest that TRPM2 could be targeted for the development of therapeutic agents effective in the treatment of dementia.

Enhanced cytotoxicity in triple-negative and estrogen receptor‑positive breast adenocarcinoma cells due to inhibition of the transient receptor potential melastatin-2 channel.

We previously demonstrated a unique protective role for the transient receptor potential, melastatin-2 (TRPM2) cation channel in breast cancer cells. In the present study, we investigated the chemotherapeutic effects elicited by inhibiting this protective role in metastatic breast adenocarcinoma cells. TRPM2 inhibition led to dose-dependent increases in MDA-MB-231 breast adenocarcinoma cell death after treatment with doxorubicin or the DNA-methylating agent, N-methyl-N'-nitro-N-nitrosoguanidine. Similar results were observed after RNAi silencing of TRPM2 in these cells after doxorubicin treatment. However, TRPM2 RNAi silencing also led to increased MCF-7 breast adenocarcinoma cell death after tamoxifen treatment, yet not in non-cancerous human mammary epithelial cells. These results thus revealed that TRPM2 inhibition selectively increased cytotoxicity in a triple-negative and an estrogen receptor-positive breast cancer cell line, with minimal deleterious effects in non-cancerous breast cells. Analysis of DNA damage revealed enhanced DNA damage levels in MCF-7 cells treated with doxorubicin due to TRPM2 inhibition. Analysis of cell death demonstrated that inhibition of apoptosis, caspase-independent cell death or autophagy failed to significantly reduce cell death induced by TRPM2 inhibition and chemotherapy. These results indicate that TRPM2 inhibition activates alternative pathways of cell death in breast cancer cells. Taken together, our results provide significant evidence that TRPM2 inhibition is a potential strategy to induce triple-negative and estrogen receptor-positive breast adenocarcinoma cell death via alternative cell death pathways. This is expected to provide a basis for inhibiting TRPM2 for the improved treatment of breast cancer, which potentially includes treating breast tumors that are resistant to chemotherapy due to their evasion of apoptosis.

PIP2 and PIP3 interact with N-terminus region of TRPM4 channel

The transient receptor potential melastatin 4 (TRPM4) is a calcium-activated non-selective ion channel broadly expressed in a variety of tissues. Receptor has been identified as a crucial modulator of numerous calcium dependent mechanisms in the cell such as immune response, cardiac conduction, neurotransmission and insulin secretion. It is known that phosphoinositide lipids (PIPs) play a unique role in the regulation of TRP channel function. However the molecular mechanism of this process is still unknown. We characterized the binding site of PIP2 and its structural analogue PIP3 in the E733–W772 proximal region of the TRPM4 N-terminus via biophysical and molecular modeling methods. The specific positions R755 and R767 in this domain were identified as being important for interactions with PIP2/PIP3 ligands. Their mutations caused a partial loss of PIP2/PIP3 binding specificity. The interaction of PIP3 with TRPM4 channels has never been described before. These findings provide new insight into the ligand binding domains of the TRPM4 channel.

Ruling out pyridine dinucleotides as true TRPM2 channel activators reveals novel direct agonist ADP-ribose-2'-phosphate.

Transient receptor potential melastatin 2 (TRPM2), a Ca(2+)-permeable cation channel implicated in postischemic neuronal cell death, leukocyte activation, and insulin secretion, is activated by intracellular ADP ribose (ADPR). In addition, the pyridine dinucleotides nicotinamide-adenine-dinucleotide (NAD), nicotinic acid-adenine-dinucleotide (NAAD), and NAAD-2'-phosphate (NAADP) have been shown to activate TRPM2, or to enhance its activation by ADPR, when dialyzed into cells. The precise subset of nucleotides that act directly on the TRPM2 protein, however, is unknown. Here, we use a heterologously expressed, affinity-purified-specific ADPR hydrolase to purify commercial preparations of pyridine dinucleotides from substantial contaminations by ADPR or ADPR-2'-phosphate (ADPRP). Direct application of purified NAD, NAAD, or NAADP to the cytosolic face of TRPM2 channels in inside-out patches demonstrated that none of them stimulates gating, or affects channel activation by ADPR, indicating that none of these dinucleotides directly binds to TRPM2. Instead, our experiments identify for the first time ADPRP as a true direct TRPM2 agonist of potential biological interest.

Inhibition of the transient receptor potential melastatin-2 channel causes increased DNA damage and decreased proliferation in breast adenocarcinoma cells.

Transient receptor potential, melastatin-2 (TRPM2) is a plasma membrane cation channel with important roles in sensory functions and promoting cell death. However, we demonstrated here that TRPM2 was present in the nuclei of MCF-7 and MDA-MB-231 human breast adenocarcinoma cells, and its pharmacologic inhibition or RNAi silencing caused decreased cell proliferation. Neither an effect on proliferation nor a localization of TRPM2 in the nucleus was observed in noncancerous HMEC and MCF-10A human mammary epithelial cells. Investigation of possible effects of TRPM2 function in the nucleus demonstrated that pharmacologic inhibition or RNAi silencing of TRPM2 in MCF-7 and MDA-MB-231 human breast adenocarcinoma cells caused up to 4-fold increases in DNA damage levels, as compared to noncancerous breast cells after equivalent treatments. These results indicate that TRPM2 has a novel nuclear function in human breast adenocarcinoma cells that facilitates the integrity of genomic DNA, a finding that is distinct from its previously reported role as a plasma membrane cation channel in noncancerous cells. In summary, we report here a novel effect promoted by TRPM2, where it functions to minimize DNA damage and thus may have a role in the protection of genomic DNA in breast cancer cells. Our study therefore provides compelling evidence that TRPM2 has a unique role in breast adenocarcinoma cells. Accordingly, these studies suggest that TRPM2 is a potential therapeutic target, where its pharmacologic inhibition may provide an innovative strategy to selectively increase DNA damage levels in breast cancer cells.

Chemical Activation of Endogenous and Recombinant TRPM4 Channels

TRPM4 (transient receptor potential melastatin 4) channels constitute Ca2+-activated non-selective cationic currents in a variety of tissues (e.g. heart and brain) and in cells of the immune system. The channels are gated by elevated cytosolic Ca2+, and channel activity is modulated by signalling molecules such as ATP and phospholipids. Activation of TRPM4 depolarises the membrane potential, which modulates the driving force for ions and the activity of ion channels. TRPM4 channel agonists and Ca2+-independent gating have not been demonstrated yet. In patch clamp experiments, the maleimide U73122, which is widely used as phospholipase C inhibitor, activated recombinant human TRPM4 and endogenous TRPM4 channels in cell lines. In contrast, U73122 inhibited TRPM3 channels and did not affect TRPM5 channel activity. TRPM4 current amplitudes were independent on phospholipid levels and, strikingly, U73122 activated TRPM4 even in absence of intracellular Ca2+. As the structural analogue U73343 was ineffective, these findings suggested covalent modification as the underlying mechanism. In fact, pre-application of N-ethylmaleimide (NEM) to prevent further covalent modification abolished the activation of TRPM4 by U73122. Taken together, we demonstrated activation of otherwise Ca2+-dependent TRPM4 channels by U73122. These findings raise the possibility that endogenous substances act as physiologically relevant TRPM4 agonists. This work was supported by a Research Grant of the University Medical Center Giessen und Marburg (UKGM) to M.G.L and by Deutsche Forschungsgemeinschaft through SFB 593 to J.O. (TPA16) and D.O. (TPA12).