Deletion of Trpm4 Alters the Function of the Nav1.5 Channel in Murine Cardiac Myocytes

Transient receptor potential melastatin member 4 (TRPM4) encodes a Ca²⁺-activated, non-selective cation channel that is functionally expressed in several tissues, including the heart. Pathogenic mutants in TRPM4 have been reported in patients with inherited cardiac diseases, including conduction blockage and Brugada syndrome. Heterologous expression of mutant channels in cell lines indicates that these mutations can lead to an increase or decrease in TRPM4 expression and function at the cell surface. While the expression and clinical variant studies further stress the importance of TRPM4 in cardiac function, the cardiac electrophysiological phenotypes in Trpm4 knockdown mouse models remain incompletely characterized. To study the functional consequences of Trpm4 deletion on cardiac electrical activity in mice, we performed perforated-patch clamp and immunoblotting studies on isolated atrial and ventricular cardiac myocytes and surfaces, as well as on pseudo- and intracardiac ECGs, either in vivo or in Langendorff-perfused explanted mouse hearts. We observed that TRPM4 is expressed in atrial and ventricular cardiac myocytes and that deletion of Trpm4 unexpectedly reduces the peak Na⁺ currents in myocytes. Hearts from Trpm4^−/− mice presented increased sensitivity towards mexiletine, a Na⁺ channel blocker, and slower intraventricular conduction, consistent with the reduction of the peak Na⁺ current observed in the isolated cardiac myocytes. This study suggests that TRPM4 expression impacts the Na⁺ current in murine cardiac myocytes and points towards a novel function of TRPM4 regulating the Na_v1.5 function in murine cardiac myocytes.     

Peripheral Ion Channel Gene Screening in Painful- and Painless-Diabetic Neuropathy

Neuropathic pain is common in diabetic peripheral neuropathy (DN), probably caused by pathogenic ion channel gene variants. Therefore, we performed molecular inversion probes-next generation sequencing of 5 transient receptor potential cation channels, 8 potassium channels and 2 calcium-activated chloride channel genes in 222 painful- and 304 painless-DN patients. Twelve painful-DN (5.4%) patients showed potentially pathogenic variants (five nonsense/frameshift, seven missense, one out-of-frame deletion) in ANO3 (n = 3), HCN1 (n = 1), KCNK18 (n = 2), TRPA1 (n = 3), TRPM8 (n = 3) and TRPV4 (n = 1) and fourteen painless-DN patients (4.6%—three nonsense/frameshift, nine missense, one out-of-frame deletion) in ANO1 (n = 1), KCNK18 (n = 3), KCNQ3 (n = 1), TRPA1 (n = 2), TRPM8 (n = 1), TRPV1 (n = 3) and TRPV4 (n = 3). Missense variants were present in both conditions, presumably with loss- or gain-of-functions. KCNK18 nonsense/frameshift variants were found in painless/painful-DN, making a causal role in pain less likely. Surprisingly, premature stop-codons with likely nonsense-mediated RNA-decay were more frequent in painful-DN. Although limited in number, painful-DN patients with ion channel gene variants reported higher maximal pain during the night and day. Moreover, painful-DN patients with TRP variants had abnormal thermal thresholds and more severe pain during the night and day. Our results suggest a role of ion channel gene variants in neuropathic pain, but functional validation is required.     

TRPM8 Channels: Advances in Structural Studies and Pharmacological Modulation

The transient receptor potential melastatin subtype 8 (TRPM8) is a cold sensor in humans, activated by low temperatures (>10, <28 °C), but also a polymodal ion channel, stimulated by voltage, pressure, cooling compounds (menthol, icilin), and hyperosmolarity. An increased number of experimental results indicate the implication of TRPM8 channels in cold thermal transduction and pain detection, transmission, and maintenance in different tissues and organs. These channels also have a repercussion on different kinds of life-threatening tumors and other pathologies, which include urinary and respiratory tract dysfunctions, dry eye disease, and obesity. This compendium firstly covers newly described papers on the expression of TRPM8 channels and their correlation with pathological states. An overview on the structural knowledge, after cryo-electron microscopy success in solving different TRPM8 structures, as well as some insights obtained from mutagenesis studies, will follow. Most recently described families of TRPM8 modulators are also covered, along with a section of molecules that have reached clinical trials. To finalize, authors provide an outline of the potential prospects in the TRPM8 field.     

Oxaliplatin Causes Transient Changes in TRPM8 Channel Activity

Oxaliplatin is a third-generation platinum-based anticancer drug that is widely used as first-line treatment for colorectal carcinoma. Patients treated with oxaliplatin develop an acute peripheral pain several hours after treatment, mostly characterized by cold allodynia as well as a long-term chronic neuropathy. These two phenomena seem to be causally connected. However, the underlying mechanisms that trigger the acute peripheral pain are still poorly understood. Here we show that the activity of the transient receptor potential melastatin 8 (TRPM8) channel but not the activity of any other member of the TRP channel family is transiently increased 1 h after oxaliplatin treatment and decreased 24 h after oxaliplatin treatment. Mechanistically, this is connected with activation of the phospholipase C (PLC) pathway and depletion of phosphatidylinositol 4,5-bisphosphate (PIP₂) after oxaliplatin treatment. Inhibition of the PLC pathway can reverse the decreased TRPM8 activity as well as the decreased PIP₂-concentrations after oxaliplatin treatment. In summary, these results point out transient changes in TRPM8 activity early after oxaliplatin treatment and a later occurring TRPM8 channel desensitization in primary sensory neurons. These mechanisms may explain the transient cold allodynia after oxaliplatin treatment and highlight an important role of TRPM8 in oxaliplatin-induced acute and neuropathic pain.     

β–Lactam TRPM8 Antagonist RGM8-51 Displays Antinociceptive Activity in Different Animal Models

Transient receptor potential melastatin subtype 8 (TRPM8) is a cation channel extensively expressed in sensory neurons and implicated in different painful states. However, the effectiveness of TRPM8 modulators for pain relief is still a matter of discussion, since structurally diverse modulators lead to different results, depending on the animal pain model. In this work, we described the antinociceptive activity of a β–lactam derivative, RGM8-51, showing good TRPM8 antagonist activity, and selectivity against related thermoTRP channels and other pain-mediating receptors. In primary cultures of rat dorsal root ganglion (DRG) neurons, RGM8-51 potently reduced menthol-evoked neuronal firing without affecting the major ion conductances responsible for action potential generation. This compound has in vivo antinociceptive activity in response to cold, in a mouse model of oxaliplatin-induced peripheral neuropathy. In addition, it reduces cold, mechanical and heat hypersensitivity in a rat model of neuropathic pain arising after chronic constriction of the sciatic nerve. Furthermore, RGM8-51 exhibits mechanical hypersensitivity-relieving activity, in a mouse model of NTG-induced hyperesthesia. Taken together, these preclinical results substantiate that this TRPM8 antagonist is a promising pharmacological tool to study TRPM8-related diseases.     

Expression of the C-Terminal Domain of Phospholipase Cβ3 Inhibits Signaling via Gαq-Coupled Receptors and Transient Receptor Potential Channels

Transient receptor potential (TRP) channels are cation channels that play a regulatory role in pain and thermosensation, insulin secretion, and neurotransmission. It has been proposed that activation of TRP channels requires phosphatidylinositol 4,5-bisphosphate, the major substrate for phospholipase C (PLC). We investigated whether inhibition of PLCβ has an impact on TRP channel signaling. A genetic approach was used to avoid off-target effects observed when using a pharmacological PLCβ inhibitor. In this study, we show that expression of PLCβ1ct and PLCβ3ct, truncated forms of PLCβ1 or PLCβ3 that contain the C-terminal membrane binding domains, almost completely blocked the signal transduction of a Gαq-coupled designer receptor, including the phosphorylation of ERK1/2. In contrast, expression of the helix-turn-helix motif (Hα1—Hα2) of the proximal C-terminal domain of PLCβ3 did not affect Gαq-coupled receptor signaling. PLCβ3ct expression impaired signaling of the TRP channels TRPM3 and TRPM8, stimulated with either prognenolone sulfate or icilin. Thus, the C-terminal domain of PLCβ3 interacts with plasma membrane targets, most likely phosphatidylinositol 4,5-bisphosphate, and in this way blocks the biological activation of TRPM3 and TRPM8, which require interaction with this phospholipid. PLCβ thus regulates TRPM3 and TRPM8 channels by masking phosphatidylinositol 4,5-bisphosphate with its C-terminal domain.     

In Vitro and In Vivo Pharmacological Characterization of a Novel TRPM8 Inhibitor Chemotype Identified by Small-Scale Preclinical Screening

Transient receptor potential melastatin type 8 (TRPM8) is a target for the treatment of different physio-pathological processes. While TRPM8 antagonists are reported as potential drugs for pain, cancer, and inflammation, to date only a limited number of chemotypes have been investigated and thus a limited number of compounds have reached clinical trials. Hence there is high value in searching for new TRPM8 antagonistic to broaden clues to structure-activity relationships, improve pharmacological properties and explore underlying molecular mechanisms. To address this, the EDASA Scientific in-house molecular library has been screened in silico, leading to identifying twenty-one potentially antagonist compounds of TRPM8. Calcium fluorometric assays were used to validate the in-silico hypothesis and assess compound selectivity. Four compounds were identified as selective TRPM8 antagonists, of which two were dual-acting TRPM8/TRPV1 modulators. The most potent TRPM8 antagonists (BB 0322703 and BB 0322720) underwent molecular modelling studies to highlight key structural features responsible for drug–protein interaction. The two compounds were also investigated by patch-clamp assays, confirming low micromolar potencies. The most potent compound (BB 0322703, IC₅₀ 1.25 ± 0.26 μM) was then profiled in vivo in a cold allodinya model, showing pharmacological efficacy at 30 μM dose. The new chemotypes identified showed remarkable pharmacological properties paving the way to further investigations for drug discovery and pharmacological purposes.     

Validation of Six Commercial Antibodies for the Detection of Heterologous and Endogenous TRPM8 Ion Channel Expression

TRPM8 is a non-selective cation channel expressed in primary sensory neurons and other tissues, including the prostate and urothelium. Its participation in different physiological and pathological processes such as thermoregulation, pain, itch, inflammation and cancer has been widely described, making it a promising target for therapeutic approaches. The detection and quantification of TRPM8 seems crucial for advancing the knowledge of the mechanisms underlying its role in these pathophysiological conditions. Antibody-based techniques are commonly used for protein detection and quantification, although their performance with many ion channels, including TRPM8, is suboptimal. Thus, the search for reliable antibodies is of utmost importance. In this study, we characterized the performance of six TRPM8 commercial antibodies in three immunodetection techniques: Western blot, immunocytochemistry and immunohistochemistry. Different outcomes were obtained for the tested antibodies; two of them proved to be successful in detecting TRPM8 in the three approaches while, in the conditions tested, the other four were acceptable only for specific techniques. Considering our results, we offer some insight into the usefulness of these antibodies for the detection of TRPM8 depending on the methodology of choice.     

The Association between TRP Channels Expression and Clinicopathological Characteristics of Patients with Pancreatic Adenocarcinoma

Pancreatic adenocarcinoma (PDAC) has low survival rates worldwide due to its tendency to be detected late and its characteristic desmoplastic reaction, which slows the use of targeted therapies. As such, the discovery of new connections between genes and the clinicopathological parameters contribute to the search for new biomarkers or targets for therapy. Transient receptor potential (TRP) channels are promising tools for cancer therapy or markers for PDAC. Therefore, in this study, we selected several genes encoding TRP proteins previously reported in cellular models, namely, Transient Receptor Potential Cation Channel Subfamily V Member 6 (TRPV6), Transient receptor potential ankyrin 1 (TRPA1), and Transient receptor potential cation channel subfamily M (melastatin) member 8 (TRPM8), as well as the TRPM8 Channel Associated Factor 1 (TCAF1) and TRPM8 Channel Associated Factor 2 (TCAF2) proteins, as regulatory factors. We analyzed the expression levels of tumors from patients enrolled in public datasets and confirmed the results with a validation cohort of PDAC patients enrolled in the Clinical Institute Fundeni, Romania. We found significantly higher expression levels of TRPA1, TRPM8, and TCAF1/F2 in tumoral tissues compared to normal tissues, but lower expression levels of TRPV6, suggesting that TRP channels have either tumor-suppressive or oncogenic roles. The expression levels were correlated with the tumoral stages and are related to the genes involved in calcium homeostasis (Calbindin 1 or S100A4) or to proteins participating in metastasis (PTPN1). We conclude that the selected TRP proteins provide new insights in the search for targets and biomarkers needed for therapeutic strategies for PDAC treatment.     

A Central Role for TRPM4 in Ca2+-Signal Amplification and Vasoconstriction

Transient receptor potential melastatin-4 (TRPM4) is activated by an increase in intracellular Ca²⁺ concentration and is expressed on smooth muscle cells (SMCs). It is implicated in the myogenic constriction of cerebral arteries. We hypothesized that TRPM4 has a general role in intracellular Ca²⁺ signal amplification in a wide range of blood vessels. TRPM4 function was tested with the TRPM4 antagonist 9-phenanthrol and the TRPM4 activator A23187 on the cardiovascular responses of the rat, in vivo and in isolated basilar, mesenteric, and skeletal muscle arteries. TRPM4 inhibition by 9-phenanthrol resulted in hypotension and a decreased heart rate in the rat. TRPM4 inhibition completely antagonized myogenic tone development and norepinephrine-evoked vasoconstriction, and depolarization (high extracellular KCl concentration) evoked vasoconstriction in a wide range of peripheral arteries. Vasorelaxation caused by TRPM4 inhibition was accompanied by a significant decrease in intracellular Ca²⁺ concentration, suggesting an inhibition of Ca²⁺ signal amplification. Immunohistochemistry confirmed TRPM4 expression in the smooth muscle cells of the peripheral arteries. Finally, TRPM4 activation by the Ca²⁺ ionophore A23187 was competitively inhibited by 9-phenanthrol. In summary, TRPM4 was identified as an essential Ca²⁺-amplifying channel in peripheral arteries, contributing to both myogenic tone and agonist responses. These results suggest an important role for TRPM4 in the circulation. The modulation of TRPM4 activity may be a therapeutic target for hypertension. Furthermore, the Ca²⁺ ionophore A23187 was identified as the first high-affinity (nanomolar) direct activator of TRPM4, acting on the 9-phenanthrol binding site.     

cADPR Does Not Activate TRPM2

cADPR is a second messenger that releases Ca²⁺ from intracellular stores via the ryanodine receptor. Over more than 15 years, it has been controversially discussed whether cADPR also contributes to the activation of the nucleotide-gated cation channel TRPM2. While some groups have observed activation of TRPM2 by cADPR alone or in synergy with ADPR, sometimes only at 37 °C, others have argued that this is due to the contamination of cADPR by ADPR. The identification of a novel nucleotide-binding site in the N-terminus of TRPM2 that binds ADPR in a horseshoe-like conformation resembling cADPR as well as the cADPR antagonist 8-Br-cADPR, and another report that demonstrates activation of TRPM2 by binding of cADPR to the NUDT9H domain raised the question again and led us to revisit the topic. Here we show that (i) the N-terminal MHR1/2 domain and the C-terminal NUDT9H domain are required for activation of human TRPM2 by ADPR and 2′-deoxy-ADPR (2dADPR), (ii) that pure cADPR does not activate TRPM2 under a variety of conditions that have previously been shown to result in channel activation, (iii) the cADPR antagonist 8-Br-cADPR also inhibits activation of TRPM2 by ADPR, and (iv) cADPR does not bind to the MHR1/2 domain of TRPM2 while ADPR does.     

TRPM8: A Therapeutic Target for Neuroinflammatory Symptoms Induced by Severe Dry Eye Disease

Dry eye disease (DED) is commonly associated with ocular surface inflammation and pain. In this study, we evaluated the effectiveness of repeated instillations of transient receptor potential melastatin 8 (TRPM8) ion channel antagonist M8-B on a mouse model of severe DED induced by the excision of extra-orbital lacrimal and Harderian glands. M8-B was topically administered twice a day from day 7 until day 21 after surgery. Cold and mechanical corneal sensitivities and spontaneous ocular pain were monitored at day 21. Ongoing and cold-evoked ciliary nerve activities were next evaluated by electrophysiological multi-unit extracellular recording. Corneal inflammation and expression of genes related to neuropathic pain and inflammation were assessed in the trigeminal ganglion. We found that DED mice developed a cold allodynia consistent with higher TRPM8 mRNA expression in the trigeminal ganglion (TG). Chronic M8-B instillations markedly reversed both the corneal mechanical allodynia and spontaneous ocular pain commonly associated with persistent DED. M8-B instillations also diminished the sustained spontaneous and cold-evoked ciliary nerve activities observed in DED mice as well as inflammation in the cornea and TG. Overall, our study provides new insight into the effectiveness of TRPM8 blockade for alleviating corneal pain syndrome associated with severe DED, opening a new avenue for ocular pain management.     

Species-Specific Regulation of TRPM2 by PI(4,5)P2 via the Membrane Interfacial Cavity

The human apoptosis channel TRPM2 is stimulated by intracellular ADR-ribose and calcium. Recent studies show pronounced species-specific activation mechanisms. Our aim was to analyse the functional effect of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂), commonly referred to as PIP₂, on different TRPM2 orthologues. Moreover, we wished to identify the interaction site between TRPM2 and PIP₂. We demonstrate a crucial role of PIP₂, in the activation of TRPM2 orthologues of man, zebrafish, and sea anemone. Utilizing inside-out patch clamp recordings of HEK-293 cells transfected with TRPM2, differential effects of PIP₂ that were dependent on the species variant became apparent. While depletion of PIP₂ via polylysine uniformly caused complete inactivation of TRPM2, restoration of channel activity by artificial PIP₂ differed widely. Human TRPM2 was the least sensitive species variant, making it the most susceptible one for regulation by changes in intramembranous PIP₂ content. Furthermore, mutations of highly conserved positively charged amino acid residues in the membrane interfacial cavity reduced the PIP₂ sensitivity in all three TRPM2 orthologues to varying degrees. We conclude that the membrane interfacial cavity acts as a uniform PIP₂ binding site of TRPM2, facilitating channel activation in the presence of ADPR and Ca²⁺ in a species-specific manner.     

Structure and Function of Ion Channels Regulating Sperm Motility—An Overview

Sperm motility is linked to the activation of signaling pathways that trigger movement. These pathways are mainly dependent on Ca²⁺, which acts as a secondary messenger. The maintenance of adequate Ca²⁺ concentrations is possible thanks to proper concentrations of other ions, such as K⁺ and Na⁺, among others, that modulate plasma membrane potential and the intracellular pH. Like in every cell, ion homeostasis in spermatozoa is ensured by a vast spectrum of ion channels supported by the work of ion pumps and transporters. To achieve success in fertilization, sperm ion channels have to be sensitive to various external and internal factors. This sensitivity is provided by specific channel structures. In addition, novel sperm-specific channels or isoforms have been found with compositions that increase the chance of fertilization. Notably, the most significant sperm ion channel is the cation channel of sperm (CatSper), which is a sperm-specific Ca²⁺ channel required for the hyperactivation of sperm motility. The role of other ion channels in the spermatozoa, such as voltage-gated Ca²⁺ channels (VGCCs), Ca²⁺-activated Cl-channels (CaCCs), SLO K⁺ channels or voltage-gated H⁺ channels (VGHCs), is to ensure the activation and modulation of CatSper. As the activation of sperm motility differs among metazoa, different ion channels may participate; however, knowledge regarding these channels is still scarce. In the present review, the roles and structures of the most important known ion channels are described in regard to regulation of sperm motility in animals.     

Phenylalanine-Derived β-Lactam TRPM8 Modulators. Configuration Effect on the Antagonist Activity

Transient receptor potential cation channel subfamily M member 8 (TRPM8) is a Ca²⁺ non-selective ion channel implicated in a variety of pathological conditions, including cancer, inflammatory and neuropathic pain. In previous works we identified a family of chiral, highly hydrophobic β–lactam derivatives, and began to intuit a possible effect of the stereogenic centers on the antagonist activity. To investigate the influence of configuration on the TRPM8 antagonist properties, here we prepare and characterize four possible diastereoisomeric derivatives of 4-benzyl-1-[(3′-phenyl-2′-dibenzylamino)prop-1′-yl]-4-benzyloxycarbonyl-3-methyl-2-oxoazetidine. In microfluorography assays, all isomers were able to reduce the menthol-induced cell Ca²⁺ entry to larger or lesser extent. Potency follows the order 3R,4R,2′R > 3S,4S,2′R ≅ 3R,4R,2′S > 3S,4S,2′S, with the most potent diastereoisomer showing a half inhibitory concentration (IC₅₀) in the low nanomolar range, confirmed by Patch-Clamp electrophysiology experiments. All four compounds display high receptor selectivity against other members of the TRP family. Furthermore, in primary cultures of rat dorsal root ganglion (DRG) neurons, the most potent diastereoisomers do not produce any alteration in neuronal excitability, indicating their high specificity for TRPM8 channels. Docking studies positioned these β-lactams at different subsites by the pore zone, suggesting a different mechanism than the known N-(3-aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)-benzamide (AMTB) antagonist.     

Peripheral Ion Channel Genes Screening in Painful Small Fiber Neuropathy

Neuropathic pain is a characteristic feature of small fiber neuropathy (SFN), which in 18% of the cases is caused by genetic variants in voltage-gated sodium ion channels. In this study, we assessed the role of fifteen other ion channels in neuropathic pain. Patients with SFN (n = 414) were analyzed for ANO1, ANO3, HCN1, KCNA2, KCNA4, KCNK18, KCNN1, KCNQ3, KCNQ5, KCNS1, TRPA1, TRPM8, TRPV1, TRPV3 and TRPV4 variants by single-molecule molecular inversion probes–next-generation sequencing. These patients did not have genetic variants in SCN3A, SCN7A-SCN11A and SCN1B-SCN4B. In twenty patients (20/414, 4.8%), a potentially pathogenic heterozygous variant was identified in an ion-channel gene (ICG). Variants were present in seven genes, for two patients (0.5%) in ANO3, one (0.2%) in KCNK18, two (0.5%) in KCNQ3, seven (1.7%) in TRPA1, three (0.7%) in TRPM8, three (0.7%) in TRPV1 and two (0.5%) in TRPV3. Variants in the TRP genes were the most frequent (n = 15, 3.6%), partly in patients with high mean maximal pain scores VAS = 9.65 ± 0.7 (n = 4). Patients with ICG variants reported more severe pain compared to patients without such variants (VAS = 9.36 ± 0.72 vs. VAS = 7.47 ± 2.37). This cohort study identified ICG variants in neuropathic pain in SFN, complementing previous findings of ICG variants in diabetic neuropathy. These data show that ICG variants are central in neuropathic pain of different etiologies and provides promising gene candidates for future research.     

Detection of TRPM6 and TRPM7 Proteins in Normal and Diseased Cardiac Atrial Tissue and Isolated Cardiomyocytes

Magnesium-sensitive transient receptor potential melastatin (TRPM) ion channels, TRPM6 and TRPM7, are present in several organs, but their roles in the heart remain unclear. Therefore, here, we studied the expression patterns of TRPM6 and TRPM7 in normal and diseased myocardium. Cardiac atrial tissue and cardiomyocytes were obtained from healthy pigs and undiseased human hearts as well as from hearts of patients with ischemic heart disease (IHD) or atrial fibrillation (AF). Immunofluorescence and ELISA were used to detect TRP proteins. TRPM6 and TRPM7 immunofluorescence signals, localized at/near the cell surface or intracellularly, were detected in pig and human atrial tissues. The TRP channel modulators carvacrol (CAR, 100 µM) or 2-aminoethoxydiphenyl borate (2-APB, 500 µM) decreased the TRPM7 signal, but enhanced that of TRPM6. At a higher concentration (2 mM), 2-APB enhanced the signals of both proteins. TRPM6 and TRPM7 immunofluorescence signals and protein concentrations were increased in atrial cells and tissues from IHD or AF patients. TRPM6 and TRPM7 proteins were both detected in cardiac atrial tissue, with relatively similar subcellular localization, but distinctive drug sensitivity profiles. Their upregulated expression in IHD and AF suggests a possible role of the channels in cardiac atrial disease.