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Transient Receptor Potential Melastatin 2 (TRPM2) cation channels contribute to immunocyte activation, insulin secretion, and central thermoregulation. TRPM2 opens upon binding cytosolic Ca 2+ 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 Ca 2+ 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 Ca 2+ alone., Competing Interests: Competing interests statement:The authors declare no competing interest.

Transient receptor potential (TRP) channels are a large and diverse family of tetrameric cation-selective channels that are activated by many different types of stimuli, including noxious heat or cold, organic ligands such as vanilloids or cooling agents, or intracellular Ca 2+ . Structures available for all subtypes of TRP channels reveal that the transmembrane domains are closely related despite their unique sensitivity to activating stimuli. Here, we use computational and electrophysiological approaches to explore the conservation of the cooling agent binding pocket identified within the S1-S4 domain of the Melastatin subfamily member TRPM8, the mammalian sensor of noxious cold, with other TRPM channel subtypes. We find that a subset of TRPM channels, including TRPM2, TRPM4, and TRPM5, contain pockets very similar to the cooling agent binding pocket in TRPM8. We then show how the cooling agent icilin modulates activation of mouse TRPM4 to intracellular Ca 2+ , enhancing the sensitivity of the channel to Ca 2+ and diminishing outward-rectification to promote opening at negative voltages. Mutations known to promote or diminish activation of TRPM8 by cooling agents similarly alter activation of TRPM4 by icilin, suggesting that icilin binds to the cooling agent binding pocket to promote opening of the channel. These findings demonstrate that TRPM4 and TRPM8 channels share related ligand binding pockets that are allosterically coupled to opening of the pore., Competing Interests: KH, MD, EO No competing interests declared, KS Reviewing editor, eLife

In order to understand protein function, the field of structural biology makes extensive use of cryogenic electron microscopy (cryo-EM), a technique that enables structure determination at atomic resolution following embedding of protein particles in vitreous ice. Considering the profound effects of temperature on macromolecule function, an important-but often neglected-question is how the frozen particles relate to the actual protein conformations at physiological temperatures. In a recent study, Hu et al. compare structures of the cation channel TRPM4 "frozen" at 4 °C versus 37 °C, revealing how temperature critically affects the binding of activating Ca 2+ ions and other channel modulators., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Thomas Voets reports financial support was provided by Research Foundation Flanders. Brett Boonen reports financial support was provided by Research Foundation Flanders. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

A key capability of ion channels is the facilitation of selective permeation of certain ionic species across cellular membranes at high rates. Due to their physiological significance, ion channels are of great pharmaceutical interest as drug targets. The polymodal signal-detecting transient receptor potential (TRP) superfamily of ion channels forms a particularly promising group of drug targets. While most members of this family permeate a broad range of cations including Ca 2+ , TRPM4 and TRPM5 are unique due to their strong monovalent selectivity and impermeability for divalent cations. Here, we investigated the mechanistic basis for their unique monovalent selectivity by in silico electrophysiology simulations of TRPM5. Our simulations reveal an unusual mechanism of cation selectivity, which is underpinned by the function of the central channel cavity alongside the selectivity filter. Our results suggest that a subtle hydrophobic barrier at the cavity entrance ("hydrophobic funnel") enables monovalent but not divalent cations to pass and occupy the cavity at physiologically relevant membrane voltages. Monovalent cations then permeate efficiently by a cooperative, distant knock-on mechanism between two binding regions in the extracellular pore vestibule and the central cavity. By contrast, divalent cations do not enter or interact favorably with the channel cavity due to its raised hydrophobicity. Hydrophilic mutations in the transition zone between the selectivity filter and the central channel cavity abolish the barrier for divalent cations, enabling both monovalent and divalent cations to traverse TRPM5., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Channel enzymes represent a class of ion channels with enzymatic activity directly or indirectly linked to their channel function. We investigated a TRPM2 chanzyme from choanoflagellates that integrates two seemingly incompatible functions into a single peptide: a channel module activated by ADP-ribose with high open probability and an enzyme module (NUDT9-H domain) consuming ADP-ribose at a remarkably slow rate. Using time-resolved cryogenic-electron microscopy, we captured a complete series of structural snapshots of gating and catalytic cycles, revealing the coupling mechanism between channel gating and enzymatic activity. The slow kinetics of the NUDT9-H enzyme module confers a self-regulatory mechanism: ADPR binding triggers NUDT9-H tetramerization, promoting channel opening, while subsequent hydrolysis reduces local ADPR, inducing channel closure. We further demonstrated how the NUDT9-H domain has evolved from a structurally semi-independent ADP-ribose hydrolase module in early species to a fully integrated component of a gating ring essential for channel activation in advanced species., (© 2024. The Author(s).)

TRPM8 is a non-selective cation channel that is expressed in several tissues and cells and also has a unique property to be activated by low-temperature. In this work, we have analyzed the conservation of amino acids that are present in the lipid-water-interface (LWI) region of TRPM8, the region which experiences a microenvironment near the membrane surface. We demonstrate that the amino acids present in the LWI region are more conserved than the transmembrane or even full-length TRPM8, suggesting strong selection pressure in these residues. TRPM8 also has several conserved cholesterol-binding motifs where cholesterol can bind in different modes and energies. We suggest that mutations and/or physiological conditions can potentially alter these TRPM8-cholesterol complexes and can lead to physiological disorders or even apparently irreversible diseases such as cancer and neurodegeneration., Competing Interests: Declarations. Conflict of Interest: All authors declare the existence of no conflict with this work. The funding bodies have no role in experiment design, data analysis, manuscript preparation or decision to publish this work. The authors declare no competing interests., (© 2024. The Author(s).)

The transient receptor potential melastatin (TRPM) tetrameric cation channels are involved in a wide range of biological functions, from temperature sensing and taste transduction to regulation of cardiac function, inflammatory pain, and insulin secretion. The structurally conserved TRPM cytoplasmic domains make up >70 % of the total protein. To investigate the mechanism by which the TRPM cytoplasmic domains contribute to gating, we employed electrophysiology and cryo-EM to study TRPM5-a channel that primarily relies on activation via intracellular Ca 2+ . Here, we show that activation of mammalian TRPM5 channels is strongly altered by Ca 2+ -dependent desensitization. Structures of rat TRPM5 identify a series of conformational transitions triggered by Ca 2+ binding, whereby formation and dissolution of cytoplasmic interprotomer interfaces appear to control activation and desensitization of the channel. This study shows the importance of the cytoplasmic assembly in TRPM5 channel function and sets the stage for future investigations of other members of the TRPM family., Competing Interests: Competing interests statement:The authors declare no competing interest.

Temperature profoundly affects macromolecular function, particularly in proteins with temperature sensitivity 1,2 . However, its impact is often overlooked in biophysical studies that are typically performed at non-physiological temperatures, potentially leading to inaccurate mechanistic and pharmacological insights. Here we demonstrate temperature-dependent changes in the structure and function of TRPM4, a temperature-sensitive Ca 2+ -activated ion channel 3-7 . By studying TRPM4 prepared at physiological temperature using single-particle cryo-electron microscopy, we identified a 'warm' conformation that is distinct from those observed at lower temperatures. This conformation is driven by a temperature-dependent Ca 2+ -binding site in the intracellular domain, and is essential for TRPM4 function in physiological contexts. We demonstrated that ligands, exemplified by decavanadate (a positive modulator) 8 and ATP (an inhibitor) 9 , bind to different locations of TRPM4 at physiological temperatures than at lower temperatures 10,11 , and that these sites have bona fide functional relevance. We elucidated the TRPM4 gating mechanism by capturing structural snapshots of its different functional states at physiological temperatures, revealing the channel opening that is not observed at lower temperatures. Our study provides an example of temperature-dependent ligand recognition and modulation of an ion channel, underscoring the importance of studying macromolecules at physiological temperatures. It also provides a potential molecular framework for deciphering how thermosensitive TRPM channels perceive temperature changes., (© 2024. The Author(s).)

G-protein coupled receptors (GPCRs) govern the physiological response to stimuli by modulating the activity of downstream effectors, including ion channels. TRPM3 is an ion channel inhibited by GPCRs through direct interaction with G protein (Gβγ) released upon their activation. This GPCR-TRPM3 signaling pathway contributes to the analgesic effect of morphine. Here, we characterized Gβγ inhibition of TRPM3 using electrophysiology and single particle cryo-electron microscopy (cryo-EM). From electrophysiology, we obtained a half inhibition constant (IC50) of ∼240 nM. Using cryo-EM, we determined structures of mouse TRPM3 expressed in human cells with and without Gβγ and with and without PIP 2 , a lipid required for TRPM3 activity, at resolutions of 2.7-4.7 Å. Gβγ-TRPM3 interfaces vary depending on PIP 2 occupancy; however, in all cases, Gβγ appears loosely attached to TRPM3. The IC50 in electrophysiology experiments raises the possibility that additional unknown factors may stabilize the TRPM3-Gβγ complex., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The cation nonselective channel TRPM8 is activated by multiple stimuli, including moderate cold and various chemical compounds (i.e., menthol and icilin [Fig. 1], among others). While research continues growing on the understanding of the physiological involvement of TRPM8 channels and their role in various pathological states, the information available on its activation mechanisms has also increased, supported by mutagenesis and structural studies. This review compiles known information on specific mutations of channel residues and their consequences on channel viability and function. Besides, the comparison of sequence of animals living in different environments, together with chimera and mutagenesis studies are helping to unravel the mechanism of adaptation to different temperatures. The results of mutagenesis studies, grouped by different channel regions, are compared with the current knowledge of TRPM8 structures obtained by cryo-electron microscopy. Trying to make this review self-explicative and highly informative, important residues for TRPM8 function are summarized in a figure, and mutants, deletions and chimeras are compiled in a table, including also the observed effects by different methods of activation and the corresponding references. The information provided by this review may also help in the design of new ligands for TRPM8, an interesting biological target for therapeutic intervention., (© 2022 The Authors. Medicinal Research Reviews published by Wiley Periodicals LLC.)

Magnesium ions (Mg 2+ ) are the most abundant divalent cations in living organisms and are essential for various physiological processes, including ATP utilization and the catalytic activity of numerous enzymes. Therefore, the homeostatic mechanisms associated with cellular Mg 2+ are crucial for both eukaryotic and prokaryotic organisms and are thus strictly controlled by Mg 2+ channels and transporters. Technological advances in structural biology, such as the expression screening of membrane proteins, in meso phase crystallization, and recent cryo-EM techniques, have enabled the structure determination of numerous Mg 2+ channels and transporters. In this review article, we provide an overview of the families of Mg 2+ channels and transporters (MgtE/SLC41, TRPM6/7, CorA/Mrs2, CorC/CNNM), and discuss the structural biology prospects based on the known structures of MgtE, TRPM7, CorA and CorC., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The transient receptor potential melastatin 8 (TRPM8) channel is the primary molecular transducer responsible for the cool sensation elicited by menthol and cold in mammals. TRPM8 activation is controlled by cooling compounds together with the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP 2 ). Our knowledge of cold sensation and the therapeutic potential of TRPM8 for neuroinflammatory diseases and pain will be enhanced by understanding the structural basis of cooling agonist- and PIP 2 -dependent TRPM8 activation. We present cryo-electron microscopy structures of mouse TRPM8 in closed, intermediate, and open states along the ligand- and PIP 2 -dependent gating pathway. Our results uncover two discrete agonist sites, state-dependent rearrangements in the gate positions, and a disordered-to-ordered transition of the gate-forming S6-elucidating the molecular basis of chemically induced cool sensation in mammals.

Transient receptor potential melastatin 2 (TRPM2) is a Ca 2+ -permeable, nonselective cation channel involved in diverse physiological processes such as immune response, apoptosis, and body temperature sensing. TRPM2 is activated by ADP-ribose (ADPR) and 2'-deoxy-ADPR in a Ca 2+ -dependent manner. While two distinct binding sites exist for ADPR that exert different functions dependent on the species, the involvement of either binding site regarding the superagonistic effect of 2'-deoxy-ADPR is not clear yet. Here, we report the crystal structure of the MHR1/2 domain of TRPM2 from zebrafish (Danio rerio), and show that both ligands bind to this domain and activate the channel. We identified a so far unrecognized Zn 2+ -binding domain that was not resolved in previous cryo-EM structures and that is conserved in most TRPM channels. In combination with patch clamp experiments we comprehensively characterize the effect of the Zn 2+ -binding domain on TRPM2 activation. Our results provide insight into a conserved motif essential for structural integrity and channel activity., (© 2022 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

To cope with temperature fluctuations, molecular thermosensors in animals play a pivotal role in accurately sensing ambient temperature. Transient receptor potential melastatin 8 (TRPM8) is the most established cold sensor. In order to understand how the evolutionary forces bestowed TRPM8 with cold sensitivity, insights into both emergence of cold sensing during evolution and the thermodynamic basis of cold activation are needed. Here, we show that the trpm8 gene evolved by forming and regulating two domains (MHR1-3 and pore domains), thus determining distinct cold-sensitive properties among vertebrate TRPM8 orthologs. The young trpm8 gene without function can be observed in the closest living relatives of tetrapods (lobe-finned fishes), while the mature MHR1-3 domain with independent cold sensitivity has formed in TRPM8s of amphibians and reptiles to enable channel activation by cold. Furthermore, positive selection in the TRPM8 pore domain that tuned the efficacy of cold activation appeared late among more advanced terrestrial tetrapods. Interestingly, the mature MHR1-3 domain is necessary for the regulatory mechanism of the pore domain in TRPM8 cold activation. Our results reveal the domain-based evolution for TRPM8 functions and suggest that the acquisition of cold sensitivity in TRPM8 facilitated terrestrial adaptation during the water-to-land transition.

Melastatin transient receptor potential (TRPM) channels belong to one of the most significant subgroups of the transient receptor potential (TRP) channel family. Here, we studied the TRPM5 member, the receptor exposed to calcium-mediated activation, resulting in taste transduction. It is known that most TRP channels are highly modulated through interactions with extracellular and intracellular agents. The binding sites for these ligands are usually located at the intracellular N- and C-termini of the TRP channels, and they can demonstrate the character of an intrinsically disordered protein (IDP), which allows such a region to bind various types of molecules. We explored the N-termini of TRPM5 and found the intracellular regions for calcium-binding proteins (CBPs) the calmodulin (CaM) and calcium-binding protein S1 (S100A1) by in vitro binding assays. Furthermore, molecular docking and molecular dynamics simulations (MDs) of the discovered complexes confirmed their known common binding interface patterns and the uniqueness of the basic residues present in the TRPM binding regions for CaM/S100A1.

An autoimmune disease is an inappropriate response to one's tissues due to a break in immune tolerance and exposure to self-antigens. It often leads to structural and functional damage to organs and systemic disorders. To date, there are no effective interventions to prevent the progression of autoimmune diseases. Hence, there is an urgent need for new treatment targets. TRPM7 is an enzyme-coupled, transient receptor ion channel of the subfamily M that plays a vital role in pathologic and physiologic conditions. While TRPM7 is constitutively activated under certain conditions, it can regulate cell migration, polarization, proliferation and cytokine secretion. However, a growing body of evidence highlights the critical role of TRPM7 in autoimmune diseases, including rheumatoid arthritis, multiple sclerosis and diabetes. Herein, we present (a) a review of the channel kinase properties of TRPM7 and its pharmacological properties, (b) discuss the role of TRPM7 in immune cells (neutrophils, macrophages, lymphocytes and mast cells) and its upstream immunoreactive substances, and (c) highlight TRPM7 as a potential therapeutic target for autoimmune diseases., (© 2021 John Wiley & Sons Ltd.)

Sex hormone steroidal drugs were reported to have modulating actions on the ion channel TRPM3. Pregnenolone sulphate (PS) presents the most potent known endogenous chemical agonist of TRPM3 and affects several gating modes of the channel. These includes a synergistic action of PS and high temperatures on channel opening and the PS-induced opening of a noncanonical pore in the presence of other TRPM3 modulators. Moreover, human TRPM3 variants associated with neurodevelopmental disease exhibit an increased sensitivity for PS. However, other steroidal sex hormones were reported to influence TRPM3 functions with activating or inhibiting capacity. Here, we aimed to answer how DHEAS, estradiol, progesterone and testosterone act on the various modes of TRPM3 function in the wild-type channel and two-channel variants associated with human disease. By means of calcium imaging and whole-cell patch clamp experiments, we revealed that all four drugs are weak TRPM3 agonists that share a common steroidal interaction site. Furthermore, they exhibit increased activity on TRPM3 at physiological temperatures and in channels that carry disease-associated mutations. Finally, all steroids are able to open the noncanonical pore in wild-type and DHEAS also in mutant TRPM3. Collectively, our data provide new valuable insights in TRPM3 gating, structure-function relationships and ligand sensitivity.

Ischemic stroke has a high lethality rate worldwide, and novel treatments are limited. Calcium overload is considered to be one of the mechanisms of cerebral ischemia. Transient receptor potential melastatin 2 (TRPM2) is a reactive oxygen species (ROS)-sensitive calcium channel. Cerebral ischemia-induced TRPM2 activation triggers abnormal intracellular Ca 2+ accumulation and cell death, which in turn causes irreversible brain damage. Thus, TRPM2 has emerged as a new therapeutic target for ischemic stroke. This review provides data on the expression, structure, and function of TRPM2 and illustrates its cellular and molecular mechanisms in ischemic stroke. Natural and synthetic TRPM2 inhibitors (both specific and nonspecific) are also summarized. The three-dimensional protein structure of TRPM2 has been identified, and we speculate that molecular simulation techniques will be essential for developing new drugs that block TRPM2 channels. These insights about TRPM2 may be the key to find potent therapeutic approaches for the treatment of ischemic stroke.

Transient receptor potential subfamily M member 7 (TRPM7), a mechanosensitive Ca 2+ channel, plays a crucial role in intracellular Ca 2+ homeostasis. However, it is currently unclear how cell mechanical cues control TRPM7 activity and its associated Ca 2+ influx at plasma membrane microdomains. Using two different types of Ca 2+ biosensors (Lyn-D3cpv and Kras-D3cpv) based on fluorescence resonance energy transfer, we investigate how Ca 2+ influx generated by the TRPM7-specific agonist naltriben is mediated at the detergent-resistant membrane (DRM) and non-DRM regions. This study reveals that TRPM7-induced Ca 2+ influx mainly occurs at the DRM, and chemically induced mechanical perturbations in the cell mechanosensitive apparatus substantially reduce Ca 2+ influx through TRPM7, preferably located at the DRM. Such perturbations include the disintegration of lipid rafts, microtubules, or actomyosin filaments; the alteration of actomyosin contractility; and the inhibition of focal adhesion and Src kinases. These results suggest that the mechanical membrane environment contributes to the TRPM7 function and activity. Thus, this study provides a fundamental understanding of how the mechanical aspects of the cell membrane regulate the function of mechanosensitive channels., (© 2021. The Author(s).)

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.

Cancer is the second leading cause of death worldwide and accounted for an estimated 9.6 million deaths, or 1 in 6 deaths, in 2018. Despite recent advances in cancer prevention, diagnosis, and treatment strategies, the burden of this disease continues to grow with each year, with dire physical, emotional, and economic consequences for all levels of society. Classic characteristics of cancer include rapid, uncontrolled cell proliferation and spread of cancerous cells to other parts of the body, a process known as metastasis. Transient receptor potential melastatin 7 (TRPM7), a Ca 2+ - and Mg 2+ -permeable nonselective divalent cation channel defined by the atypical presence of an α-kinase within its C-terminal domain, has been implicated, due to its modulation of Ca 2+ and Mg 2+ influx, in a wide variety of physiological and pathological processes, including cancer. TRPM7 is overexpressed in several cancer types and has been shown to variably increase cellular proliferation, migration, and invasion of tumour cells. However, the relative contribution of TRPM7 kinase domain activity to cancer as opposed to ion flux through its channel pore remains an area of active discovery. In this review, we describe the specific role of the TRPM7 kinase domain in cancer processes as well as mechanisms of regulation and inhibition of the kinase domain., (Copyright © 2021. Published by Elsevier Ltd.)

The transient receptor potential melastatin-subfamily member 7 (TRPM7) is a ubiquitously expressed protein that contains both an ion channel and an active kinase. TRPM7 has involved in a variety of cellular functions and critically participates in various diseases mainly including cancer and neurodegenerative disorders. However, the theme trends and knowledge structures for TRPM7 have not yet been studied bibliometrically. The main purposes of this research are to compare the scientific production in the research field of TRPM7 among countries and to evaluate the publication trend between 2004 and 2019. All publications were extracted from the Web of Science Core Collection (WoSCC) database from 2004 to 2019. Microsoft Excel 2018, Prism 6, and CiteSpace V were applied to analyze the scientific research outputs including journals, countries, territories, institutions, authors, and research hotspots. In this report, a total of 860 publications related to TRPM7 were analyzed. Biophysical Journal ranked top for publishing 31 papers. The United States of America had the largest number of publications (320) with a high citation frequency (11,298) and H-index (58). Chubanov V (38 publications) and Gudermann T (38 citations), who from Ludwig Maximilian University of Munich, were the most productive authors and had the greatest co-citation counts. Our study also combined the bibliometric study with a systematic review on TRPM7, highlighting the four research frontiers of TRPM7. This is the first study that demonstrated the trends and future development in TRPM7 publications, providing a clear and intuitive profile for the contributions in this field.

Transient receptor potential melastatin 2 (TRPM2) is a Ca 2+ - permeable nonselective cation channel that is involved in diverse biological functions as a cellular sensor for oxidative stress and temperature. It has been considered a promising therapeutic target for the treatment of ischemia/reperfusion (IR) injury, inflammation, cancer, and neurodegenerative diseases. Development of highly potent and selective TRPM2 inhibitors and validation of their use in relevant disease models will advance drug discovery. In this review, we describe the molecular structures and gating mechanism of the TRPM2 channel, and offer a comprehensive review of advances in the discovery of TRPM2 inhibitors. Furthermore, we analyze the properties of reported TRPM2 inhibitors with an emphasis on how specific inhibitors targeting this channel could be better developed., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

TRPM3 channels play important roles in the detection of noxious heat and in inflammatory thermal hyperalgesia. The activity of these ion channels in somatosensory neurons is tightly regulated by µ-opioid receptors through the signaling of Gβγ proteins, thereby reducing TRPM3-mediated pain. We show here that Gβγ directly binds to a domain of 10 amino acids in TRPM3 and solve a cocrystal structure of this domain together with Gβγ. Using these data and mutational analysis of full-length proteins, we pinpoint three amino acids in TRPM3 and their interacting partners in Gβ 1 that are individually necessary for TRPM3 inhibition by Gβγ. The 10-amino-acid Gβγ-interacting domain in TRPM3 is subject to alternative splicing. Its inclusion in or exclusion from TRPM3 channel proteins therefore provides a mechanism for switching on or off the inhibitory action that Gβγ proteins exert on TRPM3 channels., Competing Interests: The authors declare no competing interest.

Transient receptor potential melastatin 8 (TRPM8) ion channel represents a valuable pharmacological option for several therapeutic areas. Here, a series of conformationally restricted derivatives of the previously described TRPM8 antagonist N , N '-dibenzyl tryptophan 4 were prepared and characterized in vitro by Ca 2+ -imaging and patch-clamp electrophysiology assays. Molecular modeling studies led to identification of a broad and well-defined interaction network of these derivatives inside the TRPM8 binding site, underlying their antagonist activity. The (5 R ,11a S )-5-(4-chlorophenyl)-2-(4-fluorobenzyl)-5,6,11,11a-tetrahydro-1 H -imidazo[1',5':1,6]pyrido[3,4- b ]indole-1,3(2 H )-dione ( 31a ) emerged as a potent (IC 50 = 4.10 ± 1.2 nM), selective, and metabolically stable TRPM8 antagonist. In vivo, 31a showed significant target coverage in an icilin-induced WDS (at 11.5 mg/kg ip), an oxaliplatin-induced cold allodynia (at 10-30 μg sc), and CCI-induced thermal hyperalgesia (at 11.5 mg/kg ip) mice models. These results confirm the tryptophan moiety as a solid pharmacophore template for the design of highly potent modulators of TRPM8-mediated activities.

In mammals, temperature-sensitive TRP channels make membrane conductance of cells extremely temperature dependent, allowing the detection of temperature ranging from noxious cold to noxious heat. We progressively deleted the distal carboxyl terminus domain (CTD) of the cold-activated melastatin receptor channel, TRPM8. We found that the enthalpy change associated with channel gating is proportional to the length of the CTD. Deletion of the last 36 amino acids of the CTD transforms TRPM8 into a reduced temperature-sensitivity channel (Q 10 ∼4). Exposing the intracellular domain to a denaturing agent increases the energy required to open the channel indicating that cold drives channel gating by stabilizing the folded state of the CTD. Experiments in the presence of an osmoticant agent suggest that channel gating involves a change in solute-inaccessible volume in the CTD of ∼1,900 Å 3 This volume matches the void space inside the coiled coil according to the cryogenic electron microscopy structure of TRPM8. The results indicate that a folding-unfolding reaction of a specialized temperature-sensitive structure is coupled to TRPM8 gating., Competing Interests: The authors declare no competing interest.

Menthol in mints elicits coolness sensation by selectively activating TRPM8 channel. Although structures of TRPM8 were determined in the apo and liganded states, the menthol-bounded state is unresolved. To understand how menthol activates the channel, we docked menthol to the channel and systematically validated our menthol binding models with thermodynamic mutant cycle analysis. We observed that menthol uses its hydroxyl group as a hand to specifically grab with R842, and its isopropyl group as legs to stand on I846 and L843. By imaging with fluorescent unnatural amino acid, we found that menthol binding induces wide-spread conformational rearrangements within the transmembrane domains. By Φ analysis based on single-channel recordings, we observed a temporal sequence of conformational changes in the S6 bundle crossing and the selectivity filter leading to channel activation. Therefore, our study suggested a 'grab and stand' mechanism of menthol binding and how menthol activates TRPM8 at the atomic level.

TRPM8 member of the TRP superfamily of membrane proteins participates to various cellular processes ranging from Ca 2+ uptake and cold sensation to cellular proliferation and migration. TRPM8 is a large tetrameric protein with more than 70% of its residues located in the cytoplasm. TRPM8 is N-glycosylated, with a single site per subunit. This work focuses on the N-glycosylation of TRPM8 channel that was previously studied by our group in relation to proliferation and migration of tumoral cells. Here, experimental data performed with deglycosylating agents assess that the sole glycosylation site contains complex glycans with a molecular weight of 2.5 kDa. The glycosylation state of TRPM8 in cells untreated and treated with a deglycosylating agent was addressed with Terahertz (THz) spectroscopy. Results show a clear difference between cells comprising glycosylated and deglycosylated TRPM8, the first presenting an increased THz absorption. Human TRPM8 was modelled using as templates the available TRPM8 and other TRPM channels structures. Glycosylations were modelled by considering two glycan structures with molecular weight close to the experiment: shorter and branched at the first sugar unit (glc1) and longer and unbranched (glc2). Simulation of THz spectra based on the molecular dynamics of unglycosylated and the two glycosylated TRPM8 models in lipid membrane and solvation box showed that glycan structure strongly influences the THz spectrum of the channel and of other components from the simulation system. Only spectra of TRPM8 with glc1 glycans were in agreement with the experiment, leading to the validation of glc1 glycan structure., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Molecular determinants of the binding of various endogenous modulators to transient receptor potential (TRP) channels are crucial for the understanding of necessary cellular pathways, as well as new paths for rational drug designs. The aim of this study was to characterise interactions between the TRP cation channel subfamily melastatin member 4 (TRPM4) and endogenous intracellular modulators-calcium-binding proteins (calmodulin (CaM) and S100A1) and phosphatidylinositol 4, 5-bisphosphate (PIP 2 ). We have found binding epitopes at the N- and C-termini of TRPM4 shared by CaM, S100A1 and PIP 2 . The binding affinities of short peptides representing the binding epitopes of N- and C-termini were measured by means of fluorescence anisotropy (FA). The importance of representative basic amino acids and their combinations from both peptides for the binding of endogenous TRPM4 modulators was proved using point alanine-scanning mutagenesis. In silico protein-protein docking of both peptides to CaM and S100A1 and extensive molecular dynamics (MD) simulations enabled the description of key stabilising interactions at the atomic level. Recently solved cryo-Electron Microscopy (EM) structures made it possible to put our findings into the context of the entire TRPM4 channel and to deduce how the binding of these endogenous modulators could allosterically affect the gating of TRPM4. Moreover, both identified binding epitopes seem to be ideally positioned to mediate the involvement of TRPM4 in higher-order hetero-multimeric complexes with important physiological functions.

Transient receptor potential (TRP) ion channels are polymodal receptors that have been implicated in a variety of pathophysiologies, including pain, obesity, and cancer. The capsaicin and heat sensor TRPV1, and the menthol and cold sensor TRPM8, have been shown to be modulated by the membrane protein PIRT (Phosphoinositide-interacting regulator of TRP). The emerging mechanism of PIRT-dependent TRPM8 regulation involves a competitive interaction between PIRT and TRPM8 for the activating phosphatidylinositol 4,5-bisphosphate (PIP 2 ) lipid. As many PIP 2 modulated ion channels also interact with calmodulin, we investigated the possible interaction between PIRT and calmodulin. Using microscale thermophoresis (MST), we show that calmodulin binds to the PIRT C-terminal α-helix, which we corroborate with a pull-down experiment, nuclear magnetic resonance-detected binding study, and Rosetta-based computational studies. Furthermore, we identify a cholesterol-recognition amino acid consensus (CRAC) domain in the outer leaflet of the first transmembrane helix of PIRT, and with MST, show that PIRT specifically binds to a number of cholesterol-derivatives. Additional studies identified that PIRT binds to cholecalciferol and oxytocin, which has mechanistic implications for the role of PIRT regulation of additional ion channels. This is the first study to show that PIRT specifically binds to a variety of ligands beyond TRP channels and PIP 2 .

Transient receptor potential melastatin 2 (TRPM2) is a calcium-permeable channel activated by adenosine diphosphate ribose metabolites and oxidative stress. TRPM2 contributes to neuronal injury in the brain caused by stroke and cardiac arrest among other diseases including pain, inflammation, and cancer. However, the lack of specific inhibitors hinders the study of TRPM2 in brain pathophysiology. Here, we present the design of a novel TRPM2 antagonist, tatM2NX, which prevents ligand binding and TRPM2 activation. We used mutagenesis of tatM2NX to determine the structure-activity relationship and antagonistic mechanism on TRPM2 using whole-cell patch clamp and Calcium imaging in human embryonic kidney 293 cells with stable human TRPM2 expression. We show that tatM2NX inhibits over 90% of TRPM2 channel currents at concentrations as low as 2 μM. Moreover, tatM2NX is a potent antagonist with an IC 50 of 396 nM. Our results from tatM2NX mutagenesis indicate that specific residues within the tatM2NX C terminus are required to confer antagonism on TRPM2. Therefore, the peptide tatM2NX represents a new tool for the study of TRPM2 function in cell biology and enhances our understanding of TRPM2 in disease. SIGNIFICANCE STATEMENT: TatM2NX is a potent TRPM2 channel antagonist with the potential for clinical benefit in neurological diseases. This study characterizes interactions of tatM2NX with TRPM2 and the mechanism of action using structure-activity analysis., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

The ion channel TRPM8 has been identified as the primary cold sensor in humans, but insights into how cold perception is established remained elusive. This has changed now with newly published structures of TRPM8 in different states during channel gating, which help us understand why we feel cold and adapt to it., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The TRPM (transient receptor potential melastatin) family belongs to the superfamily of TRP cation channels. The TRPM subfamily is composed of eight members that are involved in diverse biological functions such as temperature sensing, inflammation, insulin secretion, and redox sensing. Since the first cloning of TRPM1 in 1998, tremendous progress has been made uncovering the function, structure, and pharmacology of this family. Complete structures of TRPM2, TRPM4, and TRPM8, as well as a partial structure of TRPM7, have been determined by cryo-EM, providing insights into their channel assembly, ion permeation, gating mechanisms, and structural pharmacology. Here we summarize the current knowledge about channel structure, emphasizing general features and principles of the structure of TRPM channels discovered since 2017. We also discuss some of the key unresolved issues in the field, including the molecular mechanisms underlying voltage and temperature dependence, as well as the functions of the TRPM channels' C-terminal domains., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Transient receptor potential melastatin 7 (TRPM7), along with the closely related TRPM6, are unique channels that have dual operations: cation permeability and kinase activity. In contrast to the limited tissue distribution of TRPM6, TRPM7 is widely expressed among tissues and is therefore implicated in a variety of cellular functions physiologically and pathophysiologically. The discovery of TRPM7's unique structure imparting dual ion channel and kinase activities shed light onto novel and peculiar biological functions, such as Mg2+ homeostasis, cellular Ca2+ flickering, and even intranuclear transcriptional regulation by a cleaved kinase domain translocated to nuclei. Interestingly, at a higher level, TRPM7 participates in several biological processes in the nervous and cardiovascular systems, in which excitatory responses in neurons and cardiomyocytes are critical for their function. Here, we review the roles of TRPM7 in cells involved in the nervous and cardiovascular systems and discuss its potential as a future therapeutic target., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Membrane proteins are targeted to the surface transmembrane after folding and assembling in the endoplasmic reticulum (ER). Misfolded- and unassembled-proteins are degraded by proteasomes following ubiquitination, termed ER-associated degradation (ERAD). Transient receptor potential melastatin 2 (TRPM2) is an oxidative stress-sensitive channel. One of the TRPM2 splicing variants, short TRPM2 (TRPM2-S) having only the N-terminus and first two transmembrane domains, was reported to prevent full-length TRPM2 (TRPM2-L) activation. Although TRPM2-S interacts with TRPM2-L, the inhibitory mechanisms of TRPM2-S are unclear. We found that TRPM2-S prevents transmembrane expression of TRPM2-L by targeting ERAD. TRPM2-S expression was lower than that of TRPM2-L, and was increased by an ERAD inhibitor. TRPM2-S was not expressed at the transmembrane. This suggests that TRPM2-S is a substrate for ERAD. Upon the simultaneous expression of TRPM2-S, the transmembrane expression of TRPM2-L was attenuated and the poly-ubiquitination of TRPM2-L was facilitated. Our study may clarify why TRPM2-S inhibits oxidative stress-induced TRPM2-L activation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Cannabinoid compounds are potential analgesics. Users of medicinal Cannabis report efficacy for pain control, clinical studies show that cannabis can be effective and opioid sparing in chronic pain, and some constituent cannabinoids have been shown to target nociceptive ion channels. Here, we explore and compare a suite of cannabinoids for their impact upon the physiology of TRPV1. The cannabinoids tested evoke differential responses in terms of kinetics of activation and inactivation. Cannabinoid activation of TRPV1 displays significant dependence on internal and external calcium levels. Cannabinoid activation of TRPV1 does not appear to induce the highly permeant, pore-dilated channel state seen with Capsaicin, even at high current amplitudes. Finally, we analyzed cannabinoid responses at nociceptive channels other than TRPV1 (TRPV2, TRPM8, and TRPA1), and report that cannabinoids differentially activate these channels. On the basis of response activation and kinetics, state-selectivity and receptor selectivity, it may be possible to rationally design approaches to pain using single or multiple cannabinoids.

As a crucial signaling molecule, calcium plays a critical role in many physiological and pathological processes by regulating ion channel activity. Recently, one study resolved the structure of the transient receptor potential melastatin 2 (TRPM2) channel from Nematostella vectensis (nvTRPM2). This identified a calcium-binding site in the S2-S3 loop, while its effect on channel gating remains unclear. Here, we investigated the role of this calcium-binding site in both nvTRPM2 and human TRPM2 (hTRPM2) by mutagenesis and patch-clamp recording. Unlike hTRPM2, nvTRPM2 cannot be activated by calcium alone. Moreover, the inactivation rate of nvTRPM2 was decreased as intracellular calcium concentration was increased. In addition, our results showed that the four key residues in the calcium-binding site of S2-S3 loop have similar effects on the gating processes of nvTRPM2 and hTRPM2. Among them, the mutations at negatively charged residues (glutamate and aspartate) substantially decreased the currents of nvTRPM2 and hTRPM2. This suggests that these sites are essential for calcium-dependent channel gating. For the charge-neutralizing residues (glutamine and asparagine) in the calcium-binding site, our data showed that glutamine mutating to alanine or glutamate did not affect the channel activity, but glutamine mutating to lysine caused loss of function. Asparagine mutating to aspartate still remained functional, while asparagine mutating to alanine or lysine led to little channel activity. These results suggest that the side chain of glutamine has a less contribution to channel gating than does asparagine. However, our data indicated that both glutamine mutating to alanine or glutamate and asparagine mutating to aspartate accelerated the channel inactivation rate, suggesting that the calcium-binding site in the S2-S3 loop is important for calcium-dependent channel inactivation. Taken together, our results uncovered the effect of four key residues in the S2-S3 loop of TRPM2 on the TRPM2 gating process.

Thermosensitive transient receptor potential (TRP) ion channels detect changes in ambient temperature to regulate body temperature and temperature-dependent cellular activity. Rodent orthologs of TRP vanilloid 2 (TRPV2) are activated by nonphysiological heat exceeding 50 °C, and human TRPV2 is heat-insensitive. TRPV2 is required for phagocytic activity of macrophages which are rarely exposed to excessive heat, but what activates TRPV2 in vivo remains elusive. Here we describe the molecular mechanism of an oxidation-induced temperature-dependent gating of TRPV2. While high concentrations of H 2 O 2 induce a modest sensitization of heat-induced inward currents, the oxidant chloramine-T (ChT), ultraviolet A light, and photosensitizing agents producing reactive oxygen species (ROS) activate and sensitize TRPV2. This oxidation-induced activation also occurs in excised inside-out membrane patches, indicating a direct effect on TRPV2. The reducing agent dithiothreitol (DTT) in combination with methionine sulfoxide reductase partially reverses ChT-induced sensitization, and the substitution of the methionine (M) residues M528 and M607 to isoleucine almost abolishes oxidation-induced gating of rat TRPV2. Mass spectrometry on purified rat TRPV2 protein confirms oxidation of these residues. Finally, macrophages generate TRPV2-like heat-induced inward currents upon oxidation and exhibit reduced phagocytosis when exposed to the TRP channel inhibitor ruthenium red (RR) or to DTT. In summary, our data reveal a methionine-dependent redox sensitivity of TRPV2 which may be an important endogenous mechanism for regulation of TRPV2 activity and account for its pivotal role for phagocytosis in macrophages., Competing Interests: The authors declare no competing interest.

The developmental and epileptic encephalopathies (DEE) are a heterogeneous group of chronic encephalopathies frequently associated with rare de novo nonsynonymous coding variants in neuronally expressed genes. Here, we describe eight probands with a DEE phenotype comprising intellectual disability, epilepsy, and hypotonia. Exome trio analysis showed de novo variants in TRPM3, encoding a brain-expressed transient receptor potential channel, in each. Seven probands were identically heterozygous for a recurrent substitution, p.(Val837Met), in TRPM3's S4-S5 linker region, a conserved domain proposed to undergo conformational change during gated channel opening. The eighth individual was heterozygous for a proline substitution, p.(Pro937Gln), at the boundary between TRPM3's flexible pore-forming loop and an adjacent alpha-helix. General-population truncating variants and microdeletions occur throughout TRPM3, suggesting a pathomechanism other than simple haploinsufficiency. We conclude that de novo variants in TRPM3 are a cause of intellectual disability and epilepsy.

The transient receptor potential melastatin 8 (TRPM8) ion channel is the primary detector of environmental cold and an important target for treating pathological cold hypersensitivity. Here, we present cryo-electron microscopy structures of TRPM8 in ligand-free, antagonist-bound, or calcium-bound forms, revealing how robust conformational changes give rise to two nonconducting states, closed and desensitized. We describe a malleable ligand-binding pocket that accommodates drugs of diverse chemical structures, and we delineate the ion permeation pathway, including the contribution of lipids to pore architecture. Furthermore, we show that direct calcium binding mediates stimulus-evoked desensitization, clarifying this important mechanism of sensory adaptation. We observe large rearrangements within the S4-S5 linker that reposition the S1-S4 and pore domains relative to the TRP helix, leading us to propose a distinct model for modulation of TRPM8 and possibly other TRP channels., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

TRPM2 is critically involved in diverse physiological processes including core temperature sensing, apoptosis, and immune response. TRPM2's activation by Ca 2+ and ADP ribose (ADPR), an NAD + -metabolite produced under oxidative stress and neurodegenerative conditions, suggests a role in neurological disorders. We provide a central concept between triple-site ligand binding and the channel gating of human TRPM2. We show consecutive structural rearrangements and channel activation of TRPM2 induced by binding of ADPR in two indispensable locations, and the binding of Ca 2+ in the transmembrane domain. The 8-Br-cADPR-an antagonist of cADPR-binds only to the MHR1/2 domain and inhibits TRPM2 by stabilizing the channel in an apo-like conformation. We conclude that MHR1/2 acts as a orthostatic ligand-binding site for TRPM2. The NUDT9-H domain binds to a second ADPR to assist channel activation in vertebrates, but not necessary in invertebrates. Our work provides insights into the gating mechanism of human TRPM2 and its pharmacology., Competing Interests: YH, BR, WL, JD No competing interests declared, (© 2019, Huang et al.)

Transient receptor potential (TRPs) channels are crucial downstream targets of calcium signalling cascades. They can be modulated either by calcium itself and/or by calcium-binding proteins (CBPs). Intracellular messengers usually interact with binding domains present at the most variable TRP regions-N- and C-cytoplasmic termini. Calmodulin (CaM) is a calcium-dependent cytosolic protein serving as a modulator of most transmembrane receptors. Although CaM-binding domains are widespread within intracellular parts of TRPs, no such binding domain has been characterised at the TRP melastatin member-the transient receptor potential melastatin 6 (TRPM6) channel. Another CBP, the S100 calcium-binding protein A1 (S100A1), is also known for its modulatory activities towards receptors. S100A1 commonly shares a CaM-binding domain. Here, we present the first identified CaM and S100A1 binding sites at the N-terminal of TRPM6. We have confirmed the L520-R535 N-terminal TRPM6 domain as a shared binding site for CaM and S100A1 using biophysical and molecular modelling methods. A specific domain of basic amino acid residues (R526/R531/K532/R535) present at this TRPM6 domain has been identified as crucial to maintain non-covalent interactions with the ligands. Our data unambiguously confirm that CaM and S100A1 share the same binding domain at the TRPM6 N-terminus although the ligand-binding mechanism is different.

Transient receptor potential melastatin 2 (TRPM2) is a non-selective cation channel that allows Ca 2+ influx across the plasma membrane and efflux from lysosomes upon opening. TRPM2 is best known as a biosensor of reactive oxygen species (ROS), which mediates some of the body's responses to oxidative stress. As such, TRPM2 is involved in a plethora of biological processes including immune response, insulin secretion, body temperature control and neuronal cell death, and represents an emerging therapeutic target for many human diseases, from diabetes to inflammatory and neurodegenerative diseases. A direct ligand of TRPM2 is ADP-ribose (ADPR), which accumulates in cells at high levels of ROS, and activates TRPM2 synergistically with intracellular calcium (Ca 2+ ). Here, we describe recent cryo-electron microscopy (cryo-EM) structures of TRPM2 and summarize the insights they provided into the gating mechanism of the channel., (© 2019 Federation of European Biochemical Societies.)

The transient receptor potential melastatin 2 (TRPM2) channel plays a key role in redox sensation in many cell types. Channel activation requires binding of both ADP-ribose (ADPR) and Ca 2+ . The recently published TRPM2 structures from Danio rerio in the ligand-free and the ADPR/Ca 2+ -bound conditions represent the channel in closed and open states, which uncovered substantial tertiary and quaternary conformational rearrangements. However, it is unclear how these rearrangements are achieved within the tetrameric channel during channel gating. Here we report the cryo-electron microscopy structures of Danio rerio TRPM2 in the absence of ligands, in complex with Ca 2+ alone, and with both ADPR and Ca 2+ , resolved to ~4.3 Å, ~3.8 Å, and ~4.2 Å, respectively. In contrast to the published results, our studies capture ligand-bound TRPM2 structures in two-fold symmetric intermediate states, offering a glimpse of the structural transitions that bridge the closed and open conformations.

The study of membrane proteins, and in particular ion channels, is crucial to understanding cellular function. Mass spectrometry-based approaches including bottom-up strategies to study membrane proteins have been successful yet still can remain challenging. In this study, we sought to evaluate the phosphorylation patterns of the ion channel TRPM7 which is involved in a range of critical physiological functions. To overcome extraction obstacles associated with analyzing membrane proteins, we incorporated the use of 5% SDS solubilization coupled with SCAD and S-Trap digestion methods to eliminate detergent interference in downstream LC-MS/MS analysis. We found that the SCAD method was more efficient, yielding 84% of the overall identified proteins; however, the variability was greater than the S-Trap method. Using both methods together with TiO 2 and Fe-NTA phospho-enrichment protocols, we successfully observed the phosphorylation pattern of TRPM7 in a transfected cell line. An average of 22 ± 6% of the TRPM7 amino acid sequence was observed. In addition to several previously reported phosphorylation sites, we identified six new phosphosites (S5, S233, S554, S824, T1265, and S1401), providing new targets for further functional analyses of TRPM7.

TRPM2 is a non-selective, Ca 2+ -permeable cation channel, which plays a role in cell death but also contributes to diverse immune cell functions. In addition, TRPM2 contributes to the control of body temperature and is involved in perception of non-noxious heat and thermotaxis. TRPM2 is regulated by many factors including Ca 2+ , ADPR, 2'-deoxy-ADPR, Ca 2+ -CaM, and temperature. However, the molecular basis for the temperature sensitivity of TRPM2 as well as the interplay between the regulatory factors is still not understood. Here we identify a novel CaM-binding site in the unique NudT9H domain of TRPM2. Using a multipronged biophysical approach we show that binding of Ca 2+ -CaM to this site occurs upon partial unfolding at temperatures >35 °C and prevents further thermal destabilization. In combination with patch-clamp measurements of full-length TRPM2 our results suggest a role of this CaM-binding site in the temperature sensitivity of TRPM2. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech., (Copyright © 2019 Elsevier B.V. All rights reserved.)

cADPR is a well-recognized signaling molecule by modulating the RyRs, but considerable debate exists regarding whether cADPR can bind to and gate the TRPM2 channel, which mediates oxidative stress signaling in diverse physiological and pathological processes. Here, we show that purified cADPR evoked TRPM2 channel currents in both whole-cell and cell-free single-channel recordings and specific binding of cADPR to the purified NUDT9-H domain of TRPM2 by surface plasmon resonance. Furthermore, by combining computational modeling with electrophysiological recordings, we show that the TRPM2 channels carrying point mutations at H1346, T1347, L1379, S1391, E1409, and L1484 possess distinct sensitivity profiles for ADPR and cADPR. These results clearly indicate cADPR is a bona fide activator at the TRPM2 channel and clearly delineate the structural basis for cADPR binding, which not only lead to a better understanding in the gating mechanism of TRPM2 channel but also shed light on a cADPR-induced RyRs-independent Ca 2+ signaling mechanism., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

The transient receptor potential melastatin subtype 8 (TRPM8) is a nonselective, multimodal ion channel, activated by low temperatures (<28 °C), pressure, and cooling compounds (menthol, icilin). Experimental evidences indicated a role of TRPM8 in cold thermal transduction, different life-threatening tumors, and other pathologies, including migraine, urinary tract dysfunction, dry eye disease, and obesity. Hence, the modulation of the TRPM8 channel could be essential in order to understand its implications in these pathologies and for therapeutic intervention. This short review will cover recent progress on the TRPM8 agonists and antagonists, describing newly reported chemotypes, and their application in the pharmacological characterization of TRPM8 in health and disease. The recently described structures of the TRPM8 channel alone or complexed with known agonists and PIP 2 are also discussed.

Objective: To construct a three-dimensional structural model for the selectivity filter in the transient receptor pontential melastatin 8 (TRPM8) ion channel., Methods: In the Rosetta computational structural biology suite, multiple rounds of de novo modeling with the kinematic loop closure algorithm were performed., Results: After nine rounds of computational modeling, we obtained the models of the selectivity filter within the TRPM8 channel with the lowest energy and high convergence. The model showed that the sidechain of D918 points were away from the central ion permeation pathway, while the sidechains of Q914, D920 and T923 pointed towards it. The glycosylation site N934 was located outside the pore region and its side chain directed to the extracellular water environment., Conclusions: A three-dimensional structural model for the selectivity filter in the TRPM8 ion channel was constructed, which provides reliable structural information for exploring the mechanism of ion selectivity.

Objective: To develop methods of extraction and purification of Cterminal NUDT9 homology domain of human transient receptor potential melastatin 2 (TRPM2) channel., Methods: After sonication and centrifuge of Escherichia coli strain Rosetta (DE3) which was induced by isopropylthio-β-D-galactoside, GST-NUDT9-H was collected after the binding of supernatant with GST beads and eluted with reduced glutathione. Then the elution buffer containing fusion protein was purified by size exclusion chromatography after concentration and centrifuge. Finally, with the cleavage of thrombin and binding with the GST beads, NUDT9-H with high purity in supernatant was collected., Results: The GST-NUDT9-H fusion protein was stabilized with lysis buffer containing 0.5% n-dodecyl -β-d-maltoside (DDM), and wash buffer containing 0.025% DDM in size-exclusion chromatography system, and finally the NUDT9-H with high purity was obtained after cleaved by thrombin (1 U/2 mg fusion protein) for 24 h., Conclusions: Due to the poor stability of NUDT9-H, it is necessary to add DDM in extraction and purification buffer to stabilize the conformation of NUDT9-H, so as to increase its yields and purity.