Conservation of the cooling agent binding pocket within the TRPM subfamily.

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 Ca2+. 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 Ca2+, enhancing the sensitivity of the channel to Ca2+ 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.

A hydrophobic funnel governs monovalent cation selectivity in the ion channel TRPM5.

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 Ca2+, 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.

A frozen portrait of a warm channel.

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 Ca2+ ions and other channel modulators.

Structural dynamics at cytosolic interprotomer interfaces control gating of a mammalian TRPM5 channel.

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 Ca2+. Here, we show that activation of mammalian TRPM5 channels is strongly altered by Ca2+-dependent desensitization. Structures of rat TRPM5 identify a series of conformational transitions triggered by Ca2+ 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.

Coupling enzymatic activity and gating in an ancient TRPM chanzyme and its molecular evolution.

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.

Physiological temperature drives TRPM4 ligand recognition and gating.

Temperature profoundly affects macromolecular function, particularly in proteins with temperature sensitivity1,2. 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 Ca2+-activated ion channel3-7. 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 Ca2+-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 temperatures10,11, 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.

Structural and functional analyses of a GPCR-inhibited ion channel TRPM3.

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 (Gßγ) released upon their activation. This GPCR-TRPM3 signaling pathway contributes to the analgesic effect of morphine. Here, we characterized Gßγ 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 Gßγ and with and without PIP2, a lipid required for TRPM3 activity, at resolutions of 2.7-4.7 Å. Gßγ-TRPM3 interfaces vary depending on PIP2 occupancy; however, in all cases, Gßγ appears loosely attached to TRPM3. The IC50 in electrophysiology experiments raises the possibility that additional unknown factors may stabilize the TRPM3-Gßγ complex.

Activation mechanism of the mouse cold-sensing TRPM8 channel by cooling agonist and PIP2.

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 (PIP2). 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 PIP2-dependent TRPM8 activation. We present cryo-electron microscopy structures of mouse TRPM8 in closed, intermediate, and open states along the ligand- and PIP2-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.

Mutations of TRPM8 channels: Unraveling the molecular basis of activation by cold and ligands.

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.

Recent Advances in the Structural Biology of Mg2+ Channels and Transporters.

Magnesium ions (Mg2+) 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 Mg2+ are crucial for both eukaryotic and prokaryotic organisms and are thus strictly controlled by Mg2+ 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 Mg2+ channels and transporters. In this review article, we provide an overview of the families of Mg2+ 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.

The acquisition of cold sensitivity during TRPM8 ion channel evolution.

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.

The crystal structure of TRPM2 MHR1/2 domain reveals a conserved Zn2+ -binding domain essential for structural integrity and channel activity.

Transient receptor potential melastatin 2 (TRPM2) is a Ca2+ -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 Ca2+ -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 Zn2+ -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 Zn2+ -binding domain on TRPM2 activation. Our results provide insight into a conserved motif essential for structural integrity and channel activity.

TRPM5 Channel Binds Calcium-Binding Proteins Calmodulin and S100A1.

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.

Immunomodulatory functions of TRPM7 and its implications in autoimmune diseases.

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.

Partial Agonistic Actions of Sex Hormone Steroids on TRPM3 Function.

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.

Distinct calcium regulation of TRPM7 mechanosensitive channels at plasma membrane microdomains visualized by FRET-based single cell imaging.

Transient receptor potential subfamily M member 7 (TRPM7), a mechanosensitive Ca2+ channel, plays a crucial role in intracellular Ca2+ homeostasis. However, it is currently unclear how cell mechanical cues control TRPM7 activity and its associated Ca2+ influx at plasma membrane microdomains. Using two different types of Ca2+ biosensors (Lyn-D3cpv and Kras-D3cpv) based on fluorescence resonance energy transfer, we investigate how Ca2+ 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 Ca2+ influx mainly occurs at the DRM, and chemically induced mechanical perturbations in the cell mechanosensitive apparatus substantially reduce Ca2+ 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.

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.

Role of TRPM7 kinase in cancer.

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 Ca2+- and Mg2+-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 Ca2+ and Mg2+ 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.

The structural basis for an on-off switch controlling Gßγ-mediated inhibition of TRPM3 channels.

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 Gßγ proteins, thereby reducing TRPM3-mediated pain. We show here that Gßγ directly binds to a domain of 10 amino acids in TRPM3 and solve a cocrystal structure of this domain together with Gßγ. Using these data and mutational analysis of full-length proteins, we pinpoint three amino acids in TRPM3 and their interacting partners in Gß1 that are individually necessary for TRPM3 inhibition by Gßγ. The 10-amino-acid Gßγ-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 Gßγ proteins exert on TRPM3 channels.

Medicinal chemistry perspective of TRPM2 channel inhibitors: where we are and where we might be heading?

Transient receptor potential melastatin 2 (TRPM2) is a Ca2+- 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.