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Transient receptor potential melastatin (TRPM) ion channels constitute the largest TRP subfamily and are involved in many physiological processes. TRPM8 is the primary cold and menthol sensor, and TRPM4 is associated with cardiovascular disorders. Yin et al. and Autzen et al. shed light on the general architecture of the TRPM subfamily by solving the structures of TRPM8 and TRPM4, respectively (see the Perspective by Bae et al. ). The three-layered architecture of the TRPM8 channel provides the framework for understanding the mechanisms of cold and menthol sensing. The two distinct closed states of TRPM4, with and without calcium, reveal a calcium-binding site and calcium-binding-induced conformational changes. Science , this issue p. 237 , p. 228 ; see also p. 160 Structures of a human cation channel revealed by single-particle cryo–electron microscopy elucidate the calcium-binding site. Transient receptor potential (TRP) melastatin 4 (TRPM4) is a widely expressed cation channel associated with a variety of cardiovascular disorders. TRPM4 is activated by increased intracellular calcium in a voltage-dependent manner but, unlike many other TRP channels, is permeable to monovalent cations only. Here we present two structures of full-length human TRPM4 embedded in lipid nanodiscs at ~3-angstrom resolution, as determined by single-particle cryo–electron microscopy. These structures, with and without calcium bound, reveal a general architecture for this major subfamily of TRP channels and a well-defined calcium-binding site within the intracellular side of the S1-S4 domain. The structures correspond to two distinct closed states. Calcium binding induces conformational changes that likely prime the channel for voltage-dependent opening.

TRPM4 is a calcium-activated, phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P 2 ) -modulated, non-selective cation channel that belongs to the family of melastatin-related transient receptor potential (TRPM) channels. Here we present the electron cryo-microscopy structures of the mouse TRPM4 channel with and without ATP. TRPM4 consists of multiple transmembrane and cytosolic domains, which assemble into a three-tiered architecture. The N-terminal nucleotide-binding domain and the C-terminal coiled-coil participate in the tetrameric assembly of the channel; ATP binds at the nucleotide-binding domain and inhibits channel activity. TRPM4 has an exceptionally wide filter but is only permeable to monovalent cations; filter residue Gln973 is essential in defining monovalent selectivity. The S1–S4 domain and the post-S6 TRP domain form the central gating apparatus that probably houses the Ca 2+ - and PtdIns(4,5)P 2 -binding sites. These structures provide an essential starting point for elucidating the complex gating mechanisms of TRPM4 and reveal the molecular architecture of the TRPM family. Electron cryo-microscopy structures of mouse TRPM4, a calcium-activated, non-selective cation channel, in the apo and ATP-bound states. Scoping out TRPM channels Melastatin-related transient receptor potential (TRPM) ion channels are the largest group of the TRP superfamily and, as such, are widespread throughout the body with diverse physiological roles including heat and taste sensation and regulating ion homeostasis. For example, TRPM4 is a Ca 2+ -activated non-selective channel expressed in many of the central organs including the brain and heart, and is involved in the cardiac rhythm, breath pacemaking and insulin secretion. In this issue of Nature , two groups report the structure of TRPM4 by electron cryo-microscopy. Wei Lü and colleagues solved the structure of human TRPM4, which shows an umbrella-like structure, bound to Ca(ɪɪ) and decavanadate. Youxing Jiang and colleagues report the structure of mouse TRPM4 with and without ATP, which inhibits channel activity. These studies provide the first structural insights into the TRPM family.

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, butwhat 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 H₂O₂ 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) residuesM528 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.

Transient receptor potential melastatin subfamily member 4 (TRPM4) is a widely distributed, calcium-activated, monovalent-selective cation channel. Mutations in human TRPM4 (hTRPM4) result in progressive familial heart block. Here, we report the electron cryomicroscopy structure of hTRPM4 in a closed, Na⁺-bound, apo state at pH 7.5 to an overall resolution of 3.7 Å. Five partially hydrated sodium ions are proposed to occupy the center of the conduction pore and the entrance to the coiled-coil domain. We identify an upper gate in the selectivity filter and a lower gate at the entrance to the cytoplasmic coiled-coil domain. Intramolecular interactions exist between the TRP domain and the S4–S5 linker, N-terminal domain, and N and C termini. Finally, we identify aromatic interactions via π–π bonds and cation–π bonds, glycosylation at an N-linked extracellular site, a pore-loop disulfide bond, and 24 lipid binding sites. We compare and contrast this structure with other TRP channels and discuss potential mechanisms of regulation and gating of human full-length TRPM4.

Transient receptor potential melastatin 2 (TRPM2) is a calcium-permeable, non-selective cation channel that has an essential role in diverse physiological processes such as core body temperature regulation, immune response and apoptosis 1 – 4 . TRPM2 is polymodal and can be activated by a wide range of stimuli 1 – 7 , including temperature, oxidative stress and NAD + -related metabolites such as ADP-ribose (ADPR). Its activation results in both Ca 2+ entry across the plasma membrane and Ca 2+ release from lysosomes 8 , and has been linked to diseases such as ischaemia-reperfusion injury, bipolar disorder and Alzheimer’s disease 9 – 11 . Here we report the cryo-electron microscopy structures of the zebrafish TRPM2 in the apo resting (closed) state and in the ADPR/Ca 2+ -bound active (open) state, in which the characteristic NUDT9-H domains hang underneath the MHR1/2 domain. We identify an ADPR-binding site located in the bi-lobed structure of the MHR1/2 domain. Our results provide an insight into the mechanism of activation of the TRPM channel family and define a framework for the development of therapeutic agents to treat neurodegenerative diseases and temperature-related pathological conditions. Structures of the transient receptor potential melastatin 2 channel in the apo resting (closed) state and in the ADP-ribose/Ca 2+ -bound active (open) state are determined by cryo-electron microscopy.

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. Menthol in mints elicits a coolness sensation by selective activation of TRPM8 ion channel. Here authors dock menthol to TRPM8 and systematically validate their menthol binding models with thermodynamic mutant cycle analysis in functional tests, and shed light on TRPM8 activation by menthol at the atomic level.

Adenosine diphosphate–ribose (ADPR) mediates calcium (Ca 2+ ) release by activating the transient receptor potential melastatin 2 (TRPM2) channel. Three structures now elucidate the conformational regulation mechanism of TRPM2 gating. Wang et al. describe cryo–electron microscopy structures of human TRPM2 in the apo, ADPR-bound, and ADPR- and Ca 2+ -bound states. In the apo state, both intra- and intersubunit interactions appeared to lock TRPM2 into a closed and autoinhibited state. ADPR binding disrupted some interactions and dramatically altered the TRPM2 conformation. Binding of Ca 2+ further primed the opening of the channel. Science , this issue p. eaav4809 Gating of the TRPM2 (transient receptor potential melastatin 2) cation channel involves transmembrane helix–linked conformational changes. Transient receptor potential (TRP) melastatin 2 (TRPM2) is a cation channel associated with numerous diseases. It has a C-terminal NUDT9 homology (NUDT9H) domain responsible for binding adenosine diphosphate (ADP)–ribose (ADPR), and both ADPR and calcium (Ca 2+ ) are required for TRPM2 activation. Here we report cryo–electron microscopy structures of human TRPM2 alone, with ADPR, and with ADPR and Ca 2+ . NUDT9H forms both intra- and intersubunit interactions with the N-terminal TRPM homology region (MHR1/2/3) in the apo state but undergoes conformational changes upon ADPR binding, resulting in rotation of MHR1/2 and disruption of the intersubunit interaction. The binding of Ca 2+ further engages transmembrane helices and the conserved TRP helix to cause conformational changes at the MHR arm and the lower gating pore to potentiate channel opening. These findings explain the molecular mechanism of concerted TRPM2 gating by ADPR and Ca 2+ and provide insights into the gating mechanism of other TRP channels.

The transient receptor potential ion channel subfamily M, member 7 (TRPM7), is a ubiquitously expressed protein that is required for mouse embryonic development. TRPM7 contains both an ion channel and an α-kinase. The channel domain comprises a nonselective cation channel with notable permeability to Mg2+ and Zn2+. Here, we report the closed state structures of the mouse TRPM7 channel domain in three different ionic conditions to overall resolutions of 3.3, 3.7, and 4.1 Å. The structures reveal key residues for an ion binding site in the selectivity filter, with proposed partially hydrated Mg2+ ions occupying the center of the conduction pore. In high [Mg2+], a prominent external disulfide bond is found in the pore helix, which is essential for ion channel function. Our results provide a structural framework for understanding the TRPM1/3/6/7 subfamily and extend the knowledge base upon which to study the diversity and evolution of TRP channels.

Transient receptor potential (TRP) melastatin 4 (TRPM4) protein is a calcium-activated monovalent cation channel associated with various genetic and cardiovascular disorders. The anthranilic acid derivative NBA is a potent and specific TRPM4 inhibitor, but its binding site in TRPM4 has been unknown, although this information is crucial for drug development targeting TRPM4. We determine three cryo-EM structures of full-length human TRPM4 embedded in native lipid nanodiscs without inhibitor, bound to NBA, and an anthranilic acid derivative, IBA. We found that the small molecules NBA and IBA were bound in a pocket formed between the S3, S4, and TRP helices and the S4-S5 linker of TRPM4. Our structural data and results from patch clamp experiments enable validation of a binding site for small molecule inhibitors, paving the way for further drug development targeting TRPM4. TRPM4 is an ion channel associated with various genetic and cardiovascular disorders. The authors utilized cryo-EM and patch clamp experiments to determine the binding sites of potent and specific TRPM4 inhibitors driving further drug development targeting TRPM4.

In humans, cold is primarily sensed by transient receptor potential melastatin member 8 (TRPM8), a calcium channel. Yin et al. present cryo–electron microscopy structures of TRPM8 with cooling agents, membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2), and calcium. Structural and functional analyses showed that the PIP2 binding site in TRPM8 is completely different from PIP2 sites in other TRP channels. The binding of PIP2 and cooling agents allosterically enhance each other and activate the channel opening. Thus, the activation mechanism of TRPM8 is distinct from that used by other TRP channels. Science , this issue p. eaav9334 Cryo-EM structures elucidate the molecular basis for cold and menthol sensing and reveal the distinctive PIP2 dependence in the TRPM8 calcium channel. Transient receptor potential melastatin member 8 (TRPM8) is a calcium ion (Ca 2+ )–permeable cation channel that serves as the primary cold and menthol sensor in humans. Activation of TRPM8 by cooling compounds relies on allosteric actions of agonist and membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP 2 ), but lack of structural information has thus far precluded a mechanistic understanding of ligand and lipid sensing by TRPM8. Using cryo–electron microscopy, we determined the structures of TRPM8 in complex with the synthetic cooling compound icilin, PIP 2 , and Ca 2+ , as well as in complex with the menthol analog WS-12 and PIP 2 . Our structures reveal the binding sites for cooling agonists and PIP 2 in TRPM8. Notably, PIP 2 binds to TRPM8 in two different modes, which illustrate the mechanism of allosteric coupling between PIP 2 and agonists. This study provides a platform for understanding the molecular mechanism of TRPM8 activation by cooling agents.