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The TRPA1 ion channel (also known as the wasabi receptor) is a detector of noxious chemical agents encountered in our environment or produced endogenously during tissue injury or drug metabolism. These include a broad class of electrophiles that activate the channel through covalent protein modification. TRPA1 antagonists hold potential for treating neurogenic inflammatory conditions provoked or exacerbated by irritant exposure. Despite compelling reasons to understand TRPA1 function, structural mechanisms underlying channel regulation remain obscure. Here we use single-particle electron cryo- microscopy to determine the structure of full-length human TRPA1 to ∼4 Å resolution in the presence of pharmacophores, including a potent antagonist. Several unexpected features are revealed, including an extensive coiled-coil assembly domain stabilized by polyphosphate co-factors and a highly integrated nexus that converges on an unpredicted transient receptor potential (TRP)-like allosteric domain. These findings provide new insights into the mechanisms of TRPA1 regulation, and establish a blueprint for structure-based design of analgesic and anti-inflammatory agents. The high-resolution electron cryo-microscopy structure of the full-length human TRPA1 ion channel is presented; the structure reveals a unique ankyrin repeat domain arrangement, a tetrameric coiled-coil in the centre of the channel that acts as a binding site for inositol hexakisphosphate, an outer poor domain with two pore helices, and a new drug binding site, findings that collectively provide mechanistic insight into TRPA1 regulation. Structure of multifunctional TRPA1 receptor TRP (transient receptor potential) channels are expressed by all eukaryotic organisms and act as sensors for a wide range of physical and chemical stimuli. This paper reports the high-resolution electron cryomicroscopy structure of full-length human TRPA1, a sensory receptor for noxious chemical agents such as wasabi. The overall structure of this membrane protein differs markedly from the previously published structure of TRPV1, as TRPA1 has many ankyrin repeat domains, a tetrameric coiled-coil in the center of the channel that appears to serve as a binding site for inositol hexakisphosphate and an outer pore domain with two pore helices. TRPA1 is associated with persistent pain, respiratory and chronic itch syndromes, so TRPA1 antagonists are of interest as potential analgesics.

Single-particle electron cryo-microscopy analysis of the mechanotransduction channel NOMPC reveals that it contains a bundle of four helical spring-shaped ankyrin repeat domains that undergo motion, potentially allowing mechanical movement of the cytoskeleton to be coupled to the opening of the channel. Molecular mechanics of sensations Mechanosensation forms the basis of many of our senses, including touch, balance, hearing and pain. Mechanically gated ion channels are responsible for transmitting mechanical force into electrical signals. However, how this occurs is not well understood at the molecular level. Here the authors report the structure of the Drosophila mechanotransduction channel NOMPC by single-particle cryo-electron microscopy. The channel contains a long, helical domain of ankyrin repeats, which appears to undergo a spring-like motion. This motion allows the mechanical movement of the cytoskeleton to be relayed into opening the channel. Mechanosensory transduction for senses such as proprioception, touch, balance, acceleration, hearing and pain relies on mechanotransduction channels, which convert mechanical stimuli into electrical signals in specialized sensory cells 1 . How force gates mechanotransduction channels is a central question in the field, for which there are two major models. One is the membrane-tension model: force applied to the membrane generates a change in membrane tension that is sufficient to gate the channel, as in the bacterial MscL channel and certain eukaryotic potassium channels 2 , 3 , 4 , 5 . The other is the tether model: force is transmitted via a tether to gate the channel. The transient receptor potential (TRP) channel NOMPC is important for mechanosensation-related behaviours such as locomotion, touch and sound sensation across different species including Caenorhabditis elegans 6 , Drosophila 7 , 8 , 9 and zebrafish 10 . NOMPC is the founding member of the TRPN subfamily 11 , and is thought to be gated by tethering of its ankyrin repeat domain to microtubules of the cytoskeleton 12 , 13 , 14 , 15 . Thus, a goal of studying NOMPC is to reveal the underlying mechanism of force-induced gating, which could serve as a paradigm of the tether model. NOMPC fulfils all the criteria that apply to mechanotransduction channels 1 , 7 and has 29 ankyrin repeats, the largest number among TRP channels. A key question is how the long ankyrin repeat domain is organized as a tether that can trigger channel gating. Here we present a de novo atomic structure of Drosophila NOMPC determined by single-particle electron cryo-microscopy. Structural analysis suggests that the ankyrin repeat domain of NOMPC resembles a helical spring, suggesting its role of linking mechanical displacement of the cytoskeleton to the opening of the channel. The NOMPC architecture underscores the basis of translating mechanical force into an electrical signal within a cell.

Transient receptor potential mucolipin 1 (TRPML1) is a Ca 2+ -releasing cation channel that mediates the calcium signalling and homeostasis of lysosomes. Mutations in TRPML1 lead to mucolipidosis type IV, a severe lysosomal storage disorder. Here we report two electron cryo-microscopy structures of full-length human TRPML1: a 3.72-Å apo structure at pH 7.0 in the closed state, and a 3.49-Å agonist-bound structure at pH 6.0 in an open state. Several aromatic and hydrophobic residues in pore helix 1, helices S5 and S6, and helix S6 of a neighbouring subunit, form a hydrophobic cavity to house the agonist, suggesting a distinct agonist-binding site from that found in TRPV1, a TRP channel from a different subfamily. The opening of TRPML1 is associated with distinct dilations of its lower gate together with a slight structural movement of pore helix 1. Our work reveals the regulatory mechanism of TRPML channels, facilitates better understanding of TRP channel activation, and provides insights into the molecular basis of mucolipidosis type IV pathogenesis. Two structures of human transient receptor potential mucolipin 1 (TRPML1), in the closed and agonist-bound open states, have been resolved by electron cryo-microscopy. Closing in on ion channels Numerous ion channels sit in the membranes of intracellular organelles and are responsible for maintaining concentration gradients and ionic signalling. The transient receptor potential mucolipin (TRPML) channels are Ca( II )-releasing channels that are crucial to endolysosomal function. While TRPML channels regulate physiological processes including membrane trafficking and exocytosis, mutations of TRPML1 cause the lysosomal storage disorder mucolipidosis type IV. Three papers in this issue of Nature report the structure of TRPML channels by cryo-electron microscopy. Seok-Yong Lee and colleagues report the structure of TRPML3, while studies from teams led by Xiaochun Li and Youxing Jiang present the structure of TRPML1. Together, these studies reveal the open and closed states of the TRPML family, indicating the regulatory mechanisms of these channels. As with most TRP channels, TRPML can be gated by specific lipids, and these studies provide insights into substrate binding and channel activation.

The structure of mouse transient receptor potential mucolipin 1 (TRPML1), a cation channel located within endosomal and lysosomal membranes, is resolved using single-particle electron cryo-microscopy. Closing in on ion channels Numerous ion channels sit in the membranes of intracellular organelles and are responsible for maintaining concentration gradients and ionic signalling. The transient receptor potential mucolipin (TRPML) channels are Ca( II )-releasing channels that are crucial to endolysosomal function. While TRPML channels regulate physiological processes including membrane trafficking and exocytosis, mutations of TRPML1 cause the lysosomal storage disorder mucolipidosis type IV. Three papers in this issue of Nature report the structure of TRPML channels by cryo-electron microscopy. Seok-Yong Lee and colleagues report the structure of TRPML3, while studies from teams led by Xiaochun Li and Youxing Jiang present the structure of TRPML1. Together, these studies reveal the open and closed states of the TRPML family, indicating the regulatory mechanisms of these channels. As with most TRP channels, TRPML can be gated by specific lipids, and these studies provide insights into substrate binding and channel activation. Transient receptor potential mucolipin 1 (TRPML1) is a cation channel located within endosomal and lysosomal membranes. Ubiquitously expressed in mammalian cells 1 , 2 , its loss-of-function mutations are the direct cause of type IV mucolipidosis, an autosomal recessive lysosomal storage disease 3 , 4 , 5 , 6 . Here we present the single-particle electron cryo-microscopy structure of the mouse TRPML1 channel embedded in nanodiscs. Combined with mutagenesis analysis, the TRPML1 structure reveals that phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P 2 ) binds to the N terminus of the channel—distal from the pore—and the helix–turn–helix extension between segments S2 and S3 probably couples ligand binding to pore opening. The tightly packed selectivity filter contains multiple ion-binding sites, and the conserved acidic residues form the luminal Ca 2+ -blocking site that confers luminal pH and Ca 2+ modulation on channel conductance. A luminal linker domain forms a fenestrated canopy atop the channel, providing several luminal ion passages to the pore and creating a negative electrostatic trap, with a preference for divalent cations, at the luminal entrance. The structure also reveals two equally distributed S4–S5 linker conformations in the closed channel, suggesting an S4–S5 linker-mediated PtdInsP 2 gating mechanism among TRPML channels 7 , 8 .

Amphipathic polymers (amphipols), such as A8-35 and SApol, are a new tool for stabilizing integral membrane proteins in detergent-free conditions for structural and functional studies. Transient receptor potential (TRP) ion channels function as tetrameric protein complexes in a diverse range of cellular processes including sensory transduction. Mammalian TRP channels share ~20 % sequence similarity and are categorized into six subfamilies: TRPC (canonical), TRPV (vanilloid), TRPA (ankyrin), TRPM (melastatin), TRPP (polycystin), and TRPML (mucolipin). Due to the inherent difficulties in purifying eukaryotic membrane proteins, structural studies of TRP channels have been limited. Recently, A8-35 was essential in resolving the molecular architecture of the nociceptor TRPA1 and led to the determination of a high-resolution structure of the thermosensitive TRPV1 channel by cryo-EM. Newly developed maltose-neopentyl glycol (MNG) detergents have also proven to be useful in stabilizing TRP channels for structural analysis. In this review, we will discuss the impacts of amphipols and MNG detergents on structural studies of TRP channels by cryo-EM. We will compare how A8-35 and MNG detergents interact with the hydrophobic transmembrane domains of TRP channels. In addition, we will discuss what these cryo-EM studies reveal on the importance of screening different types of surfactants toward determining high-resolution structures of TRP channels.

Transient receptor potential mucolipin 1 (TRPML1), a lysosomal channel, maintains the low pH and calcium levels for lysosomal function. Several small molecules modulate TRPML1 activity. ML-SA1, a synthetic agonist, binds to the pore region and phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P ), a natural lipid, stimulates channel activity to a lesser extent than ML-SA1; moreover, PtdIns(4,5)P , another natural lipid, prevents TRPML1-mediated calcium release. Notably, PtdIns(3,5)P and ML-SA1 cooperate further increasing calcium efflux. Here we report the structures of human TRPML1 at pH 5.0 with PtdIns(3,5)P , PtdIns(4,5)P , or ML-SA1 and PtdIns(3,5)P , revealing a unique lipid-binding site. PtdIns(3,5)P and PtdIns(4,5)P bind to the extended helices of S1, S2, and S3. The phosphate group of PtdIns(3,5)P induces Y355 to form a π-cation interaction with R403, moving the S4-S5 linker, thus allosterically activating the channel. Our structures and electrophysiological characterizations reveal an allosteric site and provide molecular insight into how lipids regulate TRP channels.

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.

Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disease that can lead to kidney failure. Mutations in the proteins PKD1 and PKD2 are linked to the disease, but the function of these proteins remains unclear, both in physiology and disease. PKD1 has been implicated in the sensing of chemical and mechanical force stimuli, and PKD2 is proposed to be a calcium ion channel. Su et al. show that the transmembrane regions form a PKD1-PKD2 complex assembled in a 1:3 ratio. Their high-resolution cryo–electron microscopy structure confirms that the complex adopts transient receptor potential channel architecture, with some distinctive features. Mapping disease-causing mutations onto the structure suggests that pathogenesis may come from incorrect folding or trafficking of the complex rather than from disruption of channel activity. Science , this issue p. eaat9819 This structure provides a framework for further investigations into a complex involved in polycystic kidney disease. Mutations in two genes, PKD1 and PKD2 , account for most cases of autosomal dominant polycystic kidney disease, one of the most common monogenetic disorders. Here we report the 3.6-angstrom cryo–electron microscopy structure of truncated human PKD1-PKD2 complex assembled in a 1:3 ratio. PKD1 contains a voltage-gated ion channel (VGIC) fold that interacts with PKD2 to form the domain-swapped, yet noncanonical, transient receptor potential (TRP) channel architecture. The S6 helix in PKD1 is broken in the middle, with the extracellular half, S6a, resembling pore helix 1 in a typical TRP channel. Three positively charged, cavity-facing residues on S6b may block cation permeation. In addition to the VGIC, a five–transmembrane helix domain and a cytosolic PLAT domain were resolved in PKD1. The PKD1-PKD2 complex structure establishes a framework for dissecting the function and disease mechanisms of the PKD proteins.

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.