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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.

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 .

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 Transient Receptor Potential Ankyrin 1 channel (TRPA1), is a member of the large TRP family of ion channels, and functions as a Ca 2+ permeable non-selective cation channel in many different cell processes, ranging from sensory to homeostatic tasks. TRPA1 is highly conserved across the animal kingdom. The only mammalian TRPA subfamily member, TRPA1, is widely expressed in neuronal (e.g. sensory dorsal root and trigeminal ganglia neurons)- and in non-neuronal cells (e.g. epithelial cells, hair cells). It exhibits 14–19 amino-( N -)terminal ankyrin repeats, an unusual structural feature. The TRPA1 channel is activated by noxious cold (<17 °C) as well as by a plethora of chemical compounds that includes not only electrophilic compounds and oxidants that can modify, in an alkylative or oxidative fashion, nucleophilic cysteine residues in the channel’s N -terminus, but also compounds that do not covalently bind to the channel proteins (e.g. menthol, nifedipin). Based on localization and functional properties, TRPA1 is considered a key player in acute and chronic (neuropathic) pain and inflammation. Moreover, its role in the (patho)physiology of nearly all organ systems is anticipated, and will be discussed along with the potential of TRPA1 as a drug target for the management of various pathological conditions.

Key Points The role of transient receptor potential (TRP) channels is best understood in the pain area. As TRP channels are expressed on peripheral nociceptors, where pain is generated, it is hoped that TRP channel blockers will be devoid of the side effects that limit the use of analgesic agents that act on the central nervous system. Several TRP cation channel subfamily V, member 1 (TRPV1) antagonists have advanced to clinical trials, but their side effects (which include hyperthermia and impaired noxious heat detection) have prevented any compounds from progressing beyond Phase II clinical trials. TRPV3 antagonists have shown efficacy in models of neuropathic and inflammatory pain, and one antagonist has entered Phase I clinical trials. An autosomal dominant mutation in the gene that encodes TRP cation channel subfamily A, member 1 (TRPA1) causes familial episodic pain syndrome. Indeed, TRPA1 antagonists have been shown to reduce cold hypersensitivity in rodent models of neuropathic pain without altering normal cold sensation in naive animals. Several TRP channels (such as TRPV1, TRPV4 and TRP cation channel subfamily M, member 8 (TRPM8)) are expressed in the urinary bladder, where they presumably function as sensors of stretch and chemical irritation. TRPV1 and TRPV4 antagonists improve bladder function in rodent models of cystitis. Populations of non-neuronal cells within the skin express many different types of TRP channels that are implicated in the regulation of several key cutaneous functions including skin-derived pruritus, proliferation, differentiation and inflammatory processes. TRPA1 and TRPV1 serve as polymodal sensors in the mammalian respiratory tract that integrate varied inflammatory, oxidant and hazardous irritant stimuli to produce noxious sensations (for example, breathlessness, the urge to cough and nasopharyngeal pain) and respiratory reflexes such as coughing. Several TRP channels — including members of TRP cation channel subfamily C (TRPC) and TRPV — influence the process of gas exchange by regulating airflow, blood flow and airway permeability. Mutations in at least six of the 28 members of the TRP channel superfamily are associated with heritable genetic diseases in humans. These mutations have implicated TRP channels in many pathophysiological states and expanded our understanding of the physiological role of these channels. The role of TRP channels in the brain remains to be elucidated, but it seems to be clear that some members of the superfamily are involved in neuronal excitability and neurotransmitter release. Genetic deletion of TRPC5 leads to an anxiolytic phenotype, whereas a point mutation in TRPC3 leads to ataxia. TRP channels also serve important functions in other diseases that are not fully explored in this Review. For example, cancer and metabolic diseases will be particularly interesting to watch in the future. Transient receptor potential (TRP) channels are a diverse family of cation channels. Here, the authors discuss recent developments in this area, highlight recent developments and setbacks in the field of pain research and analyse TRP channels as targets for skin, pulmonary and urological disorders. Transient receptor potential (TRP) cation channels have been among the most aggressively pursued drug targets over the past few years. Although the initial focus of research was on TRP channels that are expressed by nociceptors, there has been an upsurge in the amount of research that implicates TRP channels in other areas of physiology and pathophysiology, including the skin, bladder and pulmonary systems. In addition, mutations in genes encoding TRP channels are the cause of several inherited diseases that affect a variety of systems including the renal, skeletal and nervous system. This Review focuses on recent developments in the TRP channel-related field, and highlights potential opportunities for therapeutic intervention.

Cation channels of the transient receptor potential (TRP) family serve important physiological roles by opening in response to diverse intra- and extracellular stimuli that regulate their lower or upper gates. Despite extensive studies, the mechanism coupling these gates has remained obscure. Previous structures have failed to resolve extracellular loops, known in the TRPV subfamily as ‘pore turrets’, which are proximal to the upper gates. We established the importance of the pore turret through activity assays and by solving structures of rat TRPV2, both with and without an intact turret at resolutions of 4.0 Å and 3.6 Å, respectively. These structures resolve the full-length pore turret and reveal fully open and partially open states of TRPV2, both with unoccupied vanilloid pockets. Our results suggest a mechanism by which physiological signals, such as lipid binding, can regulate the lower gate and couple to the upper gate through a pore-turret-facilitated mechanism.

Cryo–electron microscopy, or simply cryo-EM, refers mainly to three very different yet closely related techniques: electron crystallography, single-particle cryo-EM, and electron cryotomography. In the past few years, single-particle cryo-EM in particular has triggered a revolution in structural biology and has become a newly dominant discipline. This Review examines the fascinating story of its start and evolution over the past 40-plus years, delves into how and why the recent technological advances have been so groundbreaking, and briefly considers where the technique may be headed in the future.

The redox-sensitive TRP channel TRPA1 is activated in hyperoxic and hypoxic conditions directly through modification of cysteine residues by O 2 and indirectly through prolyl hydroxylation by PHDs, enzymes related to the hypoxia-inducible factor HIF-1, thus helping to explain how O 2 is sensed by sensory and vagal neurons. Oxygen (O 2 ) is a prerequisite for cellular respiration in aerobic organisms but also elicits toxicity. To understand how animals cope with the ambivalent physiological nature of O 2 , it is critical to elucidate the molecular mechanisms responsible for O 2 sensing. Here our systematic evaluation of transient receptor potential (TRP) cation channels using reactive disulfides with different redox potentials reveals the capability of TRPA1 to sense O 2 . O 2 sensing is based upon disparate processes: whereas prolyl hydroxylases (PHDs) exert O 2 -dependent inhibition on TRPA1 activity in normoxia, direct O 2 action overrides the inhibition via the prominent sensitivity of TRPA1 to cysteine-mediated oxidation in hyperoxia. Unexpectedly, TRPA1 is activated through relief from the same PHD-mediated inhibition in hypoxia. In mice, disruption of the Trpa1 gene abolishes hyperoxia- and hypoxia-induced cationic currents in vagal and sensory neurons and thereby impedes enhancement of in vivo vagal discharges induced by hyperoxia and hypoxia. The results suggest a new O 2 -sensing mechanism mediated by TRPA1.

Mechanotransduction channels studied to date are mainly involved with sensing noxious mechanical stimuli; here NOMPC, a member of the TRP ion channel family, is identified as a pore-forming subunit of an ion channel essential to the sensation of gentle touch in Drosophila . The gentle touch of the fruitfly Like vertebrates, Drosophila can perform mechanical sensing, such as gravity sensing, hearing, proprioception, mechanical nociception and gentle-touch sensation. The mechanotransduction channels studied to date are mostly involved in sensing noxious stimuli. Now Yuh Nung Jan and colleagues identify NOMPC—a member of the transient receptor potential family of ion channels—as a pore-forming subunit of an ion channel essential to the sensation of gentle touch in Drosophila . Functional studies suggest that different mechanosensitive channels may be used to sense gentle touch and noxious mechanical stimuli. Touch sensation is essential for behaviours ranging from environmental exploration to social interaction; however, the underlying mechanisms are largely unknown 1 . In Drosophila larvae, two types of sensory neurons, class III and class IV dendritic arborization neurons, tile the body wall. The mechanotransduction channel PIEZO in class IV neurons is essential for sensing noxious mechanical stimuli but is not involved in gentle touch 2 . On the basis of electrophysiological-recording, calcium-imaging and behavioural studies, here we report that class III dendritic arborization neurons are touch sensitive and contribute to gentle-touch sensation. We further identify NOMPC (No mechanoreceptor potential C), a member of the transient receptor potential (TRP) family of ion channels, as a mechanotransduction channel for gentle touch. NOMPC is highly expressed in class III neurons and is required for their mechanotransduction. Moreover, ectopic NOMPC expression confers touch sensitivity to the normally touch-insensitive class IV neurons. In addition to the critical role of NOMPC in eliciting gentle-touch-mediated behavioural responses, expression of this protein in the Drosophila S2 cell line also gives rise to mechanosensitive channels in which ion selectivity can be altered by NOMPC mutation, indicating that NOMPC is a pore-forming subunit of a mechanotransduction channel. Our study establishes NOMPC as a bona fide mechanotransduction channel that satisfies all four criteria proposed for a channel to qualify as a transducer of mechanical stimuli 3 and mediates gentle-touch sensation. Our study also suggests that different mechanosensitive channels may be used to sense gentle touch versus noxious mechanical stimuli.