Sensory TRP Channels in Three Dimensions.

Transient receptor potential (TRP) ion channels are sophisticated signaling machines that detect a wide variety of environmental and physiological signals. Every cell in the body expresses one or more members of the extended TRP channel family, which consists of over 30 subtypes, each likely possessing distinct pharmacological, biophysical, and/or structural attributes. While the function of some TRP subtypes remains enigmatic, those involved in sensory signaling are perhaps best characterized and have served as models for understanding how these excitatory ion channels serve as polymodal signal integrators. With the recent resolution revolution in cryo-electron microscopy, these and other TRP channel subtypes are now yielding their secrets to detailed atomic analysis, which is beginning to reveal structural underpinnings of stimulus detection and gating, ion permeation, and allosteric mechanisms governing signal integration. These insights are providing a framework for designing and evaluating modality-specific pharmacological agents for treating sensory and other TRP channel-associated disorders.

The Touching Tail of a Mechanotransduction Channel.

In mechanotransduction, sensory receptors convert force into electrical signals to mediate such diverse functions as touch, pain, and hearing. In this issue of Cell, Zhang et al. present evidence that the fly NompC channel senses mechanical stimuli using its N-terminal tail as a tether between the cell membrane and microtubules.

Ankyrin Repeats Convey Force to Gate the NOMPC Mechanotransduction Channel.

How metazoan mechanotransduction channels sense mechanical stimuli is not well understood. The NOMPC channel in the transient receptor potential (TRP) family, a mechanotransduction channel for Drosophila touch sensation and hearing, contains 29 Ankyrin repeats (ARs) that associate with microtubules. These ARs have been postulated to act as a tether that conveys force to the channel. Here, we report that these N-terminal ARs form a cytoplasmic domain essential for NOMPC mechanogating in vitro, mechanosensitivity of touch receptor neurons in vivo, and touch-induced behaviors of Drosophila larvae. Duplicating the ARs elongates the filaments that tether NOMPC to microtubules in mechanosensory neurons. Moreover, microtubule association is required for NOMPC mechanogating. Importantly, transferring the NOMPC ARs to mechanoinsensitive voltage-gated potassium channels confers mechanosensitivity to the chimeric channels. These experiments strongly support a tether mechanism of mechanogating for the NOMPC channel, providing insights into the basis of mechanosensitivity of mechanotransduction channels.

TRP channels and pain.

Nociception is the process whereby primary afferent nerve fibers of the somatosensory system detect noxious stimuli. Pungent irritants from pepper, mint, and mustard plants have served as powerful pharmacological tools for identifying molecules and mechanisms underlying this initial step of pain sensation. These natural products have revealed three members of the transient receptor potential (TRP) ion channel family--TRPV1, TRPM8, and TRPA1--as molecular detectors of thermal and chemical stimuli that activate sensory neurons to produce acute or persistent pain. Analysis of TRP channel function and expression has validated the existence of nociceptors as a specialized group of somatosensory neurons devoted to the detection of noxious stimuli. These studies are also providing insight into the coding logic of nociception and how specification of nociceptor subtypes underlies behavioral discrimination of noxious thermal, chemical, and mechanical stimuli. Biophysical and pharmacological characterization of these channels has provided the intellectual and technical foundation for developing new classes of analgesic drugs.

Molecular Physiology of TRPV Channels: Controversies and Future Challenges.

The ability to detect stimuli from the environment plays a pivotal role in our survival. The molecules that allow the detection of such signals include ion channels, which are proteins expressed in different cells and organs. Among these ion channels, the transient receptor potential (TRP) family responds to the presence of diverse chemicals, temperature, and osmotic changes, among others. This family of ion channels includes the TRPV or vanilloid subfamily whose members serve several physiological functions. Although these proteins have been studied intensively for the last two decades, owing to their structural and functional complexities, a number of controversies regarding their function still remain. Here, we discuss some salient features of their regulation in light of these controversies and outline some of the efforts pushing the field forward.

Roles of Intramolecular Interactions in the Regulation of TRP Channels.

The transient receptor potential (TRP) channels, classified into six (-A, -V, -P, -C, -M, -ML, -N and -Y) subfamilies, are important membrane sensors and mediators of diverse stimuli including pH, light, mechano-force, temperature, pain, taste, and smell. The mammalian TRP superfamily of 28 members share similar membrane topology with six membrane-spanning helices (S1-S6) and cytosolic N-/C-terminus. Abnormal function or expression of TRP channels is associated with cancer, skeletal dysplasia, immunodeficiency, and cardiac, renal, and neuronal diseases. The majority of TRP members share common functional regulators such as phospholipid PIP2, 2-aminoethoxydiphenyl borate (2-APB), and cannabinoid, while other ligands are more specific, such as allyl isothiocyanate (TRPA1), vanilloids (TRPV1), menthol (TRPM8), ADP-ribose (TRPM2), and ML-SA1 (TRPML1). The mechanisms underlying the gating and regulation of TRP channels remain largely unclear. Recent advances in cryogenic electron microscopy provided structural insights into 19 different TRP channels which all revealed close proximity of the C-terminus with the N-terminus and intracellular S4-S5 linker. Further studies found that some highly conserved residues in these regions of TRPV, -P, -C and -M members mediate functionally critical intramolecular interactions (i.e., within one subunit) between these regions. This review provides an overview on (1) intramolecular interactions in TRP channels and their effect on channel function; (2) functional roles of interplays between PIP2 (and other ligands) and TRP intramolecular interactions; and (3) relevance of the ligand-induced modulation of intramolecular interaction to diseases.

Cryo-EM structure of mouse TRPML2 in lipid nanodiscs.

In mammalians, transient receptor potential mucolipin ion channels (TRPMLs) exhibit variable permeability to cations such as Ca2+, Fe2+, Zn2+, and Na+ and can be activated by the phosphoinositide PI(3,5)P2 in the endolysosomal system. Loss or dysfunction of TRPMLs has been implicated in lysosomal storage disorders, infectious diseases, and metabolic diseases. TRPML2 has recently been identified as a mechanosensitive and hypotonicity-sensitive channel in endolysosomal organelles, which distinguishes it from TRPML1 and TRPML3. However, the molecular and gating mechanism of TRPML2 remains elusive. Here, we present the cryo-EM structure of the full-length mouse TRPML2 in lipid nanodiscs at 3.14 Å resolution. The TRPML2 homotetramer structure at pH 7.4 in the apo state reveals an inactive conformation and some unique features of the extracytosolic/luminal domain and voltage sensor-like domain that have implications for the ion-conducting pathway. This structure enables new comparisons between the different subgroups of TRPML channels with available structures and provides structural insights into the conservation and diversity of TRPML channels. These comparisons have broad implications for understanding a variety of molecular mechanisms of TRPMLs in different pH conditions, including with and without bound agonists and antagonists.

Cryo-electron microscopy structure of the lysosomal calcium-permeable channel TRPML3.

The modulation of ion channel activity by lipids is increasingly recognized as a fundamental component of cellular signalling. The transient receptor potential mucolipin (TRPML) channel family belongs to the TRP superfamily and is composed of three members: TRPML1-TRPML3. TRPMLs are the major Ca2+-permeable channels on late endosomes and lysosomes (LEL). They regulate the release of Ca2+ from organelles, which is important for various physiological processes, including organelle trafficking and fusion. Loss-of-function mutations in the MCOLN1 gene, which encodes TRPML1, cause the neurodegenerative lysosomal storage disorder mucolipidosis type IV, and a gain-of-function mutation (Ala419Pro) in TRPML3 gives rise to the varitint-waddler (Va) mouse phenotype. Notably, TRPML channels are activated by the low-abundance and LEL-enriched signalling lipid phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P2), whereas other phosphoinositides such as PtdIns(4,5)P2, which is enriched in plasma membranes, inhibit TRPMLs. Conserved basic residues at the N terminus of the channel are important for activation by PtdIns(3,5)P2 and inhibition by PtdIns(4,5)P2. However, owing to a lack of structural information, the mechanism by which TRPML channels recognize PtdIns(3,5)P2 and increase their Ca2+ conductance remains unclear. Here we present the cryo-electron microscopy (cryo-EM) structure of a full-length TRPML3 channel from the common marmoset (Callithrix jacchus) at an overall resolution of 2.9 Å. Our structure reveals not only the molecular basis of ion conduction but also the unique architecture of TRPMLs, wherein the voltage sensor-like domain is linked to the pore via a cytosolic domain that we term the mucolipin domain. Combined with functional studies, these data suggest that the mucolipin domain is responsible for PtdIns(3,5)P2 binding and subsequent channel activation, and that it acts as a 'gating pulley' for lipid-dependent TRPML gating.

Human TRPML1 channel structures in open and closed conformations.

Transient receptor potential mucolipin 1 (TRPML1) is a Ca2+-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.

Electron cryo-microscopy structure of the mechanotransduction channel NOMPC.

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. 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. 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, Drosophila and zebrafish. NOMPC is the founding member of the TRPN subfamily, and is thought to be gated by tethering of its ankyrin repeat domain to microtubules of the cytoskeleton. 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 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.

Structure of mammalian endolysosomal TRPML1 channel in nanodiscs.

Transient receptor potential mucolipin 1 (TRPML1) is a cation channel located within endosomal and lysosomal membranes. Ubiquitously expressed in mammalian cells, its loss-of-function mutations are the direct cause of type IV mucolipidosis, an autosomal recessive lysosomal storage disease. 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)P2) 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 Ca2+-blocking site that confers luminal pH and Ca2+ 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 PtdInsP2 gating mechanism among TRPML channels.

Ultrastructural organization of NompC in the mechanoreceptive organelle of Drosophila campaniform mechanoreceptors.

Mechanoreceptive organelles (MOs) are specialized subcellular entities in mechanoreceptors that transform extracellular mechanical stimuli into intracellular signals. Their ultrastructures are key to understanding the molecular nature and mechanics of mechanotransduction. Campaniform sensilla detect cuticular strain caused by muscular activities or external stimuli in Drosophila Each campaniform sensillum has an MO located at the distal tip of its dendrite. Here we analyzed the molecular architecture of the MOs in fly campaniform mechanoreceptors using electron microscopic tomography. We focused on the ultrastructural organization of NompC (a force-sensitive channel) that is linked to the array of microtubules in these MOs via membrane-microtubule connectors (MMCs). We found that NompC channels are arranged in a regular pattern, with their number increasing from the distal to the proximal end of the MO. Double-length MMCs in nompC29+29ARs confirm the ankyrin-repeat domain of NompC (NompC-AR) as a structural component of MMCs. The unexpected finding of regularly spaced NompC-independent linkers in nompC3 suggests that MMCs may contain non-NompC components. Localized laser ablation experiments on mechanoreceptor arrays in halteres suggest that MMCs bear tension, providing a possible mechanism for why the MMCs are longer when NompC-AR is duplicated or absent in mutants. Finally, mechanical modeling shows that upon cuticular deformation, sensillar architecture imposes a rotational activating force, with the proximal end of the MO, where more NOMPC channels are located, being subject to larger forces than the distal end. Our analysis reveals an ultrastructural pattern of NompC that is structurally and mechanically optimized for the sensory functions of campaniform mechanoreceptors.

Structure of the TRPA1 ion channel suggests regulatory mechanisms.

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.

Computational Analysis of the Crystal and Cryo-EM Structures of P-Loop Channels with Drugs.

The superfamily of P-loop channels includes various potassium channels, voltage-gated sodium and calcium channels, transient receptor potential channels, and ionotropic glutamate receptors. Despite huge structural and functional diversity of the channels, their pore-forming domain has a conserved folding. In the past two decades, scores of atomic-scale structures of P-loop channels with medically important drugs in the inner pore have been published. High structural diversity of these complexes complicates the comparative analysis of these structures. Here we 3D-aligned structures of drug-bound P-loop channels, compared their geometric characteristics, and analyzed the energetics of ligand-channel interactions. In the superimposed structures drugs occupy most of the sterically available space in the inner pore and subunit/repeat interfaces. Cationic groups of some drugs occupy vacant binding sites of permeant ions in the inner pore and selectivity-filter region. Various electroneutral drugs, lipids, and detergent molecules are seen in the interfaces between subunits/repeats. In many structures the drugs strongly interact with lipid and detergent molecules, but physiological relevance of such interactions is unclear. Some eukaryotic sodium and calcium channels have state-dependent or drug-induced π-bulges in the inner helices, which would be difficult to predict. The drug-induced π-bulges may represent a novel mechanism of gating modulation.

Structural and Evolutionary Insights Point to Allosteric Regulation of TRP Ion Channels.

The familiar pungent taste of spicy food, the refreshing taste of mint, and many other physiological phenomena are mediated by transient receptor potential (TRP) ion channels. TRP channels are a superfamily of ion channels that are sensitive to diverse chemical and physical stimuli and play diverse roles in biology. In addition to chemical regulation, some family members also sense common physical stimuli, such as temperature or pressure. Since their discovery and cloning in the 1990s and 2000s, understanding the molecular mechanisms governing TRP channel function and polymodal regulation has been a consistent but challenging goal. Until recently, a general lack of high-resolution TRP channel structures had significantly limited a molecular understanding of their function. In the past few years, a flood of TRP channel structures have been released, made possible primarily by advances in cryo-electron microscopy (cryo-EM). The boon of many structures has unleashed unparalleled insight into TRP channel architecture. Substantive comparative studies between TRP structures provide snapshots of distinct states such as ligand-free, stabilized by chemical agonists, or antagonists, partially illuminating how a given channel opens and closes. However, the now ∼75 TRP channel structures have ushered in surprising outcomes, including a lack of an apparent general mechanism underlying channel opening and closing among family members. Similarly, the structures reveal a surprising diversity in which chemical ligands bind TRP channels. Several TRP channels are activated by temperature changes in addition to ligand binding. Unraveling mechanisms of thermosensation has proven an elusive challenge to the field. Although some studies point to thermosensitive domains in the transmembrane region of the channels, results have sometimes been contradictory and difficult to interpret; in some cases, a domain that proves essential for thermal sensitivity in one context can be entirely removed from the channel without affecting thermosensation in another context. These results are not amenable to simple interpretations and point to allosteric networks of regulation within the channel structure. TRP channels have evolved to be fine-tuned for the needs of a species in its environmental niche, a fact that has been both a benefit and burden in unlocking their molecular features. Functional evolutionary divergence has presented challenges for studying TRP channels, as orthologs from different species can give conflicting experimental results. However, this diversity can also be examined comparatively to decipher the basis for functional differences. As with structural biology, untangling the similarities and differences resulting from evolutionary pressure between species has been a rich source of data guiding the field. This Account will contextualize the existing biochemical and functional data with an eye to evolutionary data and couple these insights with emerging structural biology to better understand the molecular mechanisms behind chemical and physical regulation of TRP channels.

Steroids and TRP Channels: A Close Relationship.

Transient receptor potential (TRP) channels are remarkable transmembrane protein complexes that are essential for the physiology of the tissues in which they are expressed. They function as non-selective cation channels allowing for the signal transduction of several chemical, physical and thermal stimuli and modifying cell function. These channels play pivotal roles in the nervous and reproductive systems, kidney, pancreas, lung, bone, intestine, among others. TRP channels are finely modulated by different mechanisms: regulation of their function and/or by control of their expression or cellular/subcellular localization. These mechanisms are subject to being affected by several endogenously-produced compounds, some of which are of a lipidic nature such as steroids. Fascinatingly, steroids and TRP channels closely interplay to modulate several physiological events. Certain TRP channels are affected by the typical genomic long-term effects of steroids but others are also targets for non-genomic actions of some steroids that act as direct ligands of these receptors, as will be reviewed here.

Atomistic Insights of Calmodulin Gating of Complete Ion Channels.

Intracellular calcium is essential for many physiological processes, from neuronal signaling and exocytosis to muscle contraction and bone formation. Ca2+ signaling from the extracellular medium depends both on membrane potential, especially controlled by ion channels selective to K+, and direct permeation of this cation through specialized channels. Calmodulin (CaM), through direct binding to these proteins, participates in setting the membrane potential and the overall permeability to Ca2+. Over the past years many structures of complete channels in complex with CaM at near atomic resolution have been resolved. In combination with mutagenesis-function, structural information of individual domains and functional studies, different mechanisms employed by CaM to control channel gating are starting to be understood at atomic detail. Here, new insights regarding four types of tetrameric channels with six transmembrane (6TM) architecture, Eag1, SK2/SK4, TRPV5/TRPV6 and KCNQ1-5, and its regulation by CaM are described structurally. Different CaM regions, N-lobe, C-lobe and EF3/EF4-linker play prominent signaling roles in different complexes, emerging the realization of crucial non-canonical interactions between CaM and its target that are only evidenced in the full-channel structure. Different mechanisms to control gating are used, including direct and indirect mechanical actuation over the pore, allosteric control, indirect effect through lipid binding, as well as direct plugging of the pore. Although each CaM lobe engages through apparently similar alpha-helices, they do so using different docking strategies. We discuss how this allows selective action of drugs with great therapeutic potential.

In Vivo Pharmacokinetic Analysis Utilizing Non-Targeted and Targeted Mass Spectrometry and In Vitro Assay against Transient Receptor Potential Channels of Maobushisaishinto and Its Constituent Asiasari Radix.

The Japanese traditional medicine maobushisaishinto (MBST) has been prescribed for treating upper respiratory tract infections, such as a common cold. However, its mode of action is poorly understood, especially concerning the MBST constituent Asiasari Radix (AR). In this study, we focused on AR, with an objective of clarifying its bioavailable active ingredients and role within MBST by performing pharmacokinetic and pharmacological studies. Firstly, we performed qualitative non-targeted analysis utilizing high-resolution mass spectrometry to explore the bioavailable ingredients of AR as well as quantitative targeted analysis to reveal plasma concentrations following oral administration of MBST in rats. Secondly, we performed in vitro pharmacological study of bioavailable AR ingredients in addition to other ingredients of MBST to confirm any agonistic activities against transient receptor potential (TRP) channels. As a result, methyl kakuol and other compounds derived from AR were detected in the rat plasma and showed agonistic activity against TRPA1. This study suggests that methyl kakuol as well as other compounds have the potential to be an active ingredient in AR and thus presumably would contribute in part to the effects exerted by MBST.

Identification of a single aspartate residue critical for both fast and slow calcium-dependent inactivation of the human TRPML1 channel.

Transient receptor potential mucolipin subfamily 1 (TRPML1) is a nonselective cation channel mainly located in late endosomes and lysosomes. Mutations of the gene encoding human TRPML1 can cause severe lysosomal diseases. The activity of TRPML1 is regulated by both Ca2+ and H+, which are important for its critical physiological functions in membrane trafficking, exocytosis, autophagy, and intracellular signal transduction. However, the molecular mechanism of its dual regulation by Ca2+ and H+ remains elusive. Here, using a mutant screening method in combination with a whole-cell patch clamp technique, we identified a key TRPML1 residue, Asp-472, responsible for both fast calcium-dependent inactivation (FCDI) and slow calcium-dependent inactivation (SCDI) as well as H+ regulation. We also found that, in acidic pH, H+ can significantly delay FCDI and abolish SCDI and thereby presumably facilitate the ion conductance of the human TRPML1 channel. In summary, we have identified a key residue critical for Ca2+-induced inhibition of TRPML1 channel currents and uncovered pH-dependent regulation of this channel, providing vital information regarding the detailed mechanism of action of human TRPML1.

Identification and characterization of hydrophobic gate residues in TRP channels.

Transient receptor potential (TRP) channels, subdivided into 6 subfamilies in mammals, have essential roles in sensory physiology. They respond to remarkably diverse stimuli, comprising thermal, chemical, and mechanical modalities, through opening or closing of channel gates. In this study, we systematically substituted the hydrophobic residues within the distal fragment of pore-lining helix S6 with hydrophilic residues and, based on Xenopus oocyte and mammalian cell electrophysiology and a hydrophobic gate theory, identified hydrophobic gates in TRPV6/V5/V4/C4/M8. We found that channel activity drastically increased when TRPV6Ala616 or Met617 or TRPV5Ala576 or Met577, but not any of their adjacent residues, was substituted with hydrophilic residues. Channel activity strongly correlated with the hydrophilicity of the residues at those sites, suggesting that consecutive hydrophobic residues TRPV6Ala616-Met617 and TRPV5Ala576-Met577 form a double-residue gate in each channel. By the same strategy, we identified a hydrophobic single-residue gate in TRPV4Iso715, TRPC4Iso617, and TRPM8Val976. In support of the hydrophobic gate theory, hydrophilic substitution at the gate site, which removes the hydrophobic gate seal, substantially increased the activity of TRP channels in low-activity states but had little effect on the function of activated channels. The double-residue gate channels were more sensitive to small changes in the gate's hydrophobicity or size than single-residue gate channels. The unconventional double-reside gating mechanism in TRP channels may have been evolved to respond especially to physiologic stimuli that trigger relatively small gate conformational changes.-Zheng, W., Hu, R., Cai, R., Hofmann, L., Hu, Q., Fatehi, M., Long, W., Kong, T., Tang, J., Light, P., Flockerzi, V., Cao, Y., Chen, X.-Z. Identification and characterization of hydrophobic gate residues in TRP channels.