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The Concise Guide to PHARMACOLOGY 2017/18 is the third in this series of biennial publications. This version provides concise overviews of the key properties of nearly 1800 human drug targets with an emphasis on selective pharmacology (where available), plus links to an open access knowledgebase of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide represents approximately 400 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.13882/full. In addition to this overview, in which are identified 'Other protein targets' which fall outside of the subsequent categorisation, there are eight areas of focus: G protein-coupled receptors, ligand-gated ion channels, voltage-gated ion channels, other ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2017, and supersedes data presented in the 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature Committee of the Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

The Concise Guide to PHARMACOLOGY 2015/16 provides concise overviews of the key properties of over 1750 human drug targets with their pharmacology, plus links to an open access knowledgebase of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. The full contents can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.13347/full. This compilation of the major pharmacological targets is divided into eight areas of focus: G protein-coupled receptors, ligand-gated ion channels, voltage-gated ion channels, other ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The Concise Guide is published in landscape format in order to facilitate comparison of related targets. It is a condensed version of material contemporary to late 2015, which is presented in greater detail and constantly updated on the website www.guidetopharmacology.org, superseding data presented in the previous Guides to Receptors & Channels and the Concise Guide to PHARMACOLOGY 2013/14. It is produced in conjunction with NC-IUPHAR and provides the official IUPHAR classification and nomenclature for human drug targets, where appropriate. It consolidates information previously curated and displayed separately in IUPHAR-DB and GRAC and provides a permanent, citable, point-in-time record that will survive database updates.

Photoactivatable or "caged" pharmacological agents combine the high spatiotemporal specificity of light application with the molecular specificity of drugs. A key factor in all optopharmacology experiments is the mechanism of uncaging, which dictates the photochemical quantum yield and determines the byproducts produced by the light-driven chemical reaction. In previous work, we demonstrated that coumarin-based photolabile groups could be used to cage tertiary amine drugs as quaternary ammonium salts. Although stable, water-soluble, and useful for experiments in brain tissue, these first-generation compounds exhibit relatively low uncaging quantum yield (Φ u < 1%) and release the toxic byproduct formaldehyde upon photolysis. Here, we elucidate the photochemical mechanisms of coumarin-caged tertiary amines and then optimize the major pathway using chemical modification. We discovered that the combination of 3,3-dicarboxyazetidine and bromine substituents shift the mechanism of release to heterolysis, eliminating the formaldehyde byproduct and giving photolabile tertiary amine drugs with Φ u > 20%─a 35-fold increase in uncaging efficiency. This new "ABC" cage allows synthesis of improved photoactivatable derivatives of escitalopram and nicotine along with a novel caged agonist of the oxytocin receptor.

The severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) and SARS-CoV-1 accessory protein Orf3a colocalizes with markers of the plasma membrane, endocytic pathway, and Golgi apparatus. Some reports have led to annotation of both Orf3a proteins as viroporins. Here, we show that neither SARS-CoV-2 nor SARS-CoV-1 Orf3a form functional ion conducting pores and that the conductances measured are common contaminants in overexpression and with high levels of protein in reconstitution studies. Cryo-EM structures of both SARS-CoV-2 and SARS-CoV-1 Orf3a display a narrow constriction and the presence of a positively charged aqueous vestibule, which would not favor cation permeation. We observe enrichment of the late endosomal marker Rab7 upon SARS-CoV-2 Orf3a overexpression, and co-immunoprecipitation with VPS39. Interestingly, SARS-CoV-1 Orf3a does not cause the same cellular phenotype as SARS-CoV-2 Orf3a and does not interact with VPS39. To explain this difference, we find that a divergent, unstructured loop of SARS-CoV-2 Orf3a facilitates its binding with VPS39, a HOPS complex tethering protein involved in late endosome and autophagosome fusion with lysosomes. We suggest that the added loop enhances SARS-CoV-2 Orf3a's ability to co-opt host cellular trafficking mechanisms for viral exit or host immune evasion., Competing Interests: AM, PH, EM, DC, GL, BC, TD, SS, DM, RY, ZY, DR, SB, DC No competing interests declared

Chemical synapses between axons and dendrites mediate neuronal intercellular communication. Here, we describe a synapse between axons and primary cilia: the axo-ciliary synapse. Using enhanced focused ion beam-scanning electron microscopy on samples with optimally preserved ultrastructure, we discovered synapses between brainstem serotonergic axons and the primary cilia of hippocampal CA1 pyramidal neurons. Functionally, these cilia are enriched in a ciliary-restricted serotonin receptor, the 5-hydroxytryptamine receptor 6 (5-HTR6). Using a cilia-targeted serotonin sensor, we show that opto- and chemogenetic stimulation of serotonergic axons releases serotonin onto cilia. Ciliary 5-HTR6 stimulation activates a non-canonical G αq/11 -RhoA pathway, which modulates nuclear actin and increases histone acetylation and chromatin accessibility. Ablation of this pathway reduces chromatin accessibility in CA1 pyramidal neurons. As a signaling apparatus with proximity to the nucleus, axo-ciliary synapses short circuit neurotransmission to alter the postsynaptic neuron's epigenetic state., Competing Interests: Declaration of interests Portions of the technology described herein are covered by U.S. Patent 10,600,615 titled “enhanced FIB-SEM systems for large-volume 3D imaging,” which was issued to C.S.X. and H.F.H. and assigned to Howard Hughes Medical Institute., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Chemical neurotransmission constitutes one of the fundamental modalities of communication between neurons. Monitoring release of these chemicals has traditionally been difficult to carry out at spatial and temporal scales relevant to neuron function. To understand chemical neurotransmission more fully, we need to improve the spatial and temporal resolutions of measurements for neurotransmitter release. To address this, we engineered a chemi-sensitive, two-dimensional composite nanofilm that facilitates visualization of the release and diffusion of the neurochemical dopamine with synaptic resolution, quantal sensitivity, and simultaneously from hundreds of release sites. Using this technology, we were able to monitor the spatiotemporal dynamics of dopamine release in dendritic processes, a poorly understood phenomenon. We found that dopamine release is broadcast from a subset of dendritic processes as hotspots that have a mean spatial spread of ≈ 3.2 µm (full width at half maximum [FWHM]) and are observed with a mean spatial frequency of one hotspot per ≈ 7.5 µm of dendritic length. Major dendrites of dopamine neurons and fine dendritic processes, as well as dendritic arbors and dendrites with no apparent varicose morphology participated in dopamine release. Remarkably, these release hotspots co-localized with Bassoon, suggesting that Bassoon may contribute to organizing active zones in dendrites, similar to its role in axon terminals., Competing Interests: CB, AK, BC, WV, DW, DA, DC, AB No competing interests declared, (© 2022, Bulumulla et al.)

Teeth are composed of many tissues, covered by an inflexible and obdurate enamel. Unlike most other tissues, teeth become extremely cold sensitive when inflamed. The mechanisms of this cold sensation are not understood. Here, we clarify the molecular and cellular components of the dental cold sensing system and show that sensory transduction of cold stimuli in teeth requires odontoblasts. TRPC5 is a cold sensor in healthy teeth and, with TRPA1, is sufficient for cold sensing. The odontoblast appears as the direct site of TRPC5 cold transduction and provides a mechanism for prolonged cold sensing via TRPC5's relative sensitivity to intracellular calcium and lack of desensitization. Our data provide concrete functional evidence that equipping odontoblasts with the cold-sensor TRPC5 expands traditional odontoblast functions and renders it a previously unknown integral cellular component of the dental cold sensing system., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Recording of the electrical activity from one of the smallest cells of a mammalian organism- a sperm cell- has been a challenging task for electrophysiologists for many decades. The method known as "spermatozoan patch clamp" was introduced in 2006. It has enabled the direct recording of ion channel activity in whole-cell and cell-attached configurations and has been instrumental in describing sperm cell physiology and the molecular identity of various calcium, potassium, sodium, chloride, and proton ion channels. However, recording from single spermatozoa requires advanced skills and training in electrophysiology. This detailed protocol summarizes the step-by-step procedure and highlights several 'tricks-of-the-trade' in order to make it available to anyone who wishes to explore the fascinating physiology of the sperm cell. Specifically, the protocol describes recording from human and murine sperm cells but can be adapted to essentially any mammalian sperm cell of any species. The protocol covers important details of the application of this technique, such as isolation of sperm cells, selection of reagents and equipment, immobilization of the highly motile cells, formation of the tight (Gigaohm) seal between a recording electrode and the plasma membrane of the sperm cells, transition into the whole-spermatozoan mode (also known as break-in), and exemplary recordings of the sperm cell calcium ion channel, CatSper, from six mammalian species. The advantages and limitations of the sperm patch clamp method, as well as the most critical steps, are discussed.

NaChBac, the first bacterial voltage-gated Na + (Na v ) channel to be characterized, has been the prokaryotic prototype for studying the structure-function relationship of Na v channels. Discovered nearly two decades ago, the structure of NaChBac has not been determined. Here we present the single particle electron cryomicroscopy (cryo-EM) analysis of NaChBac in both detergent micelles and nanodiscs. Under both conditions, the conformation of NaChBac is nearly identical to that of the potentially inactivated Na v Ab. Determining the structure of NaChBac in nanodiscs enabled us to examine gating modifier toxins (GMTs) of Na v channels in lipid bilayers. To study GMTs in mammalian Na v channels, we generated a chimera in which the extracellular fragment of the S3 and S4 segments in the second voltage-sensing domain from Na v 1.7 replaced the corresponding sequence in NaChBac. Cryo-EM structures of the nanodisc-embedded chimera alone and in complex with HuwenToxin IV (HWTX-IV) were determined to 3.5 and 3.2 Å resolutions, respectively. Compared to the structure of HWTX-IV-bound human Na v 1.7, which was obtained at an overall resolution of 3.2 Å, the local resolution of the toxin has been improved from ∼6 to ∼4 Å. This resolution enabled visualization of toxin docking. NaChBac can thus serve as a convenient surrogate for structural studies of the interactions between GMTs and Na v channels in a membrane environment., Competing Interests: The authors declare no competing interest.

Targeting small-molecule fluorescent indicators using genetically encoded protein tags yields new hybrid sensors for biological imaging. Optimization of such systems requires redesign of the synthetic indicator to allow cell-specific targeting without compromising the photophysical properties or cellular performance of the small-molecule probe. We developed a bright and sensitive Ca 2+ indicator by systematically exploring the relative configuration of dye and chelator, which can be targeted using the HaloTag self-labeling tag system. Our "isomeric tuning" approach is generalizable, yielding a far-red targetable indicator to visualize Ca 2+ fluxes in the primary cilium.

The transient receptor potential canonical subfamily member 5 (TRPC5), one of seven mammalian TRPC members, is a nonselective calcium-permeant cation channel. TRPC5 is of considerable interest as a drug target in the treatment of progressive kidney disease, depression, and anxiety. Here, we present the 2.8-Å resolution cryo-electron microscopy (cryo-EM) structure of the mouse TRPC5 (mTRPC5) homotetramer. Comparison of the TRPC5 structure to previously determined structures of other TRPC and TRP channels reveals differences in the extracellular pore domain and in the length of the S3 helix. The disulfide bond at the extracellular side of the pore and a preceding small loop are essential elements for its proper function. This high-resolution structure of mTRPC5, combined with electrophysiology and mutagenesis, provides insight into the lipid modulation and gating mechanisms of the TRPC family of ion 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 Mg 2+ and Zn 2+ 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 Mg 2+ ions occupying the center of the conduction pore. In high [Mg 2+ ], 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., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Members of the transient receptor potential (TRP) ion channels conduct cations into cells. They mediate functions ranging from neuronally mediated hot and cold sensation to intracellular organellar and primary ciliary signaling. Here we report a cryo-electron microscopy (cryo-EM) structure of TRPC4 in its unliganded (apo) state to an overall resolution of 3.3 Å. The structure reveals a unique architecture with a long pore loop stabilized by a disulfide bond. Beyond the shared tetrameric six-transmembrane fold, the TRPC4 structure deviates from other TRP channels with a unique cytosolic domain. This unique cytosolic N-terminal domain forms extensive aromatic contacts with the TRP and the C-terminal domains. The comparison of our structure with other known TRP structures provides molecular insights into TRPC4 ion selectivity and extends our knowledge of the diversity and evolution of the TRP channels.

We report the near atomic resolution (3.3 Å) of the human polycystic kidney disease 2-like 1 (polycystin 2-l1) ion channel. Encoded by PKD2L1, polycystin 2-l1 is a calcium and monovalent cation-permeant ion channel in primary cilia and plasma membranes. The related primary cilium-specific polycystin-2 protein, encoded by PKD2, shares a high degree of sequence similarity, yet has distinct permeability characteristics. Here we show that these differences are reflected in the architecture of polycystin 2-l1., Competing Interests: RH, ZL, RH, JZ, DC No competing interests declared, (© 2018, Hulse et al.)

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., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Mutations in the polycystin genes, PKD1 or PKD2, results in Autosomal Dominant Polycystic Kidney Disease (ADPKD). Although a genetic basis of ADPKD is established, we lack a clear understanding of polycystin proteins' functions as ion channels. This question remains unsolved largely because polycystins localize to the primary cilium - a tiny, antenna-like organelle. Using a new ADPKD mouse model, we observe primary cilia that are abnormally long in cells associated with cysts after conditional ablation of Pkd1 or Pkd2 . Using primary cultures of collecting duct cells, we show that polycystin-2, but not polycystin-1, is a required subunit for the ion channel in the primary cilium. The polycystin-2 channel preferentially conducts K + and Na + ; intraciliary Ca 2+ , enhances its open probability. We introduce a novel method for measuring heterologous polycystin-2 channels in cilia, which will have utility in characterizing PKD2 variants that cause ADPKD., Competing Interests: XL, TV, JD, SS, PD, DC No competing interests declared, (© 2018, Liu et al.)

Background: Leucine-rich repeat containing 8A (LRRC8A) is an ubiquitously expressed transmembrane protein with 17 leucine-rich repeats (LRRs) at its C-terminal end and is an essential component of the volume-regulated anion channel (VRAC), which controls cellular volume. A heterozygous mutation in LRRC8A that truncates the 2 terminal LRRs was reported in a patient with agammaglobulinemia and absent B cells and was demonstrated to exert a dominant negative effect on T- and B-cell development in mice. Lrrc8a -/- mice have severely defective T-cell development and function. It is not known whether the T- and B-cell defects caused by LRRC8A deficiency are caused by loss of VRAC activity., Objective: We sought to determine whether VRAC activity is required for normal T-cell development and function., Methods: VRAC activity was examined by using patch-clamp analysis. Flow cytometry was used to examine T-cell development. T-cell proliferation, cytokine secretion, and antibody titers were measured by using standard techniques., Results: We demonstrate that the spontaneous mouse mutant ébouriffé (ebo/ebo) harbors a homozygous 2-bp frameshift mutation in Lrrc8a that truncates the 15 terminal LRRs of LRRC8A. The Lrrc8a ebo mutation does not affect protein expression but drastically diminishes VRAC activity in T cells. ebo/ebo mice share features with Lrrc8a -/- mice that include curly hair, infertility, reduced longevity, and kidney abnormalities. However, in contrast to Lrrc8a -/- mice, ebo/ebo mice have normal T-cell development and function and intact antibody response to T-dependent antigen., Conclusion: LRRC8A-dependent VRAC activity is dispensable for T-cell development and function., (Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

TRPM6 and TRPM7 are members of the melastatin-related transient receptor potential (TRPM) subfamily of ion channels. Deletion of either gene in mice is embryonically lethal. TRPM6/7 are the only known examples of single polypeptides containing both an ion channel pore and a serine/threonine kinase (chanzyme). Here we show that the C-terminal kinase domain of TRPM6 is cleaved from the channel domain in a cell type-specific fashion and is active. Cleavage requires that the channel conductance is functional. The cleaved kinase translocates to the nucleus, where it is strictly localized and phosphorylates specific histone serine and threonine (S/T) residues. TRPM6-cleaved kinases (M6CKs) bind subunits of the protein arginine methyltransferase 5 (PRMT5) molecular complex that make important epigenetic modifications by methylating histone arginine residues. Histone phosphorylation by M6CK results in a dramatic decrease in methylation of arginines adjacent to M6CK-phosphorylated amino acids. Knockout of TRPM6 or inactivation of its kinase results in global changes in histone S/T phosphorylation and changes the transcription of hundreds of genes. We hypothesize that M6CK associates with the PRMT5 molecular complex in the nucleus, directing M6CK to a specific genomic location and providing site-specific histone phosphorylation. M6CK histone phosphorylation, in turn, regulates transcription by attenuating the effect of local arginine methylation., Competing Interests: The authors declare no conflict of interest.

TRPM7 (transient receptor potential cation channel subfamily M member 7) regulates gene expression and stress-induced cytotoxicity and is required in early embryogenesis through organ development. Here, we show that the majority of TRPM7 is localized in abundant intracellular vesicles. These vesicles (M7Vs) are distinct from endosomes, lysosomes, and other familiar vesicles or organelles. M7Vs accumulate Zn 2+ in a glutathione-enriched, reduced lumen when cytosolic Zn 2+ concentrations are elevated. Treatments that increase reactive oxygen species (ROS) trigger TRPM7-dependent Zn 2+ release from the vesicles, whereas reduced glutathione prevents TRPM7-dependent cytosolic Zn 2+ influx. These observations strongly support the notion that ROS-mediated TRPM7 activation releases Zn 2+ from intracellular vesicles after Zn 2+ overload. Like the endoplasmic reticulum, these vesicles are a distributed system for divalent cation uptake and release, but in this case the primary divalent ion is Zn 2+ rather than Ca 2 ., Competing Interests: The authors declare no conflict of interest.

We report that the Gm7068 ( CatSpere ) and Tex40 ( CatSperz ) genes encode novel subunits of a 9-subunit CatSper ion channel complex. Targeted disruption of CatSperz reduces CatSper current and sperm rheotactic efficiency in mice, resulting in severe male subfertility. Normally distributed in linear quadrilateral nanodomains along the flagellum, the complex lacking CatSperζ is disrupted at ~0.8 μm intervals along the flagellum. This disruption renders the proximal flagellum inflexible and alters the 3D flagellar envelope, thus preventing sperm from reorienting against fluid flow in vitro and efficiently migrating in vivo . Ejaculated CatSperz -null sperm cells retrieved from the mated female uterus partially rescue in vitro fertilization (IVF) that failed with epididymal spermatozoa alone. Human CatSperε is quadrilaterally arranged along the flagella, similar to the CatSper complex in mouse sperm. We speculate that the newly identified CatSperζ subunit is a late evolutionary adaptation to maximize fertilization inside the mammalian female reproductive tract.

Mammalian cilia are ubiquitous appendages found on the apical surface of cells. Primary and motile cilia are distinct in both morphology and function. Most cells have a solitary primary cilium (9+0), which lacks the central microtubule doublet characteristic of motile cilia (9+2). The immotile primary cilia house unique signaling components and sequester several important transcription factors. In contrast, motile cilia commonly extend into the lumen of respiratory airways, fallopian tubes, and brain ventricles to move their contents and/or produce gradients. In this review, we focus on the composition of putative ion channels found in both types of cilia and in the periciliary membrane and discuss their proposed functions. Our discussion does not cover specialized cilia in photoreceptor or olfactory cells, which express many more ion channels., (© 2017 Pablo et al.)

The Polycystic Kidney Disease 2 (Pkd2) gene is mutated in autosomal dominant polycystic kidney disease (ADPKD), one of the most common human monogenic disorders. Here, we present the cryo-EM structure of PKD2 in lipid bilayers at 3.0 Å resolution, which establishes PKD2 as a homotetrameric ion channel and provides insight into potential mechanisms for its activation. The PKD2 voltage-sensor domain retains two of four gating charges commonly found in those of voltage-gated ion channels. The PKD2 ion permeation pathway is constricted at the selectivity filter and near the cytoplasmic end of S6, suggesting that two gates regulate ion conduction. The extracellular domain of PKD2, a hotspot for ADPKD pathogenic mutations, contributes to channel assembly and strategically interacts with the transmembrane core, likely serving as a physical substrate for extracellular stimuli to allosterically gate the channel. Finally, our structure establishes the molecular basis for the majority of pathogenic mutations in Pkd2-related ADPKD., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Native PKD2-L1 channel subunits are present in primary cilia and other restricted cellular spaces. Here we investigate the mechanism for the channel's unusual regulation by external calcium, and rationalize this behavior to its specialized function. We report that the human PKD2-L1 selectivity filter is partially selective to calcium ions (Ca(2+)) moving into the cell, but blocked by high internal Ca(2+)concentrations, a unique feature of this transient receptor potential (TRP) channel family member. Surprisingly, we find that the C-terminal EF-hands and coiled-coil domains do not contribute to PKD2-L1 Ca(2+)-induced potentiation and inactivation. We propose a model in which prolonged channel activity results in calcium accumulation, triggering outward-moving Ca(2+) ions to block PKD2-L1 in a high-affinity interaction with the innermost acidic residue (D523) of the selectivity filter and subsequent long-term channel inactivation. This response rectifies Ca(2+) flow, enabling Ca(2+) to enter but not leave small compartments such as the cilium.

(E)-2-alkenals are aldehydes containing an unsaturated bond between the alpha and beta carbons. 2-alkenals are produced by many organisms for defense against predators and secretions containing (E)-2-alkenals cause predators to stop attacking and allow the prey to escape. Chemical ecologists have described many alkenal compounds with 3-20 carbons common, having varied positions of double bonds and substitutions. How do these defensive alkenals act to deter predators? We have tested the effects of (E)-2-alkenals with 6-12 carbons on transient receptor potential channels (TRP) commonly found in sensory neurons. We find that (E)-2-alkenals activate transient receptor potential ankyrin subtype 1 (TRPA1) at low concentrations-EC50s 10-100 µM (in 0 added Ca(2+) external solutions). Other TRP channels were either weakly activated (TRPV1, TRPV3) or insensitive (TRPV2, TRPV4, TRPM8). (E)-2-alkenals may activate TRPA1 by modifying cysteine side chains. However, target cysteines include others beyond the 3 in the amino-terminus implicated in activation, as a channel with cysteines at 621, 641, 665 mutated to serine responded robustly. Related chemicals, including the aldehydes hexanal and decanal, and (E)-2-hexen-1-ol also activated TRPA1, but with weaker potency. Rat trigeminal nerve recordings and behavioral experiments showed (E)-2-hexenal was aversive. Our results suggest that TRPA1 is likely a major target of these commonly used defensive chemicals., (© The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Voltage-gated sodium channels are essential for electrical signalling across cell membranes. They exhibit strong selectivities for sodium ions over other cations, enabling the finely tuned cascade of events associated with action potentials. This paper describes the ion permeability characteristics and the crystal structure of a prokaryotic sodium channel, showing for the first time the detailed locations of sodium ions in the selectivity filter of a sodium channel. Electrostatic calculations based on the structure are consistent with the relative cation permeability ratios (Na(+) ≈ Li(+) ≫ K(+), Ca(2+), Mg(2+)) measured for these channels. In an E178D selectivity filter mutant constructed to have altered ion selectivities, the sodium ion binding site nearest the extracellular side is missing. Unlike potassium ions in potassium channels, the sodium ions in these channels appear to be hydrated and are associated with side chains of the selectivity filter residues, rather than polypeptide backbones., (© 2016 The Authors. Published under the terms of the CC BY 4.0 license.)

Primary cilia are solitary, generally non-motile, hair-like protrusions that extend from the surface of cells between cell divisions. Their antenna-like structure leads naturally to the assumption that they sense the surrounding environment, the most common hypothesis being sensation of mechanical force through calcium-permeable ion channels within the cilium. This Ca(2+)-responsive mechanosensor hypothesis for primary cilia has been invoked to explain a large range of biological responses, from control of left-right axis determination in embryonic development to adult progression of polycystic kidney disease and some cancers. Here we report the complete lack of mechanically induced calcium increases in primary cilia, in tissues upon which this hypothesis has been based. We developed a transgenic mouse, Arl13b-mCherry-GECO1.2, expressing a ratiometric genetically encoded calcium indicator in all primary cilia. We then measured responses to flow in primary cilia of cultured kidney epithelial cells, kidney thick ascending tubules, crown cells of the embryonic node, kinocilia of inner ear hair cells, and several cell lines. Cilia-specific Ca(2+) influxes were not observed in physiological or even highly supraphysiological levels of fluid flow. We conclude that mechanosensation, if it originates in primary cilia, is not via calcium signalling.

During the mitochondrial permeability transition, a large channel in the inner mitochondrial membrane opens, leading to the loss of multiple mitochondrial solutes and cell death. Key triggers include excessive reactive oxygen species and mitochondrial calcium overload, factors implicated in neuronal and cardiac pathophysiology. Examining the differential behavior of mitochondrial Ca(2+) overload in Drosophila versus human cells allowed us to identify a gene, MCUR1, which, when expressed in Drosophila cells, conferred permeability transition sensitive to electrophoretic Ca(2+) uptake. Conversely, inhibiting MCUR1 in mammalian cells increased the Ca(2+) threshold for inducing permeability transition. The effect was specific to the permeability transition induced by Ca(2+), and such resistance to overload translated into improved cell survival. Thus, MCUR1 expression regulates the Ca(2+) threshold required for permeability transition.

The basal fungus Allomyces macrogynus (A. macrogynus) produces motile male gametes displaying well-studied chemotaxis toward their female counterparts. This chemotaxis is driven by sirenin, a sexual pheromone released by the female gametes. The pheromone evokes a large calcium influx in the motile gametes, which could proceed through the cation channel of sperm (CatSper) complex. Herein, we report the total synthesis of sirenin in 10 steps and 8% overall yield and show that the synthetic pheromone activates the CatSper channel complex, indicated by a concentration-dependent increase in intracellular calcium in human sperm. Sirenin activation of the CatSper channel was confirmed using whole-cell patch clamp electrophysiology with human sperm. Based on this proficient synthetic route and confirmed activation of CatSper, analogues of sirenin can be designed as blockers of the CatSper channel that could provide male contraceptive agents.

The beating of motile cilia generates fluid flow over epithelia in brain ventricles, airways, and Fallopian tubes. Here, we patch clamp single motile cilia of mammalian ependymal cells and examine their potential function as a calcium signaling compartment. Resting motile cilia calcium concentration ([Ca 2+ ] ~170 nM) is only slightly elevated over cytoplasmic [Ca 2+ ] (~100 nM) at steady state. Ca 2+ changes that arise in the cytoplasm rapidly equilibrate in motile cilia. We measured Ca V 1 voltage-gated calcium channels in ependymal cells, but these channels are not specifically enriched in motile cilia. Membrane depolarization increases ciliary [Ca 2+ ], but only marginally alters cilia beating and cilia-driven fluid velocity within short (~1 min) time frames. We conclude that beating of ependymal motile cilia is not tightly regulated by voltage-gated calcium channels, unlike that of well-studied motile cilia and flagella in protists, such as Paramecia and Chlamydomonas .

The basic principles of Ca(2+) regulation emerged early in prokaryotes. Ca(2+) signaling acquired more extensive and varied functions when life evolved into multicellular eukaryotes with intracellular organelles. Animals, fungi and plants display differences in the mechanisms that control cytosolic Ca(2+) concentrations. The aim of this review is to examine recent findings from comparative genomics of Ca(2+) signaling molecules in close unicellular relatives of animals and in common unicellular ancestors of animals and fungi. Also discussed are the evolution and origins of the sperm-specific CatSper channel complex, cation/Ca(2+) exchangers and four-domain voltage-gated Ca(2+) channels. Newly identified evolutionary evidence suggests that the distinct Ca(2+) signaling machineries in animals, plants and fungi likely originated from an ancient Ca(2+) signaling machinery prior to early eukaryotic radiation., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Entry and extrusion of cations are essential processes in living cells. In alkaliphilic prokaryotes, high external pH activates voltage-gated sodium channels (Nav), which allows Na(+) to enter and be used as substrate for cation/proton antiporters responsible for cytoplasmic pH homeostasis. Here, we describe a new member of the prokaryotic voltage-gated Na(+) channel family (NsvBa; Non-selective voltage-gated, Bacillus alcalophilus) that is nonselective among Na(+), Ca(2+) and K(+) ions. Mutations in NsvBa can convert the nonselective filter into one that discriminates for Na(+) or divalent cations. Gain-of-function experiments demonstrate the portability of ion selectivity with filter mutations to other Bacillus Nav channels. Increasing pH and temperature shifts their activation threshold towards their native resting membrane potential. Furthermore, we find drugs that target Bacillus Nav channels also block the growth of the bacteria. This work identifies some of the adaptations to achieve ion discrimination and gating in Bacillus Nav channels.

The recent discovery of genes encoding the mitochondrial calcium (Ca(2+)) uniporter has revealed new opportunities for studying how abnormal Ca(2+) signals cause disease. Ca(2+) transport across the mitochondrial inner membrane is highly regulated, and the uniporter is the channel that acts as a major portal for Ca(2+) influx. Low amounts of mitochondrial Ca(2+) can boost ATP synthesis, but excess amounts, such as following cytoplasmic Ca(2+) overload in heart failure, triggers mitochondrial failure and cell death. In fact, precisely because mitochondrial Ca(2+) transport is so tightly regulated, a fundamental understanding of how the uniporter functions is necessary. Two key uniporter features allow Ca(2+) influx without mitochondrial damage during normal physiology. First, the channel is significantly more selective than other known Ca(2+) channels. This prevents the permeation of other ions and uncoupling of the electrochemical gradient. Second, the uniporter becomes active at only high Ca(2+) concentrations, preventing a resting leak of cytoplasmic Ca(2+) itself. Now possessing the identities of the various proteins forming the uniporter, we can proceed with efforts to define the molecular determinants of permeation, selectivity and Ca(2+)-regulation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Voltage-gated sodium channels are important targets for the development of pharmaceutical drugs, because mutations in different human sodium channel isoforms have causal relationships with a range of neurological and cardiovascular diseases. In this study, functional electrophysiological studies show that the prokaryotic sodium channel from Magnetococcus marinus (NavMs) binds and is inhibited by eukaryotic sodium channel blockers in a manner similar to the human Nav1.1 channel, despite millions of years of divergent evolution between the two types of channels. Crystal complexes of the NavMs pore with several brominated blocker compounds depict a common antagonist binding site in the cavity, adjacent to lipid-facing fenestrations proposed to be the portals for drug entry. In silico docking studies indicate the full extent of the blocker binding site, and electrophysiology studies of NavMs channels with mutations at adjacent residues validate the location. These results suggest that the NavMs channel can be a valuable tool for screening and rational design of human drugs.

TRPM7 is a ubiquitous ion channel and kinase, a unique "chanzyme," required for proper early embryonic development. It conducts Zn(2+), Mg(2+), and Ca(2+) as well as monovalent cations and contains a functional serine/threonine kinase at its carboxyl terminus. Here, we show that in normal tissues and cell lines, the kinase is proteolytically cleaved from the channel domain in a cell-type-specific manner. These TRPM7 cleaved kinase fragments (M7CKs) translocate to the nucleus and bind multiple components of chromatin-remodeling complexes, including Polycomb group proteins. In the nucleus, the kinase phosphorylates specific serines/threonines of histones. M7CK-dependent phosphorylation of H3Ser10 at promoters of TRPM7-dependent genes correlates with their activity. We also demonstrate that cytosolic free [Zn(2+)] is TRPM7 dependent and regulates M7CK binding to transcription factors containing zinc-finger domains. These findings suggest that TRPM7-mediated modulation of intracellular Zn(2+) concentration couples ion-channel signaling to epigenetic chromatin covalent modifications that affect gene expression patterns. PAPERCLIP:, (Copyright © 2014 Elsevier Inc. All rights reserved.)