Calcium (Ca(2+))-activated chloride channels are fundamental mediators in numerous physiological processes including transepithelial secretion, cardiac and neuronal excitation, sensory transduction, smooth muscle contraction and fertilization. Despite their physiological importance, their molecular identity has remained largely unknown. Here we show that transmembrane protein 16A (TMEM16A, which we also call anoctamin 1 (ANO1)) is a bona fide Ca(2+)-activated chloride channel that is activated by intracellular Ca(2+) and Ca(2+)-mobilizing stimuli. With eight putative transmembrane domains and no apparent similarity to previously characterized channels, ANO1 defines a new family of ionic channels. The biophysical properties as well as the pharmacological profile of ANO1 are in full agreement with native Ca(2+)-activated chloride currents. ANO1 is expressed in various secretory epithelia, the retina and sensory neurons. Furthermore, knockdown of mouse Ano1 markedly reduced native Ca(2+)-activated chloride currents as well as saliva production in mice. We conclude that ANO1 is a candidate Ca(2+)-activated chloride channel that mediates receptor-activated chloride currents in diverse physiological processes.

Capsaicin, a pungent ingredient of hot peppers, causes excitation of small sensory neurons, and thereby produces severe pain. A nonselective cation channel activated by capsaicin has been identified in sensory neurons and a cDNA encoding the channel has been cloned recently. However, an endogenous activator of the receptor has not yet been found. In this study, we show that several products of lipoxygenases directly activate the capsaicinactivated channel in isolated membrane patches of sensory neurons. Among them, 12-and 15-(S)-hydroperoxyeicosatetraenoic acids, 5-and 15-(S)-hydroxyeicosatetraenoic acids, and leukotriene B4 possessed the highest potency. The eicosanoids also activated the cloned capsaicin receptor (VR1) expressed in HEK cells. Prostaglandins and unsaturated fatty acids failed to activate the channel. These results suggest a novel signaling mechanism underlying the pain sensory transduction.

Nociceptors are a subset of small primary afferent neurons that respond to noxious chemical, thermal and mechanical stimuli. Ion channels in nociceptors respond differently to noxious stimuli and generate electrical signals in different ways. Anoctamin 1 (ANO1 also known as TMEM16A) is a Ca(2+)-activated chloride channel that is essential for numerous physiological functions. We found that ANO1 was activated by temperatures over 44 °C with steep heat sensitivity. ANO1 was expressed in small sensory neurons and was highly colocalized with nociceptor markers, which suggests that it may be involved in nociception. Application of heat ramps to dorsal root ganglion (DRG) neurons elicited robust ANO1-dependent depolarization. Furthermore, knockdown or deletion of ANO1 in DRG neurons substantially reduced nociceptive behavior in thermal pain models. These results indicate that ANO1 is a heat sensor that detects nociceptive thermal stimuli in sensory neurons and possibly mediates nociception.

Hearing in Drosophila depends on the transduction of antennal vibration into receptor potentials by ciliated sensory neurons in Johnston's organ, the antennal chordotonal organ. We previously found that a Drosophila protein in the vanilloid receptor subfamily (TRPV) channel subunit, Nanchung (NAN), is localized to the chordotonal cilia and required to generate sound-evoked potentials (Kim et al., 2003). Here, we show that the only other Drosophila TRPV protein is mutated in the behavioral mutant inactive (iav). The IAV protein forms a hypotonically activated channel when expressed in cultured cells; in flies, it is specifically expressed in the chordotonal neurons, localized to their cilia and required for hearing. IAV and NAN are each undetectable in cilia of mutants lacking the other protein, indicating that they both contribute to a heteromultimeric transduction channel in vivo. A functional green fluorescence protein-IAV fusion protein shows that the channel is restricted to the proximal cilium, constraining models for channel activation.

The capsaicin-sensitive vanilloid receptor (VR1) was recently shown to play an important role in inflammatory pain (hyperalgesia), but the underlying mechanism is unknown. We hypothesized that pain-producing inflammatory mediators activate capsaicin receptors by inducing the production of fatty acid agonists of VR1. This study demonstrates that bradykinin, acting at B2 bradykinin receptors, excites sensory nerve endings by activating capsaicin receptors via production of 12-lipoxygenase metabolites of arachidonic acid. This finding identifies a mechanism that might be targeted in the development of new therapeutic strategies for the treatment of inflammatory pain. V R1, a cloned capsaicin receptor, is a nonspecific cation channel expressed preferentially in small sensory neurons and activated by the vanilloids, capsaicin and resiniferatoxin (1). Because VR1 is also activated by heat and acid (1, 2), it is now considered to be a molecular sensor that detects a variety of painful stimuli. Indeed, recent experiments performed in mice that lack VR1 demonstrated that the receptor is essential for inflammation-induced heat hyperalgesia (3, 4). Therefore, understanding the cellular mechanisms by which capsaicin receptors are activated by inflammatory mediators may be a key to identifying novel therapeutic targets for pain treatment. Because of the presence of VR1 in sensory neurons and an apparent role in inflammatory hyperalgesia, endogenous activators of VR1 have been suspected. We recently demonstrated that products of the lipoxygenase pathway of arachidonic acid (AA) metabolism can activate capsaicin receptors (5). Among the eicosanoids tested, the 12-lipoxygenase product, 12-hydroperoxyeicosatetraenoic acid (12-HPETE), structurally similar to capsaicin, was the most potent VR1 agonist. Thus, metabolic products of lipoxygenases become candidates for the endogenous capsaicinlike substances. However, the upstream signals that stimulate lipoxygenase and activate VR1 are elusive.Bradykinin (BK) is a potent inflammatory mediator that causes pain and hyperalgesia. BK is known to activate as well as sensitize sensory neurons to other stimuli. Various signaling pathways have been suggested to mediate the sensitizing effect of BK on sensory neurons (6, 7). However, activation mechanism by BK is not known. BK is now known to stimulate the production of AA in sensory neurons (8), a key substrate of lipoxygenases. Therefore, on the basis of previous observations that products of lipoxygenase activate VR1 (5), we hypothesized that BK excites sensory neurons by opening the capsaicin receptor via production of 12-lipoxygenase products of AA metabolism. Materials and MethodsCell Culture. Experiments were carried out according to the Ethical Guidelines of the International Association for the Study of Pain and approved by the research ethics committee for the use of animals of the Seoul National University and the University of California, San Francisco. Thoracic and lumbar dorsal root ganglia (DRGs) were dissected from 1-to 2 day-...

Vanilloid receptor 1 (VR1), a capsaicin receptor, is known to play a major role in mediating inflammatory thermal nociception. Although the physiological role and biophysical properties of VR1 are known, the mechanism of its activation by ligands is poorly understood. Here we show that VR1 must be phosphorylated by Ca 2؉ -calmodulin dependent kinase II (CaMKII) before its activation by capsaicin. In contrast, the dephosphorylation of VR1 by calcineurin leads to a desensitization of the receptor. Moreover, point mutations in VR1 at two putative consensus sites for CaMKII failed to elicit capsaicin-sensitive currents and caused a concomitant reduction in VR1 phosphorylation in vivo. Such mutants also lost their high affinity binding with [ 3 H]resiniferatoxin, a potent capsaicin receptor agonist. We conclude that the dynamic balance between the phosphorylation and dephosphorylation of the VR1 channel by CaMKII and calcineurin, respectively, controls the activation/ desensitization states by regulating VR1 binding. Furthermore, because sensitization by protein kinase A and C converge at these sites, phosphorylation stress in the cell appears to control a wide range of excitabilities in response to various adverse stimuli.

Mechanosensitive (MS) ion channels are present in a variety of cells. However, very little is known about the ion channels that account for mechanical sensitivity in sensory neurons. We identified the two most frequently encountered but distinct types of MS channels in 1390 of 2962 membrane patches tested in cultured dorsal root ganglion neurons. The two MS channels exhibited different thresholds, thus named as low-threshold (LT) and high-threshold (HT) MS channels, and sensitivity to pressure. The two channels retained different single-channel conductances and current-voltage relationships: LT and HT channels elicited large- and small-channel conductance with outwardly rectifying and linear I-V relationships, respectively. Both LT and HT MS channels were permeable to monovalent cations and Ca2+ and were blocked by gadolinium, a blocker of MS channels. Colchicine and cytochalasin D markedly reduced the activities of the two MS channels, indicating that cytoskeletal elements support the mechanosensitivity. Both types of MS channels were found primarily in small sensory neurons with diameters of <30 microm. Furthermore, HT MS channels were sensitized by a well known inducer of mechanical hyperalgesia, prostaglandin E2, via the protein kinase A pathway. We identified two distinct types of MS channels in sensory neurons that probably give rise to the observed MS whole-cell currents and transduce mechanical stimuli to neural signals involved in somatosensation, including pain.

Vanilloid receptor 1 (VR1), a ligand-gated ion channel activated by vanilloids, acid, and heat, is a molecular detector that integrates multiple modes of pain. Although the function and the biophysical properties of the channel are now known, the regions of VR1 that recognize ligands are largely unknown. By the stepwise deletion of VR1 and by chimera construction using its capsaicin-insensitive homologue, VRL1, we localized key amino acids, Arg-114 and Glu-761, in the N-and C-cytosolic tails, respectively, that determine ligand binding. Point mutations of the two key residues resulted in a loss of sensitivity to capsaicin and a concomitant loss of specific binding to [ 3 H]resiniferatoxin, a potent vanilloid. Furthermore, changes in the charges of the two amino acids blocked capsaicin-sensitive currents and ligand binding without affecting current responses to heat. Thus, these two regions in the cytoplasmic tails of VR1 provide structural elements for its hydrophilic interaction with vanilloids and might constitute a long-suspected binding pocket.Capsaicin, the principal pungent ingredient of hot peppers, excites sensory neurons by opening an ion channel, the vanilloid receptor 1 (VR1), 1 thereby causing pain. VR1 is a ligand-gated, cationic channel that is present mainly in small nociceptive sensory neurons (1-3). The presence of VR1 in sensory neurons leads to questions concerning the existence of endogenous capsaicin-like substances, and various lipid metabolic products of lipoxygenases or anandamide have been suggested as candidates, because they activate VR1 and are structurally similar to capsaicin (4, 5). Accordingly, a role for lipoxygenase products in the activation of VR1 during inflammation was suggested (5), and in fact, bradykinin, a potent pain-causing inflammatory mediator, is now known to activate VR1 via the lipoxygenase/VR1 pathway (6). In addition, bradykinin also has a potential to sensitize VR1 via a phospholipase C or protein kinase C pathway (7-9).VR1 is also activated by acid or heat at over 43°C, a threshold temperature for pain (3, 10 -12). Moreover, because ischemic or inflamed tissues become acidic, the acid activation of VR1 is a pathologically relevant event (13). More direct evidence for the pathophysiological role of VR1 in the production of inflammatory pain came from knock-out experiments. In mice lacking VR1, thermal hyperalgesia evoked by inflammation is reduced (14, 15). Furthermore, hyperalgesia induced by the key inflammatory mediators, bradykinin and nerve growth factor, is reduced in mice lacking VR1 (8). Thus, VR1 is now considered a primary molecular transducer that mediates inflammatory hyperalgesia (13).The putative topology of VR1 indicates that it belongs to a class of transient receptor potential channels possessing six transmembrane domains and two cytosolic domains at each Nand C terminus (3, 16). VR1 appears to form a homotetramer when expressed heterologously (17). However, VR1 may form a heteromultimer with another temperature-sensitive channel, transient rec...