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Pain may be induced by activation of various ion channels expressed in primary afferent neurons. These channels function as molecular sensors that detect noxious chemical, temperature, or tactile stimuli and transduce them into nociceptor electrical signals. Transient receptor potential channels are good examples because they are activated by chemicals, heat, cold, and acid in nociceptors. Anion channels were little studied in nociception because of the notion that anion channels might induce hyperpolarization of nociceptors on opening. In contrast, opening of Cl- channels in dorsal root ganglion (DRG) neurons depolarizes sensory neurons, resulting in excitation of nociceptors, thereby inducing pain. Anoctamin 1(ANO1)/TMEM16A is a Ca2+-activated Cl- channel expressed mainly in small DRG neurons, suggesting a nociception role. ANO1 is a heat sensor that detects heat over 44°C. Ano1-deficient mice elicit less nocifensive behaviors to hot temperatures. In addition, mechanical allodynia and hyperalgesia induced by inflammation or nerve injury are alleviated in Ano1-/- mice. More important, Ano1 transcripts are increased in chronic pain models. Bestrophin 1 (Best1) is another Ca2+-activated Cl- channel expressed in nociceptors. Best1 is increased in axotomized DRG neurons. The role of Best1 in nociception is not clear. GABAA receptors are in the central process of DRG neurons; GABA depolarizes the primary afferents. This depolarization consists of primary afferent depolarization essential for inhibiting nociceptive input to second-order neurons in the spinal cord, regulating pain signals to the brain. Thus, although Cl- channels in nociceptors are not as numerous as TRP channels, their role in nociception is distinct and significant.

Anoctamins (ANOs) are multifunctional membrane proteins that consist of 10 homologs. ANO1 (TMEM16A) and ANO2 (TMEM16B) are anion channels activated by intracellular calcium that meditate numerous physiological functions. ANO6 is a scramblase that redistributes phospholipids across the cell membrane. The other homologs are not well characterized. We found ANO9/TMEM16J is a cation channel activated by a cAMP-dependent protein kinase A (PKA). Intracellular cAMP-activated robust currents in whole cells expressing ANO9, which were inhibited by a PKA blocker. A cholera toxin that persistently stimulated adenylate cyclase activated ANO9 as did the application of PKA. The cAMP-induced ANO9 currents were permeable to cations. The cAMP-dependent ANO9 currents were augmented by intracellular Ca2+. Ano9 transcripts were predominant in the intestines. Human intestinal SW480 cells expressed high levels of Ano9 transcripts and showed PKA inhibitor-reversible cAMP-dependent currents. We conclude that ANO9 is a cation channel activated by a cAMP/PKA pathway and could play a role in intestine function.

Background Airway responsiveness after acute inhalation of ozone is related to the concentration and duration of ozone exposure. Using barometric whole-body plethysmography and increase in enhanced pause (Penh) as an index of airway obstruction, we measured the response of BALB/c mice to acute ozone inhalation to study the time course change of pulmonary function after ozone exposure. Methods Penh was measured before and after exposure to filtered air or 0.12, 0.5, 1, or 2 ppm ozone for 3 hr (n=6/group). In addition, Penh was measured 24, 48 and 72 hr after ozone exposure. Bronchoalveolar lavage (BAL) and histopathologic examinations were performed. Results The increase in Penh after ozone exposure was significantly higher in the 0.12, 0.5, 1 and 2 ppm groups compared with the control group (all p

Anoctamin 1 (ANO1)/TMEM16A is a Cl− channel activated by intracellular Ca2+ mediating numerous physiological functions. However, little is known of the ANO1 activation mechanism by Ca2+. Here, we demonstrate that two helices, “reference” and “Ca2+ sensor” helices in the third intracellular loop face each other with opposite charges. The two helices interact directly in a Ca2+-dependent manner. Positively and negatively charged residues in the two helices are essential for Ca2+-dependent activation because neutralization of these charges change the Ca2+ sensitivity. We now predict that the Ca2+ sensor helix attaches to the reference helix in the resting state, and as intracellular Ca2+ rises, Ca2+ acts on the sensor helix, which repels it from the reference helix. This Ca2+-dependent push-pull conformational change would be a key electromechanical movement for gating the ANO1 channel. Because chemical activation of ANO1 is viewed as an alternative means of rescuing cystic fibrosis, understanding its gating mechanism would be useful in developing novel treatments for cystic fibrosis. Electronic supplementary material The online version of this article (doi:10.1007/s00424-014-1603-2) contains supplementary material, which is available to authorized users.

Capsaicin-activated channels present in sensory neurons are ligand-gated cation channels that largely account for mediating some types of pain. The cAMP-dependent protein kinase (PKA) signal pathway was suggested to mediate the prostaglandin-induced enhancement of capsaicin-evoked inward current (I(CAP)) in rat sensory neurons. It is not clear, however, whether PKA acts directly on the capsaicin-sensitive channel that is responsible for I(CAP). To address this issue, we overexpressed the cloned capsaicin receptor, VR1, in heterologous expression systems such as Xenopus oocytes or Aplysia R2 neuron and stimulated PKA pathways. As a result, activation of PKA by applying either 8-bromo-cAMP or forskolin with 3-isobutyl-1-methylxanthine or through activation of beta(2) adrenergic receptors failed to enhance I(CAP) in oocytes or R2 neurons expressing VR1. Our results raise two possibilities. (1) Direct phosphorylation of VR1 by PKA may not be responsible for the sensitization; instead, phosphorylation of regulatory proteins associated with VR1 would account for the sensitization of I(CAP) evoked by prostaglandin E(2) in dorsal root ganglion (DRG) neurons. (2) DRG neurons may have a different PKA signaling mechanism that is not replicable in Xenopus oocytes or Aplysia R2 neurons.

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 capsaicin-activated channel in isolated membrane patches of sensory neurons. Among them, 12- and 15-( S )-hydroperoxyeicosatetraenoic acids, 5- and 15-( S )-hydroxyeicosatetraenoic acids, and leukotriene B 4 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.

In the present study, the presence of an endogenous capsaicin-like substance and the role of capsaicin receptors in nociception during inflammation were assessed using Fos immunohistochemistry and the paw-withdrawal test in rats. Intradermal injection of carrageenan in the hind-paw produced inflammation in the foot pad, increased the number of cells exhibiting Fos-like immunoreactivity in the dorsal horn of the spinal cord, and decreased the paw-withdrawal latency. Intradermal injection of capsazepine, a capsaicin-receptor antagonist, significantly reduced the number of cells exhibiting Fos-like immunoreactivity, significantly increased the paw-withdrawal latency, but did not decrease inflammation induced by carrageenan injection. Intradermal injection of capsaicin or formalin also increased Fos-positive neurons. Capsaicin- or formalin-induced Fos expression was reduced in both cases by pretreatment of capsazepine, but to a much lesser extent for formalin. The capsazepine inhibition of carrageenan inflammation-induced hyperalgesic responses strongly suggests that an endogenous capsaicin-like substance is released in inflamed tissues and produces nociceptive neural impulses by acting on capsaicin receptors present on sensory neurons. Furthermore, our results indicate that capsaicin receptors take part only in generating nociceptive signals in sensory neurons, but not in activating the inflammation-promoting cells.

Intracellular Ca(2+) signal is a key regulator of axonal growth during brain development. As transient receptor potential (TRP) channels are permeable to Ca(2+) and mediate numerous brain functions, it is conceivable that many TRP channels would regulate neuronal differentiation. We therefore screened TRP channels that are involved in the regulation of neurite growth. Among the TRP channels, the Trpm2 level was inversely associated with neurite growth. TRPM2 was highly expressed in embryonic brain. Pharmacological perturbation or knockdown of TRPM2 markedly increased the axonal growth, whereas its overexpression inhibited the axonal growth. Addition of ADP ribose, an endogenous activator of TRPM2, to PC12 cells significantly repressed the axonal growth. TRPM2 was actively involved in the neuronal retraction induced by cerebrospinal fluid-rich lysophosphatidic acid (LPA). More importantly, neurons isolated from the brain of Trpm2-deficient mice have significantly longer neurites with a greater number of spines than those obtained from the brain of wild-type mice. Therefore, we conclude that TRPM2 mediates the LPA-induced suppression of axonal growth, which provides a long-sought mechanism underlying the effect of LPA on neuronal development.

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 Ca2+-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 [3H]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.

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.

Capsaicin (CAP) excites small sensory neurons, causing pain, neurogenic inflammation, and other visceral reflexes. These effects have been proposed to be the result of CAP activation of a nonselective cation current. It is generally assumed that CAP binds to an extracellular domain of the membrane receptor. However, the exact binding site is not known because of the lipophilic nature of CAP. To determine whether the binding domain is extracellular or intracellular, we tested the effect of a synthetic water-soluble CAP analog, DA-5018·HCl, on current activation. CAP activated the 45 pS (at −60 mV) nonselective cation channel from either side of the membrane. However, DA-5018·HCl, which had a greater potency and efficacy than CAP, activated the channels only from the cytosolic side of the patch membrane in a capsazepine, a CAP receptor antagonist, reversible manner. When applied extracellularly, DA-5018·HCl did not, but CAP did, activate whole-cell currents in sensory neurons, as well as in oocytes expressing vanilloid receptor 1, a recently cloned CAP receptor. Hydrogen ions, reported as a possible endogenous activator of cation current, failed to elicit any current when acidic medium (pH 5.0–6.0) was applied intracellularly, indicating that H+does not mediate the CAP effect. These results indicate that CAP and its analog bind to the cytosolic domain of the CAP receptor and suggest that an endogenous CAP-like substance other than H+may be present in the cell.