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Significance Radial glial cells (RGCs), a type of neural stem cell in the developing brain, not only generate progenitors, newly born neurons and glial cells, but also deliver neurons through its process to the appropriate cortical target layers. Thus, the function of RGCs is crucial for cortex development, in which Cl− channels are thought to play a role. Here we highlight that Anoctamin 1 (ANO1)/TMEM16A, a Ca2+-activated Cl− channel, mediates the process extension in RGCs. ANO1-null mice show a decrease in cortical thickness with disorganized cortical layers. Thus, as a Cl− channel, ANO1 is involved in the process maturation of RGCs and contributes to cortex development., Neural stem cells (NSCs) are primary progenitor cells in the early developmental stage in the brain that initiate a diverse lineage of differentiated neurons and glia. Radial glial cells (RGCs), a type of neural stem cell in the ventricular zone, are essential for nurturing and delivering new immature neurons to the appropriate cortical target layers. Here we report that Anoctamin 1 (ANO1)/TMEM16A, a Ca2+-activated chloride channel, mediates the Ca2+-dependent process extension of RGCs. ANO1 is highly expressed and functionally active in RGCs of the mouse embryonic ventricular zone. Knockdown of ANO1 suppresses RGC process extension and protrusions, whereas ANO1 overexpression stimulates process extension. Among various trophic factors, brain-derived neurotrophic factor (BDNF) activates ANO1, which is required for BDNF-induced process extension in RGCs. More importantly, Ano1-deficient mice exhibited disrupted cortical layers and reduced cortical thickness. We thus conclude that the regulation of RGC process extension by ANO1 contributes to the normal formation of mouse embryonic brain.

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.

Touch sensation or proprioception requires the transduction of mechanical stimuli into electrical signals by mechanoreceptors in the periphery. These mechanoreceptors are equipped with various transducer channels. Although Piezo1 and 2 are mechanically activated (MA) channels with rapid inactivation, MA molecules with other inactivation kinetics have not been identified. Here we report that heterologously expressed Tentonin3 (TTN3)/TMEM150C is activated by mechanical stimuli with distinctly slow inactivation kinetics. Genetic ablation of Ttn3/Tmem150c markedly reduced slowly adapting neurons in dorsal-root ganglion neurons. The MA TTN3 currents were inhibited by known blockers of mechanosensitive ion channels. Moreover, TTN3 was localized in muscle spindle afferents. Ttn3-deficient mice exhibited the loss of coordinated movements and abnormal gait. Thus, TTN3 appears to be a component of a mechanosensitive channel with a slow inactivation rate and contributes to motor coordination. Identification of this gene advances our understanding of the various types of mechanosensations, including proprioception.

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.

The transient receptor potential (TRP) channel superfamily is a set of channel genes that mediate numerous physiological functions such as sensing irritants or detecting temperature changes. Despite their functions, expressional information on TRP channels in various organs is largely elusive. Therefore, we conducted a systematic quantitative comparison of each mRNA expression level of 22 mouse TRP channels in various organs. As a result, we found that average levels of TRP channel transcripts were very low reaching ∼3% of the GAPDH transcript level. Among 22 TRP channels, TRPC1 and TRPM7 were most abundant in the majority of organs. In contrast, TRPV3, TRPV5, TRPV6, TRPC7, TRPM1, and TRPM5 elicited very low message profiles throughout the major organs. Consistent with their functions as molecular sensors for irritants and temperature changes, TRPV1, TRPM8 and TRPA1 showed exclusive expression in sensory ganglia. TRPC3 and TRPM3 were abundant in the sensory ganglia and brain. High levels of transcripts of TRPV2, TRPC6, TRPM4, and TRPM6 were observed in the lung. In addition, channel transcript levels were very low except TRPM7 in the liver. In summary, the expression profile of TRP channels in major tissues provides insight to their physiological functions and therefore application to new drug development.

Spinal muscular atrophy and hereditary motor and sensory neuropathies are characterized by muscle weakness and atrophy caused by the degenerations of peripheral motor and sensory nerves. Recent advances in genetics have resulted in the identification of missense mutations in TRPV4 in patients with these hereditary neuropathies. Neurodegeneration caused by Ca(2+) overload due to the gain-of-function mutation of TRPV4 was suggested as the molecular mechanism for the neuropathies. Despite the importance of TRPV4 mutations in causing neuropathies, the precise role of TRPV4 in the sensory/motor neurons is unknown. Here, we report that TRPV4 mediates neurotrophic factor-derived neuritogenesis in developing peripheral neurons. TRPV4 was found to be highly expressed in sensory and spinal motor neurons in early development as well as in the adult, and the overexpression or chemical activation of TRPV4 was found to promote neuritogenesis in sensory neurons as well as PC12 cells, whereas its knockdown and pharmacologic inhibition had the opposite effect. More importantly, nerve growth factor or cAMP treatment up-regulated the expression of phospholipase A(2) and TRPV4. Neurotrophic factor-derived neuritogenesis appears to be regulated by the phospholipase A(2)-mediated TRPV4 pathway. These findings show that TRPV4 mediates neurotrophic factor-induced neuritogenesis in developing peripheral nerves. Because neurotrophic factors are essential for the maintenance of peripheral nerves, these findings suggest that aberrant TRPV4 activity may lead to some types of pathology of sensory and motor nerves.

Significance Benign prostatic hyperplasia (BPH) is characterized by an enlargement of the prostate gland, a common disease in elderly men. Excessive testosterone is considered to cause BPH. However, its etiologic mechanisms are elusive. We found that ANO1, a Ca 2+ -activated Cl − channel, is essential for the testosterone-induced BPH. ANO1 was highly expressed in dihydrotestosterone (DHT)-treated prostate epithelial cells. The selective knockdown of ANO1 suppressed DHT-induced cell proliferation. Surprisingly, we found that there were three androgen-response elements in the ANO1 promoter region, which were relevant for the DHT-dependent induction of ANO1. Intraprostate treatment of Ano1 siRNA inhibited the prostate enlargement in vivo. Thus, ANO1 appears essential for the development of prostate hyperplasia and becomes a useful target for treating BPH.

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.

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.

Jumonji (JMJ) can function as a transcriptional repressor and plays critical roles in embryonic development including heart development in mice. Although JMJ has been suggested to play a role in cell growth, the molecular mechanisms have not been resolved. The present data demonstrate that JMJ interacts with the retinoblastoma protein (Rb), one of the master regulatory genes of cell cycle. JMJ potentiates the repression function of Rb on E2F activities, leading to reduced cell cycle progression. The transcriptional repression domain of JMJ is critical for the interaction with Rb as well as repression of cell cycle. The physiological relevance of the association between Rb and JMJ was assessed in cardiomyocytes. Primary cardiomyocytes cultured from homozygous jmj knock-out mouse embryos (jmj mutants) show increased cell mitosis in a cardiomyocyte-specific manner. Reporter gene analyses demonstrate that promoter activities of cyclin D1, cyclin D2, and Cdc2 are up-regulated in jmj mutant cardiomyocytes. These data suggest that JMJ down-regulates the cell growth via interaction with Rb, which would provide important insights into the cardiac defects observed in jmj mutant mice.

Expression of alpha-cardiac myosin heavy chain gene (alphaMHC) is developmentally regulated in normal embryonic hearts and down-regulated in cardiac myopathy and failing hearts. Jumonji (JMJ) has been shown to be critical for normal cardiovascular development and functions as a transcriptional repressor. Here, we demonstrate that JMJ represses alphaMHC expression through inhibition of myocyte enhancer factor 2 (MEF2) activity. In primary cardiomyocytes, overexpression of JMJ leads to marked reduction of endogenous alphaMHC expression. JMJ represses the synergistic activation of alphaMHC by MEF2 and thyroid hormone receptor (TR). Interestingly, JMJ inhibits transcriptional activities of all MEF2 isoforms, but not the TR-dependent activation. The transcriptional repression domain of JMJ interacts with the N-terminal part of MEF2A, resulting in the repression of MEF2A activities. These results suggest that JMJ represses alphaMHC expression via protein-protein interaction with MEF2A.

Cardiac development is a complex biological process requiring the integration of cell specification, differentiation, migration, proliferation, and morphogenesis. Although significant progress has been made recently in understanding the molecular basis of cardiac development, mechanisms of transcriptional control of cardiac development remain largely unknown. In search for the developmentally important genes, the jumonji gene (jmj) was identified by gene trap technology and characterized as a critical nuclear factor for mouse embryonic development. Jmj has been shown to play important roles in cardiovascular development, neural tube fusion process, hematopoiesis, and liver development in mouse embryos. The amino acid sequence of the JUMONJI protein (JMJ) reveals that JMJ belongs to the AT-rich interaction domain transcription factor family and more recently has been described as a member of the JMJ transcription factor family. Here, we review the roles of jmj in multiple organ development with a focus on cardiovascular development in mice.

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.

Ca(2+)-activated Cl(-) channels (CaCCs) are a class of Cl(-) channels activated by intracellular Ca(2+) that are known to mediate numerous physiological functions. In 2008, the molecular identity of CaCCs was found to be anoctamin 1 (ANO1/TMEM16A). Its roles have been studied in electrophysiological, histological, and genetic aspects. ANO1 is known to mediate Cl(-) secretion in secretory epithelia such as airways, salivary glands, intestines, renal tubules, and sweat glands. ANO1 is a heat sensor activated by noxious heat in somatosensory neurons and mediates acute pain sensation as well as chronic pain. ANO1 is also observed in vascular as well as airway smooth muscles, controlling vascular tone as well as airway hypersensitivity. ANO1 is upregulated in numerous types of cancers and thus thought to be involved in tumorigenesis. ANO1 is also found in proliferating cells. In addition to ANO1, involvement of its paralogs in pathophysiological conditions was also reported. ANO2 is involved in olfaction, whereas ANO6 works as a scramblase whose mutation causes a rare bleeding disorder, the Scott syndrome. ANO5 is associated with muscle and bone diseases. Recently, an X-ray crystal structure of a fungal TMEM16 was reported, which explains a precise molecular gating mechanism as well as ion conduction or phospholipid transport across the plasma membrane.

Background Various pathological conditions such as inflammation or injury can evoke pain hypersensitivity. That represents the response to innocuous stimuli or exaggerated response to noxious stimuli. The molecular mechanism based on the pain hypersensitivity is associated with changes in many of ion channels in dorsal-root ganglion (DRG) neurons. Anoctamin 1 (ANO1/TMEM16A), a Ca2+ activated chloride channel is highly visible in small DRG neurons and responds to heat. Mice with an abolished function of ANO1 in DRG neurons demonstrated attenuated pain-like behaviors when exposed to noxious heat, suggesting a role in acute thermal nociception. In this study, we further examined the function of ANO1 in mediating inflammation- or injury-induced hyperalgesia or allodynia. Results Using Advillin/Ano1 fl/fl (Adv/Ano1 fl/fl ) mice that have a functional ablation of Ano1 mainly in DRG neurons, we were able to determine its role in mediating thermal hyperalgesia and mechanical allodynia induced by inflammation or nerve injury. The thermal hyperalgesia and mechanical allodynia induced by carrageenan injection and spared-nerve injury were significantly reduced in Adv/Ano1 fl/fl mice. In addition, flinching or licking behavior after bradykinin or formalin injection was also significantly reduced in Adv/Ano1 fl/fl mice. Since pathological conditions augment nociceptive behaviors, we expected ANO1′s contribution to the excitability of DRG neurons. Indeed, the application of inflammatory mediators reduced the threshold for action potential (rheobase) or time for induction of the first action potential in DRG neurons isolated from control (Ano1 fl/fl ) mice. These parameters for neuronal excitability induced by inflammatory mediators were not changed in Adv/Ano1 fl/fl mice, suggesting an active contribution of ANO1 in augmenting the neuronal excitability. Conclusions In addition to ANO1's role in mediating acute thermal pain as a heat sensor, ANO1 is also capable of augmenting the excitability of DRG neurons under inflammatory or neuropathic conditions and thereby aggravates inflammation- or tissue injury-induced pathological pain.