Young Duk Yang

TMEM16A confers receptor-activated calcium-dependent chloride conductance.

Calcium (Ca2+)-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 Ca2+-activated chloride channel that is activated by intracellular Ca2+ and Ca2+-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 Ca2+-activated chloride currents. ANO1 is expressed in various secretory epithelia, the retina and sensory neurons. Furthermore, knockdown of mouse Ano1 markedly reduced native Ca2+-activated chloride currents as well as saliva production in mice. We conclude that ANO1 is a candidate Ca2+-activated chloride channel that mediates receptor-activated chloride currents in diverse physiological processes.

The calcium-activated chloride channel anoctamin 1 acts as a heat sensor in nociceptive neurons

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 Ca2+-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.

TRPV1 Recapitulates Native Capsaicin Receptor in Sensory Neurons in Association with Fas-Associated Factor 1

TRPV1, a cloned capsaicin receptor, is a molecular sensor for detecting adverse stimuli and a key element for inflammatory nociception and represents biophysical properties of native channel. However, there seems to be a marked difference between TRPV1 and native capsaicin receptors in the pharmacological response profiles to vanilloids or acid. One plausible explanation for this overt discrepancy is the presence of regulatory proteins associated with TRPV1. Here, we identify Fas-associated factor 1 (FAF1) as a regulatory factor, which is coexpressed with and binds to TRPV1 in sensory neurons. When expressed heterologously, FAF1 reduces the responses of TRPV1 to capsaicin, acid, and heat, to the pharmacological level of native capsaicin receptor in sensory neurons. Furthermore, silencing FAF1 by RNA interference augments capsaicin-sensitive current in native sensory neurons. We therefore conclude that FAF1 forms an integral component of the vanilloid receptor complex and that it constitutively modulates the sensitivity of TRPV1 to various noxious stimuli in sensory neurons.

Quantitative analysis of TRP channel genes in mouse organs

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.

Anoctamin 1 contributes to inflammatory and nerve-injury induced hypersensitivity.

BackgroundVarious 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.ResultsUsing Advillin/Ano1fl/fl (Adv/Ano1fl/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/Ano1fl/fl mice. In addition, flinching or licking behavior after bradykinin or formalin injection was also significantly reduced in Adv/Ano1fl/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 (Ano1fl/fl) mice. These parameters for neuronal excitability induced by inflammatory mediators were not changed in Adv/Ano1fl/fl mice, suggesting an active contribution of ANO1 in augmenting the neuronal excitability.ConclusionsIn addition to ANO1s 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.

TRPM8 mediates cold and menthol allergies associated with mast cell activation.

Exposure to low temperatures often causes allergic responses or urticaria. Similarly, menthol, a common food additive is also known to cause urticaria, asthma, and rhinitis. However, despite the obvious clinical implications, the molecular mechanisms responsible for inducing allergic responses to low temperatures and menthol have not been determined. Because a non-selective cation channel, transient receptor potential subtype M8 (TRPM8) is activated by cold and menthol, we hypothesized that this channel mediates cold- and menthol-induced histamine release in mast cells. Here, we report that TRPM8 is expressed in the basophilic leukemia mast cell line, RBL-2H3, and that exposure to menthol or low temperatures induced Ca(2+) influx in RBL-2H3 cells, which was reversed by a TRPM8 blocker. Furthermore, menthol, a TRPM8 agonist, induced the dose-dependent release of histamine from RBL-2H3 cells. When TRPM8 transcripts were reduced by siRNA (small interfering RNA), menthol- and cold-induced Ca(2+) influx and histamine release were significantly reduced. In addition, subcutaneous injection of menthol evoked scratching, a typical histamine-induced response which was reversed by a TRPM8 blocker. Thus, our findings indicate that TRPM8 mediates the menthol- and cold-induced allergic responses of mast cells, and suggest that TRPM8 antagonists be viewed as potential treatments for cold- and menthol-induced allergies.

Calcium‐activated cationic channel in rat sensory neurons

Ion channels in sensory neurons are molecular sensors that detect external stimuli and transduce them to neuronal signals. Although Ca2+‐activated nonselective cation (CAN) channels were found in many cell types, CAN channels in mammalian sensory neurons are not yet identified. In the present study, we describe an ion channel that is activated by intracellular Ca2+ in cultured rat sensory neurons. Half‐maximal concentration of Ca2+ in activating the CAN channel was approximately 780u2003µm. The current–voltage relationship of this channel was linear with a unit conductance of 28.8u2003±u20030.4u2003pS at −60u2003mV in symmetrical 140u2003mm Na+ solution. The CAN channel was permeable to monovalent cations such as Na+, K+, Cs+, and Li+, but poorly permeable to Ca2+. The CAN channel in mammalian sensory neurons was reversibly blocked by intracellular adenine nucleotides, such as ATP, ADP, and AMP. Interestingly, single‐channel currents activated by Ca2+ were blocked by fenamates, such as flufenamic acid, a class of nonsteroidal anti‐inflammatory drugs. Thus, these results suggest that CAN channels in mammalian sensory neurons would participate in modulating nociceptive neural transmission in response to ever‐changing intracellular Ca2+ in the local microenvironment.

Two helices in the third intracellular loop determine anoctamin 1 (TMEM16A) activation by calcium

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.

Different perception levels of histamine-induced itch sensation in young adult mice

Itch is an unpleasant sensation that evokes behavioral responses such as scratching the skin. Interestingly, it is conceived that the perception of itch sensation is influenced by age. Indeed, accumulating evidence supports the idea that even children or younger adults show distinctive itch sensation depending on age. This evidence implies the presence of a mechanism that regulates the perception of itch sensation in an age-dependent fashion. Therefore, the purpose of the present study was to investigate a putative mechanism for the age-dependent perception of itch sensation by comparing histamine-induced scratching behaviors in 45-day old (D45) and 75-day old male young adult mice. The results indicated that, following histamine administration, the D75 mice spent a longer time scratching than D45 mice. However, the intensity of the calcium influx induced by histamine in primary culture of dorsal root ganglia (DRG) neurons was not different between D45 and D75 mice. Moreover, no apparent difference was observed in mRNA levels of a characteristic His-related receptor and ion channel. In contrast, the mRNA levels of Toll-Like Receptor 4 (TLR4) were increased approximately by two-fold in D75 DRG compared with D45 DRG. Additionally, D75-derived DRG neurons exhibited enhanced intracellular calcium increase by lipopolysaccharide (LPS, a TLR4 agonist) than those of D45 mice. Furthermore, intensities of calcium influx induced by histamine were significantly potentiated when co-treated with LPS in D75 DRG neurons, but not in those of D45 mice. Thus, it appears that D75 mice showed enhanced histamine-induced scratching behaviors not by increased expression levels of histamine-related genes, but probably due to augmented TLR4 expression in DRG neurons. Consequently, the current study found that different perception levels of histamine-induced itch sensation are present in different age groups of young adult mice.

Nociceptive Roles of TRPM2 Ion Channel in Pathologic Pain

Pain is a protective mechanism that enables us to avoid potentially harmful environments. However, when pathologically persisted and aggravated under severely injured or inflamed conditions, pain often reduces the quality of life and thus is considered as a disease to eliminate. Inflammatory and/or neuropathic mechanisms may exaggerate interactions between damaged tissues and neural pathways for pain mediation. Similar mechanisms also promote the communication among cellular participants in synapses at spinal or higher levels, which may amplify nociceptive firing and subsequent signal transmission, deteriorating the pain sensation. In this pathology, important cellular players are afferent sensory neurons, peripheral immune cells, and spinal glial cells. Arising from damage of injury, overloaded interstitial and intracellular reactive oxygen species (ROS) and intracellular Ca2+ are key messengers in the development and maintenance of pathologic pain. Thus, an ROS-sensitive and Ca2+-permeable ion channel that is highly expressed in the participant cells might play a critical role in the pathogenesis. Transient receptor potential melastatin subtype 2 (TRPM2) is the unique molecule that satisfies all of the requirements: the sensitivity, permeability, and its expressing cells. Notable progress in delineating the role of TRPM2 in pain has been achieved during the past decade. In the present review, we summarize the important findings in the key cellular components that are involved in pathologic pain. This overview will help to understand TRPM2-mediated pain mechanisms and speculate therapeutic strategies by utilizing this updated information.