Tentonin 3 is a pore-forming subunit of a slow inactivation mechanosensitive channel.

Mechanically activating (MA) channels transduce numerous physiological functions. Tentonin 3/TMEM150C (TTN3) confers MA currents with slow inactivation kinetics in somato- and barosensory neurons. However, questions were raised about its role as a Piezo1 regulator and its potential as a channel pore. Here, we demonstrate that purified TTN3 proteins incorporated into the lipid bilayer displayed spontaneous and pressure-sensitive channel currents. These MA currents were conserved across vertebrates and differ from Piezo1 in activation threshold and pharmacological response. Deep neural network structure prediction programs coupled with mutagenetic analysis predicted a rectangular-shaped, tetrameric structure with six transmembrane helices and a pore at the inter-subunit center. The putative pore aligned with two helices of each subunit and had constriction sites whose mutations changed the MA currents. These findings suggest that TTN3 is a pore-forming subunit of a distinct slow inactivation MA channel, potentially possessing a tetrameric structure.

Amplification of olfactory signals by Anoctamin 9 is important for mammalian olfaction.

Sensing smells of foods, prey, or predators determines animal survival. Olfactory sensory neurons in the olfactory epithelium (OE) detect odorants, where cAMP and Ca2+ play a significant role in transducing odorant inputs to electrical activity. Here we show Anoctamin 9, a cation channel activated by cAMP/PKA pathway, is expressed in the OE and amplifies olfactory signals. Ano9-deficient mice had reduced olfactory behavioral sensitivity, electro-olfactogram signals, and neural activity in the olfactory bulb. In line with the difference in olfaction between birds and other vertebrates, chick ANO9 failed to respond to odorants, whereas chick CNGA2, a major transduction channel, showed greater responses to cAMP. Thus, we concluded that the signal amplification by ANO9 is important for mammalian olfactory transduction.

Lotus Seed Green Embryo Extract and a Purified Glycosyloxyflavone Constituent, Narcissoside, Activate TRPV1 Channels in Dorsal Root Ganglion Sensory Neurons.

Several studies have documented the broad-spectrum bioactivities of a lotus seed (Plumula nelumbinis [PN]) green embryo extract. However, the specific bioactive components and associated molecular mechanisms remain largely unknown. This study aimed to identify the ion channel-activating mechanisms of PN extracts. Using fluorometric imaging and patch-clamp recordings, PN extracts were screened for calcium channel activation in dorsal root ganglion (DRG) neurons. The TRPV1 channels in DRG neurons were strongly activated by the PN extract (mean amplitude of 131 ± 45 pA at 200 µg/mL) and its purified glycosyloxyflavone narcissoside (401 ± 271 pA at 100 µM). Serial treatment with a 200 µg/mL PN extract in TRPV1-overexpressing HEK293T cells induced robust desensitization to 10 ± 10% of the initial current amplitude. Thus, we propose that the PN extract and narcissoside function as TRPV1 agonists. This new finding may advance our knowledge regarding the traditional and scientific functions of PN in human health and disease.

Anoctamin 1/TMEM16A in pruritoceptors is essential for Mas-related G protein receptor-dependent itch.

ABSTRACT: Itch is an unpleasant sensation that evokes a desire to scratch. Pathologic conditions such as allergy or atopic dermatitis produce severe itching sensation. Mas-related G protein receptors (Mrgprs) are receptors for many endogenous pruritogens. However, signaling pathways downstream to these receptors in dorsal root ganglion (DRG) neurons are not yet understood. We found that anoctamin 1 (ANO1), a Ca 2+ -activated chloride channel, is a transduction channel mediating Mrgpr-dependent itch signals. Genetic ablation of Ano1 in DRG neurons displayed a significant reduction in scratching behaviors in response to acute and chronic Mrgpr-dependent itch models and the epidermal hyperplasia induced by dry skin. In vivo Ca 2+ imaging and electrophysiological recording revealed that chloroquine and other agonists of Mrgprs excited DRG neurons via ANO1. More importantly, the overexpression of Ano1 in DRG neurons of Ano1 -deficient mice rescued the impaired itching observed in Ano1 -deficient mice. These results demonstrate that ANO1 mediates the Mrgpr-dependent itch signaling in pruriceptors and provides clues to treating pathologic itch syndromes.

Tentonin 3/TMEM150C regulates glucose-stimulated insulin secretion in pancreatic ß-cells.

Glucose homeostasis is initially regulated by the pancreatic hormone insulin. Glucose-stimulated insulin secretion in ß-cells is composed of two cellular mechanisms: a high glucose concentration not only depolarizes the membrane potential of the ß-cells by ATP-sensitive K+ channels but also induces cell inflation, which is sufficient to release insulin granules. However, the molecular identity of the stretch-activated cation channel responsible for the latter pathway remains unknown. Here, we demonstrate that Tentonin 3/TMEM150C (TTN3), a mechanosensitive channel, contributes to glucose-stimulated insulin secretion by mediating cation influx. TTN3 is expressed specifically in ß-cells and mediates cation currents to glucose and hypotonic stimulations. The glucose-induced depolarization, firing activity, and Ca2+ influx of ß-cells were significantly lower in Ttn3-/- mice. More importantly, Ttn3-/- mice show impaired glucose tolerance with decreased insulin secretion in vivo. We propose that TTN3, as a stretch-activated cation channel, contributes to glucose-stimulated insulin secretion.

Tentonin 3/TMEM150C senses blood pressure changes in the aortic arch.

The baroreceptor reflex is a powerful neural feedback that regulates arterial pressure (AP). Mechanosensitive channels transduce pulsatile AP to electrical signals in baroreceptors. Here we show that tentonin 3 (TTN3/TMEM150C), a cation channel activated by mechanical strokes, is essential for detecting AP changes in the aortic arch. TTN3 was expressed in nerve terminals in the aortic arch and nodose ganglion (NG) neurons. Genetic ablation of Ttn3 induced ambient hypertension, tachycardia, AP fluctuations, and impaired baroreflex sensitivity. Chemogenetic silencing or activation of Ttn3+ neurons in the NG resulted in an increase in AP and heart rate, or vice versa. More important, overexpression of Ttn3 in the NG of Ttn3-/- mice reversed the cardiovascular changes observed in Ttn3-/- mice. We conclude that TTN3 is a molecular component contributing to the sensing of dynamic AP changes in baroreceptors.

TMEM16A expression in cholinergic neurons of the medial habenula mediates anxiety-related behaviors.

TMEM16A, a Ca2+ -activated Cl- channel, is known to modulate the excitability of various types of cells; however, its function in central neurons is largely unknown. Here, we show the specific expression of TMEM16A in the medial habenula (mHb) via RNAscope in situ hybridization, immunohistochemistry, and electrophysiology. When TMEM16A is ablated in the mHb cholinergic neurons (TMEM16A cKO mice), the slope of after-hyperpolarization of spontaneous action potentials decreases and the firing frequency is reduced. Reduced mHb activity also decreases the activity of the interpeduncular nucleus (IPN). Moreover, TMEM16A cKO mice display anxiogenic behaviors and deficits in social interaction without despair-like phenotypes or cognitive dysfunctions. Finally, chemogenetic inhibition of mHb cholinergic neurons using the DREADD (Designer Receptors Exclusively Activated by Designer Drugs) approach reveals similar behavioral phenotypes to those of TMEM16A cKO mice. We conclude that TMEM16A plays a key role in anxiety-related behaviors regulated by mHb cholinergic neurons and could be a potential therapeutic target against anxiety-related disorders.

Anoctamin 1/TMEM16A controls intestinal Cl- secretion induced by carbachol and cholera toxin.

Calcium-activated chloride channels (CaCCs) mediate numerous physiological functions and are best known for the transport of electrolytes and water in epithelia. In the intestine, CaCC currents are considered necessary for the secretion of fluid to protect the intestinal epithelium. Although genetic ablation of ANO1/TMEM16A, a gene encoding a CaCC, reduces the carbachol-induced secretion of intestinal fluid, its mechanism of action is still unknown. Here, we confirm that ANO1 is essential for the secretion of intestinal fluid. Carbachol-induced transepithelial currents were reduced in the proximal colon of Ano1-deficient mice. Surprisingly, cholera toxin-induced and cAMP-induced fluid secretion, believed to be mediated by CFTR, were also significantly reduced in the intestine of Ano1-deficient mice. ANO1 is largely expressed in the apical membranes of intestines, as predicted for CaCCs. The Ano1-deficient colons became edematous under basal conditions and had a greater susceptibility to dextran sodium sulfate-induced colitis. However, Ano1 depletion failed to affect tumor development in a model of colorectal cancer. We thus conclude that ANO1 is necessary for cAMP- and carbachol-induced Cl- secretion in the intestine, which is essential for the protection of the intestinal epithelium from colitis.

ANO1/TMEM16A regulates process maturation in radial glial cells in the developing brain.

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.

Korea Brain Initiative: Emerging Issues and Institutionalization of Neuroethics.

Neuroscience research has become a national priority for the Korean government. Korean scholars have dedicated interest in the societal ramifications of neurotechnologies; neuroethics is an integral component of the Korea Brain Initiative and to the formation of its growing neuroscience community.

EF-hand like Region in the N-terminus of Anoctamin 1 Modulates Channel Activity by Ca2+ and Voltage.

Anoctamin1 (ANO1) also known as TMEM16A is a transmembrane protein that functions as a Ca2+ activated chloride channel. Recently, the structure determination of a fungal Nectria haematococca TMEM16 (nhTMEM16) scramblase by X-ray crystallography and a mouse ANO1 by cryo-electron microscopy has provided the insight in molecular architecture underlying phospholipid scrambling and Ca2+ binding. Because the Ca2+ binding motif is embedded inside channel protein according to defined structure, it is still unclear how intracellular Ca2+ moves to its deep binding pocket effectively. Here we show that EF-hand like region containing multiple acidic amino acids at the N-terminus of ANO1 is a putative site regulating the activity of ANO1 by Ca2+ and voltage. The EF-hand like region of ANO1 is highly homologous to the canonical EF hand loop in calmodulin that contains acidic residues in key Ca2+-coordinating positions in the canonical EF hand. Indeed, deletion and Ala-substituted mutation of this region resulted in a significant reduction in the response to Ca2+ and changes in its key biophysical properties evoked by voltage pulses. Furthermore, only ANO1 and ANO2, and not the other TMEM16 isoforms, contain the EF-hand like region and are activated by Ca2+. Moreover, the molecular modeling analysis supports that EF-hand like region could play a key role during Ca2+ transfer. Therefore, these findings suggest that EF-hand like region in ANO1 coordinates with Ca2+ and modulate the activation by Ca2+ and voltage.

Trpm2 Ablation Accelerates Protein Aggregation by Impaired ADPR and Autophagic Clearance in the Brain.

TRPM2 a cation channel is also known to work as an enzyme that hydrolyzes highly reactive, neurotoxic ADP-ribose (ADPR). Although ADPR is hydrolyzed by NUT9 pyrophosphatase in major organs, the enzyme is defective in the brain. The present study questions the role of TRPM2 in the catabolism of ADPR in the brain. Genetic ablation of Trpm2 results in the disruption of ADPR catabolism that leads to the accumulation of ADPR and reduction in AMP. Trpm2-/- mice elicit the reduction in autophagosome formation in the hippocampus. Trpm2-/- mice also show aggregations of proteins in the hippocampus, aberrant structural changes and neuronal connections in synapses, and neuronal degeneration. Trpm2-/- mice exhibit learning and memory impairment, enhanced neuronal intrinsic excitability, and imbalanced synaptic transmission. These results respond to long-unanswered questions regarding the potential role of the enzymatic function of TRPM2 in the brain, whose dysfunction evokes protein aggregation. In addition, the present finding answers to the conflicting reports such as neuroprotective or neurodegenerative phenotypes observed in Trpm2-/- mice.

Anoctamin 9/TMEM16J is a cation channel activated by cAMP/PKA signal.

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.

Evidence for Mechanosensitive Channel Activity of Tentonin 3/TMEM150C.

Mechanosensation is essential for various physiological processes, and it is mediated by mechanotransduction channels. Recently, we reported that TMEM150C/Tentonin 3 (TTN3) confers mechanically activated currents with slow inactivation kinetics in several cell types, including dorsal root ganglion neurons (Hong et al., 2016). The accompanying Matters Arising by Dubin, Murthy, and colleagues confirms that naive heterologous cells demonstrate a mechanically activated current, but finds that this response is absent in CRISPR-Cas9 Piezo1 knockout cell lines and suggests that TTN3 is a modulator of Piezo1. We present and discuss evidence based on co-expression of TTN3 and Peizo1 and mutant variants of the pore region of TTN3 to support that TTN3 is a pore-forming unit, not an amplifying adaptor for Piezo1 activity. This Matters Arising Response paper, along with Zhao et al. (2017), addresses the Matters Arising from Dubin et al. (2017), published concurrently in this issue of Neuron.

Conditional genetic deletion of Ano1 in interstitial cells of Cajal impairs Ca2+ transients and slow waves in adult mouse small intestine.

Myenteric plexus interstitial cells of Cajal (ICC-MY) in the small intestine are Kit+ electrical pacemakers that express the Ano1/TMEM16A Ca2+-activated Cl- channel, whose functions in the gastrointestinal tract remain incompletely understood. In this study, an inducible Cre-LoxP-based approach was used to advance the understanding of Ano1 in ICC-MY of adult mouse small intestine. KitCreERT2/+;Ano1Fl/Fl mice were treated with tamoxifen or vehicle, and small intestines (mucosa free) were examined. Quantitative RT-PCR demonstrated ~50% reduction in Ano1 mRNA in intestines of conditional knockouts (cKOs) compared with vehicle-treated controls. Whole mount immunohistochemistry showed a mosaic/patchy pattern loss of Ano1 protein in ICC networks. Ca2+ transients in ICC-MY network of cKOs displayed reduced duration compared with highly synchronized controls and showed synchronized and desynchronized profiles. When matched, the rank order for Ano1 expression in Ca2+ signal imaged fields of view was as follows: vehicle controls>>>cKO(synchronized)>cKO(desynchronized). Maintenance of Ca2+ transients' synchronicity despite high loss of Ano1 indicates a large functional reserve of Ano1 in the ICC-MY network. Slow waves in cKOs displayed reduced duration and increased inter-slow-wave interval and occurred in regular- and irregular-amplitude oscillating patterns. The latter activity suggested ongoing interaction by independent interacting oscillators. Lack of slow waves and depolarization, previously reported for neonatal constitutive knockouts, were also seen. In summary, Ano1 in adults regulates gastrointestinal function by determining Ca2+ transients and electrical activity depending on the level of Ano1 expression. Partial Ano1 loss results in Ca2+ transients and slow waves displaying reduced duration, while complete and widespread absence of Ano1 in ICC-MY causes lack of slow wave and desynchronized Ca2+ transients.NEW & NOTEWORTHY The Ca2+-activated Cl- channel, Ano1, in interstitial cells of Cajal (ICC) is necessary for normal gastrointestinal motility. We knocked out Ano1 to varying degrees in ICC of adult mice. Partial knockout of Ano1 shortened the widths of electrical slow waves and Ca2+ transients in myenteric ICC but Ca2+ transient synchronicity was preserved. Near-complete knockout was necessary for transient desynchronization and loss of slow waves, indicating a large functional reserve of Ano1 in ICC.

Tentonin 3/TMEM150c Confers Distinct Mechanosensitive Currents in Dorsal-Root Ganglion Neurons with Proprioceptive Function.

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.

Cellular functions of TMEM16/anoctamin.

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.

TRPM2, a Susceptibility Gene for Bipolar Disorder, Regulates Glycogen Synthase Kinase-3 Activity in the Brain.

Bipolar disorder (BD) is a psychiatric disease that causes mood swings between manic and depressed states. Although genetic linkage studies have shown an association between BD and TRPM2, a Ca(2+)-permeable cation channel, the nature of this association is unknown. Here, we show that D543E, a mutation of Trpm2 that is frequently found in BD patients, induces loss of function. Trpm2-deficient mice exhibited BD-related behavior such as increased anxiety and decreased social responses, along with disrupted EEG functional connectivity. Moreover, the administration of amphetamine in wild-type mice evoked a notable increase in open-field activity that was reversed by the administration of lithium. However, the anti-manic action of lithium was not observed in the Trpm2(-/-) mice. The brains of Trpm2(-/-) mice showed a marked increase in phosphorylated glycogen synthase kinase-3 (GSK-3), a key element in BD-like behavior and a target of lithium. In contrast, activation of TRPM2 induced the dephosphorylation of GSK-3 via calcineurin, a Ca(2+)-dependent phosphatase. Importantly, the overexpression of the D543E mutant failed to induce the dephosphorylation of GSK-3. Therefore, we conclude that the genetic dysfunction of Trpm2 causes uncontrolled phosphorylation of GSK-3, which may lead to the pathology of BD. Our findings explain the long-sought etiologic mechanism underlying the genetic link between Trpm2 mutation and BD. SIGNIFICANCE STATEMENT: Bipolar disorder (BD) is a mental disorder that causes changes in mood and the etiology is still unknown. TRPM2 is highly associated with BD; however, its involvement in the etiology of BD is still unknown. We show here that TRPM2 plays a central role in causing the pathology of BD. We found that D543E, a mutation of Trpm2 frequently found in BD patients, induces the loss of function. Trpm2-deficient mice exhibited mood disturbances and impairments in social cognition. TRPM2 actively regulates the phosphorylation of GSK-3, which is a main target of lithium, a primary medicine for treating BD. Therefore, abnormal regulation of GSK-3 by hypoactive TRPM2 mutants accounts for the pathology of BD, providing the possible link between BD and TRPM2.

Protons inhibit anoctamin 1 by competing with calcium.

Cl(-) efflux through Ca(2+)-activated Cl(-) channels (CaCCs) in secretory epithelial cells plays a key role in the regulation of fluid secretion. The fluid and electrolyte secretion is closely related to intracellular pH. CaCCs have been known to be inhibited by intracellular acid. However, the molecular mechanism for the inhibition remains unknown. Anoctamin 1 (ANO1) is a Ca(2+)-activated Cl(-) channel that mediates numerous physiological functions including fluid secretion in secretory epithelia. However, little is known about whether ANO1 can be modulated by change of intracellular pH. Here, we demonstrate that Ca(2+)-induced activation of ANO1 and its homolog ANO2 are strongly inhibited by intracellular acid. Intracellular acid caused a rightward shift of the concentration-response curve of Ca(2+) in activating ANO1 and ANO2. To identify the location of the acid-induced inhibition, mutations were made on each of all histidine residues in cytoplasmic part of ANO1. However, none of the His-mutant showed the reduction in the acid-induced inhibition. Furthermore, mutation on Glu- or Asp-residues in the multiple acidic-amino acid regions was ineffective in blocking the acid-induced inhibition. Because the Ca(2+)-binding site of a fungal anoctamin (nhTMEM16) was uncovered by crystallography, mutagenesis was performed in this region. Surprisingly, mutations at Glu, Asp or Asn residues in the hydrophobic core that are known to be essential for Ca(2+)-induced activation of ANO1 blocked the acid-induced inhibition. These results suggest that protons interfere with Ca(2+) at the Ca(2+) binding site of ANO1. These findings provide a molecular mechanism underlying the acid-induced inhibition of ANO1, which may contribute to control fluid and electrolyte secretion in the secretory epithelia.