Paradoxical electro-olfactogram responses in TMEM16B knock-out mice.

The Ca2+-activated Cl¯ channel TMEM16B carries up to 90% of the transduction current evoked by odorant stimulation in olfactory sensory neurons and control the number of action potential firing and therefore the length of the train of action potentials. A loss of function approach revealed that TMEM16B is required for olfactory-driven behaviors such as tracking unfamiliar odors. Here, we used the electro-olfactogram (EOG) technique to investigate the contribution of TMEM16B to odorant transduction in the whole olfactory epithelium. Surprisingly, we found that EOG responses from Tmem16b knock out mice have a bigger amplitude compared to those of wild type. Moreover, the kinetics of EOG responses is faster in absence of TMEM16B, while the ability to adapt to repeated stimulation is altered in knock out mice. The larger EOG responses in Tmem16b knock out may be the results of the removal of the clamping and/or shunting action of the Ca2+-activated Cl¯ currents leading to the paradox of having smaller transduction current but larger generator potential.

Molecular mimicry between Anoctamin 2 and Epstein-Barr virus nuclear antigen 1 associates with multiple sclerosis risk.

Multiple sclerosis (MS) is a chronic inflammatory, likely autoimmune disease of the central nervous system with a combination of genetic and environmental risk factors, among which Epstein-Barr virus (EBV) infection is a strong suspect. We have previously identified increased autoantibody levels toward the chloride-channel protein Anoctamin 2 (ANO2) in MS. Here, IgG antibody reactivity toward ANO2 and EBV nuclear antigen 1 (EBNA1) was measured using bead-based multiplex serology in plasma samples from 8,746 MS cases and 7,228 controls. We detected increased anti-ANO2 antibody levels in MS (P = 3.5 × 10-36) with 14.6% of cases and 7.8% of controls being ANO2 seropositive (odds ratio [OR] = 1.6; 95% confidence intervals [95%CI]: 1.5 to 1.8). The MS risk increase in ANO2-seropositive individuals was dramatic when also exposed to 3 known risk factors for MS: HLA-DRB1*15:01 carriage, absence of HLA-A*02:01, and high anti-EBNA1 antibody levels (OR = 24.9; 95%CI: 17.9 to 34.8). Reciprocal blocking experiments with ANO2 and EBNA1 peptides demonstrated antibody cross-reactivity, mapping to ANO2 [aa 140 to 149] and EBNA1 [aa 431 to 440]. HLA gene region was associated with anti-ANO2 antibody levels and HLA-DRB1*04:01 haplotype was negatively associated with ANO2 seropositivity (OR = 0.6; 95%CI: 0.5 to 0.7). Anti-ANO2 antibody levels were not increased in patients from 3 other inflammatory disease cohorts. The HLA influence and the fact that specific IgG production usually needs T cell help provides indirect evidence for a T cell ANO2 autoreactivity in MS. We propose a hypothesis where immune reactivity toward EBNA1 through molecular mimicry with ANO2 contributes to the etiopathogenesis of MS.

VI-116, A Novel Potent Inhibitor of VRAC with Minimal Effect on ANO1.

Volume-regulated anion channel (VRAC) is ubiquitously expressed and plays a pivotal role in vertebrate cell volume regulation. A heterologous complex of leucine-rich repeat containing 8A (LRRC8A) and LRRC8B-E constitutes the VRAC, which is involved in various processes such as cell proliferation, migration, differentiation, intercellular communication, and apoptosis. However, the lack of a potent and selective inhibitor of VRAC limits VRAC-related physiological and pathophysiological studies, and most previous VRAC inhibitors strongly blocked the calcium-activated chloride channel, anoctamin 1 (ANO1). In the present study, we performed a cell-based screening for the identification of potent and selective VRAC inhibitors. Screening of 55,000 drug-like small-molecules and subsequent chemical modification revealed 3,3'-((2-hydroxy-3-methoxyphenyl)methylene)bis(4-hydroxy-2H-chromen-2-one) (VI-116), a novel potent inhibitor of VRAC. VI-116 fully inhibited VRAC-mediated I- quenching with an IC50 of 1.27 ± 0.18 µM in LN215 cells and potently blocked endogenous VRAC activity in PC3, HT29 and HeLa cells in a dose-dependent manner. Notably, VI-116 had no effect on intracellular calcium signaling up to 10 µM, which completely inhibited VRAC, and showed high selectivity for VRAC compared to ANO1 and ANO2. However, DCPIB, a VRAC inhibitor, significantly affected ATP-induced increases in intracellular calcium levels and Eact-induced ANO1 activation. In addition, VI-116 showed minimal effect on hERG K+ channel activity up to 10 µM. These results indicate that VI-116 is a potent and selective VRAC inhibitor and a useful research tool for pharmacological dissection of VRAC.

TMEM16B Calcium-Activated Chloride Channels Regulate Action Potential Firing in Lateral Septum and Aggression in Male Mice.

The lateral septum (LS) plays an important role in regulating aggression. It is well recognized that LS lesions lead to a dramatic increase in aggressive behaviors. A better understanding of LS neurophysiology and its functional output is therefore important to assess LS involvement in regulating aggression. The LS is a heterogeneous structure that maintains inputs and outputs with multiple brain regions, and is also divided into subregions that innervate one another. Thus, it is challenging to identify the exact cell type and projections for characterization. In this study, we determined the expression pattern of the calcium-activated chloride channel, TMEM16B, in the LS of both male and female mice. We then investigated the physiological contribution of the calcium-activated chloride channel to LS neuronal signaling. By performing whole-cell patch-clamp recording, we showed that TMEM16B alters neurotransmitter release at the hippocampal-LS synapse, and regulates spike frequency and spike frequency adaptation in subpopulations of LS neurons. We further demonstrated that loss of TMEM16B function promotes lengthened displays of aggressive behaviors by male mice during the resident intruder paradigm. In conclusion, our findings suggest that TMEM16B function contributes to neuronal excitability in subpopulations of LS neurons and the regulation of aggression in male mice.SIGNIFICANCE STATEMENT Aggression is a behavior that arose evolutionarily from the necessity to compete for limited resources and survival. One particular brain region involved in aggression is the lateral septum (LS). In this study, we characterized the expression of the TMEM16B calcium-activated chloride channel in the LS and showed that TMEM16B regulates the action potential firing frequency of LS neurons. We discovered that loss of TMEM16B function lengthens the displays of aggressive behaviors in male mice. These findings suggest that TMEM16B plays an important role in regulating LS neuronal excitability and behaviors associated with LS function, thereby contributing to our understanding of how the LS may regulate aggression.

Anoctamin2 (TMEM16B) forms the Ca2+-activated Cl- channel in the retinal pigment epithelium.

Chloride channels (Cl channels) play an essential role for the retinal pigment epithelium (RPE). They provide a plasma membrane conductance for Cl- important for transepithelial transport and volume regulation. Ca2+-dependent chloride channels (CaCC) in the RPE were found to adapt Cl- transport to specific needs by increasing intracellular free Ca2+. Although a variety of Cl channels have been identified in the RPE, the molecular identity of the CaCC remains controversial. Sagittal sections of mouse retina were stained against anoctamin2 (Ano2) and analyzed by confocal microscopy. Membrane currents from ARPE-19 cells and primary murine RPE cells were recorded in the whole-cell configuration of the patch-clamp technique. Expression of Ano2 was assessed via immunocytochemistry, PCR and western-blot and down-regulated via siRNA approaches. In the mouse retina, Ano2 was found in the basolateral membrane of the RPE. In primary mouse RPE cells, Ano2 was localized predominantly in the cell membrane. Ano2 mRNA and protein were also detected in rat and primate RPE as well as ARPE-19 cells. Whole-cell currents were elicited by increasing intracellular free Ca2+ via ATP application. These currents were identified as Cl- currents by their reversal potential and blocker sensitivity. Knock-down of Ano2 by siRNA decreased both the Ca2+ dependent chloride conductance and protein expression of Ano2. The biophysical and pharmacological properties of CaCC in ARPE-19 and primary mouse RPE cells resemble those described in previous publications using RPE cells from different species. The siRNA knock-down suggests that Ano2 contributes to Ca2+-dependent chloride conductance in the RPE.

Multiple effects of anthracene-9-carboxylic acid on the TMEM16B/anoctamin2 calcium-activated chloride channel.

Ca(2+)-activated Cl(-) currents (CaCCs) play important roles in many physiological processes. Recent studies have shown that TMEM16A/anoctamin1 and TMEM16B/anoctamin2 constitute CaCCs in several cell types. Here we have investigated for the first time the extracellular effects of the Cl(-) channel blocker anthracene-9-carboxylic acid (A9C) and of its non-charged analogue anthracene-9-methanol (A9M) on TMEM16B expressed in HEK 293T cells, using the whole-cell patch-clamp technique. A9C caused a voltage-dependent block of outward currents and inhibited a larger fraction of the current as depolarization increased, whereas the non-charged A9M produced a small, not voltage dependent block of outward currents. A similar voltage-dependent block by A9C was measured both when TMEM16B was activated by 1.5 and 13µM Ca(2+). However, in the presence of 1.5µM Ca(2+) (but not in 13µM Ca(2+)), A9C also induced a strong potentiation of tail currents measured at -100mV after depolarizing voltages, as well as a prolongation of the deactivation kinetics. On the contrary, A9M did not produce potentiation of tail currents, showing that the negative charge is required for potentiation. Our results provide the first evidence that A9C has multiple effects on TMEM16B and that the negative charge of A9C is necessary both for voltage-dependent block and for potentiation. Future studies are required to identify the molecular mechanisms underlying these complex effects of A9C on TMEM16B. Understanding these mechanisms will contribute to the elucidation of the structure and functional properties of TMEM16B channels.

Expression of calcium-activated chloride channels Ano1 and Ano2 in mouse taste cells.

Specialized Ca(2+)-dependent ion channels ubiquitously couple intracellular Ca(2+) signals to a change in cell polarization. The existing physiological evidence suggests that Ca(2+)-activated Cl(-) channels (CaCCs) are functional in taste cells. Because Ano1 and Ano2 encode channel proteins that form CaCCs in a variety of cells, we analyzed their expression in mouse taste cells. Transcripts for Ano1 and Ano2 were detected in circumvallate (CV) papillae, and their expression in taste cells was confirmed using immunohistochemistry. When dialyzed with CsCl, taste cells of the type III exhibited no ion currents dependent on cytosolic Ca(2+). Large Ca(2+)-gated currents mediated by TRPM5 were elicited in type II cells by Ca(2+) uncaging. When TRPM5 was inhibited by triphenylphosphine oxide (TPPO), ionomycin stimulated a small but resolvable inward current that was eliminated by anion channel blockers, including T16Ainh-A01 (T16), a specific Ano1 antagonist. This suggests that CaCCs, including Ano1-like channels, are functional in type II cells. In type I cells, CaCCs were prominently active, blockable with the CaCC antagonist CaCCinh-A01 but insensitive to T16. By profiling Ano1 and Ano2 expressions in individual taste cells, we revealed Ano1 transcripts in type II cells only, while Ano2 transcripts were detected in both type I and type II cells. P2Y agonists stimulated Ca(2+)-gated Cl(-) currents in type I cells. Thus, CaCCs, possibly formed by Ano2, serve as effectors downstream of P2Y receptors in type I cells. While the role for TRPM5 in taste transduction is well established, the physiological significance of expression of CaCCs in type II cells remains to be elucidated.

Neurotransmitter map of the asymmetric dorsal habenular nuclei of zebrafish.

The role of the habenular nuclei in modulating fear and reward pathways has sparked a renewed interest in this conserved forebrain region. The bilaterally paired habenular nuclei, each consisting of a medial/dorsal and lateral/ventral nucleus, can be further divided into discrete subdomains whose neuronal populations, precise connectivity, and specific functions are not well understood. An added complexity is that the left and right habenulae show pronounced morphological differences in many non-mammalian species. Notably, the dorsal habenulae of larval zebrafish provide a vertebrate genetic model to probe the development and functional significance of brain asymmetry. Previous reports have described a number of genes that are expressed in the zebrafish habenulae, either in bilaterally symmetric patterns or more extensively on one side of the brain than the other. The goal of our study was to generate a comprehensive map of the zebrafish dorsal habenular nuclei, by delineating the relationship between gene expression domains, comparing the extent of left-right asymmetry at larval and adult stages, and identifying potentially functional subnuclear regions as defined by neurotransmitter phenotype. Although many aspects of habenular organization appear conserved with rodents, the zebrafish habenulae also possess unique properties that may underlie lateralization of their functions.

TMEM16B regulates anxiety-related behavior and GABAergic neuronal signaling in the central lateral amygdala.

TMEM16B (ANO2) is the Ca2+-activated chloride channel expressed in multiple brain regions, including the amygdala. Here we report that Ano2 knockout mice exhibit impaired anxiety-related behaviors and context-independent fear memory, thus implicating TMEM16B in anxiety modulation. We found that TMEM16B is expressed in somatostatin-positive (SOM+) GABAergic neurons of the central lateral amygdala (CeL), and its activity modulates action potential duration and inhibitory postsynaptic current (IPSC). We further provide evidence for TMEM16B actions not only in the soma but also in the presynaptic nerve terminals of GABAergic neurons. Our study reveals an intriguing role for TMEM16B in context-independent but not context-dependent fear memory, and supports the notion that dysfunction of the amygdala contributes to anxiety-related behaviors.

Inferior Olivary TMEM16B Mediates Cerebellar Motor Learning.

Ca2+-activated ion channels shape membrane excitability and Ca2+ dynamics in response to cytoplasmic Ca2+ elevation. Compared to the Ca2+-activated K+ channels, known as BK and SK channels, the physiological importance of Ca2+-activated Cl- channels (CaCCs) in neurons has been largely overlooked. Here we report that CaCCs coexist with BK and SK channels in inferior olivary (IO) neurons that send climbing fibers to innervate cerebellar Purkinje cells for the control of motor learning and timing. Ca2+ influx through the dendritic high-threshold voltage-gated Ca2+ channels activates CaCCs, which contribute to membrane repolarization of IO neurons. Loss of TMEM16B expression resulted in the absence of CaCCs in IO neurons, leading to markedly diminished action potential firing of IO neurons in TMEM16B knockout mice. Moreover, these mutant mice exhibited severe cerebellar motor learning deficits. Our findings thus advance the understanding of the neurophysiology of CaCCs and the ionic basis of IO neuron excitability.

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.

Developmental expression of the calcium-activated chloride channels TMEM16A and TMEM16B in the mouse olfactory epithelium.

Calcium-activated chloride channels are involved in several physiological processes including olfactory perception. TMEM16A and TMEM16B, members of the transmembrane protein 16 family (TMEM16), are responsible for calcium-activated chloride currents in several cells. Both are present in the olfactory epithelium of adult mice, but little is known about their expression during embryonic development. Using immunohistochemistry we studied their expression in the mouse olfactory epithelium at various stages of prenatal development from embryonic day (E) 12.5 to E18.5 as well as in postnatal mice. At E12.5, TMEM16A immunoreactivity was present at the apical surface of the entire olfactory epithelium, but from E16.5 became restricted to a region near the transition zone with the respiratory epithelium, where localized at the apical part of supporting cells and in their microvilli. In contrast, TMEM16B immunoreactivity was present at E14.5 at the apical surface of the entire olfactory epithelium, increased in subsequent days, and localized to the cilia of mature olfactory sensory neurons. These data suggest different functional roles for TMEM16A and TMEM16B in the developing as well as in the postnatal olfactory epithelium. The presence of TMEM16A at the apical part and in microvilli of supporting cells is consistent with a role in the regulation of the chloride ionic composition of the mucus covering the apical surface of the olfactory epithelium, whereas the localization of TMEM16B to the cilia of mature olfactory sensory neurons is consistent with a role in olfactory signal transduction.