Channel properties of the splicing isoforms of the olfactory calcium-activated chloride channel Anoctamin 2
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PROVIDER: S-EPMC4113895 | biostudies-literature | 2014 Aug
REPOSITORIES: biostudies-literature
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Project description:Anoctamin (ANO)2 (or TMEM16B) forms a cell membrane Ca(2+)-activated Cl(-) channel that is present in cilia of olfactory receptor neurons, vomeronasal microvilli, and photoreceptor synaptic terminals. Alternative splicing of Ano2 transcripts generates multiple variants with the olfactory variants skipping exon 14 and having alternative splicing of exon 4. In the present study, 5' rapid amplification of cDNA ends analysis was conducted to characterize the 5' end of olfactory Ano2 transcripts, which showed that the most abundant Ano2 transcripts in the olfactory epithelium contain a novel starting exon that encodes a translation initiation site, whereas transcripts of the publically available sequence variant, which has an alternative and longer 5' end, were present in lower abundance. With two alternative starting exons and alternative splicing of exon 4, four olfactory ANO2 isoforms are thus possible. Patch-clamp experiments in transfected HEK293T cells expressing these isoforms showed that N-terminal sequences affect Ca(2+) sensitivity and that the exon 4-encoded sequence is required to form functional channels. Coexpression of the two predominant isoforms, one with and one without the exon 4 sequence, as well as coexpression of the two rarer isoforms showed alterations in channel properties, indicating that different isoforms interact with each other. Furthermore, channel properties observed from the coexpression of the predominant isoforms better recapitulated the native channel properties, suggesting that the native channel may be composed of two or more splicing isoforms acting as subunits that together shape the channel properties.
| S-EPMC3664704 | biostudies-literature
Project description:Anoctamin-1 (ANO1 or TMEM16A) is a homo-dimeric Ca2+-activated Cl- channel responsible for essential physiological processes. Each monomer harbours a pore and a Ca2+-binding pocket; the voltage-dependent binding of two intracellular Ca2+ ions to the pocket gates the pore. However, in the absence of intracellular Ca2+ voltage activates TMEM16A by an unknown mechanism. Here we show voltage-activated anion currents that are outwardly rectifying, time-independent with fast or absent tail currents that are inhibited by tannic and anthracene-9-carboxylic acids. Since intracellular protons compete with Ca2+ for binding sites in the pocket, we hypothesized that voltage-dependent titration of these sites would induce gating. Indeed intracellular acidification enabled activation of TMEM16A by voltage-dependent protonation, which enhanced the open probability of the channel. Mutating Glu/Asp residues in the Ca2+-binding pocket to glutamine (to resemble a permanent protonated Glu) yielded channels that were easier to activate at physiological pH. Notably, the response of these mutants to intracellular acidification was diminished and became voltage-independent. Thus, voltage-dependent protonation of glutamate/aspartate residues (Glu/Asp) located in the Ca2+-binding pocket underlines TMEM16A activation in the absence of intracellular Ca2+.
| S-EPMC7170896 | biostudies-literature
Project description:Calcium (Ca(2+))-activated chloride (Cl(-)) channels (CaCCs) play a role in the modulation of action potentials and synaptic responses in the somatodendritic regions of central neurons. In the vertebrate retina, large Ca(2+)-activated Cl(-) currents (ICl(Ca)) regulate synaptic transmission at photoreceptor terminals; however, the molecular identity of CaCCs that mediate ICl(Ca) remains unclear. The transmembrane protein, TMEM16A, also called anoctamin 1 (ANO1), has been recently validated as a CaCC and is widely expressed in various secretory epithelia and nervous tissues. Despite the fact that tmem16a was first cloned in the retina, there is little information on its cellular localization and function in the mammalian retina. In this study, we found that ANO1 was abundantly expressed as puncta in 2 synaptic layers. More specifically, ANO1 immunoreactivity was observed in the presynaptic terminals of various retinal neurons, including photoreceptors. ICl(Ca) was first detected in dissociated rod bipolar cells expressing ANO1. ICl(Ca) was abolished by treatment with the Ca(2+) channel blocker Co(2+), the L-type Ca(2+) channel blocker nifedipine, and the Cl(-) channel blockers 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and niflumic acid (NFA). More specifically, a recently discovered ANO1-selective inhibitor, T16Ainh-A01, and a neutralizing antibody against ANO1 inhibited ICl(Ca) in rod bipolar cells. Under a current-clamping mode, the suppression of ICl(Ca) by using NPPB and T16Ainh-A01 caused a prolonged Ca(2+) spike-like depolarization evoked by current injection in dissociated rod bipolar cells. These results suggest that ANO1 confers ICl(Ca) in retinal neurons and acts as an intrinsic regulator of the presynaptic membrane potential during synaptic transmission.
| S-EPMC3693959 | biostudies-literature
Project description:Ca-activated Cl channels are an important component of olfactory transduction. Odor binding to olfactory receptors in the cilia of olfactory sensory neurons (OSNs) leads to an increase of intraciliary Ca concentration by Ca entry through cyclic nucleotide-gated (CNG) channels. Ca activates a Cl channel that leads to an efflux of Cl from the cilia, contributing to the amplification of the OSN depolarization. The molecular identity of this Cl channel remains elusive. Recent evidence has indicated that bestrophins are able to form Ca-activated Cl channels in heterologous systems. Here we have analyzed the expression of bestrophins in the mouse olfactory epithelium and demonstrated that only mouse bestrophin-2 (mBest2) was expressed. Single-cell RT-PCR showed that mBest2 was expressed in OSNs but not in supporting cells. Immunohistochemistry revealed that mBest2 was expressed on the cilia of OSNs, the site of olfactory transduction, and colocalized with the main CNGA2 channel subunit. Electrophysiological properties of Ca-activated Cl currents from native channels in dendritic knob/cilia of mouse OSNs were compared with those induced by the expression of mBest2 in HEK-293 cells. We found the same anion permeability sequence, small estimated single-channel conductances, a Ca sensitivity difference of one order of magnitude, and the same side-specific blockage of the two Cl channel blockers commonly used to inhibit the odorant-induced Ca-activated Cl current in OSNs, niflumic acid, and 4-acetamido-4'-isothiocyanato-stilben-2,2'-disulfonate (SITS). Therefore, our data suggest that mBest2 is a good candidate for being a molecular component of the olfactory Ca-activated Cl channel.
| S-EPMC1568948 | biostudies-literature
Project description:For vertebrate olfactory signal transduction, a calcium-activated chloride conductance serves as a major amplification step. However, the molecular identity of the olfactory calcium-activated chloride channel (CaCC) is unknown. Here we report a proteomic screen for cilial membrane proteins of mouse olfactory sensory neurons (OSNs) that identified all the known olfactory transduction components as well as Anoctamin 2 (ANO2). Ano2 transcripts were expressed specifically in OSNs in the olfactory epithelium, and ANO2::EGFP fusion protein localized to the OSN cilia when expressed in vivo using an adenoviral vector. Patch-clamp analysis revealed that ANO2, when expressed in HEK-293 cells, forms a CaCC and exhibits channel properties closely resembling the native olfactory CaCC. Considering these findings together, we propose that ANO2 constitutes the olfactory calcium-activated chloride channel.
| S-EPMC2702256 | biostudies-literature
Project description:The recently described human anion channel Anoctamin (ANO) protein family comprises at least ten members, many of which have been shown to correspond to calcium-activated chloride channels. To date, the only reported human mutations in this family of genes are dominant mutations in ANO5 (TMEM16E, GDD1) in the rare skeletal disorder gnathodiaphyseal dysplasia. We have identified recessive mutations in ANO5 that result in a proximal limb-girdle muscular dystrophy (LGMD2L) in three French Canadian families and in a distal non-dysferlin Miyoshi myopathy (MMD3) in Dutch and Finnish families. These mutations consist of a splice site, one base pair duplication shared by French Canadian and Dutch cases, and two missense mutations. The splice site and the duplication mutations introduce premature-termination codons and consequently trigger nonsense-mediated mRNA decay, suggesting an underlining loss-of-function mechanism. The LGMD2L phenotype is characterized by proximal weakness, with prominent asymmetrical quadriceps femoris and biceps brachii atrophy. The MMD3 phenotype is associated with distal weakness, of calf muscles in particular. With the use of electron microscopy, multifocal sarcolemmal lesions were observed in both phenotypes. The phenotypic heterogeneity associated with ANO5 mutations is reminiscent of that observed with Dysferlin (DYSF) mutations that can cause both LGMD2B and Miyoshi myopathy (MMD1). In one MMD3-affected individual, defective membrane repair was documented on fibroblasts by membrane-resealing ability assays, as observed in dysferlinopathies. Though the function of the ANO5 protein is still unknown, its putative calcium-activated chloride channel function may lead to important insights into the role of deficient skeletal muscle membrane repair in muscular dystrophies.
| S-EPMC2820170 | biostudies-literature
Project description:Background and purposeHigh-throughput screening of compound libraries using genetically encoded fluorescent biosensors has identified several second-generation. low MW inhibitors of the calcium-activated chloride channel anoctamin 1 (CaCC/Ano1). Here we have (i) examined the effects of these Ano1 inhibitors on gastric and intestinal pacemaker activity; (ii) compared the effects of these inhibitors with those of the more classical CaCC inhibitor, 5-nitro-2-(3-phenylpropylalanine) benzoate (NPPB); (ii) examined the mode of action of these compounds on the waveform of pacemaker activity; and (iii) compared differences in the sensitivity between gastric and intestinal pacemaker activity to the Ano1 inhibitors.Experimental approachUsing intracellular microelectrode recordings of gastric and intestinal muscle preparations from C57BL/6 mice, the dose-dependent effects of Ano1 inhibitors were examined on spontaneous electrical slow waves.Key resultsThe efficacy of second-generation Ano1 inhibitors on gastric and intestinal pacemaker activity differed significantly. Antral slow waves were more sensitive to these inhibitors than intestinal slow waves. CaCCinh -A01 and benzbromarone were the most potent at inhibiting slow waves in both muscle preparations and more potent than NPPB. Dichlorophene and hexachlorophene were equally potent at inhibiting slow waves. Surprisingly, slow waves were relatively insensitive to T16Ainh -A01 in both preparations.Conclusions and implicationsWe have identified several second-generation Ano1 inhibitors, blocking gastric and intestinal pacemaker activity. Different sensitivities to Ano1 inhibitors between stomach and intestine suggest the possibility of different splice variants in these two organs or the involvement of other conductances in the generation and propagation of pacemaker activity in these tissues.
| S-EPMC4940814 | biostudies-literature
Project description:Calcium-activated chloride channels of the anoctamin (alias TMEM16) protein family fulfill critical functions in epithelial fluid transport, smooth muscle contraction and sensory signal processing. Little is known, however, about their contribution to information processing in the central nervous system. Here we examined the recent finding that a calcium-dependent chloride conductance impacts on GABAergic synaptic inhibition in Purkinje cells of the cerebellum. We asked whether anoctamin channels may underlie this chloride conductance. We identified two anoctamin channel proteins, ANO1 and ANO2, in the cerebellar cortex. ANO1 was expressed in inhibitory interneurons of the molecular layer and the granule cell layer. Both channels were expressed in Purkinje cells but, while ANO1 appeared to be retained in the cell body, ANO2 was targeted to the dendritic tree. Functional studies confirmed that ANO2 was involved in a calcium-dependent mode of ionic plasticity that reduces the efficacy of GABAergic synapses. ANO2 channels attenuated GABAergic transmission by increasing the postsynaptic chloride concentration, hence reducing the driving force for chloride influx. Our data suggest that ANO2 channels are involved in a Ca2+-dependent regulation of synaptic weight in GABAergic inhibition. Thus, in balance with the chloride extrusion mechanism via the co-transporter KCC2, ANO2 appears to regulate ionic plasticity in the cerebellum.
| S-EPMC4641602 | biostudies-literature
Project description:ObjectiveTo determine the presence of calcium activated chloride channels anoctamin 1 (ANO1) and 2 (ANO2) in human and murine uterine smooth muscle (MUSM) and evaluate the physiologic role for these ion channels in murine myometrial contractility.Study designWe performed reverse transcription polymerase chain reaction to determine whether ANO1 and 2 are expressed in human and murine uterine tissue to validate the study of this protein in mouse models. Immunohistochemical staining of ANO1 and 2 was then performed to determine protein expression in murine myometrial tissue. The function of ANO1 and 2 in murine uterine tissue was evaluated using electrophysiologic studies, organ bath, and calcium flux experiments.ResultsANO1 and 2 are expressed in human and MUSM cells. Functional studies show that selective antagonism of these channels promotes relaxation of spontaneous MUSM contractions. Blockade of ANO1 and 2 inhibits both agonist-induced and spontaneous transient inward currents and abolishes G-protein coupled receptor (oxytocin) mediated elevations in intracellular calcium.ConclusionThe calcium activated chloride channels ANO1 and 2 are present in human and murine myometrial tissue and may provide novel potential therapeutic targets to achieve effective tocolysis.
| S-EPMC4253652 | biostudies-literature
Project description:The newly discovered Ca(2+)-activated Cl(-) channel (CaCC), Anoctamin 1 (Ano1 or TMEM16A), has been implicated in vital physiological functions including epithelial fluid secretion, gut motility, and smooth muscle tone. Overexpression of Ano1 in HEK cells or Xenopus oocytes is sufficient to generate Ca(2+)-activated Cl(-) currents, but the details of channel composition and the regulatory factors that control channel biology are incompletely understood. We used a highly sensitive quantitative SILAC proteomics approach to obtain insights into stoichiometric protein networks associated with the Ano1 channel. These studies provide a comprehensive footprint of putative Ano1 regulatory networks. We find that Ano1 associates with the signaling/scaffolding proteins ezrin, radixin, moesin, and RhoA, which link the plasma membrane to the cytoskeleton with very high stoichiometry. Ano1, ezrin, and moesin/radixin colocalize apically in salivary gland epithelial cells, and overexpression of moesin and Ano1 in HEK cells alters the subcellular localization of both proteins. Moreover, interfering RNA for moesin modifies Ano1 current without affecting its surface expression level. Another network associated with Ano1 includes the SNARE and SM proteins VAMP3, syntaxins 2 and -4, and syntaxin-binding proteins munc18b and munc18c, which are integral to translocation of vesicles to the plasma membrane. A number of other regulatory proteins, including GTPases, Ca(2+)-binding proteins, kinases, and lipid-interacting proteins are enriched in the Ano1 complex. These data provide stoichiometrically prioritized information about mechanisms regulating Ano1 function and trafficking to polarized domains of the plasma membrane.
| S-EPMC3387097 | biostudies-literature