Intracellular Loop in the Brain Isoforms of Anoctamin 2 Channels Regulates Calcium-dependent Activation

이동수,Lim Hocheol,Lee Jungryun,하고은,No Kyoung Tai,정은지 한국뇌신경과학회 2023 Experimental Neurobiology Vol.32 No.3

Anoctamin 2 (ANO2 or TMEM16B), a calcium-activated chloride channel (CaCC), performs diverse roles in neurons throughout the central nervous system. In hippocampal neurons, ANO2 narrows action potential width and reduces postsynaptic depolarization with high sensitivity to Ca2+ at relatively fast kinetics. In other brain regions, including the thalamus, ANO2 mediates activity-dependent spike frequency adaptations with low sensitivity to Ca2+ at relatively slow kinetics. How this same channel can respond to a wide range of Ca2+ levels remains unclear. We hypothesized that splice variants of ANO2 may contribute to its distinct Ca2+ sensitivity, and thus its diverse neuronal functions. We identified two ANO2 isoforms expressed in mouse brains and examined their electrophysiological properties: isoform 1 (encoded by splice variants with exons 1a, 2, 4, and 14) was expressed in the hippocampus, while isoform 2 (encoded by splice variants with exons 1a, 2, and 4) was broadly expressed throughout the brain, including in the cortex and thalamus, and had a slower calcium-dependent activation current than isoform 1. Computational modeling revealed that the secondary structure of the first intracellular loop of isoform 1 forms an entrance cavity to the calcium-binding site from the cytosol that is relatively larger than that in isoform 2. This difference provides structural evidence that isoform 2 is involved in accommodating spike frequency, while isoform 1 is involved in shaping the duration of an action potential and decreasing postsynaptic depolarization. Our study highlights the roles and molecular mechanisms of specific ANO2 splice variants in modulating neuronal functions.

Deficiency of Anoctamin 5/TMEM16E causes nuclear positioning defect and impairs Ca2+ signaling of differentiated C2C12 myotubes

Tam Thi Thanh Phuong,Jieun An,Sun Hwa Park,Ami Kim,Hyun Bin Choi,Tong Mook Kang 대한약리학회 2019 The Korean Journal of Physiology & Pharmacology Vol.23 No.6

Anoctamin 5 (ANO5)/TMEM16E belongs to a member of the ANO/ TMEM16 family member of anion channels. However, it is a matter of debate whether ANO5 functions as a genuine plasma membrane chloride channel. It has been recognized that mutations in the ANO5 gene cause many skeletal muscle diseases such as limb girdle muscular dystrophy type 2L (LGMD2L) and Miyoshi muscular dystrophy type 3 (MMD3) in human. However, the molecular mechanisms of the skeletal myopathies caused by ANO5 defects are poorly understood. To understand the role of ANO5 in skeletal muscle development and function, we silenced the ANO5 gene in C2C12 myoblasts and evaluated whether it impairs myogenesis and myotube function. ANO5 knockdown (ANO5-KD) by shRNA resulted in clustered or aggregated nuclei at the body of myotubes without affecting differentiation or myotube formation. Nuclear positioning defect of ANO5-KD myotubes was accompanied with reduced expression of Kif5b protein, a kinesin-related motor protein that controls nuclear transport during myogenesis. ANO5-KD impaired depolarization-induced [Ca2+]i transient and reduced sarcoplasmic reticulum (SR) Ca2+ storage. ANO5-KD resulted in reduced protein expression of the dihydropyridine receptor (DHPR) and SR Ca2+-ATPase subtype 1. In addition, ANO5-KD compromised co-localization between DHPR and ryanodine receptor subtype 1. It is concluded that ANO5-KD causes nuclear positioning defect by reduction of Kif5b expression, and compromises Ca2+ signaling by downregulating the expression of DHPR and SERCA proteins.

Deficiency of Anoctamin 5/TMEM16E causes nuclear positioning defect and impairs Ca²⁺ signaling of differentiated C2C12 myotubes

Phuong, Tam Thi Thanh,An, Jieun,Park, Sun Hwa,Kim, Ami,Choi, Hyun Bin,Kang, Tong Mook The Korean Society of Pharmacology 2019 The Korean Journal of Physiology & Pharmacology Vol.23 No.6

Anoctamin 5 (ANO5)/TMEM16E belongs to a member of the ANO/TMEM16 family member of anion channels. However, it is a matter of debate whether ANO5 functions as a genuine plasma membrane chloride channel. It has been recognized that mutations in the ANO5 gene cause many skeletal muscle diseases such as limb girdle muscular dystrophy type 2L (LGMD2L) and Miyoshi muscular dystrophy type 3 (MMD3) in human. However, the molecular mechanisms of the skeletal myopathies caused by ANO5 defects are poorly understood. To understand the role of ANO5 in skeletal muscle development and function, we silenced the ANO5 gene in C2C12 myoblasts and evaluated whether it impairs myogenesis and myotube function. ANO5 knockdown (ANO5-KD) by shRNA resulted in clustered or aggregated nuclei at the body of myotubes without affecting differentiation or myotube formation. Nuclear positioning defect of ANO5-KD myotubes was accompanied with reduced expression of Kif5b protein, a kinesin-related motor protein that controls nuclear transport during myogenesis. ANO5-KD impaired depolarization-induced $[Ca2^{+}]_i$ transient and reduced sarcoplasmic reticulum (SR) $Ca^{2+}$ storage. ANO5-KD resulted in reduced protein expression of the dihydropyridine receptor (DHPR) and SR $Ca^{2+}-ATPase$ subtype 1. In addition, ANO5-KD compromised co-localization between DHPR and ryanodine receptor subtype 1. It is concluded that ANO5-KD causes nuclear positioning defect by reduction of Kif5b expression, and compromises $Ca^{2+}$ signaling by downregulating the expression of DHPR and SERCA proteins.

Developmental Role of Anoctamin-1/TMEM16A in Ca2+-Dependent Volume Change in Supporting Cells of the Mouse Cochlea

이은영,이재광,이창준 한국뇌신경과학회 2013 Experimental Neurobiology Vol.22 No.4

Mammalian cochlea undergoes morphological and functional changes during the postnatal period, around the hearing onset. Majorchanges during the initial 2 postnatal weeks of mouse include maturation of sensory hair cells and supporting cells, and acquisitionof afferent and efferent innervations. During this period, supporting cells in the greater epithelial ridge (GER) of the cochlea exhibitspontaneous and periodic activities which involves ATP, increase in intracellular Ca2+, and cell volume change. This Ca2+-dependentvolume change has been proposed to involve chloride channels or transporters. We found that the spontaneous volume changeswere eliminated by anion channel blocker, 100 μM NPPB. Among candidates, expression of Anoctamin-1 (Ano1 or TMEM16A),bestriphin-1 and NKCC1 were investigated in whole-mount cochlea of P9-10 mice. Immunolabeling indicated high level of Ano1expression in the GER, but not of betrophin-1 or NKCC1. Double-labeling with calretinin and confocal image analysis furtherelucidated the cellular localization of Ano1 immunoreactivity in supporting cells. It was tested if the Ano1 expression exhibits similartime course to the spontaneous activities in postnatal cochlear supporting cells. Cochlear preparations from P2-3, P5-6, P9-10, P15-16 mice were subjected to immunolabeling. High level of Ano1 immunoreactivity was observed in the GER of P2-3, P5-6, P9-10cochleae, but not of P15-17 cochleae. Taken together, the localization and time course in Ano1 expression pattern correlates with thespontaneous, periodic volume changes recorded in postnatal cochlear supporting cells. From these results we propose that Ano1 isthe pacemaker of spontaneous activities in postnatal cochlea.

Calcium-activated chloride channels: a new target to control the spiking pattern of neurons

( Go Eun Ha ),( Eunji Cheong ) 생화학분자생물학회(구 한국생화학분자생물학회) 2017 BMB Reports Vol.50 No.3

The nature of encoded information in neural circuits is determined by neuronal firing patterns and frequencies. This paper discusses the molecular identity and cellular mecha-nisms of spike-frequency adaptation in the central nervous system (CNS). Spike-frequency adaptation in thalamocortical (TC) and CA1 hippocampal neurons is mediated by the Ca²⁺-activated Cl^- channel (CACC) anoctamin-2 (ANO2). Knockdown of ANO2 in these neurons results in increased number of spikes, in conjunction with significantly reduced spike-frequency adaptation. No study has so far demonstrated that CACCs mediate afterhyperpolarization currents, which result in the modulation of neuronal spike patterns in the CNS. Our study therefore proposes a novel role for ANO2 in spike-frequency adaptation and transmission of information in the brain. [BMB Reports 2017; 50(3): 109-110]

Spike Frequency Adaptation in Neurons of the Central Nervous System

Ha, Go Eun,Cheong, Eunji The Korean Society for Brain and Neural Science 2017 Experimental Neurobiology Vol.26 No.4

<P>Neuronal firing patterns and frequencies determine the nature of encoded information of the neurons. Here we discuss the molecular identity and cellular mechanisms of spike-frequency adaptation in central nervous system (CNS) neurons. Calcium-activated potassium (K<SUB>Ca</SUB>) channels such as BK<SUB>Ca</SUB> and SK<SUB>Ca</SUB> channels have long been known to be important mediators of spike adaptation via generation of a large afterhyperpolarization when neurons are hyper-activated. However, it has been shown that a strong hyperpolarization via these K<SUB>Ca</SUB> channels would cease action potential generation rather than reducing the frequency of spike generation. In some types of neurons, the strong hyperpolarization is followed by oscillatory activity in these neurons. Recently, spike-frequency adaptation in thalamocortical (TC) and CA1 hippocampal neurons is shown to be mediated by the Ca<SUP>2+</SUP>-activated Cl- channel (CACC), anoctamin-2 (ANO2). Knockdown of ANO2 in these neurons results in significantly reduced spike-frequency adaptation accompanied by increased number of spikes without shifting the firing mode, which suggests that ANO2 mediates a genuine form of spike adaptation, finely tuning the frequency of spikes in these neurons. Based on the finding of a broad expression of this new class of CACC in the brain, it can be proposed that the ANO2-mediated spike-frequency adaptation may be a general mechanism to control information transmission in the CNS neurons.</P>

Expression of anoctamin 7 (ANO7) is associated with poor prognosis and mucin 2 (MUC2) in colon adenocarcinoma: a study based on TCGA data

Chen, Chen,Siripat Aluksanasuwan,Keerakarn Somsuan Korea Genome Organization 2023 Genomics & informatics Vol.21 No.4

Colon adenocarcinoma (COAD) is the predominant type of colorectal cancer. Early diagnosis and treatment can significantly improve the prognosis of COAD patients. Anoctamin 7 (ANO7), an anion channel protein, has been implicated in prostate cancer and other types of cancer. In this study, we analyzed the expression of ANO7 and its correlation with clinicopathological characteristics among COAD patients using the Gene Expression Profiling Interactive Analysis 2 (GEPIA2) and the University of Alabama at Birmingham CANcer (UALCAN) databases. The GEPIA2, Kaplan-Meier plotter, and the Survival Genie platform were employed for survival analysis. The co-expression network and potential function of ANO7 in COAD were analyzed using GeneFriends, the Database for Annotation, Visualization and Integrated Discovery (DAVID), GeneMANIA, and Pathway Studio. Our data analysis revealed a significant reduction in ANO7 expression levels within COAD tissues compared to normal tissues. Additionally, ANO7 expression was found to be associated with race and histological subtype. The COAD patients exhibiting low ANO7 expression had lower survival rates compared to those with high ANO7 expression. The genes correlated with ANO7 were significantly enriched in proteolysis and mucin type O-glycan biosynthesis pathway. Furthermore, ANO7 demonstrated a direct interaction and a positive co-expression correlation with mucin 2 (MUC2). In conclusion, our findings suggest that ANO7 might serve as a potential prognostic biomarker and potentially plays a role in proteolysis and mucin biosynthesis in the context of COAD.

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

Malysz, John,Gibbons, Simon J.,Saravanaperumal, Siva A.,Du, Peng,Eisenman, Seth T.,Cao, Chike,Oh, Uhtaek,Saur, Dieter,Klein, Sabine,Ordog, Tamas,Farrugia, Gianrico American Physiological Society 2017 American journal of physiology, Gastrointestinal a Vol.312 No.3

<P>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. Kit(CreERT2/+); Ano1(Fl/Fl) mice were treated with tamoxifen or vehicle, and small intestines (mucosa free) were examined. Quantitative RTPCR demonstrated similar to 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.</P>