RESUMO
Enhancer-gene communication is dependent on topologically associating domains (TADs) and boundaries enforced by the CCCTC-binding factor (CTCF) insulator, but the underlying structures and mechanisms remain controversial. Here, we investigate a boundary that typically insulates fibroblast growth factor (FGF) oncogenes but is disrupted by DNA hypermethylation in gastrointestinal stromal tumors (GISTs). The boundary contains an array of CTCF sites that enforce adjacent TADs, one containing FGF genes and the other containing ANO1 and its putative enhancers, which are specifically active in GIST and its likely cell of origin. We show that coordinate disruption of four CTCF motifs in the boundary fuses the adjacent TADs, allows the ANO1 enhancer to contact FGF3, and causes its robust induction. High-resolution micro-C maps reveal specific contact between transcription initiation sites in the ANO1 enhancer and FGF3 promoter that quantitatively scales with FGF3 induction such that modest changes in contact frequency result in strong changes in expression, consistent with a causal relationship.
Assuntos
Cromatina , Elementos Facilitadores Genéticos , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Cromatina/genética , Oncogenes , DNA/químicaRESUMO
Smooth muscle cells (SMCs), Interstitial cells of Cajal (ICC) and Platelet-derived growth factor receptor α positive (PDGFRα+) cells form an integrated, electrical syncytium within the gastrointestinal (GI) muscular tissues known as the SIP syncytium. Immunohistochemical analysis of gastric corpus muscles showed that c-KIT+/ANO1+ ICC-IM and PDGFRα+ cells were closely apposed to one another in the same anatomical niches. We used intracellular microelectrode recording from corpus muscle bundles to characterize the roles of intramuscular ICC and PDGFRα+ cells in conditioning membrane potentials of gastric muscles. In muscle bundles, that have a relatively higher input impedance than larger muscle strips or sheets, we recorded an ongoing discharge of stochastic fluctuations in membrane potential, previously called unitary potentials or spontaneous transient depolarizations (STDs) and spontaneous transient hyperpolarizations (STHs). We reasoned that STDs should be blocked by antagonists of ANO1, the signature conductance of ICC. Activation of ANO1 has been shown to generate spontaneous transient inward currents (STICs), which are the basis for STDs. Ani9 reduced membrane noise and caused hyperpolarization, but this agent did not block the fluctuations in membrane potential quantitatively. Apamin, an antagonist of small conductance Ca2+-activated K+ channels (SK3), the signature conductance in PDGFRα+ cells, further reduced membrane noise and caused depolarization. Reversing the order of channel antagonists reversed the sequence of depolarization and hyperpolarization. These experiments show that the ongoing discharge of STDs and STHs by ICC and PDGFRα+ cells, respectively, exerts conditioning effects on membrane potentials in the SIP syncytium that would effectively regulate the excitability of SMCs.
Assuntos
Células Gigantes , Células Intersticiais de Cajal , Potenciais da Membrana , Receptor alfa de Fator de Crescimento Derivado de Plaquetas , Animais , Células Intersticiais de Cajal/fisiologia , Células Intersticiais de Cajal/metabolismo , Camundongos , Potenciais da Membrana/fisiologia , Células Gigantes/metabolismo , Células Gigantes/fisiologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Anoctamina-1/metabolismo , Estômago/fisiologia , Estômago/citologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/fisiologia , Proteínas Proto-Oncogênicas c-kit/metabolismo , Masculino , Camundongos Endogâmicos C57BLRESUMO
Liver diseases include all types of viral hepatitis, alcoholic liver disease (ALD), nonalcoholic fatty liver disease (NAFLD), cirrhosis, liver failure (LF) and hepatocellular carcinoma (HCC). Liver disease is now one of the leading causes of disease and death worldwide, which compels us to better understand the mechanisms involved in the development of liver diseases. Anoctamin 1 (ANO1), a calcium-activated chloride channel (CaCC), plays an important role in epithelial cell secretion, proliferation and migration. ANO1 plays a key role in transcriptional regulation as well as in many signalling pathways. It is involved in the genesis, development, progression and/or metastasis of several tumours and other diseases including liver diseases. This paper reviews the role and molecular mechanisms of ANO1 in the development of various liver diseases, aiming to provide a reference for further research on the role of ANO1 in liver diseases and to contribute to the improvement of therapeutic strategies for liver diseases by regulating ANO1.
Assuntos
Anoctamina-1 , Hepatopatias , Humanos , Anoctamina-1/metabolismo , Anoctamina-1/genética , Hepatopatias/metabolismo , Hepatopatias/patologia , Hepatopatias/genética , Animais , Transdução de Sinais , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Regulação da Expressão GênicaRESUMO
Ca2+ signalling plays a crucial role in determining lymphatic muscle cell excitability and contractility through its interaction with the Ca2+-activated Cl- channel anoctamin 1 (ANO1). In contrast, the large-conductance (BK) Ca2+-activated K+ channel (KCa) and other KCa channels have prominent vasodilatory actions by hyperpolarizing vascular smooth muscle cells. Here, we assessed the expression and contribution of the KCa family to mouse and rat lymphatic collecting vessel contractile function. The BK channel was the only KCa channel consistently expressed in fluorescence-activated cell sorting-purified mouse lymphatic muscle cell lymphatic muscle cells. We used a pharmacological inhibitor of BK channels, iberiotoxin, and small-conductance Ca2+-activated K+ channels, apamin, to inhibit KCa channels acutely in ex vivo isobaric myography experiments and intracellular membrane potential recordings. In basal conditions, BK channel inhibition had little to no effect on either mouse inguinal-axillary lymphatic vessel (MIALV) or rat mesenteric lymphatic vessel contractions or action potentials (APs). We also tested BK channel inhibition under loss of ANO1 either by genetic ablation (Myh11CreERT2-Ano1 fl/fl, Ano1ismKO) or by pharmacological inhibition with Ani9. In both Ano1ismKO MIALVs and Ani9-pretreated MIALVs, inhibition of BK channels increased contraction amplitude, increased peak AP and broadened the peak of the AP spike. In rat mesenteric lymphatic vessels, BK channel inhibition also abolished the characteristic post-spike notch, which was exaggerated with ANO1 inhibition, and significantly increased the peak potential and broadened the AP spike. We conclude that BK channels are present and functional on mouse and rat lymphatic muscle cells but are otherwise masked by the dominance of ANO1. KEY POINTS: Mouse and rat lymphatic muscle cells express functional BK channels. BK channels make little contribution to either rat or mouse lymphatic collecting vessel contractile function in basal conditions across a physiological pressure range. ANO1 limits the peak membrane potential achieved in the action potential and sets a plateau potential limiting the voltage-dependent activation of BK. BK channels are activated when ANO1 is absent or blocked and slightly impair contractile strength by reducing the peak membrane potential achieved in the action potential spike and accelerating the post-spike repolarization.
Assuntos
Potenciais de Ação , Anoctamina-1 , Canais de Potássio Ativados por Cálcio de Condutância Alta , Vasos Linfáticos , Animais , Anoctamina-1/metabolismo , Anoctamina-1/genética , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , Camundongos , Ratos , Potenciais de Ação/fisiologia , Masculino , Vasos Linfáticos/fisiologia , Vasos Linfáticos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Contração Muscular/fisiologia , Ratos Sprague-Dawley , Feminino , Miócitos de Músculo Liso/fisiologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacosRESUMO
The epididymal duct exhibits spontaneous phasic contractions (SPCs) to store and transport sperm. Here, we explored molecular identification of pacemaker cells driving SPCs in the caudal epididymal duct and also investigated properties of pacemaker currents underlying SPCs focusing on ANO1 Ca2+-activated Cl- channels (CaCCs). Immunohistochemistry was performed to visualise the distribution of platelet-derived growth factor receptor α (PDGFRα)- or ANO1-positive cells in the rat caudal epididymal duct. Perforated whole-cell patch clamp technique was applied to enzymatically isolated epididymal cells, while SPCs were recorded with video edge-tracking technique. Immunohistochemistry revealed the distribution of α-smooth muscle actin (α-SMA)-positive cells co-expressing both PDGFRα and ANO1 in the innermost smooth muscle layer. Approximately one-third of isolated epididymis cells exhibited spontaneous transient inward currents (STICs) at the holding potential -60 mV. The reversal potential for STICs was close to the calculated chloride equivalent potential depending on intracellular Cl- concentrations. Ani9 (3 µM), the ANO1 specific inhibitor, decreased both amplitude and frequency of STICs, while cyclopiazonic acid (CPA, 30 µM), a sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor, abolished STICs. Ani9 (3 or 10 µM) reduced the frequency of SPCs without changing their amplitude. Thus, PDGFRα+, ANO1+ specialised smooth muscle cells (SMCs) appear to function as pacemaker cells to electrically drive epididymal SPCs by generating ANO1-dependnet STICs. STICs arising from spontaneous Ca2+ release from intracellular Ca2+ store and subsequent opening of ANO1 result in depolarisations that spread into adjacent SMCs where L-type voltage-dependent Ca2+ channels are activated to develop SPCs.
Assuntos
Anoctamina-1 , Epididimo , Miócitos de Músculo Liso , Receptor alfa de Fator de Crescimento Derivado de Plaquetas , Animais , Masculino , Anoctamina-1/metabolismo , Epididimo/metabolismo , Epididimo/citologia , Miócitos de Músculo Liso/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Ratos , Canais de Cloreto/metabolismo , Ratos Sprague-Dawley , Ratos WistarRESUMO
Transient receptor potential vanilloid 4 (TRPV4) is a widely expressed cation channel that plays an important role in many physiological and pathological processes. However, most TRPV4 drugs carry a risk of side effects. Moreover, existing screening methods are not suitable for the high-throughput screening (HTS) of drugs. In this study, a cell model and HTS method for targeting TRPV4 channel drugs were established based on a calcium-activated chloride channel protein 1 Anoctamin 1 (ANO1) and a double mutant (YFP-H148Q/I152L) of the yellow fluorescent protein (YFP). Patch-clamp experiments and fluorescence quenching kinetic experiments were used to verify that the model could sensitively detect changes in intracellular Ca2+ concentration. The functionality of the TRPV4 cell model was examined through temperature variations and different concentrations of TRPV4 modulators, and the performance of the model in HTS was also evaluated. The model was able to sensitively detect changes in the intracellular Ca2+ concentration and also excelled at screening TRPV4 drugs, and the model was more suitable for HTS. We successfully constructed a drug cell screening model targeting the TRPV4 channel, which provides a tool to study the pathophysiological functions of TRPV4 in vitro.
Assuntos
Ensaios de Triagem em Larga Escala , Canais de Cátion TRPV , Canais de Cátion TRPV/metabolismo , Anoctamina-1 , Cálcio/metabolismoRESUMO
OBJECTIVE: To explore the expressions of zinc homeostasis-related proteins, G protein-coupled receptor 39 (GPR39) and ANO1 mRNA in the sperm of patients with asthenozoospermia (AS), and analyze their correlation with sperm motility. METHODS: We collected semen samples from 82 male subjects with PR+NP < 40%, PR < 32% and sperm concentration > 15×106/ml (the AS group, n = 40) or PR+NP ≥ 40%, PR ≥ 32% and sperm concentration > 15×106/ml (the normal control group, n = 42). We analyzed the routine semen parameters and measured the zinc content in the seminal plasma using the computer-assisted sperm analysis system, detected the expressions of zinc transporters (ZIP13, ZIP8 and ZNT10), metallothioneins (MT1G, MT1 and MTF), GPR39, and calcium-dependent chloride channel protein (ANO1) in the sperm by real-time quantitative PCR (RT qPCR), examined free zinc distribution in the sperm by laser confocal microscopy, and determined the expressions of GPR39 and MT1 proteins in the sperm by immunofluorescence staining, followed by Spearman rank correlation analysis of their correlation with semen parameters. RESULTS: There was no statistically significant difference in the zinc concentration in the seminal plasma between the AS and normal control groups (P>0.05). Compared with the controls, the AS patients showed a significantly reduced free zinc level (P<0.05), relative expressions of MT1G, MTF, ZIP13, GPR39 and ANO1 mRNA (P<0.05), and that of the GPR39 protein in the AS group (P<0.05). No statistically significant differences were observed in the relative expression levels of ZIP8, ZNT10 and MT1 mRNA between the two groups (P>0.05). The relative expression levels of GPR39, ANO1, MT1G and MTF mRNA were positively correlated with sperm motility and the percentage of progressively motile sperm (P<0.05). CONCLUSION: The expressions of zinc homeostasis proteins (MT1G, MTF and ZIP13), GPR39 and ANO1 mRNA are downregulated in the sperm of asthenozoospermia patients, and positively correlated with sperm motility.
Assuntos
Anoctamina-1 , Astenozoospermia , Proteínas de Transporte de Cátions , RNA Mensageiro , Receptores Acoplados a Proteínas G , Motilidade dos Espermatozoides , Espermatozoides , Zinco , Humanos , Masculino , Astenozoospermia/metabolismo , Astenozoospermia/genética , Anoctamina-1/metabolismo , Anoctamina-1/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Zinco/metabolismo , Espermatozoides/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Transporte de Cátions/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Metalotioneína/metabolismo , Metalotioneína/genética , Homeostase , Adulto , Análise do Sêmen , Relevância Clínica , Proteínas de NeoplasiasRESUMO
Gastrointestinal (GI) organs display spontaneous, non-neurogenic electrical, and mechanical rhythmicity that underlies fundamental motility patterns, such as peristalsis and segmentation. Electrical rhythmicity (aka slow waves) results from pacemaker activity generated by interstitial cells of Cajal (ICC). ICC express a unique set of ionic conductances and Ca2+ handling mechanisms that generate and actively propagate slow waves. GI smooth muscle cells lack these conductances. Slow waves propagate actively within ICC networks and conduct electrotonically to smooth muscle cells via gap junctions. Slow waves depolarize smooth muscle cells and activate voltage-dependent Ca2+ channels (predominantly CaV1.2), causing Ca2+ influx and excitation-contraction coupling. The main conductances responsible for pacemaker activity in ICC are ANO1, a Ca2+ -activated Cl- conductance, and CaV3.2. The pacemaker cycle, as currently understood, begins with spontaneous, localized Ca2+ release events in ICC that activate spontaneous transient inward currents due to activation of ANO1 channels. Depolarization activates CaV 3.2 channels, causing the upstroke depolarization phase of slow waves. The upstroke is transient and followed by a long-duration plateau phase that can last for several seconds. The plateau phase results from Ca2+ -induced Ca2+ release and a temporal cluster of localized Ca2+ transients in ICC that sustains activation of ANO1 channels and clamps membrane potential near the equilibrium potential for Cl- ions. The plateau phase ends, and repolarization occurs, when Ca2+ stores are depleted, Ca2+ release ceases and ANO1 channels deactivate. This review summarizes key mechanisms responsible for electrical rhythmicity in gastrointestinal organs.
RESUMO
Expression of transmembrane protein 16 A (TMEM16A), a calcium activated chloride channel, is elevated in some human cancers and impacts tumor cell proliferation, metastasis, and patient outcome. Evidence presented here uncovers a molecular synergy between TMEM16A and mechanistic/mammalian target of rapamycin (mTOR), a serine-threonine kinase that is known to promote cell survival and proliferation in cholangiocarcinoma (CCA), a lethal cancer of the secretory cells of bile ducts. Analysis of gene and protein expression in human CCA tissue and CCA cell line detected elevated TMEM16A expression and Cl- channel activity. The Cl- channel activity of TMEM16A impacted the actin cytoskeleton and the ability of cells to survive, proliferate, and migrate as revealed by pharmacological inhibition studies. The basal activity of mTOR, too, was elevated in the CCA cell line compared with the normal cholangiocytes. Molecular inhibition studies provided further evidence that TMEM16A and mTOR were each able to influence the regulation of the other's activity or expression respectively. Consistent with this reciprocal regulation, combined TMEM16A and mTOR inhibition produced a greater loss of CCA cell survival and migration than their individual inhibition alone. Together these data reveal that the aberrant TMEM16A expression and cooperation with mTOR contribute to a certain advantage in CCA.NEW & NOTEWORTHY This study points to the dysregulation of transmembrane protein 16 A (TMEM16A) expression and activity in cholangiocarcinoma (CCA), the inhibition of which has functional consequences. Dysregulated TMEM16A exerts an influence on the regulation of mechanistic/mammalian target of rapamycin (mTOR) activity. Moreover, the reciprocal regulation of TMEM16A by mTOR demonstrates a novel connection between these two protein families. These findings support a model in which TMEM16A intersects the mTOR pathway to regulate cell cytoskeleton, survival, proliferation, and migration in CCA.
Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Humanos , Neoplasias dos Ductos Biliares/metabolismo , Ductos Biliares Intra-Hepáticos/metabolismo , Ductos Biliares Intra-Hepáticos/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células/genética , Sobrevivência Celular , Colangiocarcinoma/patologia , Transdução de Sinais , Sirolimo/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
BACKGROUND: Anoctamin-1 (ANO1) was identified as an unfavorable prognostic marker in pancreatic cancer. However, the exact implication of ANO1 in pancreatic cancer is still poorly understood. Here we investigated the effect of ANO1 in pancreatic cancer progression under the context of oncogenic KRAS, aiming at finding a new therapeutic target. METHODS: Knockdown and overexpression of oncogenic KRAS as well as ANO1 in PDAC cell lines were performed by lentivirus infection. Cell proliferation and migration assay, RNA seq analysis were performed in PDAC cells bearing different status of ANO1 and KRAS. In vivo mice model was used to investigate the xenograft tumor growth with different status of KRAS and ANO1. RESULTS: Our results showed that ANO1 expression level is elevated in poorly differentiated cancer cells. Overexpression of ANO1 in PDAC cancer cells was found to promote cancer cell proliferation in vitro and in vivo, which synergized with the introduction of oncogenic KRAS. Consistently, knockdown of ANO1 expression was found to suppress cancer growth in vitro and in vivo. RNA seq analysis revealed that the observed synergistic cancer-promoting effect from ANO1 and oncogenic KRAS is likely due to concurrent activating key genes involved in lipid metabolism including HMGCS1. CONCLUSION: The outcome from our study suggests that ANO1 plays an important role in promoting pancreatic cancer development, especially at the presence of oncogenic KRAS. Considering the prevalence of KRAS mutation in pancreatic cancer patients, suppression ANO1 may represent a potential effective therapeutic measure in pancreatic cancer treatment.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Animais , Camundongos , Carcinoma Ductal Pancreático/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Anoctamina-1/genética , Neoplasias Pancreáticas/metabolismo , Proliferação de Células/genética , Linhagem Celular Tumoral , Proteínas de Neoplasias/metabolismo , Neoplasias PancreáticasRESUMO
Extragastrointestinal stromal tumors (EGISTs) carry the same morphological, immunohistochemical and molecular features as gastrointestinal stromal tumors (GISTs) and involve extragastrointestinal tract soft tissue. The majority of reported EGIST cases arise from intraabdominal, retroperitoneal, or pelvic soft tissue. A significant subset of such tumors originates from the gastrointestinal muscle layer, grows in an exophytic manner, then loses attachment to the gastrointestinal tract. Consequently, true EGISTs are exceedingly rare. Herein, we are reporting a case of a vulvar EGIST. A 77-year-old woman presented with a painless subcutaneous nodule on the right perineum. An excisional biopsy showed a fairly circumscribed bland spindle cell lesion in the dermis. The tumor cells were positive for CD117 and ANO1/DOG-1 and negative for smooth muscle myosin, smooth muscle actin, STAT6, low- and high-molecular-weight cytokeratins, SOX10, MART-1, CD10, S-100 protein, and estrogen and progesterone receptors. A diagnosis of EGIST was made and complete excision was recommended. Superficial/subcutaneous EGISTs are extremely rare, and it is important for dermatopathologists to be aware of this entity as it can be misdiagnosed as more common spindle cell neoplasms, both benign and malignant, including but not limited to smooth muscle neoplasms (leiomyoma/leiomyosarcoma), spindle cell melanoma, and sarcomatoid squamous cell carcinoma.
Assuntos
Tumores do Estroma Gastrointestinal , Leiomiossarcoma , Humanos , Tumores do Estroma Gastrointestinal/diagnóstico , Tumores do Estroma Gastrointestinal/patologia , Imuno-Histoquímica , Proteínas Proto-Oncogênicas c-kitRESUMO
Dynamic chloride (Cl-) regulation is critical for synaptic inhibition. In mature neurons, Cl- influx and extrusion are primarily controlled by ligand-gated anion channels (GABAA and glycine receptors) and the potassium chloride cotransporter K+-Cl- cotransporter 2 (KCC2), respectively. Here, we report for the first time, to our knowledge, a presence of a new source of Cl- influx in striatal neurons with properties similar to chloride voltage-gated channel 1 (ClC-1). Using whole cell patch-clamp recordings, we detected an outwardly rectifying voltage-dependent current that was impermeable to the large anion methanesulfonate (MsO-). The anionic current was sensitive to the ClC-1 inhibitor 9-anthracenecarboxylic acid (9-AC) and the nonspecific blocker phloretin. The mean fractions of anionic current inhibition by MsO-, 9-AC, and phloretin were not significantly different, indicating that anionic current was caused by active ClC-1-like channels. In addition, we found that Cl- current was not sensitive to the transmembrane protein 16A (TMEM16A; Ano1) inhibitor Ani9 and that the outward Cl- rectification was preserved even at a very high intracellular Ca2+ concentration (2 mM), indicating that TMEM16B (Ano2) did not contribute to the total current. Western blotting and immunohistochemical analyses confirmed the presence of ClC-1 channels in the striatum mainly localized to the somata of striatal neurons. Finally, we found that 9-AC decreased action potential firing frequencies and increased excitability in medium spiny neurons (MSNs) expressing dopamine type 1 (D1) and type 2 (D2) receptors in the brain slices, respectively. We conclude that ClC-1-like channels are preferentially located at the somata of MSNs, are functional, and can modulate neuronal excitability.
Assuntos
Cloretos , Corpo Estriado , Canais de Cloreto/metabolismo , Cloretos/metabolismo , Corpo Estriado/metabolismo , Neurônios/metabolismo , Técnicas de Patch-Clamp , Floretina/metabolismo , Floretina/farmacologia , Receptores de Dopamina D2/metabolismoRESUMO
Anoctamin-1 (ANO1) (TMEM16A) is a calcium-activated chloride channel that plays critical roles in diverse physiological processes, such as sensory transduction and epithelial secretion. ANO1 levels have been shown to be altered under physiological and pathological conditions, although the molecular mechanisms that control ANO1 protein levels remain unclear. The ubiquitin-proteasome system is known to regulate the levels of numerous ion channels, but little information is available regarding whether and how ubiquitination regulates levels of ANO1. Here, we showed that two E3 ligases, TRIM23 and TRIM21, physically interact with the C terminus of ANO1. In vitro and in vivo assays demonstrated that whereas TRIM23 ubiquitinated ANO1 leading to its stabilization, TRIM21 ubiquitinated ANO1 and induced its degradation. Notably, ANO1 regulation by TRIM23 and TRIM21 is involved in chemical-induced pain sensation, salivary secretion, and heart-rate control in mice, and TRIM23 also mediates ANO1 upregulation induced by epidermal growth factor treatment. Our results suggest that these two antagonistic E3 ligases act together to control ANO1 expression and function. Our findings reveal a previously unrecognized mechanism for regulating ANO1 protein levels and identify a potential molecular link between ANO1 regulation, epidermal growth factor, and other signaling pathways.
Assuntos
Anoctamina-1/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Neoplasias/metabolismo , Ribonucleoproteínas/metabolismo , Células HEK293 , Humanos , Estabilidade Proteica , Proteólise , UbiquitinaçãoRESUMO
Various human tissues express the calcium-activated chloride channel Anoctamin 1 (ANO1), also known as TMEM16A. ANO1 allows the passive chloride flux that controls different physiological functions ranging from muscle contraction, fluid and hormone secretion, gastrointestinal motility, and electrical excitability. Overexpression of ANO1 is associated with pathological conditions such as hypertension and cancer. The molecular cloning of ANO1 has led to a surge in structural, functional, and physiological studies of the channel in several tissues. ANO1 is a homodimer channel harboring two pores - one in each monomer - that work independently. Each pore is activated by voltage-dependent binding of two intracellular calcium ions to a high-affinity-binding site. In addition, the binding of phosphatidylinositol 4,5-bisphosphate to sites scattered throughout the cytosolic side of the protein aids the calcium activation process. Furthermore, many pharmacological studies have established ANO1 as a target of promising compounds that could treat several illnesses. This chapter describes our current understanding of the physiological roles of ANO1 and its regulation under physiological conditions as well as new pharmacological compounds with potential therapeutic applications.
RESUMO
Years ago gastrointestinal motility was thought to be due to interactions between enteric nerves and smooth muscle cells (SMCs) in the tunica muscularis. Thus, regulatory mechanisms controlling motility were either myogenic or neurogenic. Now we know that populations of interstitial cells, c-Kit+ (interstitial cells of Cajal or ICC), and PDGFRα+ cells (formerly "fibroblast-like" cells) are electrically coupled to SMCs, forming the SIP syncytium. Pacemaker and neurotransduction functions are provided by interstitial cells through Ca2+ release from the endoplasmic reticulum (ER) and activation of Ca2+-activated ion channels in the plasma membrane (PM). ICC express Ca2+-activated Cl- channels encoded by Ano1. When activated, Ano1 channels produce inward current and, therefore, depolarizing or excitatory effects in the SIP syncytium. PDGFRα+ cells express Ca2+-activated K+ channels encoded by Kcnn3. These channels generate outward current when activated and hyperpolarizing or membrane-stabilizing effects in the SIP syncytium. Inputs from enteric and sympathetic neurons regulate Ca2+ transients in ICC and PDGFRα+ cells, and currents activated in these cells conduct to SMCs and regulate contractile behaviors. ICC also serve as pacemakers, generating slow waves that are the electrophysiological basis for gastric peristalsis and intestinal segmentation. Pacemaker types of ICC express voltage-dependent Ca2+ conductances that organize Ca2+ transients, and therefore Ano1 channel openings, into clusters that define the amplitude and duration of slow waves. Ca2+ handling mechanisms are at the heart of interstitial cell function, yet little is known about what happens to Ca2+ dynamics in these cells in GI motility disorders.
Assuntos
Células Intersticiais de Cajal , Células Intersticiais de Cajal/fisiologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas , Músculo Liso/fisiologia , Trato Gastrointestinal/fisiologia , Intestino Delgado/metabolismoRESUMO
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.
Assuntos
Sinalização do Cálcio , Proteínas de Membrana , Ânions , Anoctamina-1/metabolismo , Células HeLa , Humanos , Proteínas de Membrana/metabolismo , Proteínas de NeoplasiasRESUMO
KEY POINTS: Taste transduction occurs in taste buds in the tongue epithelium. The Ca2+ -activated Cl- channels TMEM16A and TMEM16B play relevant physiological roles in several sensory systems. Here, we report that TMEM16A, but not TMEM16B, is expressed in the apical part of taste buds. Large Ca2+ -activated Cl- currents blocked by Ani-9, a selective inhibitor of TMEM16A, are measured in type I taste cells but not in type II or III taste cells. ATP indirectly activates Ca2+ -activated Cl- currents in type I cells through TMEM16A channels. These results indicate that TMEM16A is functional in type I taste cells and contribute to understanding the largely unknown physiological roles of these cells. ABSTRACT: The Ca2+ -activated Cl- channels TMEM16A and TMEM16B have relevant roles in many physiological processes including neuronal excitability and regulation of Cl- homeostasis. Here, we examined the presence of Ca2+ -activated Cl- channels in taste cells of mouse vallate papillae by using immunohistochemistry and electrophysiological recordings. By using immunohistochemistry we showed that only TMEM16A, and not TMEM16B, was expressed in taste bud cells where it largely co-localized with the inwardly rectifying K+ channel KNCJ1 in the apical part of type I cells. By using whole-cell patch-clamp recordings in isolated cells from taste buds, we measured an average current of -1083 pA at -100 mV in 1.5 µm Ca2+ and symmetrical Cl- in type I cells. Ion substitution experiments and blockage by Ani-9, a specific TMEM16A channel blocker, indicated that Ca2+ activated anionic currents through TMEM16A channels. We did not detect any Ca2+ -activated Cl- currents in type II or III taste cells. ATP is released by type II cells in response to various tastants and reaches type I cells where it is hydrolysed by ecto-ATPases. Type I cells also express P2Y purinergic receptors and stimulation of type I cells with extracellular ATP produced large Ca2+ -activated Cl- currents blocked by Ani-9, indicating a possible role of TMEM16A in ATP-mediated signalling. These results provide a definitive demonstration that TMEM16A-mediated currents are functional in type I taste cells and provide a foundation for future studies investigating physiological roles for these often-neglected taste cells.
Assuntos
Anoctamina-1/metabolismo , Papilas Gustativas , Animais , Cálcio/metabolismo , Canais de Cloreto , Camundongos , Técnicas de Patch-Clamp , Receptores Purinérgicos P2Y , Papilas Gustativas/metabolismoRESUMO
Calcium-activated chloride channels (CaCCs) are widespread chloride channels which rely on calcium activation to perform their functions. In 2008, TMEM16A (also known as anoctamin1, ANO1) was identified as the molecular basis of the CaCCs, which provided the possibility to study the physiological function of CaCCs. TMEM16A is widely expressed in various cells and controls basic physiological functions, including neuronal and cardiac excitability, nerve transduction, smooth muscle contraction, epithelial Cl- secretion and fertilization. However, the abnormal function of TMEM16A may cause a variety of diseases, including asthma, gastrointestinal motility disorder and various cancers. Therefore, TMEM16A is a putative drug target for many diseases, and it is important to determine specific and efficient modulators of TMEM16A channel. In recent years, we and others have screened several natural modulators of TMEM16A against cancers and gastrointestinal motility dysfunction. This article reviews the screening methods, efficacy of TMEM16A modulators and pharmacological effects of TMEM16A modulators on different diseases. GRAPHIC ABSTACT.
Assuntos
Cálcio , Canais de Cloreto , Anoctamina-1/genética , Cálcio/metabolismo , Canais de Cloreto/genética , Motilidade GastrointestinalRESUMO
TMEM16A (anoctamin 1) is an important calcium-activated chloride channel in airway smooth muscle (ASM). We have previously shown that TMEM16A antagonists such as benzbromarone relax ASM and have proposed TMEM16A antagonists as novel therapies for asthma treatment. However, TMEM16A is also expressed on airway epithelium, and TMEM16A agonists are being investigated as novel therapies for cystic fibrosis. There are theoretical concerns that agonism of TMEM16A on ASM could lead to bronchospasm, making them detrimental as airway therapeutics. The TMEM16A agonist Eact induced a significant contraction of human ASM and guinea pig tracheal rings in an ex vivo organ bath model. Pretreatment with two different TMEM16A antagonists, benzbromarone or T16Ainh-A01, completely attenuated these Eact-induced contractions. Pretreatment with Eact alone augmented the maximum acetylcholine contraction. Pretreatment of A/J mice in vivo with nebulized Eact caused an augmentation of methacholine-induced increases in airway resistance measured by the forced oscillatory technique (flexiVent). Pretreatment with the TMEM16A antagonist benzbromarone significantly attenuated methacholine-induced increases in airway resistance. In in vitro cellular studies, TMEM16A was found to be expressed more abundantly in ASM compared with epithelial cells in culture (8-fold higher in ASM). Eact caused an increase in intracellular calcium in human ASM cells that was completely attenuated by pretreatment with benzbromarone. Eact acutely depolarized the plasma membrane potential of ASM cells, which was attenuated by benzbromarone or nifedipine. The TMEM16A agonist Eact modulates ASM contraction in both ex vivo and in vivo models, suggesting that agonism of TMEM16A may lead to clinically relevant bronchospasm.
Assuntos
Anoctamina-1/agonistas , Anoctamina-1/metabolismo , Pulmão/metabolismo , Tono Muscular , Músculo Liso/metabolismo , Proteínas de Neoplasias/agonistas , Proteínas de Neoplasias/metabolismo , Acetilcolina/farmacologia , Animais , Anoctamina-1/genética , Hiper-Reatividade Brônquica/fisiopatologia , Broncoconstrição/efeitos dos fármacos , Cálcio/metabolismo , Células Cultivadas , Cobaias , Humanos , Fosfatos de Inositol/biossíntese , Cloreto de Metacolina/farmacologia , Contração Muscular/efeitos dos fármacos , Tono Muscular/efeitos dos fármacos , Proteínas de Neoplasias/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Long non-coding RNAs (lncRNAs) have a particular expression in the testicular tissue and exhibit a regulatory function on the reproduction system. ANO1-AS2 (linc02584), as an lncRNA is located near the anoctamin1 (ANO1) gene. ANO1 is an important component of the transmembrane system exhibiting expression modifications in the idiopathic infertile men. Therefore, the present study was conducted to investigate the relationship between ANO1-AS2 and ANO1 gene expression with sperm motility and morphology in the patients with asthenozoospermia (AZ) and terato- asthenozoospermia (TAZ). The study population included 32 patients with AZ, 35 patients with TAZ, and 34 people with normozoospermia (NZ, control). The expression levels of ANO1 gene and ANO1-AS2 in the spermatozoa were measured by the quantitative real-time polymerase chain reaction (PCR). Docking analysis was performed to investigate the interactions of the ANO1 gene promoter and intermediate elements with ANO1-AS2. ANO1 gene expression was significantly (P < 0.05) downregulated in the patients however; ANO1-AS2 expression was significantly upregulated (P < 0.05). The subsequent analysis confirmed the inverse correlation between ANO1 and ANO1-AS2. ANO1 gene expression level was significantly positively correlated with sperm motility and morphology (P < 0.05). Moreover, ANO1-AS2 expression showed an inverse correlation with sperm motility and morphology (P < 0.05). Docking analysis confirmed that ANO1-AS2 could stably interact with ANO1 gene promoter. In conclusion, ANO1-AS2 is likely to downregulate the ANO1 gene by interacting with ANO1 gene promoter, which can influence the sperm motility and morphology.