Hypoxia augments the calcium-activated chloride current carried by anoctamin-1 in cardiac vascular endothelial cells of neonatal mice.
Wu Ming-Ming,Lou Jie,Song Bin-Lin,Gong Yuan-Feng,Li Yan-Chao,Yu Chang-Jiang,Wang Qiu-Shi,Ma Tian-Xing,Ma Ke,Hartzell H Criss,Duan Dayue Darrel,Zhao Dan,Zhang Zhi-Ren
British journal of pharmacology
BACKGROUND AND PURPOSE:The molecular identity of calcium-activated chloride channels (CaCCs) in vascular endothelial cells remains unknown. This study sought to identify whether anoctamin-1 (Ano1, also known as TMEM16A) functions as a CaCC and whether hypoxia alters the biophysical properties of Ano1 in mouse cardiac vascular endothelial cells (CVECs). EXPERIMENTAL APPROACH:Western blot, quantitative real-time PCR, confocal imaging analysis and patch-clamp analysis combined with pharmacological approaches were used to determine whether Ano1 was expressed and functioned as CaCC in CVECs. KEY RESULTS:Ano1 was expressed in CVECs. The biophysical properties of the current generated in the CVECs, including the Ca(2+) and voltage dependence, outward rectification, anion selectivity and the pharmacological profile, are similar to those described for CaCCs. The density of ICl ( C a) detected in CVECs was significantly inhibited by T16Ainh -A01, an Ano1 inhibitor, and a pore-targeting, specific anti-Ano1 antibody, and was markedly decreased in Ano1 gene knockdown CVECs. The density of ICl ( C a) was significantly potentiated in CVECs exposed to hypoxia, and this hypoxia-induced increase in the density of ICl ( C a) was inhibited by T16Ainh -A01 or anti-Ano1 antibody. Hypoxia also increased the current density of ICl ( C a) in Ano1 gene knockdown CVECs. CONCLUSIONS AND IMPLICATIONS:Ano1 formed CaCC in CVECs of neonatal mice. Hypoxia enhances Ano1-mediated ICl ( C a) density via increasing its expression, altering the ratio of its splicing variants, sensitivity to membrane voltage and to Ca(2+) . Ano1 may play a role in the pathophysiological processes during ischaemia in heart, and therefore, Ano1 might be a potential therapeutic target to prevent ischaemic damage.
10.1111/bph.12730
Arctigenin Attenuates Vascular Inflammation Induced by High Salt through TMEM16A/ESM1/VCAM-1 Pathway.
Biomedicines
Salt-sensitive hypertension is closely related to inflammation, but the mechanism is barely known. Transmembrane member 16A (TMEM16A) is the Ca-activated chloride channel in epithelial cells, smooth muscle cells, and sensory neurons. It can promote inflammatory responses by increasing proinflammatory cytokine release. Here, we identified a positive role of TMEM16A in vascular inflammation. The expression of TMEM16A was increased in high-salt-stimulated vascular smooth muscle cells (VSMCs), whereas inhibiting TMEM16A or silencing TMEM16A with small interfering RNA (siRNA) can abolish this effect in vitro or in vivo. Transcriptome analysis of VSMCs revealed some differential downstream genes of TMEM16A related to inflammation, such as endothelial cell-specific molecule 1 (ESM1) and CXC chemokine ligand 16 (CXCL16). Overexpression of TMEM16A in VSMCs was accompanied by high levels of ESM1, CXCL16, intercellular adhesion molecule-1 (ICAM-1), and vascular adhesion molecule-1 (VCAM-1). We treated VSMCs cultured with high salt and arctigenin (ARC), T16Ainh-A01 (T16), and TMEM16A siRNA (siTMEM16A), leading to greatly decreased ESM1, CXCL16, VCAM-1, and ICAM-1. Beyond that, silencing ESM1, the expression of VCAM-1 and ICAM-1, and CXCL16 was attenuated. In conclusion, our results outlined a signaling scheme that increased TMEM16 protein upregulated ESM1, which possibly activated the CXCL16 pathway and increased VCAM-1 and ICAM-1 expression, which drives VSMC inflammation. Beyond that, arctigenin, as a natural inhibitor of TMEM16A, can reduce the systolic blood pressure (SBP) of salt-sensitive hypertension mice and alleviate vascular inflammation.
10.3390/biomedicines10112760
The TMEM16A anion channel as a versatile regulator of vascular tone.
Science signaling
The TMEM16A channel represents a key depolarizing mechanism in arterial smooth muscle and contractile pericytes, where it is activated by several endogenous contractile agonists. In this issue of , Mata-Daboin demonstrate a previously unidentified role for TMEM16A in endothelial cells for acetylcholine-mediated vasorelaxation. Collectively, TMEM16A serves as a transducer of vasoactive stimuli to enable fine modulation of vessel tone.
10.1126/scisignal.adk5661
TMEM16A Contributes to Endothelial Dysfunction by Facilitating Nox2 NADPH Oxidase-Derived Reactive Oxygen Species Generation in Hypertension.
Ma Ming-Ming,Gao Min,Guo Kai-Min,Wang Mi,Li Xiang-Yu,Zeng Xue-Lin,Sun Lu,Lv Xiao-Fei,Du Yan-Hua,Wang Guan-Lei,Zhou Jia-Guo,Guan Yong-Yuan
Hypertension (Dallas, Tex. : 1979)
Ca-activated Cl channels play a crucial role in various physiological processes. However, the role of TMEM16A in vascular endothelial dysfunction during hypertension is unclear. In this study, we investigated the specific involvement of TMEM16A in regulating endothelial function and blood pressure and the underlying mechanism. Reverse transcription-polymerase chain reaction, Western blotting, coimmunoprecipitation, confocal imaging, patch-clamp recordings, and TMEM16A endothelial-specific transgenic and knockout mice were used. We found that TMEM16A was expressed abundantly and functioned as a Ca-activated Cl channel in endothelial cells. Angiotensin II induced endothelial dysfunction with an increase in TMEM16A expression. The knockout of endothelial-specific TMEM16A significantly lowered the blood pressure and ameliorated endothelial dysfunction in angiotensin II-induced hypertension, whereas the overexpression of endothelial-specific TMEM16A resulted in the opposite effects. These results were related to the increased reactive oxygen species production, Nox2-containing NADPH oxidase activation, and Nox2 and p22phox protein expression that were facilitated by TMEM16A on angiotensin II-induced hypertensive challenge. Moreover, TMEM16A directly bound with Nox2 and reduced the degradation of Nox2 through the proteasome-dependent degradation pathway. Therefore, TMEM16A is a positive regulator of endothelial reactive oxygen species generation via Nox2-containing NADPH oxidase, which induces endothelial dysfunction and hypertension. Modification of TMEM16A may be a novel therapeutic strategy for endothelial dysfunction-associated diseases.
10.1161/HYPERTENSIONAHA.116.08874
Vasodilators activate the anion channel TMEM16A in endothelial cells to reduce blood pressure.
Science signaling
Systemic blood pressure is acutely controlled by total peripheral resistance as determined by the diameter of small arteries and arterioles, the contractility of which is regulated by endothelial cells lining the lumen of blood vessels. We investigated the physiological functions of the chloride (Cl) channel TMEM16A in endothelial cells. TMEM16A channels generated calcium (Ca)-activated Cl currents in endothelial cells from control () mice that were absent in those from mice with tamoxifen-inducible, endothelial cell-specific knockout of TMEM16A ( ecKO). TMEM16A currents in endothelial cells were activated by the muscarinic receptor agonist acetylcholine and an agonist of the Ca channel TRPV4, which localized in nanoscale proximity with TMEM16A as assessed by single-molecule localization imaging of endothelial cells. Acetylcholine stimulated TMEM16A currents by activating Ca influx through surface TRPV4 channels without altering the nanoscale properties of TMEM16A and TRPV4 surface clusters or their colocalization. In pressurized arteries, activation of TMEM16A channels in endothelial cells induced by acetylcholine; TRPV4 channel stimulation; or intraluminal ATP, another vasodilator, produced hyperpolarization and dilation. Furthermore, deficiency of TMEM16A channels in endothelial cells resulted in increased systemic blood pressure in conscious mice. These data indicate that vasodilators stimulate TRPV4 channels, leading to Ca-dependent activation of nearby TMEM16A channels in endothelial cells to produce arterial hyperpolarization, vasodilation, and reduced blood pressure. Thus, TMEM16A is an anion channel in endothelial cells that regulates arterial contractility and blood pressure.
10.1126/scisignal.adh9399
The Ca2+-gated channel TMEM16A amplifies capillary pericyte contraction and reduces cerebral blood flow after ischemia.
The Journal of clinical investigation
Pericyte-mediated capillary constriction decreases cerebral blood flow in stroke after an occluded artery is unblocked. The determinants of pericyte tone are poorly understood. We show that a small rise in cytoplasmic Ca2+ concentration ([Ca2+]i) in pericytes activated chloride efflux through the Ca2+-gated anion channel TMEM16A, thus depolarizing the cell and opening voltage-gated calcium channels. This mechanism strongly amplified the pericyte [Ca2+]i rise and capillary constriction evoked by contractile agonists and ischemia. In a rodent stroke model, TMEM16A inhibition slowed the ischemia-evoked pericyte [Ca2+]i rise, capillary constriction, and pericyte death; reduced neutrophil stalling; and improved cerebrovascular reperfusion. Genetic analysis implicated altered TMEM16A expression in poor patient recovery from ischemic stroke. Thus, pericyte TMEM16A is a crucial regulator of cerebral capillary function and a potential therapeutic target for stroke and possibly other disorders of impaired microvascular flow, such as Alzheimer's disease and vascular dementia.
10.1172/JCI154118
Knockdown of TMEM16A suppressed MAPK and inhibited cell proliferation and migration in hepatocellular carcinoma.
Deng Liang,Yang Jihong,Chen Hongwu,Ma Bo,Pan Kangming,Su Caikun,Xu Fengfeng,Zhang Jihong
OncoTargets and therapy
TMEM16A plays an important role in cell proliferation in various cancers. However, less was known about the expression and role of TMEM16A in hepatocellular carcinoma. We screened the expression of TMEM16A in patients' hepatocellular carcinoma tissues, and also analyzed the biological function of hepatocellular carcinoma cells by knockdown of TMEM16A, as well as the expression of MAPK signaling proteins, including p38, p-p38, ERK1/2, p-ERK1/2, JNK, and p-JNK, and cell cycle regulatory protein cyclin D1 in TMEM16A siRNA-transfected SMMC-7721 cells by Western blot. Our results showed that TMEM16A was overexpressed in hepatocellular carcinoma tissues. Inhibition of TMEM16A suppressed the cell proliferation, migration, and invasion, and cell cycle progression but did not influence the cell apoptosis. TMEM16A siRNA-suppressed cancer cell proliferation and tumor growth were accompanied by a reduction of p38 and ERK1/2 activation and cyclin D1 induction, and were not influenced by other tested MAPK signaling proteins. In addition, inhibition of TMEM16A suppressed tumorigenicity in vivo. TMEM16A is overexpressed in hepatocellular carcinoma, and that inhibition of TMEM16A suppressed MAPK and growth of hepatocellular carcinoma. TMEM16A could be a potentially novel therapeutic target for human cancers, including hepatocellular carcinoma.
10.2147/OTT.S95985
TMEM16A/B associated CaCC: structural and functional insights.
Pang Chunli,Yuan Hongbo,Ren Shuxi,Chen Yafei,An Hailong,Zhan Yong
Protein and peptide letters
Calcium-activated chloride channels (CaCCs) play fundamental roles in numerous physiological processes. Transmembrane proteins 16A and 16B (TMEM16A/B) were identified to be the best molecular identities of CaCCs to date. This makes molecular investigation of CaCCs become possible. This review discusses the latest findings of TMEM16A/B associated CaCCs, the calcium and voltage dual dependence，the reorganization of Ca(2+)-binding site, the mechanisms of direct or indirect activation, the structure-functional relationship, and the possible stereoscopic structure. TMEM16A and other members of the family are associated with several kinds of cancers and other chloride channelopathies. An understanding of TMEM16 associated channel proteins will shed some light on their role in oncology and in pharmacology development.
Functional swapping between transmembrane proteins TMEM16A and TMEM16F.
The Journal of biological chemistry
The transmembrane proteins TMEM16A and -16F each carry eight transmembrane regions with cytoplasmic N and C termini. TMEM16A carries out Ca(2+)-dependent Cl(-) ion transport, and TMEM16F is responsible for Ca(2+)-dependent phospholipid scrambling. Here we established assay systems for the Ca(2+)-dependent Cl(-) channel activity using 293T cells and for the phospholipid scramblase activity using TMEM16F(-/-) immortalized fetal thymocytes. Chemical cross-linking analysis showed that TMEM16A and -16F form homodimers in both 293T cells and immortalized fetal thymocytes. Successive deletion from the N or C terminus of both proteins and the swapping of regions between TMEM16A and -16F indicated that their cytoplasmic N-terminal (147 amino acids for TMEM16A and 95 for 16F) and C-terminal (88 amino acids for TMEM16A and 68 for 16F) regions were essential for their localization at plasma membranes and protein stability, respectively, and could be exchanged. Analyses of TMEM16A and -16F mutants with point mutations in the pore region (located between the fifth and sixth transmembrane regions) indicated that the pore region is essential for both the Cl(-) channel activity of TMEM16A and the phospholipid scramblase activity of TMEM16F. Some chemicals such as epigallocatechin-3-gallate and digallic acid inhibited the Cl(-) channel activity of TMEM16A and the scramblase activity of TMEM16F with an opposite preference. These results indicate that TMEM16A and -16F use a similar mechanism for sorting to plasma membrane and protein stabilization, but their functional domains significantly differ.
10.1074/jbc.M113.542324
TMEM16A Protein: Calcium-Binding Site and its Activation Mechanism.
Ji Wanying,Shi Donghong,Shi Sai,Yang Xiao,Chen Yafei,An Hailong,Pang Chunli
Protein and peptide letters
TMEM16A mediates the calcium-activated transmembrane flow of chloride ions and a variety of physiological functions. The binding of cytoplasmic calcium ions of TMEM16A and the consequent conformational changes of it are the key issues to explore the structure-function relationship. In recent years, researchers have explored this issue through electrophysiological experiments, structure resolving, molecular dynamic simulation, and other methods. The structures of TMEM16 family members determined by cryo-Electron microscopy (cryo-EM) and X-ray crystallization provide the primary basis for the investigation of the molecular mechanism of TMEM16A. However, the binding and activation mechanism of calcium ions in TMEM16A are still unclear and controversial. This mini-review discusses four Ca sensing sites of TMEM16A and analyzes activation properties of TMEM16A by them, which will help understand the structure-function relationship of TMEM16A and throw light on the molecular design targeting the TMEM16A channel.
10.2174/0929866528666211105112131
Recent advances in TMEM16A: Structure, function, and disease.
Ji Qiushuang,Guo Shuai,Wang Xuzhao,Pang Chunli,Zhan Yong,Chen Yafei,An Hailong
Journal of cellular physiology
TMEM16A (also known as anoctamin 1, ANO1) is the molecular basis of the calcium-activated chloride channels, with ten transmembrane segments. Recently, atomic structures of the transmembrane domains of mouse TMEM16A (mTMEM16A) were determined by single-particle electron cryomicroscopy. This gives us a solid ground to discuss the electrophysiological properties and functions of TMEM16A. TMEM16A is reported to be dually regulated by Ca and voltage. In addition, the dysfunction of TMEM16A has been found to be involved in many diseases including cystic fibrosis, various cancers, hypertension, and gastrointestinal motility disorders. TMEM16A is overexpressed in many cancers, including gastrointestinal stromal tumors, gastric cancer, head and neck squamous cell carcinoma (HNSCC), colon cancer, pancreatic ductal adenocarcinoma, and esophageal cancer. Furthermore, overexpression of TMEM16A is related to the occurrence, proliferation, and migration of tumor cells. To date, several studies have shown that many natural compounds and synthetic compounds have regulatory effects on TMEM16A. These small molecule compounds might be novel drugs for the treatment of diseases caused by TMEM16A dysfunction in the future. In addition, recent studies have shown that TMEM16A plays different roles in different diseases through different signal transduction pathways. This review discusses the topology, electrophysiological properties, modulators and functions of TMEM16A in mediates nociception, gastrointestinal dysfunction, hypertension, and cancer and focuses on multiple regulatory mechanisms regarding TMEM16A.
10.1002/jcp.27865
Hypoxia increases the proliferation of brain capillary endothelial cells via upregulation of TMEM16A Ca-activated Cl channels.
Suzuki Takahisa,Suzuki Yoshiaki,Asai Kiyofumi,Imaizumi Yuji,Yamamura Hisao
Journal of pharmacological sciences
The blood-brain barrier (BBB) is mainly formed by brain capillary endothelial cells (BCECs) and is exposed to hypoxic environments under pathological conditions. The effects of hypoxia on the expression and activity of Ca-activated Cl (Cl) channels, TMEM16A, were examined in bovine brain endothelial t-BBEC117 cells and mouse BCECs. The expression of TMEM16A was upregulated by hypoxia. Whole-cell Cl currents increased under hypoxia. Hypoxia also increased cell proliferation and trans-endothelial permeability, which were attenuated by Cl channel blockers or TMEM16A siRNA. These findings are useful for elucidating the pathological role of TMEM16A Cl channels in the BBB during cerebral ischemia.
10.1016/j.jphs.2021.03.002
The diverse roles of TMEM16A Ca-activated Cl channels in inflammation.
Bai Weiliang,Liu Mei,Xiao Qinghuan
Journal of advanced research
Background:Transmembrane protein 16A (TMEM16A) Ca-activated Cl channels have diverse physiological functions, such as epithelial secretion of Cl and fluid and sensation of pain. Recent studies have demonstrated that TMEM16A contributes to the pathogenesis of infectious and non-infectious inflammatory diseases. However, the role of TMEM16A in inflammation has not been clearly elucidated. Aim of review:In this review, we aimed to provide comprehensive information regarding the roles of TMEM16A in inflammation by summarizing the mechanisms underlying TMEM16A expression and activation under inflammatory conditions, in addition to exploring the diverse inflammatory signaling pathways activated by TMEM16A. This review attempts to develop the idea that TMEM16A plays a diverse role in inflammatory processes and contributes to inflammatory diseases in a cellular environment-dependent manner. Key scientific concepts of review:Multiple inflammatory mediators, including cytokines (e.g., interleukin (IL)-4, IL-13, IL-6), histamine, bradykinin, and ATP/UTP, as well as bacterial and viral infections, promote TMEM16A expression and/or activity under inflammatory conditions. In addition, TMEM16A activates diverse inflammatory signaling pathways, including the IPR-mediated Ca signaling pathway, the NF-κB signaling pathway, and the ERK signaling pathway, and contributes to the pathogenesis of many inflammatory diseases. These diseases include airway inflammatory diseases, lipopolysaccharide-induced intestinal epithelial barrier dysfunction, acute pancreatitis, and steatohepatitis. TMEM16A also plays multiple roles in inflammatory processes by increasing vascular permeability and leukocyte adhesion, promoting inflammatory cytokine release, and sensing inflammation-induced pain. Furthermore, TMEM16A plays its diverse pathological roles in different inflammatory diseases depending on the disease severity, proliferating status of the cells, and its interacting partners. We herein propose cellular environment-dependent mechanisms that explain the diverse roles of TMEM16A in inflammation.
10.1016/j.jare.2021.01.013
Molecular mechanism of CaCC-A01 inhibiting TMEM16A channel.
Shi Sai,Guo Shuai,Chen Yafei,Sun Fude,Pang Chunli,Ma Biao,Qu Chang,An Hailong
Archives of biochemistry and biophysics
TMEM16A is a calcium-activated chloride channel that is associate with several diseases, including pulmonary diseases, hypertension, diarrhea and cancer. The CaCC-A01 (A01) is widely recognized as an efficient blocker of TMEM16A and has been used as a tool drug to inhibit TMEM16A currents in the laboratory. A01 also has excellent pharmacokinetic properties and can be developed as a drug to target TMEM16A. However, the molecular mechanism how A01 inhibits TMEM16A is still elusive, which slows down its drug development process. Here, calculations identified that the binding pocket of A01 was located above the pore, and it was also discovered that the binding of A01 to TMEM16A not only blocked the pore but also led to its collapse. The interaction model analysis predicted that R515/K603/E623 were crucial residues for the binding between TMEM16A and A01, and the site-directed mutagenesis studies confirmed the above results. The binding mode and quantum chemical calculations showed that the carboxyl and the amide oxygen atom of A01 were the key interaction sites between TMEM16A and A01. Therefore, our study proposed the inhibitory mechanism of TMEM16A current by A01 and revealed how A01 inhibits TMEM16A at the molecular level. These findings will shed light on both the development of A01 as a potential drug for TMEM16A dysfunction-related disorders and drug screening targeting the pocket.
10.1016/j.abb.2020.108650
Downregulation of Ca-Activated Cl Channel TMEM16A Mediated by Angiotensin II in Cirrhotic Portal Hypertensive Mice.
Kondo Rubii,Furukawa Nami,Deguchi Akari,Kawata Naoki,Suzuki Yoshiaki,Imaizumi Yuji,Yamamura Hisao
Frontiers in pharmacology
Portal hypertension is defined as an increased pressure in the portal venous system and occurs as a major complication in chronic liver diseases. The pathological mechanism underlying the pathogenesis and development of portal hypertension has been extensively investigated. Vascular tone of portal vein smooth muscles (PVSMs) is regulated by the activities of several ion channels, including Ca-activated Cl (Cl) channels. TMEM16A is mainly responsible for Cl channel conductance in vascular smooth muscle cells, including portal vein smooth muscle cells (PVSMCs). In the present study, the functional roles of TMEM16A channels were examined using two experimental portal hypertensive models, bile duct ligation (BDL) mice with cirrhotic portal hypertension and partial portal vein ligation (PPVL) mice with non-cirrhotic portal hypertension. Expression analyses revealed that the expression of TMEM16A was downregulated in BDL-PVSMs, but not in PPVL-PVSMs. Whole-cell Cl currents were smaller in BDL-PVSMCs than in sham- and PPVL-PVSMCs. The amplitude of spontaneous contractions was smaller and the frequency was higher in BDL-PVSMs than in sham- and PPVL-PVSMs. Spontaneous contractions sensitive to a specific inhibitor of TMEM16A channels, T16A-A01, were reduced in BDL-PVSMs. Furthermore, in normal PVSMs, the downregulation of TMEM16A expression was mimicked by the exposure to angiotensin II, but not to bilirubin. This study suggests that the activity of Cl channels is attenuated by the downregulation of TMEM16A expression in PVSMCs associated with cirrhotic portal hypertension, which is partly mediated by increased angiotensin II in cirrhosis.
10.3389/fphar.2022.831311
Function and Regulation of the Calcium-Activated Chloride Channel Anoctamin 1 (TMEM16A).
Handbook of experimental pharmacology
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.
10.1007/164_2022_592
Emerging Modulators of TMEM16A and Their Therapeutic Potential.
The Journal of membrane biology
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.
10.1007/s00232-021-00188-9
TMEM16A Ca-Activated Cl Channel Regulates the Proliferation and Migration of Brain Capillary Endothelial Cells.
Suzuki Takahisa,Yasumoto Miki,Suzuki Yoshiaki,Asai Kiyofumi,Imaizumi Yuji,Yamamura Hisao
Molecular pharmacology
The blood-brain barrier (BBB) is essential for the maintenance of homeostasis in the brain. Brain capillary endothelial cells (BCECs) comprise the BBB, and thus a delicate balance between their proliferation and death is required. Although the activity of ion channels in BCECs is involved in BBB functions, the underlying molecular mechanisms remain unclear. In the present study, the molecular components of Ca-activated Cl (Cl) channels and their physiological roles were examined using mouse BCECs (mBCECs) and a cell line derived from bovine BCECs, t-BBEC117. Expression analyses revealed that TMEM16A was strongly expressed in mBCECs and t-BBEC117 cells. In t-BBEC117 cells, whole-cell Cl currents were sensitive to the Cl channel blockers, 100 μM niflumic acid and 10 μM T16A-A01, and were also reduced markedly by small-interfering RNA (siRNA) knockdown of TMEM16A. Importantly, block of Cl currents with Cl channel blockers or TMEM16A siRNA induced membrane hyperpolarization. Moreover, treatment with TMEM16A siRNA caused an increase in resting cytosolic Ca concentration ([Ca]). T16A-A01 reduced cell viability in a concentration-dependent manner. Either Cl channel blockers or TMEM16A siRNA also curtailed cell proliferation and migration. Furthermore, Cl channel blockers attenuated the trans-endothelial permeability. In combination, these results strongly suggest that TMEM16A contributes to Cl channel conductance and can regulate both the resting membrane potential and [Ca] in BCECs. Our data also reveal how these BCECs may be involved in the maintenance of BBB functions, as both the proliferation and migration are altered following changes in channel activity. SIGNIFICANCE STATEMENT: In brain capillary endothelial cells (BCECs) of the blood-brain barrier (BBB), TMEM16A is responsible for Ca-activated Cl channels and can regulate both the resting membrane potential and cytosolic Ca concentration, contributing to the proliferation and migration of BCECs. The present study provides novel information on the molecular mechanisms underlying the physiological functions of BCECs in the BBB and a novel target for therapeutic drugs for disorders associated with dysfunctions in the BBB.
10.1124/mol.119.118844
Reduced Expression of TMEM16A Impairs Nitric Oxide-Dependent Cl Transport in Retinal Amacrine Cells.
Frontiers in cellular neuroscience
Postsynaptic cytosolic Cl concentration determines whether GABAergic and glycinergic synapses are inhibitory or excitatory. We have shown that nitric oxide (NO) initiates the release of Cl from acidic internal stores into the cytosol of retinal amacrine cells (ACs) thereby elevating cytosolic Cl. In addition, we found that cystic fibrosis transmembrane conductance regulator (CFTR) expression and Ca elevations are necessary for the transient effects of NO on cytosolic Cl levels, but the mechanism remains to be elucidated. Here, we investigated the involvement of TMEM16A as a possible link between Ca elevations and cytosolic Cl release. TMEM16A is a Ca-activated Cl channel that is functionally coupled with CFTR in epithelia. Both proteins are also expressed in neurons. Based on this and its Ca dependence, we test the hypothesis that TMEM16A participates in the NO-dependent elevation in cytosolic Cl in ACs. Chick retina ACs express TMEM16A as shown by Western blot analysis, single-cell PCR, and immunocytochemistry. Electrophysiology experiments demonstrate that TMEM16A functions in amacrine cells. Pharmacological inhibition of TMEM16A with T16inh-AO1 reduces the NO-dependent Cl release as indicated by the diminished shift in the reversal potential of GABA receptor-mediated currents. We confirmed the involvement of TMEM16A in the NO-dependent Cl release using CRISPR/Cas9 knockdown of TMEM16A. Two different modalities targeting the gene for TMEM16A () were tested in retinal amacrine cells: an all-in-one plasmid vector and crRNA/tracrRNA/Cas9 ribonucleoprotein. The all-in-one CRISPR/Cas9 modality did not change the expression of TMEM16A protein and produced no change in the response to NO. However, TMEM16A-specific crRNA/tracrRNA/Cas9 ribonucleoprotein effectively reduces both TMEM16A protein levels and the NO-dependent shift in the reversal potential of GABA-gated currents. These results show that TMEM16A plays a role in the NO-dependent Cl release from retinal ACs.
10.3389/fncel.2022.937060
Insights into the function and regulation of the calcium-activated chloride channel TMEM16A.
Cell calcium
The TMEM16A channel, a member of the TMEM16 protein family comprising chloride (Cl) channels and lipid scramblases, is activated by the free intracellular Ca increments produced by inositol 1,4,5-trisphosphate (IP3)-induced Ca release after GqPCRs or Ca entry through cationic channels. It is a ubiquitous transmembrane protein that participates in multiple physiological functions essential to mammals' lives. TMEM16A structure contains two identical 10-segment monomers joined at their transmembrane segment 10. Each monomer harbours one independent hourglass-shaped pore gated by Ca ligation to an orthosteric site adjacent to the pore and controlled by two gates. The orthosteric site is created by assembling negatively charged glutamate side chains near the pore´s cytosolic end. When empty, this site generates an electrostatic barrier that controls channel rectification. In addition, an isoleucine-triad forms a hydrophobic gate at the boundary of the cytosolic vestibule and the inner side of the neck. When the cytosolic Ca rises, one or two Ca ions bind to the orthosteric site in a voltage (V)-dependent manner, thus neutralising the electrostatic barrier and triggering an allosteric gating mechanism propagating via transmembrane segment 6 to the hydrophobic gate. These coordinated events lead to pore opening, allowing the Cl flux to ensure the physiological response. The Ca-dependent function of TMEM16A is highly regulated. Anions with higher permeability than Cl facilitate V dependence by increasing the Ca sensitivity, intracellular protons can replace Ca and induce channel opening, and phosphatidylinositol 4,5-bisphosphate bound to four cytosolic sites likely maintains Ca sensitivity. Additional regulation is afforded by cytosolic proteins, most likely by phosphorylation and protein-protein interaction mechanisms.
10.1016/j.ceca.2024.102891
Ca-activated Cl channel TMEM16A inhibition by cholesterol promotes angiogenesis in endothelial cells.
Ma Ke,Liu Sitong,Liang Hongyue,Wang Guan,Wang Tianyu,Luo Shuya,Gao Kuan,Wang Hui,Liu Mei,Bai Lichuan,Xiao Qinghuan
Journal of advanced research
Introduction:Ca-activated Cl channel TMEM16A is expressed in endothelial cells, and contributes to many diseases such as hypertension, blood-brain barrier dysfunction, and pulmonary hypertension. It remains unclear whether TMEM16A regulates endothelial angiogenesis, which participates in many physiological and pathological processes. Cholesterol regulates many ion channels including TMEM16A, and high cholesterol levels contribute to endothelial dysfunction. It remains to be determined whether cholesterol regulates TMEM16A expression and function in endothelial cells. Objective:This study aimed to investigate whether cholesterol regulated TMEM16A expression and function in endothelial angiogenesis. Methods:Whole-cell patch clamp techniques were used to record Ca-activated Cl currents in human aortic endothelial cells (HAECs) and HEK293 cells transfected with TMEM16A-overexpressing plasmids. Western blot was used to examine the expression of TMEM16A and DNA methyltransferase 1 (DNMT1) in HAECs. CCK-8 assay, would healing assay, and tube formation assay were used to test endothelial cell proliferation, migration and angiogenesis, respectively. Results:TMEM16A mediates the Ca-activated Cl channel in HAECs. Cholesterol treatment inhibited TMEM16A expression via upregulation of DNMT1 in HAECs, and the inhibitory effect of cholesterol on TMEM16A expression was blocked by 5-aza, the DNMT1 inhibitor. In addition, direct application of cholesterol inhibited TMEM16A currents in heterologous HEK293 cells with an IC of 0.1209 μM. Similarly, cholesterol directly inhibited TMEM16A currents in HAECs. Furthermore, TMEM16A knockdown increased tube formation, cell migration and proliferation of HAECs, and TMEM16A overexpression produced the opposite effect. Conclusion:This study reveals a novel mechanism of cholesterol-mediated TMEM16A inhibition, by which cholesterol reduces TMEM16A expression via DNMT1-mediated methylation and directly inhibits channel activities. TMEM16A channel inhibition promotes endothelial cell angiogenesis.
10.1016/j.jare.2020.09.003
Endothelial Dysfunction Following Enhanced TMEM16A Activity in Human Pulmonary Arteries.
Skofic Maurer Davor,Zabini Diana,Nagaraj Chandran,Sharma Neha,Lengyel Miklós,Nagy Bence M,Frank Saša,Klepetko Walter,Gschwandtner Elisabeth,Enyedi Péter,Kwapiszewska Grazyna,Olschewski Horst,Olschewski Andrea
Cells
Endothelial dysfunction is one of the hallmarks of different vascular diseases, including pulmonary arterial hypertension (PAH). Ion channelome changes have long been connected to vascular remodeling in PAH, yet only recently has the focus shifted towards Ca-activated Cl channels (CaCC). The most prominent member of the CaCC TMEM16A has been shown to contribute to the pathogenesis of idiopathic PAH (IPAH) in pulmonary arterial smooth muscle cells, however its role in the homeostasis of healthy human pulmonary arterial endothelial cells (PAECs) and in the development of endothelial dysfunction remains underrepresented. Here we report enhanced TMEM16A activity in IPAH PAECs by whole-cell patch-clamp recordings. Using adenoviral-mediated TMEM16A increase in healthy primary human PAECs in vitro and in human pulmonary arteries ex vivo, we demonstrate the functional consequences of the augmented TMEM16A activity: alterations of Ca dynamics and eNOS activity as well as decreased NO production, PAECs proliferation, wound healing, tube formation and acetylcholine-mediated relaxation of human pulmonary arteries. We propose that the ERK1/2 pathway is specifically affected by elevated TMEM16A activity, leading to these pathological changes. With this work we introduce increased TMEM16A activity in the cell membrane of human PAECs for the development of endothelial dysfunction in PAH.
10.3390/cells9091984
The role of Transmembrane Protein 16A (TMEM16A) in pulmonary hypertension.
Cardiovascular pathology : the official journal of the Society for Cardiovascular Pathology
Transmembrane protein 16A (TMEM16A), a member of the TMEM16 family, is the molecular basis of Ca2+-activated chloride channels (CaCCs) and is involved in a variety of physiological and pathological processes. Previous studies have focused more on respiratory-related diseases and tumors. However, recent studies have identified an important role for TMEM16A in cardiovascular diseases, especially in pulmonary hypertension. TMEM16A is expressed in both pulmonary artery smooth muscle cells and pulmonary artery endothelial cells and is involved in the development of pulmonary hypertension. This paper presents the structure and function of TMEM16A, the pathogenesis of pulmonary hypertension, and highlights the role and mechanism of TMEM16A in pulmonary hypertension, summarizing the controversies in this field and taking into account hypertension and portal hypertension, which have similar pathogenesis. It is hoped that the unique role of TMEM16A in pulmonary hypertension will be illustrated and provide ideas for research in this area.
10.1016/j.carpath.2023.107525
Vasodilators activate TMEM16A channels in endothelial cells to reduce blood pressure.
bioRxiv : the preprint server for biology
Endothelial cells (ECs) regulate vascular contractility to control regional organ blood flow and systemic blood pressure. Several cation channels are expressed in ECs which regulate arterial contractility. In contrast, the molecular identity and physiological functions of anion channels in ECs is unclear. Here, we generated tamoxifen-inducible, EC-specific knockout ( ecKO) mice to investigate the functional significance of this chloride (Cl ) channel in the resistance vasculature. Our data demonstrate that TMEM16A channels generate calcium-activated Cl currents in ECs of control ( ) mice that are absent in ECs of ecKO mice. Acetylcholine (ACh), a muscarinic receptor agonist, and GSK101, a TRPV4 agonist, activate TMEM16A currents in ECs. Single molecule localization microscopy data indicate that surface TMEM16A and TRPV4 clusters locate in very close nanoscale proximity, with ∼18% exhibiting overlap in ECs. ACh stimulates TMEM16A currents by activating Ca influx through surface TRPV4 channels without altering the size or density of TMEM16A or TRPV4 surface clusters, their spatial proximity or colocalization. ACh-induced activation of TMEM16A channels in ECs produces hyperpolarization in pressurized arteries. ACh, GSK101 and intraluminal ATP, another vasodilator, all dilate pressurized arteries through TMEM16A channel activation in ECs. Furthermore, EC-specific knockout of TMEM16A channels elevates systemic blood pressure in conscious mice. In summary, these data indicate that vasodilators stimulate TRPV4 channels, leading to Ca -dependent activation of nearby TMEM16A channels in ECs to produce arterial hyperpolarization, vasodilation and a reduction in blood pressure. We identify TMEM16A as an anion channel present in ECs that regulates arterial contractility and blood pressure. One sentence summary:Vasodilators stimulate TRPV4 channels, leading to calcium-dependent activation of nearby TMEM16A channels in ECs to produce arterial hyperpolarization, vasodilation and a reduction in blood pressure.
10.1101/2023.06.02.543450
The TMEM16A channel as a potential therapeutic target in vascular disease.
Current opinion in nephrology and hypertension
PURPOSE OF REVIEW:The transmembrane protein 16A (TMEM16A) Ca 2+ -activated Cl - channel constitutes a key depolarising mechanism in vascular smooth muscle and contractile pericytes, while in endothelial cells the channel is implicated in angiogenesis and in the response to vasoactive stimuli. Here, we offer a critical analysis of recent physiological investigations and consider the potential for targeting TMEM16A channels in vascular disease. RECENT FINDINGS:Genetic deletion or pharmacological inhibition of TMEM16A channels in vascular smooth muscle decreases artery tone and lowers systemic blood pressure in rodent models. Inhibition of TMEM16A channels in cerebral cortical pericytes protects against ischemia-induced tissue damage and improves microvascular blood flow in rodent stroke models. In endothelial cells, the TMEM16A channel plays varied roles including modulation of cell division and control of vessel tone through spread of hyperpolarisation to the smooth muscle cells. Genetic studies implicate TMEM16A channels in human disease including systemic and pulmonary hypertension, stroke and Moyamoya disease. SUMMARY:The TMEM16A channel regulates vascular function by controlling artery tone and capillary diameter as well as vessel formation and histology. Preclinical and clinical investigations are highlighting the potential for therapeutic exploitation of the channel in a range of maladaptive states of the (micro)circulation.
10.1097/MNH.0000000000000967