Podchanart Wanitchakool

Molecular functions of anoctamin 6 (TMEM16F): a chloride channel, cation channel, or phospholipid scramblase?

Anoctamin 6 (Ano6; TMEM16F gene) is a ubiquitous protein; the expression of which is defective in patients with Scott syndrome, an inherited bleeding disorder based on defective scrambling of plasma membrane phospholipids. For Ano6, quite diverse functions have been described: (1) it can form an outwardly rectifying, Ca2+-dependent and a volume-regulated Cl− channel; (2) it was claimed to be a Ca2+-regulated nonselective cation channel permeable for Ca2+; (3) it was shown to be essential for Ca2+-mediated scrambling of membrane phospholipids; and (4) it can regulate cell blebbing and microparticle shedding. Deficiency of Ano6 in blood cells from Scott patients or Ano6 null mice appears to affect all of these cell responses. Furthermore, Ano6 deficiency in mice impairs the mineralization of osteoblasts, resulting in reduced skeletal development. These diverse results have been obtained under different experimental conditions, which may explain some of the contradictions. This review therefore aims to summarize the currently available information on the diverse roles of Ano6 and tries to clear up some of the existing controversies.

Anoctamin 6 mediates effects essential for innate immunity downstream of P2X7 receptors in macrophages

Purinergic P2X7 receptors (P2X7R) are fundamental to innate immune response. In macrophages, transient stimulation of P2X7R activates several transport mechanisms and induces the scrambling of phospholipids with subsequent membrane blebbing and apoptosis. These processes support phagocytosis and subsequent killing of phagocytosed bacteria. Here we demonstrate that the stimulation of P2X7 receptors activates anoctamin 6 (ANO6, TMEM16F), a protein that functions as Ca(2+) dependent phospholipid scramblase and Ca(2+)-activated Cl(-) channel. Inhibition or knockdown of ANO6 attenuates ATP-induced cell shrinkage, cell migration and phospholipid scrambling. In mouse macrophages, Ano6 produces large ion currents by stimulation of P2X7 receptors and contributes to ATP-induced membrane blebbing and apoptosis, which is largely reduced in macrophages from Ano6-/- mice. ANO6 supports bacterial phagocytosis and killing by mouse and human THP-1 macrophages. Our data demonstrate that anoctamin 6 is an essential component of the immune defense by macrophages.

Role of anoctamins in cancer and apoptosis

Anoctamin 1 (TMEM16A, Ano1) is a recently identified Ca2+-activated chloride channel and a member of a large protein family comprising 10 paralogues. Before Ano1 was identified as a chloride channel protein, it was known as the cancer marker DOG1. DOG1/Ano1 is expressed in gastrointestinal stromal tumours (GIST) and particularly in head and neck squamous cell carcinoma, at very high levels never detected in other tissues. It is now emerging that Ano1 is part of the 11q13 locus, amplified in several types of tumour, where it is thought to augment cell proliferation, cell migration and metastasis. Notably, Ano1 is upregulated through histone deacetylase (HDAC), corresponding to the known role of HDAC in HNSCC. As Ano1 does not enhance proliferation in every cell type, its function is perhaps modulated by cell-specific factors, or by the abundance of other anoctamins. Thus Ano6, by regulating Ca2+-induced membrane phospholipid scrambling and annexin V binding, supports cellular apoptosis rather than proliferation. Current findings implicate other cellular functions of anoctamins, apart from their role as Ca2+-activated Cl− channels.

Anoctamins support calcium-dependent chloride secretion by facilitating calcium signaling in adult mouse intestine

Intestinal epithelial electrolyte secretion is activated by increase in intracellular cAMP or Ca2+ and opening of apical Cl− channels. In infants and young animals, but not in adults, Ca2+-activated chloride channels may cause secretory diarrhea during rotavirus infection. While detailed knowledge exists concerning the contribution of cAMP-activated cystic fibrosis transmembrane conductance regulator (CFTR) channels, analysis of the role of Ca2+-dependent Cl− channels became possible through identification of the anoctamin (TMEM16) family of proteins. We demonstrate expression of several anoctamin paralogues in mouse small and large intestines. Using intestinal-specific mouse knockout models for anoctamin 1 (Ano1) and anoctamin 10 (Ano10) and a conventional knockout model for anoctamin 6 (Ano6), we demonstrate the role of anoctamins for Ca2+-dependent Cl− secretion induced by the muscarinic agonist carbachol (CCH). Ano1 is preferentially expressed in the ileum and large intestine, where it supports Ca2+-activated Cl− secretion. In contrast, Ano10 is essential for Ca2+-dependent Cl− secretion in jejunum, where expression of Ano1 was not detected. Although broadly expressed, Ano6 has no role in intestinal cholinergic Cl− secretion. Ano1 is located in a basolateral compartment/membrane rather than in the apical membrane, where it supports CCH-induced Ca2+ increase, while the essential and possibly only apical Cl− channel is CFTR. These results define a new role of Ano1 for intestinal Ca2+-dependent Cl− secretion and demonstrate for the first time a contribution of Ano10 to intestinal transport.

Modulating Ca2+ signals: a common theme for TMEM16, Ist2, and TMC

Since the discovery of TMEM16A (anoctamin 1, ANO1) as Ca2+-activated Cl− channel, the protein was found to serve different physiological functions, depending on the type of tissue. Subsequent reports on other members of the anoctamin family demonstrated a broad range of yet poorly understood properties. Compromised anoctamin function is causing a wide range of diseases, such as hearing loss (ANO2), bleeding disorder (ANO6), ataxia and dystonia (ANO3, 10), persistent borrelia and mycobacteria infection (ANO10), skeletal syndromes like gnathodiaphyseal dysplasia and limb girdle muscle dystrophy (ANO5), and cancer (ANO1, 6, 7). Animal models demonstrate CF-like airway disease, asthma, and intestinal hyposecretion (ANO1). Although present data indicate that ANO1 is a Ca2+-activated Cl− channel, it remains unclear whether all anoctamins form plasma membrane-localized or intracellular chloride channels. We find Ca2+-activated Cl− currents appearing by expression of most anoctamin paralogs, including the Nectria haematococca homologue nhTMEM16 and the yeast homologue Ist2. As recent studies show a role of anoctamins, Ist2, and the related transmembrane channel-like (TMC) proteins for intracellular Ca2+ signaling, we will discuss the role of these proteins in generating compartmentalized Ca2+ signals, which may give a hint as to the broad range of cellular functions of anoctamins.

A Coding Variant of ANO10, Affecting Volume Regulation of Macrophages, Is Associated with Borrelia Seropositivity

In a first genome-wide association study (GWAS) approach to anti-Borrelia seropositivity, we identified two significant single nucleotide polymorphisms (SNPs) (rs17850869, P = 4.17E-09; rs41289586, P = 7.18E-08). Both markers, located on chromosomes 16 and 3, respectively, are within or close to genes previously connected to spinocerebellar ataxia. The risk SNP rs41289586 represents a missense variant (R263H) of anoctamin 10 (ANO10), a member of a protein family encoding Cl− channels and phospholipid scramblases. ANO10 augments volume-regulated Cl− currents (IHypo) in Xenopus oocytes, HEK293 cells, lymphocytes and macrophages and controls volume regulation by enhancing regulatory volume decrease (RVD). ANO10 supports migration of macrophages and phagocytosis of spirochetes. The R263H variant is inhibitory on IHypo, RVD and intracellular Ca2+ signals, which may delay spirochete clearance, thereby sensitizing adaptive immunity. Our data demonstrate for the first time that ANO10 has a central role in innate immune defense against Borrelia infection.

Control of TMEM16A by INO-4995 and other inositolphosphates

Ca2+‐dependent Cl− secretion (CaCC) in airways and other tissues is due to activation of the Cl− channel TMEM16A (anoctamin 1). Earlier studies suggested that Ca2+‐activated Cl− channels are regulated by membrane lipid inositol phosphates, and that 1‐O‐octyl‐2‐O‐butyryl‐myo‐inositol 3,4,5,6‐tetrakisphosphate octakis(propionoxymethyl) ester (INO‐4995) augments CaCC. Here we examined whether TMEM16A is the target for INO‐4995 and if the channel is regulated by inositol phosphates.

Cellular volume regulation by anoctamin 6: Ca2+, phospholipase A2 and osmosensing

During cell swelling, Cl− channels are activated to lower intracellular Cl− concentrations and to reduce cell volume, a process termed regulatory volume decrease (RVD). We show that anoctamin 6 (ANO6; TMEM16F) produces volume-regulated anion currents and controls cell volume in four unrelated cell types. Volume regulation is compromised in freshly isolated intestinal epithelial cells from Ano6−/− mice and also in lymphocytes from a patient lacking expression of ANO6. Ca2+ influx is activated and thus ANO6 is stimulated during cell swelling by local Ca2+ increase probably in functional nanodomains near the plasma membrane. This leads to stimulation of phospholipase A2 (PLA2) and generation of plasma membrane lysophospholipids, which activates ANO6. Direct application of lysophospholipids also activates an anion current that is inhibited by typical ANO6 blocker. An increase in intracellular Ca2+ supports activation of ANO6, but is not required when PLA2 is fully activated, while re-addition of arachidonic acid completely blocked ANO6. Moreover, ANO6 is activated by low intracellular Cl− concentrations and may therefore operate as a cellular osmosensor. High intracellular Cl− concentration inhibits ANO6 and activation by PLA2. Taken together, ANO6 supports volume regulation and volume activation of anion currents by action as a Cl− channel or by scrambling membrane phospholipids. Thereby, it may support the function of LRRC8 proteins.

Non-essential contribution of LRRC8A to volume regulation

Volume regulation is an essential property of any living cell and needs to be tightly controlled. While different types of K+ channels have been found to participate in the regulation of cell volume, the newly identified volume-regulated anion channel (VRAC) LRRC8 has been claimed to be essential for volume regulation. In unbiased genome-wide small interfering RNA (siRNA) screens, two independent studies identified LRRC8A/Swell1 as an essential component of VRAC, thus being indispensable for cellular volume regulation. We reanalyzed the role of LRRC8A for VRAC and regulatory volume decrease (RVD) in several cell types and under various conditions. While the role of LRRC8A for VRAC and its contribution to RVD is confirmed, we find that it is not essential for swelling-activated anion currents or cellular volume regulation, or apoptotic cell shrinkage. The contribution of LRRC8A is variable and largely depending on the cell type.

Cl(-) channels in apoptosis.

A remarkable feature of apoptosis is the initial massive cell shrinkage, which requires opening of ion channels to allow release of K+, Cl−, and organic osmolytes to drive osmotic water movement and cell shrinkage. This article focuses on the role of the Cl− channels LRRC8, TMEM16/anoctamin, and cystic fibrosis transmembrane conductance regulator (CFTR) in cellular apoptosis. LRRC8A-E has been identified as a volume-regulated anion channel expressed in many cell types. It was shown to be required for regulatory and apoptotic volume decrease (RVD, AVD) in cultured cell lines. Its presence also determines sensitivity towards cytostatic drugs such as cisplatin. Recent data point to a molecular and functional relationship of LRRC8A and anoctamins (ANOs). ANO6, 9, and 10 (TMEM16F, J, and K) augment apoptotic Cl− currents and AVD, but it remains unclear whether these anoctamins operate as Cl− channels or as regulators of other apoptotic Cl− channels, such as LRRC8. CFTR has been known for its proapoptotic effects for some time, and this effect may be based on glutathione release from the cell and increase in cytosolic reactive oxygen species (ROS). Although we find that CFTR is activated by cell swelling, it is possible that CFTR serves RVD/AVD through accumulation of ROS and activation of independent membrane channels such as ANO6. Thus activation of ANO6 will support cell shrinkage and induce additional apoptotic events, such as membrane phospholipid scrambling.