Volume regulations by Anoctamins, Bestrophin1 and Transmembrane Channel like proteins

Volume regulation is a basic cellular property, which is required in every living organism. Cells that are subjected to hypo-osmotic shock will start to swell and subsequently will release K+ and Cl- through K+ and Cl- channels in order to restore their original volume. In 2014, two independent groups identified LRRC8A as a major component of Volume Regulated Anion Channels (VRAC). This thesis provides evidences, that in addition to LRRC8A other chloride channels, namely anoctamins or TMEM16 and bestrophin 1 are involved in volume regulation and offers a putative mechanic model for VRAC activation. Additionally, regarding the high sequence similarity between Anoctamins and TMC (Transmembrane channel like proteins), the role of TMC in volume regulation and Ca2+ signaling is investigated. Patch clamp analysis of ANO6 (Anoctamin 6 or TMEM16F) overexpressing HEK 293 cells shows a two fold increase in swelling activated whole cell currents compared to control cells. These currents are inhibited by a potent inhibitor of VRAC as well as a number of inhibitors of Anoctamins. VRAC activity and RVD (Regulatory Volume Decrease) are strongly reduced when Ca2+ is removed and the mechanisms are depending on the activation of ANO6. In fact membrane stretch due to cell swelling activates TRP (Transient Receptor Potential) channels, therefore allowing Ca2+ to enter the cells. Simultaneously the intracellular Cl- concentration is diluted due to water influx and releases ANO6 from Cl- inhibition. In addition Ca2+ entering cells during cell swelling may further activate PLA2 (phospholipase A2). This phospholipase cleaves fatty acid from the plasma membrane and the subsequent accumulation of lysophospholipids leads to membrane tension which - together with increased Ca2+ levels - activates ANO6 at the junctional zone. In addition to ANO6, ANO10 (Anoctamin 10 or TMEM16K) also plays a critical role in volume regulation. This function is identified in Xenopus laevis oocytes and HEK293 cells. It appears that the expression of ANO10 produces large outwardly rectifying whole cell currents when cells are subjected to hypotonic solution. On the other hands, expression of R263H-ANO10, a coding variant of ANO10, fails to produce swelling-activated whole cell currents. ANO10 also augments Ca2+ signaling in HEK293 overexpressing cells, while Ca2+ signaling is reduced when R263H-ANO10 is expressed. These results are in agreement to the localization of ANO10 which we found to be mainly in the endoplasmic reticulum (ER). Therefore we suggest that ANO10 may facilitate volume regulation by changing Ca2+ signaling. Since volume regulation is known to be involved in cell migration, the role of ANO10 on macrophage migration and on their phagocytosis activity is investigated. The study shows that both migration and phagocytotic activity are reduced in the absence of ANO10. These results suggest that ANO10 is a new player in the innate immune system, controlling volume regulation and macrophage function. Bestrophin 1 is known to be a Ca2+ activated Cl- channel in the retinal pigment epithelial cells (RPE). Its role in volume regulation was also investigated. Interestingly, Best1 knock out mice show impairment related to volume regulation such as sperm morphology and motility. A study conducted in RPE cells isolated from patients with an inherited from of macular dystrophy, carrying heterozygous mutations in Best1, shows absence of swelling-activated whole cell currents, while knockdown of LRRC8A (a putative major component of VRAC) in healthy RPE cells does not affect swelling-activated whole cell currents. Therefore Best1 is suggested to be an essential component of VRAC in human RPE cells and mouse sperm. Since TMC proteins show sequence similarities to anoctamins, we examined their role in volume regulation. However, none of the TMCs can augment swelling-activated whole cell currents. Surprisingly TMC8 largely reduced swelling-activated whole cell currents in overexpressing HEK293 cells. Not only the swelling-activated currents, but also RVD is largely compromised in the presence of TMC8. Ca2+ measurements reveal that TMC8 inhibits ATP-induced Ca2+ release without affecting Ca2+ level in the ER store or SOCE (Store Operated Calcium Entry). Chelation of intracellular Zn2+ largely inhibited ATP-induced Ca2+ release as well as ATP-activated ANO1 currents. These and further results suggest that upon stimulation with ATP, Zn2+ is co-released with Ca2+ and further augment Ca2+ release from the store. The presence of TMC8 interferes with this process, since it is known that TMC8 interacts with ZnT1-transporter and regulate Zn2+ re-uptake into the ER via ZnT-1

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 A(2) (PLA(2)) 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 PLA(2) 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 PLA(2). 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

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

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

Cellular defects by deletion of ANO10 are due to deregulated local calcium signaling

TMEM16K (ANO10) belongs to a family of ion channels and phospholipid scramblases. Mutations in ANO10 cause neurological and immunological defects, and abrogated ion transport. Here we show that Ano10 knockout in epithelial cells leads to defective ion transport, attenuated volume regulation and deranged Ca2+ signaling. Intestinal epithelial cells from Ano10 null mice are reduced in size and demonstrate an almost abolished spontaneous and TNF alpha-induced apoptosis. Similar defects were found in mouse peritoneal Ano10 null macrophages and in human THP1 macrophages with reduced ANO10 expression. A cell cycle dependent colocalization of Ano10 with acetylated tubulin, centrioles, and a submembranous tubulin containing compartment was observed in Fisher rat thyroid cells. Axs, the Drosophila ortholog of ANO10 is known for its role in mitotic spindle formation and association with the endoplasmic reticulum and Ca2+ signaling. We therefore propose that mutations in ANO10 cause cellular defects and genetic disorders through deranged local Ca2+ signaling. (C) 2016 Elsevier Inc. All rights reserved

Relationship between TMEM16A/anoctamin 1 and LRRC8A

TMEM16A/anoctamin 1/ANO1 and VRAC/LRRC8 are independent chloride channels activated either by increase in intracellular Ca2+ or cell swelling, respectively. In previous studies, we observed overlapping properties for both types of channels. (i) TMEM16A/ANO1 and LRRC8 are inhibited by identical compounds, (ii) the volume-regulated anion channel VRAC requires compartmentalized Ca2+ increase to be fully activated, (iii) anoctamins are activated by cell swelling, (iv) both channels have a role for apoptotic cell death, (v) both channels are possibly located in lipid rafts/caveolae like structures, and (vi) VRAC and anoctamin 1 currents are not additive when each are fully activated. In the present study, we demonstrate in different cell types that loss of LRRC8A expression not only inhibited VRAC, but also attenuated Ca2+ activated Cl- currents. Moreover, expression of LRRC8A enhanced Ca2+ activated Cl- currents, and both LRRC8A and ANO1 could be coimmunoprecipitated. We found that LRRC8A becomes accessible to biotinylation upon exposure to hypotonic bath solution, while membrane capacitance was not enhanced. When intracellular Ca2+ was increased in ANO1-expressing cells, the membrane capacitance was enhanced and increased binding of FM4-64 to the membrane was observed. As this was not seen in cells lacking ANO1 expression, a role of ANO1 for exocytosis was suggested. We propose that ANO1 and LRRC8A are activated in parallel. Thus, ionomycin or purinergic stimulation will not only activate ANO1 but also LRRC8 currents. Cell swelling will not only activate LRRC8/VRAC, but also stimulate ANO1 currents by enhancing compartmentalized Ca2+ increase and/or through swelling induced autocrine release of ATP

Ca2+ signals, cell membrane disintegration, and activation of TMEM16F during necroptosis

Activated receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain like (MLKL) are essential components of the necroptotic pathway. Phosphorylated MLKL (pMLKL) is thought to induce membrane leakage, leading to cell swelling and disintegration of the cell membrane. However, the molecular identity of the necroptotic membrane pore remains unclear, and the role of pMLKL for membrane permeabilization is currently disputed. We observed earlier that the phospholipid scramblase and ion channel TMEM16F/anoctamin 6 cause large membrane currents, cell swelling, and cell death when activated by a strong increase in intracellular Ca2+. We, therefore, asked whether TMEM16F is also central to necroptotic cell death and other cellular events during necroptosis. Necroptosis was induced by TNF alpha, smac mimetic, and Z-VAD (TSZ) in NIH3T3 fibroblasts and the four additional cell lines HT29, 16HBE, H441, and L929. Time-dependent changes in intracellular Ca2+, cell morphology, and membrane currents were recorded. TSZ induced a small and only transient oscillatory rise in intracellular Ca2+, which was paralleled by the activation of outwardly rectifying Cl- currents, which were typical for TMEM16F/ANO6. Ca2+ oscillations were due to Ca2+ release from endoplasmic reticulum, and were independent of extracellular Ca2+. The initial TSZ-induced cell swelling was followed by cell shrinkage. Using typical channel blockers and siRNA-knockdown, the Cl- currents were shown to be due to the activation of ANO6. However, the knockdown of ANO6 or inhibitors of ANO6 did not inhibit necroptotic cell death. The present data demonstrate the activation of ANO6 during necroptosis, which, however, is not essential for cell death

Differential effects of anoctamins on intracellular calcium signals

The Ca2+-activated Cl- channel TMEM16A[anoctamin(ANO)1] is homologous to yeast Ist2 and has been shown to tether the cortical endoplasmic reticulum(ER) to the plasma membrane. We therefore examined whether ANO1 and other members of the ANO family affect intracellular Ca2+ ([Ca2+](i)) signals. It is shown that expression of ANO1 augments Ca2+ store release upon stimulation of GPCRs, whereas knockdown of ANO1, or lack of Ano1 expression in Ano1(-/-) animals, as shown in an earlier report, inhibits Ca2+ release. ANO6, and -10 show similar effects, whereas expression of ANO4, -8, and -9 attenuate filling of the ER store. The impact of ANO1 and -4 were examined in more detail. ANO1 colocalized and interacted with IP3R, where as ANO4 colocalized with SERCA Ca2+ pumps and interacted with ORAI-1 channels, respectively. ANO1 Cl currents were rapidly activated exclusively through Ca2+ store release, and remained untouched by influx of extracellular Ca2+. In contrast expression of ANO4 caused a delayed activation of membrane-localized ANO6 channels, solely through store-operated Ca2+ entry via ORAI. Ca2+ signals were inhibited by knocking down expression of endogenous ANO1, -5, -6, and -10 and were also reduced in epithelial cells from Ano10(-/-) mice. The data suggest that ANOs affect compartmentalized [Ca2+](i) signals, which may explain some of the cellular defects related to ANO mutations.-Cabrita, I., Benedetto, R., Fonseca, A., Wanitchakool, P., Sirianant, L., Skryabin, B. V., Schenk, L. K., Pavenstadt, H., Schreiber, R., Kunzelmann, K. Differential effects of anoctamins on intracellular calcium signals