Functions of volume-sensitive and calcium-activated chloride channels

Authors


Abstract

The review describes molecular and functional properties of the volume regulated anion channel and Ca2+-dependent Cl channels belonging to the anoctamin family with emphasis on physiological importance of these channels in regulation of cell volume, cell migration, cell proliferation, and programmed cell death. Finally, we discuss the role of Cl channels in various diseases. © 2014 IUBMB Life, 66(4):257–267, 2014

Introduction

Anion channels are present in the plasma membrane as well as membranes of organelles and vesicles. Chloride is the anion present at highest concentration in cells and the anion channels are therefore often referred to simply as chloride channels, even though they may be permeable for other anions such as bicarbonate, bromide, iodide, thiocyanate, and small organic osmolytes, for example, taurine.

Based on gating characteristics, voltage- and time-dependence, permeability sequence, single-channel conductance, and sensitivity to chloride channel blockers, chloride channels have been classified into five functional groups: (i) extracellular ligand-gated channels (GlyR and GABAAR), (ii) voltage-gated chloride channels (ClC-family), (iii) cAMP-PKA activated channel (CFTR), (iv) volume-regulated anion channel (VRAC), and (v) calcium-activated chloride channels (CaCCs) [1]. A number of chloride channel families have been identified at the molecular level and as the newest example the Anoctamin/TMEM16 family was cloned and ANO1, ANO2, and ANO6 have been demonstrated to be CaCCs [2-8]. The protein nomenclature for human anoctamin, that is, ANO is used for anoctamins off all origins throughout the article. The molecular identity of several Cl channels, for example, VRAC is still unknown.

In this review, we focus on the physiological role of CaCCs of the Anoctamin family (ANO), VRAC, and the volume sensitive organic anion channel (VSOAC) in cell volume regulation, cell proliferation, and cell death. Table 1 is a pharmacological characterization of VRAC and VSOAC and Table 2 compares biophysical and biological features of VRAC and CaCCs. For GlyR and GABAAR, which are activated by the neurotransmitters glycine and γ-aminobutyric acid (GABA), respectively, see ref. [1]. For the ClC family see refs. [9-15]. For CFTR, which is a large, integral membrane glycoprotein belonging to the ATP-binding cassette transporter superfamily see refs. [16-18].

Table 1. Comparison of VSOAC with VRAC
 VSOACVRAC
Volume sensitivity [19]Activated by osmotic cell swelling and inhibited by osmotic cell shrinkage
Osmotic set-point [65]VRAC < VSOAC
Time course for swelling induced activation [66, 67]Slow activation and inactivationFast activation and inactivation
Sensitivity to membrane potential [19]Inhibited by depolarizationOutward rectification—varying degree of inactivation at positive potentials
Modulation of volume sensitivity by RhoA [27]NoYes
Inhibition 5-LO inhibitors(NDGA, ETH615-139) [68-70]YesYes
LTD4Stimulates [71]No effect [72]
Reactive oxygen speciesModulates [60]Activates in adherent cells [31, 32, 73]
DIDSStrong inhibition even at low concentrations [61]Weak inhibition and only at positive potentials [74]
NS3728Inhibits [55]Inhibits [46]
Downregulated in MDR [54]YesYes
Cholesterol depletionInhibits [75]Potentiates [28]
Arachidonic acid [61]ActivatesInhibits
Table 2. Comparison of anoctamins with VRAC
 AnoctaminsVRAC
Permeability sequenceType I Eisenman
 SCN > I > NO3 > Br > Cl > F > gluconate
RectificationOutward rectification
Anion channel inhibitor, NS3728 [46]Inhibits (ANO1)Inhibits
Arachidonic acid [128]No effect on CACCInhibits
Isovolumic activation by a decrease in intracellular ionic strength [124]NoYes
Isovolumic activation by perfusion with GTPγS [124]NoYes
Response to a V-step into the positive region of membrane potentialsslowly activated [129]slowly deactivated [19, 81]
Activation of current during RVD in the absence of Ca2+ [64]NoYes
Role in RVD in the absence of extracellular Ca2+ [64]NoYes
Role in RVD in the presence of extracellular Ca2+Yes (ANO6) [64, 123]Yes
 No (ANO6) [8] 

Volume-Regulated Anion Channel

Cell volume regulation is a fundamental homeostatic mechanism. When cells are exposed to a hypo-osmotic shock, they initially swell but subsequently restore their initial cell volume—a process termed regulatory volume decrease (RVD) [19]. This is partly due to a transiently increased Cl permeability, which was first described in Ehrlich cells and later in lymphocytes [20]. Patch clamp experiments have shown the presence of an anion current (ICl.Swell) with outward rectification of the current–voltage relationship, which is activated by cell swelling and inhibited by cell shrinkage in almost all cell types investigated. As few cell-types are exposed to the kind of osmotic shock commonly used in many experimental protocols to activate VRAC, its ubiquitous presence suggests other roles for VRAC and the channel has accordingly been shown to be important for both cell proliferation and apoptosis [19]. VRAC has a biophysical fingerprint that is conserved between different cell-types: (i) a type I Eisenman permeability sequence (I > Br > Cl), (ii) outward rectification, and (iii) slow deactivation at positive (>40 mV) potentials [19, 21-23]. The current can be activated in the absence of free Ca2+ but does require ATP [24].

It has been proposed that a decrease in the intracellular ionic strength is a main signal for VRAC activation [25]. Furthermore, the small G-protein RhoA has been shown to play a role in the activation of VRAC [26, 27], presumably by modulation of F-actin polymerization, which is likewise affected by osmotic cell swelling [28]. Various lipids have been shown to affect VRAC activity, that is, arachidonic acid directly inhibits VRAC in most cell types, whereas PIP3 might stimulate VRAC [29]. The role of phosphorylation in the regulation of VRAC is still unclear as results are contradictory. However, pharmacological investigations indicate a role for tyrosine kinases [19]. Finally, reactive oxygen species (ROS), which are released upon cell swelling in some cell types, also seem to stimulate VRAC [30-32].

The molecular identity of VRAC is still undefined. Several proteins have been suggested to be VRAC but have been excluded because their electrical profile was different, there was no effect on VRAC in knock-out mice [19], and/or other properties were different from VRAC [1]. More recently members of the Anoctamin family have been shown not to be VRAC candidates (see below).

Role of VRAC in Proliferation and Apoptosis: Pathophysiological Significance

Various physiological functions have been attributed to VRAC, for example, VRAC modulates cardiac electrical activity, glucose sensing in the pancreatic β-cell, aqueous humour secretion, as well as vectorial transcellular Cl transport [22, 33]. In addition VRAC has been assigned a role in cell proliferation and apoptosis, which will be described below.

VRAC and Cell Cycle Progression/Proliferation

Ionic movements and resulting osmotic swelling or shrinkage have been shown essential for regulating cell proliferation [34, 35] and the role for ion channels in cell proliferation is well documented in many cell types [36]. The functional importance of volume changes during the cell cycle is best understood in glia cells, thanks to an excellent series of studies by Habela and Sontheimer [37-39]. Chloride has an essential role in cell proliferation and a role for Cl and Cl channels can be speculated into all parts of the cell cycle. In particular Cl seems important for the G1/S phase transition as many cell types are arrested in G1 by Cl channel inhibitors [14, 40-42], whereas in glia and CHO-K1 cells inhibition of ClC-3 and CLIC1, respectively, inhibits cell division in the M-phase [38, 43]. According to Sontheimer and coworkers an additional Cl channel contributes to cell proliferation, but the molecular identity has not yet been established [44]. Hence, based on our current knowledge it is very difficult to pinpoint one Cl channel with importance for proliferation, although VRAC is most likely required for cell proliferation, as VRAC inhibitors have been shown to inhibit proliferation in many cell-types, normally by cell cycle arrest in the G0–G1 phase [45-49]. It should be noted that conclusions based only on VRAC inhibitors may be affected by their low selectivity. VRAC activity was found to be cell-cycle dependent in Ehrlich Lettré ascites tumor cells (ELA), for example, decreasing from G0 to G1, and increased again from G1 to S [46]. A mammalian cell in G0 thus has two possibilities it can either decrease VRAC activity and progress into G1 or increase VRAC activity and progress into apoptosis. The decreased VRAC activity observed in multidrug resistant Ehrlich ascites tumor cells (MDR EATC) cells thus both favors cell cycle progression and prevents apoptosis [50]. It is noted that Cl channels play multiple roles in cell cycle progression in ELA cells, that is, CIC-1 and ClC-2 are downregulated at the plasma membrane in G1-S leading to hyperpolarization of the plasma membrane and hence facilitation of Ca2+ oscillations, whereas VRAC is upregulated in the S-phase which secures the volume-regulatory capacity necessary for further cell cycle progression [51].

VRAC and Apoptosis

VRAC activation has been shown in the early phase of apoptosis in a broad range of cell types [52]. This is deducted from the electrical and pharmacological profiles of VRAC and the anion current activated during apoptosis [52]. The classical hallmarks of apoptosis include an initial cell shrinkage termed apoptotic volume decrease (AVD), resulting from net loss of KCl, which is followed by caspase activation and DNA break down. VRAC and the volume sensitive organic anion channel (VSOAC, see below) contribute to AVD and inhibition of VRAC and VSOAC activities can block AVD and cell death [52-55]. As VRAC is normally silent under isotonic conditions a shift in its volume set-point towards a lower value by apoptotic stimuli is required for activation [31]. The volume set-point is reduced by tyrosine phosphatase inhibitors in, for example, EATC [19] and as ROS are released during apoptosis [31], a ROS induced oxidation of a cysteine in the catalytic site and hence inactivation of protein tyrosine phosphatases could explain a reduction in the set-point for VRAC. In agreement with the increased Cl conductance during AVD the cells are depolarized which will increase the driving force for the K+ loss [52]. Recently it has become clear that multi drug resistance to chemotherapy involves down regulation in the activity of ion channels and hence limitation in the initial cell shrinkage and initiation of apoptosis [50, 56]. Reduction in VRAC current has been correlated with drug resistance in several cell lines [54, 57, 58]. In EATC, it has been shown that inhibition of VRAC abrogates AVD and makes wild-type cells as resistant to the chemotherapeutic drug cisplatin as the multidrug resistant MDR EATC [54].

Volume Sensitive Organic Anion Channel

Taurine, amino ethane sulfonic acid, and other organic osmolytes are released from or accumulated in mammalian cells to restore cell volume following cell swelling and cell shrinkage, respectively. The volume sensitive release pathway for organic osmolytes, designated VSOAC, is permeable to various compounds (non-essential amino acids, taurine, sorbitol, choline, and thymidine) [59] and activation of VSOAC following cell swelling has in several cell lines been demonstrated to require phospholipase A2/5-lipoxygenase activity whereas the activity of VSOAC, once it has been evoked, is modulated by ROS and enzymes involved in growth factor mediated cell signaling [55, 60].

VRAC Versus VSOAC

VSOAC has not been cloned and based on differences on the time course for activation and inactivation following hypotonic cell swelling, sensitivity to anion channel blockers, fatty acids, and cholesterol (Table 1) it is currently assumed that VSOAC and VRAC are different although VSOAC has been suggested to contribute to the total Cl current under hypotonic conditions [61]. In accordance volume-sensitive taurine release has been demonstrated in the absence of I release (tracer for Cl) in rat mammary tissue [62], whereas volume-sensitive anion current and I release have been demonstrated in the absence of taurine release in human biliary cells following cell swelling [63]. Furthermore, members of the anoctamin (ANO 1 and ANO6) family have also been excluded as VSOAC candidates [64].

VSOAC and Apotosis

Cell release taurine during apoptosis [54, 76, 77] and as reduction in cellular taurine loss via VSOAC reduces caspase activity during hypoxia in, for example, A549 cells [55], it is assumed that VSOAC contributes to loss of amino acids during AVD [54]. VSOAC activity is similar to the VRAC activity reduced in MDR EATC compared to WT EATC [54].

Anoctamins: ANO1, ANO6, and ANO8

Identification: Phylogeny

In 2008, three independent groups showed that a putative transmembrane (TM) protein, ANO1, traffics to the plasma membrane, and gives rise to a current with the same characteristics as classical Ca2+-dependent Cl current (ICl.Ca), when expressed in a number of heterologous expression systems [2-4]. The three groups used different approaches: Yang et al. used a bioinformatics approach searching public databases for putative transporter-like genes with more than two TM domains and multiple isoforms [4]. Caputo and coworkers observed that interleukin-4 stimulation of bronchial epithelial cells caused a marked upregulation of ICl.Ca as measured by short circuit current recordings. A global gene expression analysis identified a number of genes up-regulated on mRNA level including ANO1 [2]. Schroeder and coworkers used an elegant expression cloning approach by introducing size-fractionated mRNA isolated from Xenopus oocytes into Axolotl (Ambystoma mexicanum) oocytes. Oocytes from the later species are polyspermic and do not have an endogenous ICl.Ca [3].

ANO1 belongs to the anoctamin family, which is conserved across the eukaryotic kingdom. The family is best represented in higher vertebrates with the mammalian anoctamin family being the largest family consisting of 10 members [78]. The ANO1/ANO2, ANO3/ANO4, ANO5/ANO6, and ANO8/ANO10 constitute distinct subfamilies within the anoctamin family. ANO1 shows a global ∼60% sequence identity with ANO2, but only 20–30% with the remaining anoctamins. The TM domains show significantly higher conservation. The anoctamin family does not share significant homology with CFTR, ClC, and GABAAR, but may be evolutionary related to the transmembrane channel-like (TMC) family [79]. ANO1 and ANO2 readily traffic to the plasma membrane and produce Ca2+ activated anion current. Many of the remaining anoctamin members do not traffic to the plasma membrane precluding their electrophysiological characterization [80]. This raises the possibility that other anoctamin family members might be 1) intracellular chloride channels, 2) channels or transporters of other substances, for example, phospholipids, or 3) have non-channel functions. Duran and coworkers concluded that in particular ANO3, ANO4, ANO5, ANO6, and ANO7 are intracellular. Evidence suggests however that at least ANO6 is a CaCC [6, 8, 81]. Furthermore, ANO6 has scramblase activity [82] and Suzuki et al. have demonstrated that also other anoctamins work as scramblases with different preference to lipid substrates [83].

The anoctamin family shows tissue-specific expression [7]. Using real-time polymerase chain reaction (RT-PCR) the mRNA abundance of all members was assayed in murine tissue. ANO2, ANO3, and ANO4 were primarily expressed in neuronal tissues, whereas ANO6, ANO7, and ANO8 were expressed in all tissues examined. ANO5 showed strong expression in skeletal muscle and the thyroid gland [7]. ANO1 is prominently expressed in airway epithelium, salivary glands, and biliary ducts [84].

CaCCs are often activated by G-protein coupled receptors (GPCR: endothelin receptor type A, angiotensin II receptor, muscarinic receptor, histamine receptor, and purinergic receptor), which mobilizes Ca2+ through through phospholipase C (PLCβ) and IP3. Co-transfection of ANO1 with these G-protein coupled receptors led to ICl.Ca after addition of the appropriate agonists [4], showing that ANO1 is the CaCC activated by GPCRs signaling through elevation of [Ca2+]i [4].

Topology

The anoctamin name was given to reflect the anion conductance and eight (octa) TM domains with the N- and C-termini located in the cytosol [4]. The region between TM5 and TM6 is highly conserved and expected to form a re-entrance loop containing the pore of the channel [4], although new experimental results [85] indicate the re-entrance loop may have a more complex topology. The region between TM6 and TM7 seems to be important for the regulation of the protein activity by Ca2+ [85]. Mouse ANO1 (mANO1) is expected to be N-glycosylated on at least three residues [86]. ANO1–6 all contain at least one putative glycosylation site in the first ectoloop, and 375N in mANO1 has been shown to be glycosylated [86]. Human ANO7 (hANO7) is glycosylated on two sites in the last ectoloop and these sequences are conserved in mANO1 [87]. ANO1 contains six extracellular-located cysteines, that is, five in the first ectoloop and one in the last. Mutating any of these cysteines leads to complete loss of current [85]. The cysteines are highly conserved within the anoctamin family and across species. Data showed that the oligomeric structure of functional ANO1 is a dimer [86] and that the dimerization happens en route to the plasma membrane making ANO1 an obligate dimer [86, 88].

ANO1

Activation

ANO1 is activated by a rise in cytosolic [Ca2+]i within a physiological relevant range, but is not simply a Ca2+-gated channel, because the gating of ANO1 is controlled by a complex interplay between [Ca2+]i, and the membrane potential (Vm). Under physiological conditions, ANO1 is likely regulated by changes in both Vm and [Ca2+]i as well as indirectly by modifications, for example, alternative splicing and post-translational modifications that alter the apparent Ca2+ and Vm sensitivity. ANO1 thus emerges as a plasma membrane hub that integrates signal from metabotropic and ionotropic inputs.

The kinetics of activation and deactivation of endogenous CaCCs have been carefully investigated in Xenopus oocytes and activation of CaCCs was found to be mainly Ca2+-dependent, whereas closing was essentially [Ca2+]i-independent, but strongly accelerated at hyperpolarizing Vm [89]. ANO1 shifts from being voltage sensitive (outwardly rectifying) at submicromolar [Ca2+]i to voltage insensitive (linear) at micromolar [Ca2+]i. Concomitantly, the time-dependent component of ANO1 activation disappears [2-4]. ANO1 contains no previous characterized Ca2+ binding motif, for example, EF hand and whether Ca2+-dependent gating is controlled by direct binding to ANO1, to an accessory component or a combination is still not completely clear. The effect of Ca2+ could be through binding to calmodulin, that is, Kunzelmann and coworkers reported calmodulin binding to and activation of a specific isoform of ANO1 [90] and Vocke et al. recently reported that calmodulin is absolutely required for ANO1 and ANO2 activation [91]. However, Min Goo Lee and collaborators reported that calmodulin affects ion permeability (the PHCO3/PCl ratio) of ANO1 but not its activity [92]. Furthermore, recent papers seriously questioned the role of calmodulin [93, 94]. Thus, the regulation by calmodulin is still a controversial issue. The first intracellular loop of ANO1 has attracted attention, because it contains a stretch of five consecutive, negatively charged glutamic acids, which resembles the “Ca2+ bowl” of BK channels [78]. However, molecular biology indicates that the first intracellular loop is unlikely to form a major Ca2+ binding site, but could couple voltage and Ca2+-binding to channel opening [95].

Addition of ATP diphosphohydrolase (apyrase) to the pipette solution in whole cell patch clamp experiments abolished ANO1 current activated by ionomycin or extracellular-applied ATP in HEK 293 cells. However, protein phosphorylation seems not to be required for full activation [90]. Two intracellular, C-terminal located sites for ERK have been identified and shown to be important for receptor-mediated activation of ANO1, indicating Ca2+ independent regulation of ANO1 [96].

Many ion channels interact with the actin-based cytoskeleton. The interaction may be through direct binding, or indirectly through actin-binding proteins and scaffolding proteins. ANO1 interacts with very high stoichiometry with the ezrin–radixin–moesin (ERM) network of actin-binding proteins, and knockdown of moesin reduced ANO1 current by >50% without affecting the surface expression [97].

Physiological Functions. Secretory epithelia

ANO1 is required for the normal development and function of the murine trachea. ANO1−/− mice develop extensive cartilage defects in the tracheal rings and it has been proposed that these defects are secondary to defects in the epithelium or tracheal muscle [98]. CFTR−/− and ANO1−/− mice display the same congenital cartilaginous defect, which indicates that defective Cl secretion may be the underlying cause in both cases [99]. Epithelial fluid secretion is driven by trans-epithelial movement of Cl. Cl is accumulated by transporters located in the basolateral membrane and secreted by the combined action of CFTR and CaCCs located in the apical (luminal) membrane [96]. VRAC, conversely, is suggested to by located at the basolateral membrane and activation of VRAC decreases the driving force for Cl across the apical membrane and hence Cl secretion [100]. Compelling evidence supports a major role for ANO1 in apical chloride secretion in a number of mouse secretory epithelia including airway epithelium, which may explain the lack of a CF lung phenotype in CFTR−/− mice. Experiments from Kunzelmanns group suggest that CFTR controls Ca2+ dependent secretion by inhibition of ANO1 [101]. ANO1 is also detected in the apical membrane of acinar cells in the submandibular mouse gland and ANO1−/− mice as well as mice treated with siRNA against ANO1 showed reduced salivary secretion upon cholinergic stimulation [101]. Finally, using inhibitors as well as knock down of ANO1 it was demonstrated that Ca2+ activated Cl secretion by ANO1 in an embryonic kidney cyst model is crucial for renal cyst growth [102].

Smooth muscle cells. ANO1, ANO6, and ANO10 were prominently detected in rat pulmonary and cerebral artery smooth muscle cells, and siRNA targeting ANO1 reduced the CaCC [103, 104]. In vascular smooth muscle cells, CaCCs are involved in contraction and regulation of vascular tone. The contraction is initiated by Ca2+ released from intracellular stores, and enhanced via influx through voltage-gated Ca2+ channels in the plasma membrane. As the equilibrium potential for Cl (ECl) is around −30 mV in these cells, opening of CaCCs will lead to depolarization, which may enhance Ca2+ influx and hence vasoconstriction [105]. It has been shown that membrane stretch activates ANO1in arterial smooth muscle cells and thereby regulate arterial smooth muscle cell membrane potential and hence vasoconstriction [106].

Gut and oviduct mobility. ANO1 is highly expressed in interstitial cells of Cajal, which generate the electrical pacemaker activity (slow waves) of gastrointestinal smooth muscles, and hence control gastric peristalsis. ANO1−/− mice fail to develop these slow waves [107]. ANO1 is also expressed in the smooth muscle cells of the oviduct where it is involved in the electrical slow waves and coordinated contractions [108].

Neurons and the olfactory epithelium. ANO1 in dorsal root ganglion neurons are heat sensitive and appears to mediate or amplify thermal pain perception [109]. ANO1 and ANO2 are both present in mice olfactory epithelium and it has been suggested that ANO1 plays a role in regulation of the chloride ionic composition of the mucus covering the apical surface of the olfactory epithelium, whereas ANO2 plays a role in olfactory signal transduction [110].

Chemical Modulators of ANO1 Current

Based on the physiological role of ANO1 in epithelial secretion and smooth muscle contraction, ANO1 antagonists may potentially be used as antidiarrheal and antihypertensives. The upregulation of ANO1 in some cancers also suggests that ANO1 antagonists could be potential anticancer agents. Screening a selection of herbal medicines led to the identification of eugenol, the main constituent of clove oil, as an ANO1-antagonist with an IC50 ∼ 150 µM [111]. Tannic acid and gallotannins, which are found in red wine and green tea, also potently inhibit ANO1, which may explain the cardioprotective benefits of red wine and tea [112]. Namkung et al. have identified two ANO1 inhibitors (T16inh-A01 and CaCCinh-A01) [113], which have been used in several studies although their specificity has been questioned [114]. ANO1 agonists, conversely, are potential drug candidates for treatment of cystic fibrosis, gland dysfunctions, and intestinal hypomobility.

Anoctamin 5

Expression and Subcellular Localization

ANO5 is highly expressed in cardial and skeletal muscle tissues and ANO5 is upregulated during myogenesis in a number of myoblast cell-lines [115]. ANO5 is further expressed in numerous other tissues including chondrocytes and osteoblasts [7]. Hence, ANO5 could play an essential role in the developments of the musculoskeletal system. ANO5 is subjected to complex alternative splicing, and some of the splice-variants are likely associated with changes in membrane topology [116]. The cellular localization of ANO5 is not clear. In differentiated myotubes and extracts of skeletal muscle tissue, ANO5 is predominantly located in what appears to be intracellular vesicles of unknown origin, even though some may also resides in the plasma membrane [115]. In transiently transfected COS-7 cells, hANO5 co-localized with the ER marker calreticulin [80, 117].

Physiological and Pathophysiological Roles

Bone and muscle diseases

ANO5 is expressed in both growth-plate chondrocytes and osteoblasts indicating a direct functional role for ANO5 in ossification [115]. Mutations in ANO5 are currently linked to the human diseases gnathodiaphyseal dysplasia (GDD), and muscular dystrophies (LGMD2L, MMD3). GDD is a rare, skeletal syndrome characterized by bone fragility and frequent bone fractures, sclerosis of tubular bones, infection and pus discharge from the jaw, and mobility of the teeth. The GDD patients show no apparent abnormalities in nonskeletal tissues [117]. Linkage analysis, performed on two unrelated families suffering from autosomal dominant inherited GDD, revealed mutations in ANO5 (loss of cysteine residues), indicating that loss of cysteine residue underlies GDD [117].

Muscular dystrophies are a group of genetic diseases characterized by progressive degeneration of skeletal muscle. LGMD2L is characterized by atrophy and MMD3 by myopathy [118-121]. Electron-microscopic examination of muscle biopsies from patients revealed multifocal disruptions of the sarcolemmal membrane, but no sub-sarcolemmal accumulation of vesicles [118, 119]. Isolated fibroblasts from one patient also showed defective membrane repair [119, 121]. The described mutations in ANO5 are found throughout the gene and involve both nonsense mutations that lead to premature stop codons and subsequent translation-coupled nonsense-mediated RNA decay, but also missense mutations [121]. As the dystrophies can result from either being homozygote for nonsense mutations or compound heterozygote, this suggests an underlying loss-of-function disease-mechanism.

Anoctamin 6

ANO6 is a pore-forming subunit of an anion channel or a channel itself [6]. Kunzelmann and colleagues have proposed that ANO6 is an essential component of outwardly rectifying chloride channels [81] and Grubb and coworkers found that ANO6 is a Ca2+-activated anion channel, with a relatively high cation permeability (PNa = 0.3 PCl), that locates to the plasma membrane [6]. Whether anions and cations move through the same channel is currently unknown. ANO6 has a significantly higher EC50 for Ca2+ compared to ANO1and a considerably delayed Ca2+ activated Cl current [6, 8]. As described below ANO6 has been assigned a role in: scrambling of phospholipids (externalization of phosphatidyl serine) during apoptosis [81, 122], cell volume regulation [123], and cell migration [22].

Physiological and Pathophysiological Roles

ANO6, the closest relative of ANO5, has been implicated in Ca2+-dependent phosphatidyl serine (PtdSer) exposure on the cell surface of a murine B cell-line [122]. The continued Ca2+-dependent PtdSer exposure was due to a mutation in ANO6, indicating that the mutation sensitizes ANO6 to the normal level of [Ca2+]i. A patient with Scott syndrome, which is a rare inheritable bleeding disorder caused by a defect in phospholipid scrambling activity, was homozygote for non-sense mutations in ANO6 [122]. Scott patients all lacked Ca2+-induced phospholipid scrambling in platelets, red blood cells, and lymphocytes [124]. The scramblase defect is only partial after apoptosis induction in platelets and lymphocytes from Scott patients especially when apoptosis is induced by Fas ligand [124], indicating that ANO6 is not likely the scramblase per se [125]. Hence, the relation between scramblase activity and ion transport is not clear [124]. Studies with ANO6 knock-out mice indicate a role of ANO6 involvement in development of skeletal tissues [126], whereas alternative splicing of human ANO6 in breast cancer associate ANO6 with development of metastasis [127]. More recently, Suzuki et al. reported scramblase activity of other anoctamins although with different phospholipid specificity [83].

Role of Anoctamins in Cell Volume Control, Proliferation, Migration, and Apoptosis: Pathophysiological Significance

Cell Volume Control

In 2009, Almaça and coworkers investigated the role of ANO1 and other ANO's including ANO6 in volume-regulated chloride currents and cell volume regulation [123]. They found that anoctamin knock-down significantly reduced ICl,swell and impaired RVD and suggested that anoctamins as candidates for VRAC [123]. However, ANO1, ANO6, and ANO10 do not contribute to the volume-activated current in ANO-overexpressing HEK293 cells in the absence of Ca2+ [64]. Moreover, based on the differences between ANO6 and VRAC (see Table 2) several groups reject ANO6 as a VRAC candidate [8, 64, 124] although ANO6 can contribute to RVD in the presence of extracellular Ca2+ [64].

Proliferation

Chloride channel inhibitors can attenuate cell growth suggesting a possible mechanistic link between cell cycle progression and the capacity for conductive Cl transport [46]. ANO1 is overexpressed in several carcinomas and described as a critical player in cellular proliferation [130, 131], where it controls proliferation at the G1/S transition [132]. The role of ANO1 in cell proliferation varies among different cancer cells, that is, ANO1 is pro-proliferative in most cancer cells [130, 131, 133], whereas it is anti-proliferative in other cancer cells [134].

Apotosis

TMEM16 proteins appear to play a role in apoptotic cell death [81, 123, 135]. In ELA cells [64] and lung epithelial cells [81] it has been shown that ANO6 plays an important role during cisplatin-induced apoptosis. In Hela cells, conversely, Shimizu et al., found ANO6 knock down does not affect staurosporine-induced AVD in HeLa cells [8].

Cancer

ANO1 is also known as DOG-1 (discovered on gastrointestinal stromal tumor), ORAOV2 (oral cancer overexpressed), and TAOS2 (tumor amplified and overexpressed sequence). These names reflect that ANO1 is upregulated in various cancers and may be considered as a diagnostic biomarker for certain types of tumors [136-138]. In oral and head and neck squamous cell carcinomas, increased ANO1 expression correlates with increased risk of developing metastases [139]. ANO1 might support metastasis by stimulating cell migration via control of the direction of migration as demonstrated in ELA cells [129] (see below).

Migration

Volume activated and Ca2+ activated channels have been assigned a role in cell migration in various cell types (see ref. [140]). According to Schwarb and coworkers migration involves local swelling at the cell front during the protrusion of the lamellipodium and local cell shrinkage at the rear part during its retraction [140]. The latter shrinkage involves concomitant activation of Ca2+-dependent K+ channels (KCa3.1) and Cl channels [140]. Volume-regulated anion channels were proposed to cooperate with KCa3.1 channels on the basis of inhibition of VRAC with a VRAC inhibitor NS3728 [34], which however is found also to inhibit CaCC [46]. As cell migration is Ca2+ dependent [141] it is likely that CaCCs are involved in the process. ANO1 [129, 134] as well as ANO6 [129] are involved in tumor cell migration. Knock-down of ANO1 and ANO6 in ELA cells followed by migration analysis showed that ANO1 is involved in directional migration whereas ANO6 determines the rate of cell migration [129]. ANO6 also mediates Ca2+-dependent phospholipid scrambling [122] and thereby regulate the phospholipid distribution in the plasma membrane. Inhibition of scramblases or flippases is known to inhibit migration [142], that is, the inhibition of migration following ANO6 knock down in ELA cells [129] could result from impaired phospholipid translocation.

Acknowledgment

This work was supported by the Augustinus Foundation and Brødrene Hartmanns Fond.

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