Rainer Schreiber

The amiloride-inhibitable Na+ conductance is reduced by the cystic fibrosis transmembrane conductance regulator in normal but not in cystic fibrosis airways.

Cystic fibrosis (CF) airway cells, besides their well-known defect in cAMP-dependent Cl- conductance, are characterized by an enhanced Na+ conductance. In this study we have examined the Na+ conductance in human respiratory tract by measuring transepithelial voltage and resistance (Vte, Rte) and by assessing membrane voltages (Vm) of freshly isolated airway epithelial cells from CF and non-CF patients. Basal amiloride inhibitable (10 micromol/liter) equivalent short circuit current (Isc = Vte/Rte) was significantly increased in CF compared with non-CF tissues. After stimulation by forskolin (10 micromol/liter) a significant depolarization of Vm corresponding to the cAMP-dependent activation of a Cl- conductance was observed in non-CF but not in CF airway cells. In non-CF tissue but not in CF tissue the effects of amiloride and N-methyl-D-glucamine on Vm were attenuated in the presence of forskolin. Also the amiloride-inhibitable Isc was significantly reduced by forskolin (1 micromol/liter) and isobutylmethylxanthine (IBMX; 100 micromol/liter) only in non-CF tissue. We conclude that cystic fibrosis transmembrane conductance regulator acts as a downregulator of epithelial Na+ channels in human airways. This downregulation of epithelial Na+ channels is absent in CF airways, leading to hyperabsorption and to the characteristic increase in mucus viscosity.

Loss of TMEM16A causes a defect in epithelial Ca2+-dependent chloride transport.

Molecular identification of the Ca2+-dependent chloride channel TMEM16A (ANO1) provided a fundamental step in understanding Ca2+-dependent Cl− secretion in epithelia. TMEM16A is an intrinsic constituent of Ca2+-dependent Cl− channels in cultured epithelia and may control salivary output, but its physiological role in native epithelial tissues remains largely obscure. Here, we demonstrate that Cl− secretion in native epithelia activated by Ca2+-dependent agonists is missing in mice lacking expression of TMEM16A. Ca2+-dependent Cl− transport was missing or largely reduced in isolated tracheal and colonic epithelia, as well as hepatocytes and acinar cells from pancreatic and submandibular glands of TMEM16A−/− animals. Measurement of particle transport on the surface of tracheas ex vivo indicated largely reduced mucociliary clearance in TMEM16A−/− mice. These results clearly demonstrate the broad physiological role of TMEM16A−/− for Ca2+-dependent Cl− secretion and provide the basis for novel treatments in cystic fibrosis, infectious diarrhea, and Sjöegren syndrome.

Expression and Function of Epithelial Anoctamins

The calcium-activated chloride channel anoctamin1 (ANO1; TMEM16A) is fundamental for the function of epithelial organs. Mice lacking ANO1 expression exhibit transport defects and a pathology similar to cystic fibrosis. They also show a general defect of epithelial electrolyte transport. Here we analyzed expression of all ten members (ANO1–ANO10) in a broad range of murine tissues and detected predominant expression of ANO1, 6, 7, 8, 9, 10 in epithelial tissues, while ANO2, 3, 4, 5 are common in neuronal and muscle tissues. When expressed in Fisher Rat Thyroid (FTR) cells, all ANO proteins localized to the plasma membrane but only ANO1, 2, 6, and 7 produced Ca2+-activated Cl− conductance, as analyzed by ATP-induced iodide quenching of YFP fluorescence. In contrast ANO9 and ANO10 suppressed baseline Cl− conductance and coexpression of ANO9 with ANO1 inhibited ANO1 activity. Patch clamping of ANO-expressing FRT cells indicated that apart from ANO1 also ANO6 and 10 produced chloride currents, albeit with very different Ca2+ sensitivity and activation time. We conclude that each tissue expresses a set of anoctamins that form cell- and tissue-specific Ca2+-dependent Cl− channels.

TMEM16A, induces MAPK and contributes directly to tumorigenesis and cancer progression

Frequent gene amplification of the receptor-activated calcium-dependent chloride channel TMEM16A (TAOS2 or ANO1) has been reported in several malignancies. However, its involvement in human tumorigenesis has not been previously studied. Here, we show a functional role for TMEM16A in tumor growth. We found TMEM16A overexpression in 80% of head and neck squamous cell carcinoma (SCCHN), which correlated with decreased overall survival in patients with SCCHN. TMEM16A overexpression significantly promoted anchorage-independent growth in vitro, and loss of TMEM16A resulted in inhibition of tumor growth both in vitro and in vivo. Mechanistically, TMEM16A-induced cancer cell proliferation and tumor growth were accompanied by an increase in extracellular signal-regulated kinase (ERK)1/2 activation and cyclin D1 induction. Pharmacologic inhibition of MEK/ERK and genetic inactivation of ERK1/2 (using siRNA and dominant-negative constructs) abrogated the growth effect of TMEM16A, indicating a role for mitogen-activated protein kinase (MAPK) activation in TMEM16A-mediated proliferation. In addition, a developmental small-molecule inhibitor of TMEM16A, T16A-inh01 (A01), abrogated tumor cell proliferation in vitro. Together, our findings provide a mechanistic analysis of the tumorigenic properties of TMEM16A, which represents a potentially novel therapeutic target. The development of small-molecule inhibitors against TMEM16A may be clinically relevant for treatment of human cancers, including SCCHN.

TMEM16 proteins produce volume regulated chloride currents that are reduced in mice lacking TMEM16A

All vertebrate cells regulate their cell volume by activating chloride channels of unknown molecular identity, thereby activating regulatory volume decrease. We show that the Ca2+-activated Cl− channel TMEM16A together with other TMEM16 proteins are activated by cell swelling through an autocrine mechanism that involves ATP release and binding to purinergic P2Y2 receptors. TMEM16A channels are activated by ATP through an increase in intracellular Ca2+ and a Ca2+-independent mechanism engaging extracellular-regulated protein kinases (ERK1/2). The ability of epithelial cells to activate a Cl− conductance upon cell swelling, and to decrease their cell volume (regulatory volume decrease) was dependent on TMEM16 proteins. Activation of ICl,swell was reduced in the colonic epithelium and in salivary acinar cells from mice lacking expression of TMEM16A. Thus TMEM16 proteins appear to be a crucial component of epithelial volume-regulated Cl− channels and may also have a function during proliferation and apoptotic cell death.

Purinergic inhibition of the epithelial Na+ transport via hydrolysis of PIP2

Stimulation of purinergic receptors inhibits amiloride‐sensitive Na+ transport in epithelial tissues by an unknown mechanism. Because previous studies excluded the role of intracellular Ca2+ or protein kinase C, we examined whether purinergic regulation of Na+ absorption occurs via hydrolysis of phospholipid such as phosphatidylinositol‐bisphosphates (PIP2). Inhibition of amiloride‐sensitive short‐circuit currents (Isc‐Amil) by adenine 5′‐triphosphate (ATP) in native tracheal epithelia and M1 collecting duct cells was suppressed by binding neomycin to PIP2, and recovery from ATP inhibition was abolished by blocking phosphatidylinositol‐4‐kinase or diacylglycerol kinase. Stimulation by ATP depleted PIP2 from apical membranes, and PIP2 co‐ immunoprecipitated the β subunit of ENaC. ENaC was inhibited by ATP stimulation of P2Y2 receptors in Xenopus oocytes. Mutations in the PIP2 binding domain of βENaC but not γENaC reduced ENaC currents without affecting surface expression. Collectively, these data supply evidence for a novel and physiologically relevant regulation of ENaC in epithelial tissues. Although surface expression is controlled by its C terminus, N‐terminal binding of βENaC to PIP2 determines channel activity.

Anoctamins are a family of Ca2+-activated Cl- channels.

Summary Anoctamin 1 (Ano1; TMEM16A) and anoctamin 2 (Ano2; TMEM16B) are novel Cl− channels transiently activated by an increase in intracellular Ca2+. These channels are essential for epithelial Cl− secretion, smooth muscle peristalsis and olfactory signal transduction. They are central to inherited diseases and cancer and can act as heat sensors. Surprisingly, another member of this protein family, Ano6, operates as a Ca2+-activated phospholipid scramblase, and others were reported as intracellular proteins. It is therefore unclear whether anoctamins constitute a family of Ca2+-activated Cl− channels, or are proteins with heterogeneous functions. Using whole-cell patch clamping we demonstrate that Ano4–10 are all able to produce transient Ca2+-activated Cl− currents when expressed in HEK293 cells. Although some anoctamins (Ano1, 2, 4, 6, 7) were found to be well expressed in the plasma membrane, others (Ano8, 9, 10) show rather poor membrane expression and were mostly retained in the cytosol. The transient nature of the Cl− currents was demonstrated to be independent of intracellular Ca2+ levels. We show that inactivation of Ano1 currents occurs in the continuous presence of elevated Ca2+ concentrations, possibly by calmodulin-dependent kinase. The present results demonstrate that anoctamins are a family of Ca2+-activated Cl− channels, which also induce permeability for cations. They may operate as Cl− channels located in the plasma membrane or in intracellular compartments. These results increase our understanding of the physiological significance of anoctamins and their role in disease.

Anoctamin 6 is an essential component of the outwardly rectifying chloride channel.

Outwardly rectifying chloride channels (ORCC, ICOR) of intermediate single-channel conductance of around 50 pS, are ubiquitously expressed, but have remained a mystery since their description more than 25 y ago. These channels have been shown to be activated on membrane excision and depolarization of the membrane voltage and by cAMP in the presence of the cystic fibrosis transmembrane conductance regulator. We show that anoctamin 6 (Ano6), a member of the recently identified family of putative Cl− channels, is the crucial component of ORCC single-channel and whole-cell currents in airway epithelial cells and Jurkat T lymphocytes. Cystic fibrosis transmembrane conductance regulator augmented ORCC produced by Ano6 in A549 airway epithelial cells. Ano6 is activated during membrane depolarization or apoptosis of Jurkat T lymphocytes and epithelial cells, and is inhibited by 5-nitro-2-(3-phenylpropylamino) benzoic acid, 4,4′-diisothio-cyanostilbene-2,2′-disulfonic acid, or AO1. Ano6 belongs to the basic equipment of any cell type, including colonic surface epithelial cells. It forms the essential component of ORCC and seems to have a role for cell shrinkage and programmed cell death.

CFTR, A Regulator of Channels

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl channel that is defective in cystic fibrosis. This statement is found in most of the articles somehow dealing with CFTR. However, the initial characterization of this puzzling protein as a conductance regulator is probably more appropriate. As we have learned over the past few years, CFTR controls the function of various other membrane conductances and the list of putative interactions of CFTR with other channels and cellular functions is continuously growing. Although it has been demonstrated in many cell types that CFTR forms a Cl channel, we may speculate that, at least in some tissues, CFTR rather acts through regulation of other membrane conductances or cellular functions. The recent findings on CFTR dependent regulation of other ion channels will challenge scientists to search for the mechanisms underlying the interaction between CFTR and other membrane proteins. In that respect, identification of consensus sites known to be important for protein interaction and isolation of putative cofactors involved in this process will be essential in order to uncover complex signaling cascades. The effects of CFTR on various cellular functions create a rather complicated pathophysiological scenario for the inherited disease cystic fibrosis. It is discussed that in addition to the well described defect of cAMP dependent CFTR Cl conductance, several other membrane conductances are prone to participate in the impaired epithelial electrolyte transport in cystic fibrosis.

Calmodulin-dependent activation of the epithelial calcium-dependent chloride channel TMEM16A

TMEM16A (anoctamin 1, Ano1), a member of a family of 10 homologous proteins, has been shown to form an essential component of Ca2+‐activated CU channels. TMEM16A‐null mice exhibit severe defects in epithelial transport along with tracheomalacia and death within 1 mo after birth. Despite its outstanding physiological significance, the mechanisms for activation of TMEM16A remain obscure. TMEM16A is activated on increase in intracellular Ca2+, but it is unclear whether Ca2+ binds directly to the channel or whether additional components are required. We demonstrate that TMEM16A is strictly membrane localized and requires cytoskeletal interactions to be fully activated. Despite the need for cytosolic ATP for full activation, phosphorylation by protein kinases is not required. In contrast, the Ca2+ binding protein calmodulin appears indispensable and interacts physically with TMEM16A. Openers of small‐ and intermediate‐conductance Ca2+‐activated potassium channels known to interact with calmodulin, such as 1‐EBIO, DCEBIO, or riluzole, also activated TMEM16A. These results reinforce the use of these compounds for activation of electrolyte secretion in diseases such as cystic fibrosis.—Tian, Y., Kongsuphol, P., Hug, M., Ousingsawat, J., Witzgall, R., Schreiber, R., Kunzelmann, K. Calmodulin‐dependent activation of the epithelial calcium‐dependent chloride channel TMEM16A. FASEB J. 25, 1058–1068 (2011). www.fasebj.org