Gain of function of TMEM16E/ANO5 scrambling activity caused by a mutation associated with gnathodiaphyseal dysplasia.

Mutations in the human TMEM16E (ANO5) gene are associated both with the bone disease gnathodiaphyseal dysplasia (GDD; OMIM: 166260) and muscle dystrophies (OMIM: 611307, 613319). However, the physiological function of TMEM16E has remained unclear. We show here that human TMEM16E, when overexpressed in mammalian cell lines, displayed partial plasma membrane localization and gave rise to phospholipid scrambling (PLS) as well as non-selective ionic currents with slow time-dependent activation at highly depolarized membrane potentials. While the activity of wild-type TMEM16E depended on elevated cytosolic Ca2+ levels, a mutant form carrying the GDD-causing T513I substitution showed PLS and large time-dependent ion currents even at low cytosolic Ca2+ concentrations. Contrarily, mutation of the homologous position in the Ca2+-activated Cl- channel TMEM16B paralog hardly affected its function. In summary, these data provide the first direct demonstration of Ca2+-dependent PLS activity for TMEM16E and suggest a gain-of-function phenotype related to a GDD mutation.

Functions of volume-sensitive and calcium-activated chloride channels.

The review describes molecular and functional properties of the volume regulated anion channel and Ca(2+)-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.

The tether function of the anoctamins.

The core functions of the anoctamins are Cl- channel activity and phosphatidylserine (and perhaps other lipids) scrambling. These functions have been extensively studied in various tissues and cells. However, another function of the anoctamins that is less recognized and minimally explored is as tethers at membrane contact sites. This short review aims to examine evidence supporting the localization of the anoctamins at membrane contact sites, their tether properties, and their functions as tethers.

In or out of the groove? Mechanisms of lipid scrambling by TMEM16 proteins.

Phospholipid scramblases mediate the rapid movement of lipids between membrane leaflets, a key step in establishing and maintaining membrane homeostasis of the membranes of all eukaryotic cells and their organelles. Thus, impairment of lipid scrambling can lead to a variety of pathologies. How scramblases catalyzed the transbilayer movement of lipids remains poorly understood. Despite the availability of direct structural information on three unrelated families of scramblases, the TMEM16s, the Xkrs, and ATG-9, a unifying mechanism has failed to emerge thus far. Among these, the most extensively studied and best understood are the Ca2+ activated TMEM16s, which comprise ion channels and/or scramblases. Early work supported the view that these proteins provided a hydrophilic, membrane-exposed groove through which the lipid headgroups could permeate. However, structural, and functional experiments have since challenged this mechanism, leading to the proposal that the TMEM16s distort and thin the membrane near the groove to facilitate lipid scrambling. Here, we review our understanding of the structural and mechanistic underpinnings of lipid scrambling by the TMEM16s and discuss how the different proposals account for the various experimental observations.

Identification of determinants of lipid and ion transport in TMEM16/anoctamin proteins through a Bayesian statistical analysis.

The TMEM16/Anoctamin protein family (TMEM16x) is composed of members with different functions; some members form Ca2+-activated chloride channels, while others are lipid scramblases or combine the two functions. TMEM16x proteins are typically activated in response to agonist-induced rises of intracellular Ca2+; thus, they couple Ca2+-signalling with cell electrical activity or plasmalemmal lipid homeostasis. The structural domains underlying these functions are not fully defined. We used a Naïve Bayes classifier to gain insights into these domains. The method enabled identification of regions involved in either ion or lipid transport, and suggested domains for possible pharmacological exploitation. The method allowed the prediction of the transport property of any given TMEM16x. We envisage this strategy could be exploited to illuminate the structure-function relationship of any protein family composed of members playing different molecular roles.

Influence of Anoctamin-4 and -9 on ADAM10 and ADAM17 Sheddase Function.

Ca2+-activated Cl- channels (TMEM16, also known as anoctamins) perform important functions in cell physiology, including modulation of cell proliferation and cancer growth. Many members, including TMEM16F/ANO6, additionally act as Ca2+-activated phospholipid scramblases. We recently presented evidence that ANO6-dependent surface exposure of phosphatidylserine (PS) is pivotal for the disintegrin-like metalloproteases ADAM10 and ADAM17 to exert their sheddase function. Here, we compared the influence of seven ANO family members (ANO1, 4, 5, 6, 7, 9, and 10) on ADAM sheddase activity. Similar to ANO6, overexpression of ANO4 and ANO9 led to increased release of ADAM10 and ADAM17 substrates, such as betacellulin, TGFα, and amphiregulin (AREG), upon ionophore stimulation in HEK cells. Inhibitor experiments indicated that ANO4/ANO9-mediated enhancement of TGFα-cleavage broadened the spectrum of participating metalloproteinases. Annexin V-staining demonstrated increased externalisation of PS in ANO4/ANO9-overexpressing cells. Competition experiments with the soluble PS-headgroup phosphorylserine indicated that the ANO4/ANO9 effects were due to increased PS exposure. Overexpression of ANO4 or ANO9 in human cervical cancer cells (HeLa), enhanced constitutive shedding of the growth factor AREG and increased cell proliferation. We conclude that ANO4 and ANO9, by virtue of their scramblase activity, may play a role as important regulators of ADAM-dependent cellular functions.

Anoctamins and Calcium Signalling: An Obstacle to EGFR Targeted Therapy in Glioblastoma?

Glioblastoma is the most common form of high-grade glioma in adults and has a poor survival rate with very limited treatment options. There have been no significant advancements in glioblastoma treatment in over 30 years. Epidermal growth factor receptor is upregulated in most glioblastoma tumours and, therefore, has been a drug target in recent targeted therapy clinical trials. However, while many inhibitors and antibodies for epidermal growth factor receptor have demonstrated promising anti-tumour effects in preclinical models, they have failed to improve outcomes for glioblastoma patients in clinical trials. This is likely due to the highly plastic nature of glioblastoma tumours, which results in therapeutic resistance. Ion channels are instrumental in the development of many cancers and may regulate cellular plasticity in glioblastoma. This review will explore the potential involvement of a class of calcium-activated chloride channels called anoctamins in brain cancer. We will also discuss the integrated role of calcium channels and anoctamins in regulating calcium-mediated signalling pathways, such as epidermal growth factor signalling, to promote brain cancer cell growth and migration.

ANO4 (Anoctamin 4) Is a Novel Marker of Zona Glomerulosa That Regulates Stimulated Aldosterone Secretion.

Microarray comparison of the transcriptomes of human adrenal zona glomerulosa (ZG) and zona fasciculata found several ZG-specific genes that negatively regulate aldosterone secretion. The third and most significantly upregulated ZG-gene (19.9-fold compared with zona fasciculata, P=6.58×10-24) was ANO4, a putative Ca2+-activated chloride channel. We have investigated the role of ANO4 in human adrenal, and whether it functions like the prototype anoctamin, ANO1. We evaluated ANO4 mRNA and protein expression in human adrenal by qPCR and immunohistochemistry, compared the effects of ANO4 and ANO1 overexpression on baseline and stimulated aldosterone secretion and cell proliferation in H295R cells, and analyzed ANO4 activity as a Ca2+-activated chloride channel in comparison with other anoctamins by a fluorescence-based functional assay. The expression of ANO4 in ZG was confirmed by qPCR as 23.21-fold upregulated compared with zona fasciculata (n=18; P=4.93×10-7). Immunohistochemistry found cytoplasmic, ZG-selective expression of ANO4 (anoctamin 4) protein. ANO4 overexpression in H295R cells attenuated calcium-mediated aldosterone secretion and cell proliferation in comparison to controls. The latter effects were in a different direction to those of ANO1. The functional assay showed that, in contrast to ANO1, ANO4 expression results in low levels of calcium-dependent anion transport. In conclusion, ANO4 is one of the most highly expressed genes in ZG. It attenuates stimulated aldosterone secretion and cell proliferation. Although belonging to a family of Ca2+-activated chloride channels, it does not generate significant plasma membrane chloride channel activity.

Anoctamin 9/TMEM16J is a cation channel activated by cAMP/PKA signal.

Anoctamins (ANOs) are multifunctional membrane proteins that consist of 10 homologs. ANO1 (TMEM16A) and ANO2 (TMEM16B) are anion channels activated by intracellular calcium that meditate numerous physiological functions. ANO6 is a scramblase that redistributes phospholipids across the cell membrane. The other homologs are not well characterized. We found ANO9/TMEM16J is a cation channel activated by a cAMP-dependent protein kinase A (PKA). Intracellular cAMP-activated robust currents in whole cells expressing ANO9, which were inhibited by a PKA blocker. A cholera toxin that persistently stimulated adenylate cyclase activated ANO9 as did the application of PKA. The cAMP-induced ANO9 currents were permeable to cations. The cAMP-dependent ANO9 currents were augmented by intracellular Ca2+. Ano9 transcripts were predominant in the intestines. Human intestinal SW480 cells expressed high levels of Ano9 transcripts and showed PKA inhibitor-reversible cAMP-dependent currents. We conclude that ANO9 is a cation channel activated by a cAMP/PKA pathway and could play a role in intestine function.

Calcium-dependent electrostatic control of anion access to the pore of the calcium-activated chloride channel TMEM16A.

TMEM16A is a ligand-gated anion channel that is activated by intracellular Ca2+. This channel comprises two independent pores and closely apposed Ca2+ binding sites that are contained within each subunit of a homodimeric protein. Previously we characterized the influence of positively charged pore-lining residues on anion conduction (Paulino et al., 2017a). Here, we demonstrate the electrostatic control of permeation by the bound calcium ions in mouse TMEM16A using electrophysiology and Poisson-Boltzmann calculations. The currents of constitutively active mutants lose their outward rectification as a function of Ca2+ concentration due to the alleviation of energy barriers for anion conduction. This phenomenon originates from Coulombic interactions between the bound Ca2+ and permeating anions and thus demonstrates that an electrostatic gate imposed by the vacant binding site present in the sterically open pore, is released by Ca2+ binding to enable an otherwise sub-conductive pore to conduct with full capacity.

Role of volume-regulated and calcium-activated anion channels in cell volume homeostasis, cancer and drug resistance.

Volume-regulated channels for anions (VRAC) / organic osmolytes (VSOAC) play essential roles in cell volume regulation and other cellular functions, e.g. proliferation, cell migration and apoptosis. LRRC8A, which belongs to the leucine rich-repeat containing protein family, was recently shown to be an essential component of both VRAC and VSOAC. Reduced VRAC and VSOAC activities are seen in drug resistant cancer cells. ANO1 is a calcium-activated chloride channel expressed on the plasma membrane of e.g., secretory epithelia. ANO1 is amplified and highly expressed in a large number of carcinomas. The gene, encoding for ANO1, maps to a region on chromosome 11 (11q13) that is frequently amplified in cancer cells. Knockdown of ANO1 impairs cell proliferation and cell migration in several cancer cells. Below we summarize the basic biophysical properties of VRAC, VSOAC and ANO1 and their most important cellular functions as well as their role in cancer and drug resistance.