• MD simulation
• TMEM16s
• membrane deformation
• scramblases

• R01 GM137109 NIGMS NIH HHS

• Anoctamins
• Ion channels
• Membrane
• Scramblases
• TMEM16s

• Humans
• Anoctamins/metabolism
• Anoctamins/chemistry
• Animals
• Phospholipid Transfer Proteins/metabolism
• Phospholipid Transfer Proteins/chemistry

• R01 AI178180 NIAID NIH HHS
• R01 GM106717 NIGMS NIH HHS
• R35 GM152012 NIGMS NIH HHS

• Calcium
• Chloride channel
• Function
• Regulation
• Structure
• TMEM16A

• Humans
• Animals
• Anoctamin-1/metabolism
• Calcium/metabolism
• Chloride Channels/metabolism
• Ion Channel Gating

• ANO1
• HCC
• anoctamin 1
• chloride channels
• liver diseases

• Humans
• Anoctamin-1/metabolism
• Anoctamin-1/genetics
• Liver Diseases/metabolism
• Liver Diseases/pathology
• Liver Diseases/genetics
• Animals
• Signal Transduction
• Neoplasm Proteins/metabolism
• Neoplasm Proteins/genetics
• Liver Neoplasms/metabolism
• Liver Neoplasms/genetics
• Liver Neoplasms/pathology
• Carcinoma, Hepatocellular/metabolism
• Carcinoma, Hepatocellular/genetics
• Carcinoma, Hepatocellular/pathology
• Gene Expression Regulation

• ANO1
• Activators
• Inhibitors
• Pharmacology
• Physiology
• TMEM16A

• Humans
• Chloride Channels/metabolism
• Anoctamin-1/metabolism
• Calcium/metabolism
• Binding Sites

• Calcium-activated chloride channels
• Gating
• Permeation
• Phospholipid scramblases
• TMEM16

• Humans
• Animals
• Chloride Channels/chemistry
• Chloride Channels/metabolism
• Phospholipid Transfer Proteins/metabolism
• Anoctamins/metabolism
• Ion Transport
• Phospholipids/metabolism
• Calcium/chemistry
• Mammals/metabolism

• Ca2+ activated Cl- channel
• Channel regulation
• KCNE1
• KCNQ1
• TMEM16A
• Voltage dependence

• TMEM16A channels
• Xenopus oocytes
• asbestos fibers
• crocidolite
• voltage clamp

• none Italian League for the Fight Against Cancer (Lega Italiana Lotta contro i Tumori, LILT, Gorizia section, Italy, Bando Ricerca Sanitaria 2017-programma 5 per mille anno 2015), Regione Friuli Venezia Giulia (Contributi alle Aziende Sanitarie per la realizza

Anoctamin 5 (ANO5)/transmembrane protein 16E belongs to the ANO/ transmembrane protein 16 anion channel family. ANOs comprise a family of plasma membrane proteins that mediate ion transport and phospholipid scrambling and regulate other membrane proteins in numerous cell types. Previous studies have elucidated the roles and mechanisms of ANO5 activation in various cancer types. However, it remains unclear whether ANO5 acts as a plasma membrane chloride channel, and its expression and functions in gastric cancer (GC) have not been investigated.

To examine the role of ANO5 in the regulation of tumor progression and clinicopathological significance of its expression in GC.

Knockdown experiments using ANO5 small interfering RNA were conducted in human GC cell lines, and changes in cell proliferation, cell cycle progression, apoptosis, and cellular movement were assessed. The gene expression profiles of GC cells were investigated following ANO5 silencing by microarray analysis. Immunohistochemical staining of ANO5 was performed on 195 primary tumor samples obtained from patients with GC who underwent curative gastrectomy between 2011 and 2013 at our department.

Reverse transcription-quantitative polymerase chain reaction (PCR) and western blotting demonstrated high ANO5 mRNA and protein expression, respectively, in NUGC4 and MKN45 cells. In these cells, ANO5 silencing inhibited cell proliferation and induced apoptosis. In addition, the knockdown of ANO5 inhibited G₁-S phase progression, invasion, and migration. The results of the microarray analysis revealed changes in the expression levels of several cyclin-associated genes, such as CDKN1A, CDK2/4/6, CCNE2, and E2F1, in ANO5-depleted NUGC4 cells. The expression of these genes was verified using reverse transcription-quantitative PCR. Immunohistochemical staining revealed that high ANO5 expression levels were associated with a poor prognosis. Multivariate analysis identified high ANO5 expression as an independent prognostic factor for 5-year survival in patients with GC (P = 0.0457).

ANO5 regulates the cell cycle progression by regulating the expression of cyclin-associated genes and affects the prognosis of patients with GC. These results may provide insights into the role of ANO5 as a key mediator in tumor progression and/or promising prognostic biomarker for GC.

• Anoctamin 5
• Gastric cancer
• Cell cycle
• G1/S checkpoint
• Cell proliferation

• Anoctamins/genetics
• Anoctamins/metabolism
• Biomarkers
• Cell Cycle
• Cell Line, Tumor
• Cell Movement
• Cell Proliferation/genetics
• Chloride Channels/genetics
• Cyclins/genetics
• Cyclins/metabolism
• Gene Expression Regulation, Neoplastic
• Humans
• Membrane Proteins/genetics
• Phospholipids
• Prognosis
• RNA, Messenger/genetics
• RNA, Small Interfering/metabolism
• Stomach Neoplasms/pathology

In smooth muscle tissues, calcium-activated chloride channels (CaCC) provide the major anionic channel. Opening of these channels leads to chloride efflux and depolarization of the myocyte membrane. In this way, activation of the channels by a rise of intracellular [Ca²⁺], from a variety of sources, produces increased excitability and can initiate action potentials and contraction or increased tone. We now have a good mechanistic understanding of how the channels are activated and regulated, due to identification of TMEM16A (ANO1) as the molecular entity of the channel, but key questions remain. In reviewing these channels and comparing two distinct smooth muscles, myometrial and vascular, we expose the differences that occur in their activation mechanisms, properties, and control. We find that the myometrium only expresses “classical,” Ca²⁺-activated, and voltage sensitive channels, whereas both tonic and phasic blood vessels express classical, and non-classical, cGMP-regulated CaCC, which are voltage insensitive. This translates to more complex activation and regulation in vascular smooth muscles, irrespective of whether they are tonic or phasic. We therefore tentatively conclude that although these channels are expressed and functionally important in all smooth muscles, they are probably not part of the mechanisms governing phasic activity. Recent knockdown studies have produced unexpected functional results, e.g. no effects on labour and delivery, and tone increasing in some but decreasing in other vascular beds, strongly suggesting that there is still much to be explored concerning CaCC in smooth muscle.

• Anoctamin 1
• TMEM16A
• calcium
• calcium-activated chloride channels
• excitability
• myometrial smooth muscle
• scramblases
• vascular smooth muscle

• ANO1
• CaMKII
• Calcium-activated chloride channel
• PIP2
• TMEM16A
• anoctamin-1
• calcium binding
• phosphorylation
• regulation
• structure

• Anoctamin-1
• Anoctamins
• Calcium/metabolism
• Calmodulin
• Chloride Channels/genetics

• P20 GM130459 NIGMS NIH HHS
• R01 EY014852 NEI NIH HHS
• R01 GM132598 NIGMS NIH HHS
• R01 HL146054 NHLBI NIH HHS
• British Heart Foundation

Cystic fibrosis (CF) is the most common of rare hereditary diseases in Caucasians, and it is estimated to affect 75,000 patients globally. CF is a complex disease due to the multiplicity of mutations found in the CF transmembrane conductance regulator (CFTR) gene causing the CFTR protein to become dysfunctional. Correctors and potentiators have demonstrated good clinical outcomes for patients with specific gene mutations; however, there are still patients for whom those treatments are not suitable and require alternative CFTR-independent strategies. Although CFTR is the main chloride channel in the lungs, others could, e.g., anoctamin-1 (ANO1 or TMEM16A), compensate for the deficiency of CFTR. This review summarizes the current knowledge on calcium-activated chloride channel (CaCC) ANO1 and presents ANO1 as an exciting target in CF.

• CFTR-independent therapy
• anoctamin-1
• calcium-activated chloride channel
• cystic fibrosis

• Ion channel
• Liposome
• Membrane transport
• Oligomerization
• Poisson distribution
• Reconstitution
• Statistical capture

• dual function
• gating mechanism
• membrane deformation
• membrane protein
• structure–function relationship

• TMEM16F
• anoctamin 6
• calcium sensitivity
• calcium-binding domain

• Amino Acid Sequence
• Anoctamins/chemistry
• Anoctamins/genetics
• Anoctamins/metabolism
• Calcium/metabolism
• EF Hand Motifs
• HEK293 Cells
• Humans
• Models, Biological
• Molecular Dynamics Simulation
• Mutation/genetics
• Phospholipid Transfer Proteins/chemistry
• Phospholipid Transfer Proteins/genetics
• Phospholipid Transfer Proteins/metabolism
• Protein Domains
• Structure-Activity Relationship

• Active transporters
• Chloride
• Ion channels
• Purification
• Reconstitution

Intracellular divalent cations control the molecular function of transmembrane protein 16 (TMEM16) family members. Both anion channels (such as TMEM16A) and phospholipid scramblases (such as TMEM16F) in this family are activated by intracellular Ca²⁺ in the low µM range. In addition, intracellular Ca²⁺ or Co²⁺ at mM concentrations have been shown to further potentiate the saturated Ca²⁺-activated current of TMEM16A. In this study, we found that all alkaline earth divalent cations in mM concentrations can generate similar potentiation effects in TMEM16A when applied intracellularly, and that manipulations thought to deplete membrane phospholipids weaken the effect. In comparison, mM concentrations of divalent cations minimally potentiate the current of TMEM16F but significantly change its cation/anion selectivity. We suggest that divalent cations may increase local concentrations of permeant ions via a change in pore electrostatic potential, possibly acting through phospholipid head groups in or near the pore. Monovalent cations appear to exert a similar effect, although with a much lower affinity. Our findings resolve controversies regarding the ion selectivity of TMEM16 proteins. The physiological role of this mechanism, however, remains elusive because of the nearly constant high cation concentrations in cytosols.

• TMEM16A
• TMEM16F
• divalent cations
• permeability ratio
• phospholipids

• CaCCs
• Drug target
• Ion channel
• Molecular mechanism
• Structure
• TMEM16A

• chloride channel
• molecular dynamics
• phospholipid
• protein–lipid interaction
• structure–function

• computational biophysics
• ion transport

• Anoctamin-1/drug effects
• Anoctamin-1/metabolism
• Binding Sites
• Calcium/metabolism
• Chloride Channel Agonists/metabolism
• Chloride Channels/drug effects
• HEK293 Cells
• Humans
• Ion Channel Gating/drug effects
• Ion Transport/drug effects
• Models, Molecular
• Phosphatidylinositol 4,5-Diphosphate/pharmacology

• DP2 GM126898 NIGMS NIH HHS
• U.S. Department of Health & Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)
• U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)
• U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)

• Ca-activated chloride channels
• CaCC
• Nociception
• TMEM16
• Therapeutic target

• S. cerevisiae
• Scrambling
• membrane channels
• membrane structure
• molecular biophysics
• phospholipids
• structural biology

• Amino Acid Sequence
• Aspergillus fumigatus/drug effects
• Aspergillus fumigatus/metabolism
• Binding Sites
• Biological Transport/drug effects
• Calcium/pharmacology
• Ceramides/pharmacology
• Fungal Proteins/chemistry
• Fungal Proteins/metabolism
• Ligands
• Lipids/chemistry
• Membrane Lipids/metabolism
• Models, Molecular
• Nanoparticles/chemistry
• Phospholipid Transfer Proteins/chemistry
• Phospholipid Transfer Proteins/metabolism
• Protein Conformation

• TL1 TR002386 NCATS NIH HHS
• P41 GM103310 NIGMS NIH HHS
• R01GM106717 NIGMS NIH HHS
• R01 GM124451 NIGMS NIH HHS
• R21 NS104512 NINDS NIH HHS
• R21NS10451 NINDS NIH HHS
• 1R01GM124451-02 NIGMS NIH HHS
• R01 GM021342 NIGMS NIH HHS
• T32 GM008539 NIGMS NIH HHS
• R01 GM106717 NIGMS NIH HHS
• GM103310 NIGMS NIH HHS
• S10 OD019994 NIH HHS
• National Institute of General Medical Sciences
• Irma T. Hirschl Trust
• Margaret and Herman Sokol Fellowship
• National Research Foundation of Korea
• Agouron Institute
• Simons Foundation
• Ministry of Science, ICT and Future Planning
• National Institute of Neurological Disorders and Stroke

• NBD
• Reconstitution
• Scrambling assay
• Sodium dithionite
• TMEM16/Anoctamin

• Anoctamins/isolation & purification
• Anoctamins/metabolism
• Phospholipid Transfer Proteins/isolation & purification
• Phospholipid Transfer Proteins/metabolism
• Proteolipids/chemistry
• Proteolipids/metabolism

• TMEM16A
• cancer
• gastrointestinal (GI) dysfunction
• hypertension
• signal transduction
• topological structure

• Acetylcholine
• Ca2+ responses
• intravital imaging
• muscarinic receptors
• salivary secretion

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.

• anoctamins
• electrophysiology
• electrostatics
• ion conduction
• ligand-gated ion channel
• molecular biophysics
• mouse
• structural biology

• Ano1
• TMEM16A
• albuminuria
• kidney
• nephron number

• anoctamin 1
• chloride channel
• magnetogenetic
• magnetothermal
• remote silencing

• R01 MH094730 NIMH NIH HHS
• R01 MH111872 NIMH NIH HHS

• Ca2+-activated Cl- channel
• deafness
• ion channel pore
• ion permeation
• lipid scramblase
• mechanosensation
• molecular biophysics
• mouse
• structural biology

• channel
• membrane
• opsin
• phospholipids
• scrambling

• Anoctamins/genetics
• Anoctamins/metabolism
• Biological Transport, Active/physiology
• HEK293 Cells
• Humans
• Lipid Metabolism/physiology
• Phospholipid Transfer Proteins/genetics
• Phospholipid Transfer Proteins/metabolism
• Receptors, G-Protein-Coupled/genetics
• Receptors, G-Protein-Coupled/metabolism
• Saccharomyces cerevisiae

• P41 GM103310 NIGMS NIH HHS
• R21 EY028314 NEI NIH HHS
• T32 GM008539 NIGMS NIH HHS
• R01 GM106717 NIGMS NIH HHS
• S10 OD019994 NIH HHS

Falzone et al. interpret the mechanisms underlying the activity of TMEM16 family members from recent structural and functional work.

The TMEM16 family of membrane proteins is composed of both Ca²⁺-gated Cl⁻ channels and Ca²⁺-dependent phospholipid scramblases. The functional diversity of TMEM16s underlies their involvement in numerous signal transduction pathways that connect changes in cytosolic Ca²⁺ levels to cellular signaling networks. Indeed, defects in the function of several TMEM16s cause a variety of genetic disorders, highlighting their fundamental pathophysiological importance. Here, we review how our mechanistic understanding of TMEM16 function has been shaped by recent functional and structural work. Remarkably, the recent determination of near-atomic-resolution structures of TMEM16 proteins of both functional persuasions has revealed how relatively minimal rearrangements in the substrate translocation pathway are sufficient to precipitate the dramatic functional differences that characterize the family. These structures, when interpreted in the light of extensive functional analysis, point to an unusual mechanism for Ca²⁺-dependent activation of TMEM16 proteins in which substrate permeation is regulated by a combination of conformational rearrangements and electrostatics. These breakthroughs pave the way to elucidate the mechanistic bases of ion and lipid transport by the TMEM16 proteins and unravel the molecular links between these transport activities and their function in human pathophysiology.

As an oxidative stress sensor, transient receptor potential melastatin 2 (TRPM2) channel is involved in many physiological and pathological processes including warmth sensing, ischemia injury, inflammatory diseases and diabetes. Intracellular calcium is critical for TRPM2 channel activation and the IQ-like motif in the N-terminus has been shown to be important by mediating calmodulin binding. Sequence analysis predicted two potential EF-loops in the N-terminus of TRPM2. Site-directed mutagenesis combining with functional assay showed that substitution with alanine of several residues, most of which are conserved in the typical EF-loop, including D267, D278, D288, and E298 dramatically reduced TRPM2 channel currents. By further changing the charges or side chain length of these conserved residues, our results indicate that the negative charge of D267 and the side chain length of D278 are critical for calcium-induced TRPM2 channel activation. G272I mutation also dramatically reduced the channel currents, suggesting that this site is critical for calcium-induced TRPM2 channel activation. Furthermore, D267A mutant dramatically reduced the currents induced by calcium alone compared with that by ADPR, indicating that D267 residue in D267–D278 motif is the most important site for calcium sensitivity of TRPM2. In addition, inside-out recordings showed that mutations at D267, G272, D278, and E298 had no effect on single-channel conductance. Taken together, our data indicate that D267–D278 motif in the N-terminus as a novel EF-loop is critical for calcium-induced TRPM2 channel activation.

• CaM
• EF-loop
• TRPM2
• alanine screen
• calcium
• channel activation

• CLCA1
• Ca2+
• Ca2+-activated Cl− channels
• Cl− channels
• TMEM16A
• epithelium

• Anoctamin-1/chemistry
• Anoctamin-1/genetics
• Calcium/chemistry
• Calcium Channel Agonists/chemistry
• Calcium Signaling/genetics
• Chloride Channels/chemistry
• Chloride Channels/genetics
• Chlorides/chemistry
• Epithelium/chemistry
• Epithelium/metabolism
• Humans
• Neoplasm Proteins/chemistry
• Neoplasm Proteins/genetics

• R01 DK104791 NIDDK NIH HHS

Our laboratory has developed bioinformatic strategies for identifying distant phylogenetic relationships and characterizing families and superfamilies of transport proteins. Results using these tools suggest that the Anoctamin Superfamily of cation and anion channels, as well as lipid scramblases, includes three functionally characterized families: the Anoctamin (ANO), Transmembrane Channel (TMC) and Ca²⁺-permeable Stress-gated Cation Channel (CSC) families; as well as four families of functionally uncharacterized proteins, which we refer to as the Anoctamin-like (ANO-L), Transmembrane Channel-like (TMC-L), and CSC-like (CSC-L1 and CSC-L2) families. We have constructed protein clusters and trees showing the relative relationships among the seven families. Topological analyses suggest that the members of these families have essentially the same topologies. Comparative examination of these homologous families provides insight into possible mechanisms of action, indicates the currently recognized organismal distributions of these proteins, and suggests drug design potential for the disease-related channel proteins.

• Anoctamins/chemistry
• Anoctamins/genetics
• Computational Biology
• Humans
• Multigene Family
• Phylogeny
• Sequence Analysis, Protein

• R01 GM077402 NIGMS NIH HHS
• GM077402 NIH HHS
• National Institutes of Health
• National Science Foundation

• Anoctamin
• Fluorescence microscopy
• Lipids
• Patch clamp
• Poly-unsaturated fatty acids
• Regulation
• TMEM16A

• TMEM16A
• electrophysiology
• ion channels

• anoctamin
• calcium-activated chloride channel
• chloride dynamic
• chloride regulation
• cystic fibrosis

• TMEM16A
• calcium-activated chloride channel
• calcium-activated chloride channel regulator 1 (CLCA1)
• chloride channel
• cystic fibrosis
• drug development
• magnesium
• metal ion-dependent adhesion site (MIDAS)
• protein-protein interaction

TMEM16A and TMEM16B encode for Ca²⁺-activated Cl⁻ channels (CaCC) and are expressed in many cell types and play a relevant role in many physiological processes. Here, I performed a site-directed mutagenesis study to understand the molecular mechanisms of ion permeation of TMEM16B. I mutated two positive charged residues R573 and K540, respectively located at the entrance and inside the putative channel pore and I measured the properties of wild-type and mutant TMEM16B channels expressed in HEK-293 cells using whole-cell and excised inside-out patch clamp experiments. I found evidence that R573 and K540 control the ion permeability of TMEM16B depending both on which side of the membrane the ion substitution occurs and on the level of channel activation. Moreover, these residues contribute to control blockage or activation by permeant anions. Finally, R573 mutation abolishes the anomalous mole fraction effect observed in the presence of a permeable anion and it alters the apparent Ca²⁺-sensitivity of the channel. These findings indicate that residues facing the putative channel pore are responsible both for controlling the ion selectivity and the gating of the channel, providing an initial understanding of molecular mechanism of ion permeation in TMEM16B.

• Anions/metabolism
• Anoctamin-1
• Anoctamins
• Calcium/metabolism
• Chloride Channels/metabolism
• Electrophysiology
• HEK293 Cells
• Humans
• Ion Channel Gating
• Membrane Proteins/metabolism
• Mutagenesis, Site-Directed
• Mutation
• Neoplasm Proteins/metabolism
• Patch-Clamp Techniques
• Permeability

• EU Marie Curie Reintegration Grant

The TMEM16 family contains dimeric membrane proteins activated by intracellular Ca²⁺. Realizing that lipid scramblase family members contain two independently activated subunits, Lim et al. use concatenated TMEM16A subunits to show that ion channel members contain two independently activated pores.

The TMEM16 proteins constitute a family of membrane proteins with unusual functional breadth, including lipid scramblases and Cl⁻ channels. Members of both these branches are activated by Ca²⁺, acting from the intracellular side, and probably share a common architecture, which was defined in the recent structure of the lipid scramblase nhTMEM16. The structural features of subunits and the arrangement of Ca²⁺-binding sites in nhTMEM16 suggest that the dimeric protein harbors two locations for catalysis that are independent with respect to both activation and lipid conduction. Here, we ask whether a similar independence is observed in the Ca²⁺-activated Cl⁻ channel TMEM16A. For this purpose, we generated concatenated constructs containing subunits with distinct activation and permeation properties. Our biochemical investigations demonstrate the integrity of concatemers after solubilization and purification. During investigation by patch-clamp electrophysiology, the functional behavior of constructs containing either two wild-type (WT) subunits or one WT subunit paired with a second subunit with compromised activation closely resembles TMEM16A. This resemblance extends to ion selectivity, conductance, and the concentration and voltage dependence of channel activation by Ca²⁺. Constructs combining subunits with different potencies for Ca²⁺ show a biphasic activation curve that can be described as a linear combination of the properties of its constituents. The functional independence is further supported by mutation of a putative pore-lining residue that changes the conduction properties of the mutated subunit. Our results strongly suggest that TMEM16A contains two ion conduction pores that are independently activated by Ca²⁺ binding to sites that are embedded within the transmembrane part of each subunit.

• ACLT
• Anion channel
• Apoptosis
• Cartilage
• Caspase
• Chloride channel
• Ion channels
• Osteoarthritis

• bicarbonate
• epithelial cell
• exocytosis
• ion channel
• pancreas
• secretion
• transporter

• Ca2+-activated Cl− channels
• Cl− channel blocker
• anion channel
• ion channel