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Electron cryo-microscopy density maps of mouse TMEM16A reconstituted in nanodiscs or solubilized in detergent reveal two functional states of calcium-activated chloride channels. TMEM16A structure solved The diverse TMEM16 membrane protein family contains Ca( II )-activated chloride channels, lipid scramblases and cation channels. TMEM16A mediates chloride-ion permeation, which controls neuronal signalling, muscle contraction and numerous other physiological functions. In this issue of Nature , two groups have solved the structure of TMEM16A by using cryo-electron microscopy, providing insights into the function of this channel. Unlike other ligand-gated ion channels, the Ca( II ) ion interacts with the pore directly, where a glycine residue acts as a flexible hinge to adjust calcium sensitivity. Raimund Dutzler and colleagues report the structure of the protein in both Ca( II )-free and Ca( II )-bound states, which shows how calcium binding facilitates the structural rearrangements involved in channel activation. In the second Letter, Lily Jan and colleagues present two functional states of TMEM16A in the glycolipid LMNG and in nanodiscs, with one and two Ca( II ) ions bound, respectively. The closed conformation observed in nanodiscs is proposed to show channel rundown after prolonged Ca( II ) activation. Calcium-activated chloride channels (CaCCs) encoded by TMEM16A 1 , 2 , 3 control neuronal signalling, smooth muscle contraction, airway and exocrine gland secretion, and rhythmic movements of the gastrointestinal system 4 , 5 , 6 , 7 . To understand how CaCCs mediate and control anion permeation to fulfil these physiological functions, knowledge of the mammalian TMEM16A structure and identification of its pore-lining residues are essential. TMEM16A forms a dimer with two pores 8 , 9 . Previous CaCC structural analyses have relied on homology modelling of a homologue (nhTMEM16) from the fungus Nectria haematococca that functions primarily as a lipid scramblase 10 , 11 , 12 , as well as subnanometre-resolution electron cryo-microscopy 12 . Here we present de novo atomic structures of the transmembrane domains of mouse TMEM16A in nanodiscs and in lauryl maltose neopentyl glycol as determined by single-particle electron cryo-microscopy. These structures reveal the ion permeation pore and represent different functional states. The structure in lauryl maltose neopentyl glycol has one Ca 2+ ion resolved within each monomer with a constricted pore; this is likely to correspond to a closed state, because a CaCC with a single Ca 2+ occupancy requires membrane depolarization in order to open (C.J.P. et al ., manuscript submitted). The structure in nanodiscs has two Ca 2+ ions per monomer and its pore is in a closed conformation; this probably reflects channel rundown, which is the gradual loss of channel activity that follows prolonged CaCC activation in 1 mM Ca 2+ . Our mutagenesis and electrophysiological studies, prompted by analyses of the structures, identified ten residues distributed along the pore that interact with permeant anions and affect anion selectivity, as well as seven pore-lining residues that cluster near pore constrictions and regulate channel gating. Together, these results clarify the basis of CaCC anion conduction.

The binding of cytoplasmic Ca 2+ to the anion-selective channel TMEM16A triggers a conformational change around its binding site that is coupled to the release of a gate at the constricted neck of an hourglass-shaped pore. By combining mutagenesis, electrophysiology, and cryo-electron microscopy, we identified three hydrophobic residues at the intracellular entrance of the neck as constituents of this gate. Mutation of each of these residues increases the potency of Ca 2+ and results in pronounced basal activity. The structure of an activating mutant shows a conformational change of an α-helix that contributes to Ca 2+ binding as a likely cause for the basal activity. Although not in physical contact, the three residues are functionally coupled to collectively contribute to the stabilization of the gate in the closed conformation of the pore, thus explaining the low open probability of the channel in the absence of Ca 2+ . The binding of cytoplasmic Ca 2+ to the anion-selective channel TMEM16A triggers a conformational change around its binding site that is coupled to the release of a gate at the constricted neck. Here authors use cryo-EM and electrophysiology to identify three hydrophobic residues at the intracellular entrance of the neck as constituents of this gate.

The anion channel TMEM16A is activated by intracellular Ca 2+ in a highly cooperative process. By combining electrophysiology and autocorrelation analysis, we investigated the mechanism of channel activation and the concurrent rearrangement of the gate in the narrow part of the pore. Features in the fluctuation characteristics of steady-state current indicate the sampling of intermediate conformations that are successively occupied during gating. The initial step is related to conformational changes induced by Ca 2+ binding, which is ensued by rearrangements that open the pore. Mutations in the gate shift the equilibrium of transitions in a manner consistent with a progressive destabilization of this region during pore opening. We come up with a mechanism of channel activation where the binding of Ca 2+ induces conformational changes in the protein that, in a sequential manner, propagate from the binding site and couple to the gate in the narrow pore to allow ion permeation. The anion channel TMEM16A is activated by intracellular Ca 2+ in a highly cooperative process. Here authors combine electrophysiology and autocorrelation analysis to observe the sampling of intermediate conformations during gating.

Altered peristaltic and ciliary dysfunction is a feature of females with endometriosis. To further explore this premise, we examined the ampulla of rhesus macaques (Macaca mulatta) with and without spontaneous endometriosis for the expression of adenylate kinase 7 (AK7), a mitochondrial-dwelling nucleotide converting enzyme with critical roles in cellular kinesis, forkhead protein box J1 (FOXJ1), a marker of cilia abundance, and Anoctamin 1 (ANO1) as a marker of both smooth muscle contraction and ciliogenesis. We further performed an in vitro experiment that treated ampullary segments with peritoneal fluid from animals with and without endometriosis. We report significantly downregulated expression of ANO1 in the ampulla of monkeys with endometriosis (in vivo), and in the ampullary segments exposed to peritoneal fluid of animals with endometriosis. We did not observe statistically significant differences in the expression of AK7 or FOXJ1 both in vivo and in vitro. This highlights potentially essential roles of ANO1 in the oviduct, the dampening of which may lead to a specific subtype of endometriosis-caused subfertility.

Transmembrane protein 16F (TMEM16F) is an enigmatic Ca 2+ -activated phospholipid scramblase (CaPLSase) that passively transports phospholipids down their chemical gradients and mediates blood coagulation, bone development and viral infection. Despite recent advances in the structure and function understanding of TMEM16 proteins, how mammalian TMEM16 CaPLSases open and close, or gate their phospholipid permeation pathways remains unclear. Here we identify an inner activation gate, which is established by three hydrophobic residues, F518, Y563 and I612, in the middle of the phospholipid permeation pathway of TMEM16F-CaPLSase. Disrupting the inner gate profoundly alters TMEM16F phospholipid permeation. Lysine substitutions of F518 and Y563 even lead to constitutively active CaPLSases that bypass Ca 2+ -dependent activation. Strikingly, an analogous lysine mutation to TMEM16F-F518 in TMEM16A (L543K) is sufficient to confer CaPLSase activity to the Ca 2+ -activated Cl − channel (CaCC). The identification of an inner activation gate can help elucidate the gating and permeation mechanism of TMEM16 CaPLSases and channels. TMEM16F is an enigmatic Ca 2 +  -activated phospholipid scramblase (CaPLSase) that passively transports phospholipids. Here authors identify an inner activation gate and its disruption profoundly alters TMEM16F phospholipid permeation.

The calcium-activated chloride channel TMEM16A is a member of a conserved protein family that comprises ion channels and lipid scramblases. Although the structure of the scramblase nhTMEM16 has defined the architecture of the family, it was unknown how a channel has adapted to cope with its distinct functional properties. Here we have addressed this question by the structure determination of mouse TMEM16A by cryo-electron microscopy and a complementary functional characterization. The protein shows a similar organization to nhTMEM16, except for changes at the site of catalysis. There, the conformation of transmembrane helices constituting a membrane-spanning furrow that provides a path for lipids in scramblases has changed to form an enclosed aqueous pore that is largely shielded from the membrane. Our study thus reveals the structural basis of anion conduction in a TMEM16 channel and it defines the foundation for the diverse functional behavior in the TMEM16 family. Cell membranes are made up of two layers of oily molecules, called lipids, embedded with a variety of proteins. Each type of membrane protein carries out a particular activity for the cell, and many are involved in transporting other molecules from one side of the membrane to the other. The TMEM16 proteins are a large family of membrane proteins. Most are known as lipid scramblases and move lipids between the two layers of the membrane. However, some TMEM16 proteins transport ions in or out of the cell, and are instead called ion channels. TMEM16 proteins are found in animals, plants and fungi but not bacteria, and play key roles in many biological activities that keep these organisms alive. For example, in humans, ion channels belonging to the TMEM16 family help keep the lining of the lung moist, and allow muscles in the gut to contract. The structure of a scramblase shows that two protein units interact, with each unit containing a furrow that spans the membrane, through which lipids can move from one layer to the other. However, to date, the shape of a TMEM16 ion channel has not been determined. It was therefore not clear how a protein with features that let it transport large, oily molecules like lipids had evolved to transport small, charged particles instead. TMEM16A is a member of the TMEM16 family that transports negatively charged chloride ions. Using a technique called cryo-electron microscopy, Paulino et al. have determined the three-dimensional shape of the version of TMEM16A from a mouse. Overall, TMEM16A is organized similarly to the lipid scramblase. However, some parts of the TMEM16A protein have undergone rearrangements such that the membrane-exposed furrow that provides a path for lipids in scramblases is now partially sealed in TMEM16A. This results in an enclosed pore that is largely shielded from the oily membrane and through which ions can pass. Additionally, biochemical analysis suggests that TMEM16A forms a narrow pore that may widen towards the side facing the inside of the cell, though further work is needed to understand if this is relevant to the protein’s activity. The three-dimensional structure of TMEM16A reveals how the protein’s architecture differs from other family members working as lipid scramblases. It also gives insight into how TMEM16 proteins might work as ion channels. These findings can now form a strong basis for future studies into the activity of TMEM16 proteins.

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is the secretory chloride/bicarbonate channel in airways and intestine that is activated through ATP binding and phosphorylation by protein kinase A, but fails to operate in cystic fibrosis (CF). TMEM16A (also known as anoctamin 1, ANO1) is thought to function as the Ca 2+ activated secretory chloride channel independent of CFTR. Here we report that tissue specific knockout of the TMEM16A gene in mouse intestine and airways not only eliminates Ca 2+ -activated Cl − currents, but unexpectedly also abrogates CFTR-mediated Cl − secretion and completely abolishes cAMP-activated whole cell currents. The data demonstrate fundamentally new roles of TMEM16A in differentiated epithelial cells: TMEM16A provides a mechanism for enhanced ER Ca 2+ store release, possibly engaging Store Operated cAMP Signaling (SOcAMPS) and activating Ca 2+ regulated adenylyl cyclases. TMEM16A is shown to be essential for proper activation and membrane expression of CFTR. This intimate regulatory relationship is the cause for the functional overlap of CFTR and Ca 2+ -dependent chloride transport.

Pancreatic cancer typically spreads rapidly and has poor survival rates. Here, we report that the calcium-activated chloride channel TMEM16A is a biomarker for pancreatic cancer with a poor prognosis. TMEM16A is up-regulated in 75% of cases of pancreatic cancer and high levels of TMEM16A expression are correlated with low patient survival probability. TMEM16A up-regulation is associated with the ligand-dependent EGFR signaling pathway. In vitro, TMEM16A is required for EGF-induced store-operated calcium entry essential for pancreatic cancer cell migration. TMEM16A also has a profound impact on phosphoproteome remodeling upon EGF stimulation. Moreover, molecular actors identified in this TMEM16A-dependent EGFR-induced calcium signaling pathway form a gene set that makes it possible not only to distinguish neuro-endocrine tumors from other forms of pancreatic cancer, but also to subdivide the latter into three clusters with distinct genetic profiles that could reflect their molecular underpinning.

The calcium-activated chloride channel TMEM16A is a promising drug target for treating hypertension, secretory diarrheas, and various cancers, including head and neck cancer. Despite its potential, no FDA-approved drugs have provided the structural basis for directly inhibiting TMEM16A. This study aims to identify a novel pore-blocker of TMEM16A by integrating virtual screening, molecular dynamics simulations, and in vitro studies. Using the calcium-bound structure of TMEM16A with and without the pore-blocker 1PBC, we performed virtual screening on nearly 90,000 compounds from the ChemDiv database. Approximately 67% of these compounds demonstrated better binding affinity than 1PBC. Among the top 20 compounds selected for short-circuit current assays using human lung adenocarcinoma cells (Calu-3), compounds N066-0059, N066-0060, and N066-0067 inhibited TMEM16A activity with IC 50 values of 0.24 µM, 0.41 µM, and 0.48 µM, respectively, which was lower than that of a positive control Ani9 (9 µM). Due to its highest potency in electrophysiological assays, N066-0059 was subjected to mechanistic studies. Molecular dynamics simulations elucidated its binding stability and strength, showing superior performance to 1PBC over 500 ns with 3 replicates. This study advances TMEM16A-targeted drug development, offering new insights for anticancer therapies.

Anoctamin-1 Ca 2+ -activated Cl − channels (ANO1) are proposed to modulate contractility of urethra smooth muscle cells (USMC), but their cellular expression and contribution to agonist/neural evoked activity is unclear. ANO1 is implicated as a potential target to treat incontinence, thus this is an important issue to resolve. We sought to clarify roles of ANO1 in contractility of mouse USMC. We found expression of Ano1 transcripts in murine urethra, with no difference between male and females. Immunolabelling revealed ANO1 was expressed in USMC and not in specialized populations of interstitial cells (c-kit + interstitial of Cajal-like cells (ICC-LC) and PDGFRα + cells). However, a specific ANO1 channel inhibitor, Ani9, failed to affect urethral contractions elicited by phenylephrine, arginine vasopressin or electrical field stimulation of intrinsic nerves. CaCC inh A01 also failed to affect urethral contractions. In addition, Ani9 had no effect on Ca 2+ signals generated by USMC in situ. In contrast, Ani9 effectively reduced spontaneous contractions and Ca 2+ signals of mouse proximal colon. In addition, Ani9 inhibited ANO1 currents recorded in HEK 293 cells, at concentrations 30 times less than those used in organ bath experiments. Our data suggest that despite expression of ANO1 in USMC, these channels do not contribute to basal Ca 2+ signalling, or agonist and neurally-evoked contractions in murine urethra.