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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.

Significance TMEM16A is a calcium-activated chloride channel, meaning that it is a protein found at the surface of a variety of cells that permits chloride to enter when internal calcium levels rise. TMEM16A has been implicated in a variety of biological processes, including epithelial fluid secretion, smooth muscle contraction, neuron firing, and cancer cell proliferation. However, it is unclear how chloride ions are guided across the cell membrane by this protein. Here, we have modified and tested positively charged amino acids of TMEM16A and found four that modify chloride flux. We also tested potential blockers from a high-throughput screen and describe two that appear to block chloride’s path, which should contribute to future studies attempting to further analyze TMEM16A’s function. TMEM16A (transmembrane protein 16) (Anoctamin-1) forms a calcium-activated chloride channel (CaCC) that regulates a broad array of physiological properties in response to changes in intracellular calcium concentration. Although known to conduct anions according to the Eisenman type I selectivity sequence, the structural determinants of TMEM16A anion selectivity are not well-understood. Reasoning that the positive charges on basic residues are likely contributors to anion selectivity, we performed whole-cell recordings of mutants with alanine substitution for basic residues within the putative pore region and identified four residues on four different putative transmembrane segments that significantly increased the permeability of the larger halides and thiocyanate relative to that of chloride. Because TMEM16A permeation properties are known to shift with changes in intracellular calcium concentration, we further examined the calcium dependence of anion selectivity. We found that WT TMEM16A but not mutants with alanine substitution at those four basic residues exhibited a clear decline in the preference for larger anions as intracellular calcium was increased. Having implicated these residues as contributing to the TMEM16A pore, we scrutinized candidate small molecules from a high-throughput CaCC inhibitor screen to identify two compounds that act as pore blockers. Mutations of those four putative pore-lining basic residues significantly altered the IC ₅₀ of these compounds at positive voltages. These findings contribute to our understanding regarding anion permeation of TMEM16A CaCC and provide valuable pharmacological tools to probe the channel pore.

Mucous cell hyperplasia and airway smooth muscle (ASM) hyperresponsiveness are hallmark features of inflammatory airway diseases, including asthma. Here, we show that the recently identified calcium-activated chloride channel (CaCC) TMEM16A is expressed in the adult airway surface epithelium and ASM. The epithelial expression is increased in asthmatics, particularly in secretory cells. Based on this and the proposed functions of CaCC, we hypothesized that TMEM16A inhibitors would negatively regulate both epithelial mucin secretion and ASM contraction. We used a high-throughput screen to identify small-molecule blockers of TMEM16A-CaCC channels. We show that inhibition of TMEM16A-CaCC significantly impairs mucus secretion in primary human airway surface epithelial cells. Furthermore, inhibition of TMEM16A-CaCC significantly reduces mouse and human ASM contraction in response to cholinergic agonists. TMEM16A-CaCC blockers, including those identified here, may positively impact multiple causes of asthma symptoms.

TMEM16A forms calcium-activated chloride channels (CaCCs) that regulate physiological processes such as the secretions of airway epithelia and exocrine glands, the contraction of smooth muscles, and the excitability of neurons. Notwithstanding intense interest in the mechanism behind TMEM16A-CaCC calcium-dependent gating, comprehensive surveys to identify and characterize potential calcium sensors of this channel are still lacking. By aligning distantly related calcium-activated ion channels in the TMEM16 family and conducting systematic mutagenesis of all conserved acidic residues thought to be exposed to the cytoplasm, we identify four acidic amino acids as putative calcium-binding residues. Alterations of the charge, polarity, and size of amino acid side chains at these sites alter the ability of different divalent cations to activate the channel. Furthermore, TMEM16A mutant channels containing double cysteine substitutions at these residues are sensitive to the redox potential of the internal solution, providing evidence for their physical proximity and solvent accessibility. Every cell in the body is surrounded by a barrier called the cell membrane. There are, however, a number of ways that molecules can pass through this membrane to either enter or leave the cell. Calcium-activated channels are a group of proteins that are embedded within the cell membrane and that allow different ions to pass through the membrane. These proteins are involved in a number of processes in a variety of tissues, for example in the gut, lungs and nervous system. A family of proteins called TMEM16 includes a number of calcium-activated channels that have been recently identified. However, it is not clear how these TMEM16 channel proteins detect the calcium ions that cause them to open. Two ideas have been suggested: the calcium ions might be detected by a protein called calmodulin, which then forces the channel to open; alternatively, the calcium ions might be detected by the channel protein itself. Tien, Peters et al. have now tested both of these ideas by focusing on a calcium-activated channel protein called TMEM16A, which allows chloride ions to pass through membranes. The possible role of calmodulin was tested in several ways, such as by preventing it from binding to the TMEM16A protein or from binding to calcium. However, none of these changes affected the opening of the channel; so Tien, Peters et al. concluded that calmodulin is not involved in these channels being activated by calcium ions. Next, Tien, Peters et al. tested specific parts of the TMEM16A channel protein itself to see if they were involved in calcium detection instead. Proteins are made from smaller building blocks called amino acids, and it is known that some amino acids are more likely to bind to calcium ions than others. There are 38 of these amino acids in the TMEM16A channel that are also found in other members of the TMEM16 family in both fruit flies and mammals. Tien, Peters et al. found that replacing five of these with other amino acids made the channel less sensitive to calcium. Further experiments suggested that four of these five amino acids are clustered at the site where a calcium ion might bind to the TMEM16A channel protein, which suggests that the protein itself can detect calcium directly. The next challenge will be to understand how calcium ions binding to the site on the TMEM16A channel protein can cause the channel to open to allow the chloride ions to pass through.

While the signals and complexes that coordinate the heartbeat are well established, how the heart maintains its electromechanical rhythm over a lifetime remains an open question with significant implications to human health. Reasoning that this homeostatic challenge confronts all pulsatile organs, we developed a high resolution imaging and analysis toolset for measuring cardiac function in intact, unanesthetized Drosophila melanogaster. We demonstrate that, as in humans, normal aging primarily manifests as defects in relaxation (diastole) while preserving contractile performance. Using this approach, we discovered that a pair of two-pore potassium channel (K2P) subunits, largely dispensable early in life, are necessary for terminating contraction (systole) in aged animals, where their loss culminates in fibrillatory cardiac arrest. As the pumping function of its heart is acutely dispensable for survival, Drosophila represents a uniquely accessible model for understanding the signaling networks maintaining cardiac performance during normal aging.

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.

In the United States, the production of many fruits and vegetables (F&Vs) is concentrated in a few key regions of the country, and it may not be possible to maintain or increase output from these areas due to natural resource constraints and climate change. Rather, developing new and more geographically diverse production systems may be necessary to meet food needs and adapt to changing conditions. However, there is currently a limited understanding of the availability and location of suitable land where future production could occur. In this project, we developed geospatial models to identify and quantify land suitable for producing F&V crops across the contiguous United States based on biophysical constraints. Suitability was based on key criteria related to land cover and use, topography, soil properties, and climate. We found tremendous potential to expand F&V production at the national level, identifying 333 million hectares of suitable land, 47% of which is currently used for agriculture. Suitable land to produce most F&Vs exists across the United States, indicating that there is significant potential to geographically diversify production in the future, though tropical and sub‐tropical fruits are restricted to the southern areas of the country. These results provide a starting point for identifying opportunities to expand F&V production, reduce geographic concentration, and establish new supply chains. Core Ideas Geospatial models were used to identify land biophysically suitable for producing fruit and vegetable crops. Three hundred and thirty‐three million hectares of suitable land were identified across the contiguous United States. Forty‐seven percent of suitable land is currently used for agriculture. Results indicate significant potential to geographically diversify production in the future.

Providing a wholesome and adequate food supply is the most basic tenet of agricultural sustainability. However, sharp increases in global food prices have occurred in the past 2 years, bringing the real price of food to the highest level seen in 30 years (FAO, 2008). This dramatic shift is a fundamental concern. The role of ‘local food’ in contributing to the solution of underlying problems is currently being debated, and the debate raises a critical question: To what degree can society continue to rely on large-scale, long-distance transportation of food? Growing concerns about climate change, the longevity of fossil fuel supplies and attempts to produce energy from agriculture suggest that energy efficiency will be critical to adapting to resource constraints and mitigating climate impacts. Moreover, these problems are urgent because energy prices, biofuel production and weather-related crop failures are partially responsible for the current world food price situation. Tools are needed to determine how the environmental impact and vulnerability of the food system are related to where food is produced in relation to where it is consumed. To this end, analyses of foodsheds, the geographic areas that feed population centers, can provide useful and unique insights.

Public concern about the environmental consequences of our diets is growing and regional food systems have emerged as one strategy to reduce these impacts. However, the potential environmental benefits of regional food systems are still largely untested and more empirical evidence is needed. Land and water are critical inputs to agriculture, but natural resource requirements and associated environmental impacts are highly dependent crop production location. This study used statistical analyses of crop yield and water footprint data to examine regional variability in land and water requirements and location‐specific tradeoffs between these resources. Regional land requirements and water requirements differed among most of the 17 fruit and vegetable crops we examined. Our results also showed inverse relationships between land and irrigation water requirements for eight crops, indicating that location‐specific tradeoffs between land and water resources exist for these crops. Understanding these regional differences and tradeoffs in natural resource requirements can help us evaluate the environmental implications of a more regionalized food system. Core Ideas Land and irrigation water requirements of 17 fruit and vegetable crops were analyzed. Regional land requirements vary significantly for all crops. Regional water requirements vary significantly for all crops, except snap beans. Location‐specific tradeoffs between land and water requirements exist for eight crops. These findings can help us evaluate the environmental impacts of regionalized food systems.

Increased fruit and vegetable (F&V) consumption would have important health benefits but would also have significant impacts on the U.S. agricultural system. The United States is currently a net importer of these foods but global agricultural systems face growing pressure to feed more people with fewer resources while also adapting to climate change. Thus, increasing self‐reliance may become a key strategy to ensuring a stable supply of nutrient‐dense foods in the United States. However, the capacity to increase the production of specific foods to accommodate shifts towards healthier dietary patterns or increase self‐reliance is not well documented. We estimated the extent to which the United States could meet current and recommended F&V consumption through domestic production based on biophysical capacity at the national and regional levels. Land suitability maps from a previous study were combined with state‐level yield data to estimate biophysical capacity and food availability data were used to estimate F&V consumption. A net‐balance analysis was conducted to compare production capacity to food consumption under both diet scenarios. Our results indicate that the United States could meet current and recommended F&V needs of Americans through domestic production and that each region could meet regional F&V consumption. However, while self‐reliance is biophysically possible, it would require substantial changes in dietary patterns and land use. These findings provide insight into the feasibility and agricultural implications of self‐reliance at the national and regional levels. Core ideas A net‐balance analysis was used to compare production capacity to food consumption. United States could meet current and recommended F&V needs of Americans through domestic production. Each region could be self‐sufficient but changes in dietary patterns would be necessary. Self‐reliance is biophysically possible but would require substantial changes in land use.