Synthesis of α3ß4 Nicotinic Acetylcholine Receptor Modulators Derived from Aristoquinoline That Reduce Reinstatement of Cocaine-Seeking Behavior.

Growing evidence suggests that inhibition of the α3ß4 nicotinic acetylcholine receptor (nAChR) represents a promising therapeutic strategy to treat cocaine use disorder. Recently, aristoquinoline (1), an alkaloid from Aristotelia chilensis, was identified as an α3ß4-selective nAChR inhibitor. Here, we prepared 22 derivatives of 1 and evaluated their ability to inhibit the α3ß4 nAChR. These studies revealed structure-activity trends and several compounds with increased potency compared to 1 with few off-target liabilities. Additional mechanistic studies indicated that these compounds inhibit the α3ß4 nAChR noncompetitively, but do not act as channel blockers, suggesting they are negative allosteric modulators. Finally, using a cocaine-primed reinstatement paradigm, we demonstrated that 1 significantly attenuates drug-seeking behavior in an animal model of cocaine relapse. The results from these studies further support a role for the α3ß4 nAChR in the addictive properties of cocaine and highlight the possible utility of aristoquinoline derivatives in treating cocaine use disorder.

Mapping U.S. Food System Localization Potential: The Impact of Diet on Foodsheds.

In the long term, food systems must heed natural resource limits. Localized production and dietary changes are often suggested as potential solutions. However, no U.S. analyses fully evaluate the feasibility to scale localization across a range of diets. We therefore modeled the biophysical capacity for regional food systems based on agricultural land area and productivity, population, and 7 diet scenarios ranging in meat-intensity, from current consumption to vegan. We estimated foodshed size, colloquially known as "food miles" for 378 U.S. metropolitan centers, in a hypothetical nationwide closed system that prioritizes localized food. We found that foodshed size (weighted average distance traveled) for three land types ranged from 351-428 km (cultivated cropland), 80-492 km (perennial forage cropland), and 117-799 km (grazing land). Localized potential varies regionally: foodsheds are generally larger in the populous Northeast, Southeast, and Southwest than in the Northwest and the center of the country. However, depending on consumption of animal-based foods, a sizable proportion of the population could meet its food needs within 250km: from 35%-53% (cultivated cropland), 39%-94% (perennial forage cropland, 100% for vegan), and 26%-88% (grazing land, 100% for ovolacto-vegetarian and vegan). All seven scenarios leave some land unused. This reserve capacity might be used to supply food to the global market, grow bioenergy crops, or for conservation.

Paralytic, the Drosophila voltage-gated sodium channel, regulates proliferation of neural progenitors.

Proliferating cells, typically considered "nonexcitable," nevertheless, exhibit regulation by bioelectric signals. Notably, voltage-gated sodium channels (VGSC) that are crucial for neuronal excitability are also found in progenitors and up-regulated in cancer. Here, we identify a role for VGSC in proliferation of Drosophila neuroblast (NB) lineages within the central nervous system. Loss of paralytic (para), the sole gene that encodes Drosophila VGSC, reduces neuroblast progeny cell number. The type II neuroblast lineages, featuring a population of transit-amplifying intermediate neural progenitors (INP) similar to that found in the developing human cortex, are particularly sensitive to para manipulation. Following a series of asymmetric divisions, INPs normally exit the cell cycle through a final symmetric division. Our data suggests that loss of Para induces apoptosis in this population, whereas overexpression leads to an increase in INPs and overall neuroblast progeny cell numbers. These effects are cell autonomous and depend on Para channel activity. Reduction of Para expression not only affects normal NB development, but also strongly suppresses brain tumor mass, implicating a role for Para in cancer progression. To our knowledge, our studies are the first to identify a role for VGSC in neural progenitor proliferation. Elucidating the contribution of VGSC in proliferation will advance our understanding of bioelectric signaling within development and disease states.

TMEM16B Calcium-Activated Chloride Channels Regulate Action Potential Firing in Lateral Septum and Aggression in Male Mice.

The lateral septum (LS) plays an important role in regulating aggression. It is well recognized that LS lesions lead to a dramatic increase in aggressive behaviors. A better understanding of LS neurophysiology and its functional output is therefore important to assess LS involvement in regulating aggression. The LS is a heterogeneous structure that maintains inputs and outputs with multiple brain regions, and is also divided into subregions that innervate one another. Thus, it is challenging to identify the exact cell type and projections for characterization. In this study, we determined the expression pattern of the calcium-activated chloride channel, TMEM16B, in the LS of both male and female mice. We then investigated the physiological contribution of the calcium-activated chloride channel to LS neuronal signaling. By performing whole-cell patch-clamp recording, we showed that TMEM16B alters neurotransmitter release at the hippocampal-LS synapse, and regulates spike frequency and spike frequency adaptation in subpopulations of LS neurons. We further demonstrated that loss of TMEM16B function promotes lengthened displays of aggressive behaviors by male mice during the resident intruder paradigm. In conclusion, our findings suggest that TMEM16B function contributes to neuronal excitability in subpopulations of LS neurons and the regulation of aggression in male mice.SIGNIFICANCE STATEMENT Aggression is a behavior that arose evolutionarily from the necessity to compete for limited resources and survival. One particular brain region involved in aggression is the lateral septum (LS). In this study, we characterized the expression of the TMEM16B calcium-activated chloride channel in the LS and showed that TMEM16B regulates the action potential firing frequency of LS neurons. We discovered that loss of TMEM16B function lengthens the displays of aggressive behaviors in male mice. These findings suggest that TMEM16B plays an important role in regulating LS neuronal excitability and behaviors associated with LS function, thereby contributing to our understanding of how the LS may regulate aggression.

TMEM16A controls EGF-induced calcium signaling implicated in pancreatic cancer prognosis.

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.

Perspective: Understanding the Intersection of Climate/Environmental Change, Health, Agriculture, and Improved Nutrition - A Case Study: Type 2 Diabetes.

Efforts to promote health through improved diet and nutrition demand an appreciation of the nutritional ecology that accounts for the intersection of agriculture, food systems, health, disease and a changing environment. The complexity and implications of this ecology is exemplified by current trends and efforts to address nutrition-related non-communicable diseases (NCDs), most prominently type 2 diabetes. The global prevalence of type 2 diabetes continues to rise unabated. Of particular concern is how to address the unhealthy dietary patterns that are contributing to this pandemic in a changing environment. A multi- disciplinary approach is required that will engage those communities that comprise the continuum of effort from research to translation and implementation of evidence-informed interventions, programs and policies. Using the prevention of type 2 diabetes by increasing fruit and vegetable consumption as an exemplar, we argue that the ability to effect positive change in this and other persistent nutrition-related problems can be achieved by moving away from siloed approaches that limit the integration of key components of the diet-health continuum. Ultimately the impact of preventing type 2 diabetes via increased fruit and vegetable consumption will depend on how the entire diet changes, not just fruits and vegetables. In addition, the rapidly changing physical environment that will confront our food production system going forward will also shape the interventions that are possible. Nonetheless, the proposed "team science" approach that accounts for all the elements of the nutrition ecology will better position us to achieve public health goals through safe and sustainable food systems.

Greenhouse Gas Emissions in the United States Food System: Current and Healthy Diet Scenarios.

We estimate the impact on greenhouse gas emissions (GHGE) of shifting from the current average United States diet to four alternative diets that meet the 2010 Dietary Guidelines for Americans (DGA). In contrast to prior studies, which rely on process-based life-cycle-analysis GHGE estimates from the literature for particular food items, we combine a diet model, an environmentally extended input-output model of energy use in the U.S. food system, and a biophysical model of land use for crops and livestock to estimate food system GHGE from the combustion of fossil fuels and from biogenic sources, including enteric fermentation, manure management, and soil management. We find that an omnivore diet that meets the DGA while constraining cost leaves food system GHGE essentially unchanged relative to the current baseline diet (985 000 000 tons of CO2 eq or 3191 kilograms of CO2 eq per capita per year), while a DGA-compliant vegetarian and a DGA-compliant omnivore diet that minimizes energy consumption in the food system reduce GHGE by 32% and 22%, respectively. These emission reductions were achieved mainly through quantity and composition changes in the meat, poultry, fish; dairy; and caloric sweeteners categories. Shifting from current to healthy diets as defined by the DGA does not necessarily reduce GHGE in the U.S. food system, although there are diets, including two presented here and by inference many others, which can achieve a reduction in GHGE.

Capacity of the US Food System to Accommodate Improved Diet Quality: A Biophysical Model Projecting to 2030.

BACKGROUND: Increasing Americans' diet quality will require changes to the food supply. Due to the complex nature of the food system, this is not as straightforward as simply increasing the production of healthy foods and decreasing the production of unhealthy foods. Little is known about whether the US food system can produce enough food, given finite agricultural resources, to support shifts toward healthier eating patterns. OBJECTIVE: The aim of this study was to model the capacity of the US food system to accommodate a shift toward a healthier diet by 2030. METHODS: A biophysical simulation model estimated the proportion of the US population that could be fed a given diet based on food system constraints, currently and projected to 2030. The model accepted data inputs on food intake, crop yields, and population size. Linear and nonlinear regression models were used to estimate projected food intake and crop yields based on recent historical data (1980-2014). Diet quality was estimated using the Healthy Eating Index-2015. RESULTS: The US agricultural system can produce enough food to feed 146% of the population by 2030. A greater proportion of the population can be fed a high-quality diet than a low-quality diet (178% compared to 119%). To accommodate increased diet quality, substantial increases in cropland acreage would be needed for fruits (P < 0.001), vegetables (P = 0.002), legumes (P = 0.002), and nuts (P = 0.007); and decreased cropland acreage would be needed for grains (P = 0.002) and sweeteners (P < 0.001). CONCLUSIONS: The US can produce more than enough food to accommodate a shift toward a healthier diet pattern, but even moderate shifts in diet quality would require major transitions in cropland use. The success of this transition is dependent on several factors, like individuals' ease of entry into the agricultural sector, producers' ability to shift production to other crops, and modifications to the food supply chain.

The Sixth Transmembrane Segment Is a Major Gating Component of the TMEM16A Calcium-Activated Chloride Channel.

Calcium-activated chloride channels (CaCCs) formed by TMEM16A or TMEM16B are broadly expressed in the nervous system, smooth muscles, exocrine glands, and other tissues. With two calcium-binding sites and a pore within each monomer, the dimeric CaCC exhibits voltage-dependent calcium sensitivity. Channel activity also depends on the identity of permeant anions. To understand how CaCC regulates neuronal signaling and how CaCC is, in turn, modulated by neuronal activity, we examined the molecular basis of CaCC gating. Here, we report that voltage modulation of TMEM16A-CaCC involves voltage-dependent occupancy of calcium- and anion-binding site(s) within the membrane electric field as well as a voltage-dependent conformational change intrinsic to the channel protein. These gating modalities all critically depend on the sixth transmembrane segment.

Cryo-EM structures of the TMEM16A calcium-activated chloride channel.

Calcium-activated chloride channels (CaCCs) encoded by TMEM16A control neuronal signalling, smooth muscle contraction, airway and exocrine gland secretion, and rhythmic movements of the gastrointestinal system. 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. 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, as well as subnanometre-resolution electron cryo-microscopy. 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 Ca2+ ion resolved within each monomer with a constricted pore; this is likely to correspond to a closed state, because a CaCC with a single Ca2+ occupancy requires membrane depolarization in order to open (C.J.P. et al., manuscript submitted). The structure in nanodiscs has two Ca2+ 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 Ca2+. 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.

Characterizing trends in fruit and vegetable intake in the USA by self-report and by supply-and-disappearance data: 2001-2014.

OBJECTIVE: To examine the comparability of fruit and vegetable (F&V) intake data in the USA from 2001 to 2014 between data acquired from two national data collection programmes. DESIGN: Cross-sectional analysis. Linear regression models estimated trends in daily per capita intake of total F&V. Pooled differences in intake of individual F&V (n 109) were examined by processing form (fresh, frozen, canned, dried and juice). SETTING: What We Eat in America (WWEIA, 2001-2014) and Loss-Adjusted Food Availability data series (LAFA, 2001-2014). RESULTS: No temporal trends were observed in daily per capita intake of total F&V from 2001 to 2014 using WWEIA and LAFA. Modest differences between WWEIA and LAFA were observed in mean pooled intake of most individual F&V. CONCLUSIONS: WWEIA and LAFA produced similar estimates of F&V intake. However, WWEIA may be best suited for monitoring intake at the national level because it allows for the identification of individual F&V in foods with multiple ingredients, and it is structured for sub-population analysis and covariate control. LAFA does retain advantages for other research protocols, specifically by providing the only nationally representative estimates of food losses at various points in the food system, which makes it useful for examining the adequacy of the food supply at the agricultural, retail and consumer levels.

Agricultural Capacity to Increase the Production of Select Fruits and Vegetables in the US: A Geospatial Modeling Analysis.

The capacity of US agriculture to increase the output of specific foods to accommodate increased demand is not well documented. This research uses geospatial modeling to examine the capacity of the US agricultural landbase to increase the per capita availability of an example set of nutrient-dense fruits and vegetables. These fruits and vegetables were selected based on nutrient content and an increasing trend of domestic production and consumption. Geographic information system models were parameterized to identify agricultural land areas meeting crop-specific growing requirements for monthly precipitation and temperature; soil depth and type; cropland availability; and proximity to existing production centers. The results of these analyses demonstrate that crop production can be expanded by nearly 144,000 ha within existing national production centers, generating an additional 0.05 cup-equivalents of fruits and vegetables per capita per day, representing a 1.7% increase above current total F&V availability. Expanding the size of national crop production centers can further increase the availability of all F&V by 2.5%-5.4%, which is still less than the recommended amount. Challenges to increasing F&V production in the US include lack of labor availability, barriers to adoption among producers, and threats to crop yields from environmental concerns.

Age-dependent diastolic heart failure in an in vivo Drosophila model.

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.

TAOK2 Kinase Mediates PSD95 Stability and Dendritic Spine Maturation through Septin7 Phosphorylation.

Abnormalities in dendritic spines are manifestations of several neurodevelopmental and psychiatric diseases. TAOK2 is one of the genes in the 16p11.2 locus, copy number variations of which are associated with autism and schizophrenia. Here, we show that the kinase activity of the serine/threonine kinase encoded by TAOK2 is required for spine maturation. TAOK2 depletion results in unstable dendritic protrusions, mislocalized shaft-synapses, and loss of compartmentalization of NMDA receptor-mediated calcium influx. Using chemical-genetics and mass spectrometry, we identified several TAOK2 phosphorylation targets. We show that TAOK2 directly phosphorylates the cytoskeletal GTPase Septin7, at an evolutionary conserved residue. This phosphorylation induces translocation of Septin7 to the spine, where it associates with and stabilizes the scaffolding protein PSD95, promoting dendritic spine maturation. This study provides a mechanistic basis for postsynaptic stability and compartmentalization via TAOK2-Sept7 signaling, with implications toward understanding the potential role of TAOK2 in neurological deficits associated with the 16p11.2 region.

Four basic residues critical for the ion selectivity and pore blocker sensitivity of TMEM16A calcium-activated chloride channels.

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 IC50 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.

A comprehensive search for calcium binding sites critical for TMEM16A calcium-activated chloride channel activity.

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.

Subdued, a TMEM16 family Ca²âº-activated Cl⁻channel in Drosophila melanogaster with an unexpected role in host defense.

TMEM16A and TMEM16B are calcium-activated chloride channels (CaCCs) with important functions in mammalian physiology. Whether distant relatives of the vertebrate TMEM16 families also form CaCCs is an intriguing open question. Here we report that a TMEM16 family member from Drosophila melanogaster, Subdued (CG16718), is a CaCC. Amino acid substitutions of Subdued alter the ion selectivity and kinetic properties of the CaCC channels heterologously expressed in HEK 293T cells. This Drosophila channel displays characteristics of classic CaCCs, thereby providing evidence for evolutionarily conserved biophysical properties in the TMEM16 family. Additionally, we show that knockout flies lacking subdued gene activity more readily succumb to death caused by ingesting the pathogenic bacteria Serratia marcescens, suggesting that subdued has novel functions in Drosophila host defense. DOI: http://dx.doi.org/10.7554/eLife.00862.001.

Allosteric coupling of the inner activation gate to the outer pore of a potassium channel.

In potassium channels, functional coupling of the inner and outer pore gates may result from energetic interactions between residues and conformational rearrangements that occur along a structural path between them. Here, we show that conservative mutations of a residue near the inner activation gate of the Shaker potassium channel (I470) modify the rate of C-type inactivation at the outer pore, pointing to this residue as part of a pathway that couples inner gate opening to changes in outer pore structure and reduction of ion flow. Because they remain equally sensitive to rises in extracellular potassium, altered inactivation rates of the mutant channels are not secondary to modified binding of potassium to the outer pore. Conservative mutations of I470 also influence the interaction of the Shaker N-terminus with the inner gate, which separately affects the outer pore.

Calcium-activated chloride channel TMEM16A modulates mucin secretion and airway smooth muscle contraction.

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.

Mechanism of accelerated current decay caused by an episodic ataxia type-1-associated mutant in a potassium channel pore.

In Kv1.1, single point mutants found below the channel activation gate at residue V408 are associated with human episodic ataxia type-1, and impair channel function by accelerating decay of outward current during periods of membrane depolarization and channel opening. This decay is usually attributed to C-type inactivation, but here we provide evidence that this is not the case. Using voltage-clamp fluorimetry in Xenopus oocytes, and single-channel patch clamp in mouse ltk- cells, of the homologous Shaker channel (with the equivalent mutation V478A), we have determined that the mutation may cause current decay through a local effect at the activation gate, by destabilizing channel opening. We demonstrate that the effect of the mutant is similar to that of trapped 4-aminopyridine in antagonizing channel opening, as the mutation and 10 mm 4-AP had similar, nonadditive effects on fluorescence recorded from the voltage-sensitive S4 helix. We propose a model where the Kv1.1 activation gate fails to enter a stabilized open conformation, from which the channel would normally C-type inactivate. Instead, the lower pore lining helix is able to enter an activated-not-open conformation during depolarization. These results provide an understanding of the molecular etiology underlying episodic ataxia type-1 due to V408A, as well as biophysical insights into the links between the potassium channel activation gate, the voltage sensor and the selectivity filter.