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 calciumactivated 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.A sthma is a significant cause of morbidity and mortality worldwide, and the prevalence of this disease is increasing among all age, sex, and racial groups. Characteristic features of asthma include inflammation, subepithelial fibrosis, hyperplasia of mucus-producing cells, accumulation of mucus within airway lumens, hyperplasia of airway smooth muscle (ASM), and ASM hyperresponsiveness. Together, these symptoms impair lung function by limiting the flow of gases to and from the alveoli in the distal lung.The current standard of care for asthma involves inhaled corticosteroids for the management of inflammation combined with long-acting agonists of β2-adrenergic receptors. Despite this treatment, lung function is not improved in 30-45% of asthmatic patients, and exacerbations continue to be a major problem (reviewed in ref. 1). Asthma can be divided into at least two distinct molecular phenotypes defined by the degree of Th2 inflammation (2, 3). Cytokines, including IL-4 and IL-13, promote airway epithelial mucous cell metaplasia, subepithelial fibrosis, and hyperplasia of smooth muscle in Th2-high asthmatics, and these patients generally show improved lung function with inhaled corticosteroid therapy. A greater understanding of this heterogeneity and the molecular and physiological events that lead to airway remodeling might lead to improved diagnosis and treatment.Calcium-activated chloride channels (CaCCs) have been ascribed numerous cellular functions (reviewed in refs. 4 and 5), among these are epithelial fluid secretion and smooth muscle contraction, both of which contribute to the progression and severity of asthma. Moreover, calcium-activated chloride currents in the airway epithelium are enhanced by the Th2 cytokines IL-4 and IL-13, as well as IFN-γ (6). For these reasons, CaCC is an attractive potential therapeutic target for asthma (7). However, the study of CaCC was impeded by lack of information about the gene(s) encoding this channel. It was only relatively recently that TMEM16A (transmembrane p...
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.DOI: http://dx.doi.org/10.7554/eLife.02772.001
Effectiveness of vegetative buffer strips for herbicide retention from agricultural runoff was evaluated in a twoyear natural rainfall study. A source area of 0.41 ha (mainly Canisteo silty clay loam soil), having an average slope of 3%, was fall chisel-plowed, spring disked, and planted to corn. Three herbicides (atrazine, metolachlor, and cyanazine) were applied to the source area in each spring. Six vegetative buffer strips, 1.52 m wide ¥ 20.12 m long, were isolated with metal borders downslope of the source area in a well established bromegrass (Bromus inermis) waterway. These strips provided for three replications of two drainage to buffer area ratio treatments of 15:1 and 30:1. Herbicide retention was dependent on the antecedent moisture conditions of the strips. These retentions ranged from 11 to 100% for atrazine, 16 to 100% for metolachlor, and 8 to 100% for cyanazine. Herbicide retention by the buffer strips for the two treatments were not significantly different for the observed storm events. Herbicide concentrations in solution in outflow from the strips were less than the inflow concentrations for all the three herbicides. Infiltration was the key process for herbicide retention by the buffer strips, although there was some adsorption to in-place soil and/or vegetation. Metolachlor concentrations in sediment increased in outflow for the two treatments; however, the opposite was true for atrazine and cyanazine. Herbicide retention by sediment deposition in the strip represented about 5% of the total herbicide retention by the buffer strips. The buffer strips were found to have high percent sediment retention, ranging from 40 to 100%; thus, the strips would be more effective for retaining strongly adsorbed herbicides.
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