The calcium-activated chloride channel anoctamin 1 (ANO1) is located within the 11q13 amplicon, one of the most frequently amplified chromosomal regions in human cancer, but its functional role in tumorigenesis has remained unclear. The 11q13 region is amplified in ∼15% of breast cancers. Whether ANO1 is amplified in breast tumors, the extent to which gene amplification contributes to ANO1 overexpression, and whether overexpression of ANO1 is important for tumor maintenance have remained unknown. We have found that ANO1 is amplified and highly expressed in breast cancer cell lines and primary tumors. Amplification of ANO1 correlated with disease grade and poor prognosis. Knockdown of ANO1 in ANO1-amplified breast cancer cell lines and other cancers bearing 11q13 amplification inhibited proliferation, induced apoptosis, and reduced tumor growth in established cancer xenografts. Moreover, ANO1 chloride channel activity was important for cell viability. Mechanistically, ANO1 knockdown or pharmacological inhibition of its chloride-channel activity reduced EGF receptor (EGFR) and calmodulin-dependent protein kinase II (CAMKII) signaling, which subsequently attenuated AKT, v-src sarcoma viral oncogene homolog (SRC), and extracellular signal-regulated kinase (ERK) activation in vitro and in vivo. Our results highlight the involvement of the ANO1 chloride channel in tumor progression and provide insights into oncogenic signaling in human cancers with 11q13 amplification, thereby establishing ANO1 as a promising target for therapy in these highly prevalent tumor types.ion channel | CaCCinh-A01 | TMEM16A | HNSCC | ESCC

Many commonly used, structurally diverse, drugs block the human ether-a-go-go-related gene (hERG) K(+) channel to cause acquired long QT syndrome, which can lead to sudden death via lethal cardiac arrhythmias. This undesirable side effect is a major hurdle in the development of safe drugs. To gain insight about the structure of hERG and the nature of drug block we have produced structural models of the channel pore domain, into each of which we have docked a set of 20 hERG blockers. In the absence of an experimentally determined three-dimensional structure of hERG, each of the models was validated against site-directed mutagenesis data. First, hERG models were produced of the open and closed channel states, based on homology with the prokaryotic K(+) channel crystal structures. The modeled complexes were in partial agreement with the mutagenesis data. To improve agreement with mutagenesis data, a KcsA-based model was refined by rotating the four copies of the S6 transmembrane helix half a residue position toward the C-terminus, so as to place all residues known to be involved in drug binding in positions lining the central cavity. This model produces complexes that are consistent with mutagenesis data for smaller, but not larger, ligands. Larger ligands could be accommodated following refinement of this model by enlarging the cavity using the inherent flexibility about the glycine hinge (Gly648) in S6, to produce results consistent with the experimental data for the majority of ligands tested.

Inhibition of airway epithelial sodium channel (ENaC) function enhances mucociliary clearance (MCC). ENaC is positively regulated by channel-activating proteases (CAPs), and CAP inhibitors are therefore predicted to be beneficial in diseases associated with impaired MCC. The aims of the present study were to 1) identify low-molecular-weight inhibitors of airway CAPs and 2) to establish whether such CAP inhibitors would translate into a negative regulation of ENaC function in vivo, with a consequent enhancement of MCC. To this end, camostat, a trypsin-like protease inhibitor, provided a potent (IC 50 ϳ50 nM) and prolonged attenuation of ENaC function in human airway epithelial cell models that was reversible upon the addition of excess trypsin. In primary human bronchial epithelial cells, a potency order of placental bikunin Ͼ camostat Ͼ 4-guanidinobenzoic acid 4-carboxymethyl-phenyl ester Ͼ aprotinin Ͼ Ͼ soybean trypsin inhibitor ϭ ␣1-antitrypsin, was largely consistent with that observed for inhibition of prostasin, a molecular candidate for the airway CAP. In vivo, topical airway administration of camostat induced a potent and prolonged attenuation of ENaC activity in the guinea pig trachea (ED 50 ϭ 3 g/kg). When administered by aerosol inhalation in conscious sheep, camostat enhanced MCC out to at least 5 h after inhaled dosing. In summary, camostat attenuates ENaC function and enhances MCC, providing an opportunity for this approach toward the negative regulation of ENaC function to be tested therapeutically.

Potassium (K (+)) channels can regulate ionic conduction through their pore by a mechanism, involving the selectivity filter, known as C-type inactivation. This process is rapid in the hERG K (+) channel and is fundamental to its physiological role. Although mutations within hERG are known to remove this process, a structural basis for the inactivation mechanism has yet to be characterized. Using MD simulations based on homology modeling, we observe that the carbonyl of the filter aromatic, Phe627, forming the S 0 K (+) binding site, swiftly rotates away from the conduction axis in the wild-type channel. In contrast, in well-characterized non-inactivating mutant channels, this conformational change occurs less frequently. In the non-inactivating channels, interactions with a water molecule located behind the selectivity filter are critical to the enhanced stability of the conducting state. We observe comparable conformational changes in the acid sensitive TASK-1 channel and propose a common mechanism in these channels for regulating efflux of K (+) ions through the selectivity filter.