CRACM ion channel function in human lung mast cells. (360G-Wellcome-087499_Z_08_Z)

We hypothesise that CRACM ion channels contribute to the Ca2+ influx pathway in human lung mast cells (HLMC) and may therefore serve as novel targets for the treatment of asthma and related diseases. We wish to strengthen this hypothesis by defining: 1. The mRNA and protein expression of CRACM channels in HLMC using quantitative RT-PCR, western blotting and flow cytometry, and their functional expression examined on single cells using patch clamp electrophysiology. 2. The functional role of CRACM channels in mast cell Ca2+ influx, secretion, migration, proliferation, survival and adhesion as determined by both pharmacological blockade and knock-down experiments using RNA interference and by overexpression of dominant negative pore mutants. 3. The factors regulating the gene expression and trafficking of CRACM channels to the mast cell plasma membrane determined by real-time PCR and live cell confocal microscopy. 4. The ability of CRACM channels to form signalling complexes with the Ca2+-activated K+ channel KCa3.1 and 2-adrenoceptor, determined by pharmacological manipulation, over-expression of epitope-tagged native and mutant channels, shRNA knockdown, and co-immunoprecipitation. 5. The effects of CRACM blockade on cell activation within cultured bronchial tissue explants. These studies will significantly enhance our understanding of the electrical mechanisms controlling HLMC function.