We report the application of a targetable green fluorescent protein-based cellular halide indicator. Fluorescence titrations of the purified recombinant yellow fluorescent protein YFP-H148Q indicated a pK a of 7.14 in the absence of Cl

ASL ͉ chloride ͉ trachea ͉ ratio imaging T he air-facing surface of respiratory airways is lined by a layer of surface epithelial cells whose mucosa is bathed by a thin film of fluid called the airway surface liquid (ASL) (1-5). The airways also contain submucosal glands that secrete fluid and macromolecules onto the ASL (6-9). The submucosal glands contain serous tubules and acini that secrete salt, water, and various antimicrobial proteins. The serous secretions pass through mucous tubules, where viscous glycoproteins are added, and then into a collecting duct and onto the airway surface. Active salt and water secretion by serous epithelial cells is believed to involve Cl Ϫ transport by the cystic fibrosis transmembrane conductance regulator (CFTR) protein (10-12) driving water transport through AQP4 and AQP5 water channels (13-15). Submucosal gland secretions are proposed to be important for generation of ASL fluid and for creation of an environment that inhibits bacterial colonization.Abnormalities in submucosal gland secretions have been proposed to contribute to the airway pathophysiology in cystic fibrosis (CF). CFTR is expressed in serous epithelial cells of submucosal glands more strongly than in other tissues of the airways and lung (16,17 ], pH and viscosity of freshly secreted fluid from human submucosal airway glands to determine: (i) whether the composition of fluid secreted from glands is similar to that of the ASL, and (ii) whether gland fluid composition differs in normal vs. CF human airways. We recently developed fluorescent probes and ratio imaging microscopy methods to measure [Na MethodsAirway Preparations. Fragments of normal and CF human airways were obtained after lung transplantation and consisted of scrap airway trimmings from normal donor lungs and larger tissue fragments from discarded CF lungs. Tissues were placed in cold Physiosol (Abbott) within 30 min after removal for transport to the laboratory and initial dissection. Tissues were then transferred to ice-cold HCO 3 Ϫ -buffered Krebs solution (see below) and continuously gassed with 95% O 2 and 5% CO 2 . Tissues were cut into rectangles of Ϸ2 cm 2 , and a 1.5-to 2-mm thick layer containing the mucosa and submucosa was dissected away from the underlying cartilage. Two different procedures were used to mount the tissues. In the first procedure, tissues were placed over a wire mesh in a tissue culture well with HCO 3 Ϫ -containing media bathing the serosa and left in a cell culture incubator for 30 min. The airway fragment was then mounted by using pins on a sponge soaked in HCO 3 Ϫ -containing Kreb's buffer (120 mM NaCl͞25 mM NaHCO 3 ͞3.3 mM KH 2 PO 4 ͞0.8 mM K 2 HPO 4 ͞1.2 mM MgCl 2 ͞1.2 mM CaCl 2 ͞10 mM glucose, pH 7.4) (on the serosal side) and held in a perfusion chamber with This paper was submitted directly (Track II) to the PNAS office.Abbreviations: CF, cystic fibrosis; ASL, airway surface liquid; BCECF, 2Ј,7Ј-bis-(2-carboxyethyl)-5-(and-6)-carboxy fluorescein; CFTR, cystic fibrosis transmembrane conductance regulator. § ...

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Drug discovery by high-throughput screening is a promising approach to develop new therapies for the most common lethal genetic disease, cystic fibrosis. Because disease-causing mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) protein produce epithelial cells with reduced or absent Cl(-) permeability, the goal of screening is to identify compounds that restore cell Cl(-) transport. We have developed a rapid, quantitative screening procedure for analysis of CFTR-mediated halide transport in cells with the use of a conventional fluorescence plate reader. Doubly transfected cell lines were generated that express wild-type or mutant CFTR together with a yellow fluorescent protein (YFP)-based halide sensor. CFTR function was assayed from the time course of cell fluorescence in response to extracellular addition of 100 mM I(-) followed by forskolin, resulting in decreased YFP fluorescence due to CFTR-mediated I(-) entry. Cell lines were chosen, and conditions were optimized to minimize basal halide transport to maximize assay sensitivity. In cells cultured on 96-well plastic dishes, the assay gave reproducible halide permeabilities from well to well and could reliably detect a 2% activation of CFTR-dependent halide transport produced by low concentrations of forskolin. Applications of the assay are shown, including comparative dose-dependent CFTR activation by genistein, apigenin, 8-cyclopentyl-1,3-dipropylxanthine, IBMX, 8-methoxypsoralen, and milrinone as well as activation of alternative Cl(-) channels. The fluorescence assay and cell lines should facilitate the screening of novel CFTR activators and the characterization of alternative Cl(-) channels and transporters.