title: Microfluidics platform for single-shot dose-response analysis of chloride channel-modulating compounds.
abstract: We previously developed cell-based kinetics assays of chloride channel modulators utilizing genetically encoded yellow fluorescent proteins. Fluorescence platereader-based high-throughput screens yielded small-molecule activators and inhibitors of the cAMP-activated chloride channel CFTR and calcium-activated chloride channels, including TMEM16A. Here, we report a microfluidics platform for single-shot determination of concentration-activity relations in which a 1.5 × 1.5 mm square area of adherent cultured cells is exposed for 5-10 min to a pseudo-logarithmic gradient of test compound generated by iterative, two-component channel mixing. Cell fluorescence is imaged following perfusion with an iodide-containing solution to give iodide influx rate at each location in the image field, thus quantifying modulator effects over a wide range of concentrations in a single measurement. IC50 determined for CFTR and TMEM16A activators and inhibitors by single-shot microfluidics were in agreement with conventional plate reader measurements. The microfluidics approach developed here may accelerate the discovery and characterization of chloride channel-targeted drugs.
authors.affiliation: Departments of Medicine and Physiology, University of California, San Francisco, CA 94143, USA. Alan.Verkman@ucsf.edu.
authors.forename: Byung-Ju
authors.initials: BJ
authors.lastname: Jin
authors.forename: Eun-A
authors.initials: EA
authors.lastname: Ko
authors.forename: Wan
authors.initials: W
authors.lastname: Namkung
authors.forename: A S
authors.initials: AS
authors.lastname: Verkman
mesh_terms: D000818:Animals; D000075369:Anoctamin-1; D002460:Cell Line; D018118:Chloride Channels / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D057166:High-Throughput Screening Assays; D006801:Humans; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D051381:Rats; D054852:Small Molecule Libraries / Q000494:pharmacology*
chemical_list: C476213:ANO1 protein, human; D000075369:Anoctamin-1; D018118:Chloride Channels; D009363:Neoplasm Proteins; D054852:Small Molecule Libraries; D019005:Cystic Fibrosis Transmembrane Conductance Regulator
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