Transepithelial secretion of Cl- is of major importance in the normal physiology and pathology of a number of organs, most notably the gastrointestinal tract and the airways. The discovery of the Cystic Fibrosis Transmembrane Conductance Regulator, CFTR, as a key apical membrane Cl-channel has been a major advance in the study of Cl-secretion. The goal of this project is to study the regulation and pharmacological modulation of CFTR in the context of a secreting epithelial monolayer utilizing the non- invasive methods of fluctuation and impedance analysis. Preliminary results will demonstrate the feasibility of performing fluctuation and impedance analysis on human colonic epithelial cells (T84 cells) and patient derived primary cultures of airway cells expressing wt and mutant forms of CFTR. Three hypotheses will be investigated: (1) CFTR is the major, if not sole, apical membrane Cl-channel mediating the secretion of Cl- and that regulation of the activity of CFTR is mediated by altering N and not P/o. (2) Benzimidazolone and psoralen stimulation of Cl-secretion is mediated by the activation of CFTR and that these agents act to cause a unique state of activation of CFTR. (3) Regulation and modulation of CFTR are cell specific. Studies for Specific Aims 1 and 2 will be performed on T84 cells and the results compared with those obtained with airway cells, Specific Aim 3.
Specific Aim 1 will determine th4e contribution of CFTR and other potential Cl-channels to transepithelial Cl- secretion, identify those kinases and phosphatases involved in their regulation and determined by what mechanism (N versus P/o) they are regulated. Sp3ecific Aim 2 will determine whether CFTR is the Cl-channel activated by the above pharmacological agents, how they regulated CFTR's channel properties and whether they cause a state of activation different from that caused by cAMP-mediated agonists.
Specific Aim 3 will determine the contribution and properties of CFTR in airway cell Cl-secretion, the kinases and phosphatases specifically involved in airway cell CFTR regulation, the influence of the above pharmacological agents on CFTR in airway cells and determine the properties, regulation and modulation of mutant forms of CFTR.
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