The present research proposal deals with the biochemical definition of conductive chloride channels in airway epithelia of several animal species and of human nasal tissue. The ultimate goal is to gain understanding of the possible structural changes in the channel components which underlie the deranged activity in epithelia of cystic fibrosis patients. Several basic approaches are proposed to be used for the identification and structural characterization of the putative channel components: (i) affinity labeling based on the use of covalent binding and photoreactive binding analogs of phenyl-anthranilate-related drugs which have demonstrable inhibitory activity on the channels. (ii) immunodetection of the channel components by antiidiotypic antibodies raised against the channel blockers-coupled to carrier proteins. (iii) isolation of the putative components by immunoaffinity chromatography and/or immunoprecipitation of detergent solubilized membranes. (iv) reconstitution of the isolated components into proteoliposomes of defined lipid composition. (v) assessment of the protein activity in the reconstituted state by novel transport techniques of high temporal and detection sensitivity. (vi) assessment of possible modulatory role of ionic and hormonic effectors on the transport function in isolated and reconstituted vesicles in relation to the native function activity. These studies are aimed at providing basic information on chloride channel structure and function and ana experimental model system for evaluating the role played by them in secretory processes of respiratory tissue in both physiological and pathological conditions.
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