Secretory Cl channels are present in airway, colonic, pancreatic, and sweat coil epithelia. Recent compelling data from several laboratories studying a variety of this Cl channel represents the underlying defect in this disease. At this point in time, virtually nothing is known about the molecular mechanisms underlying the regulation of this important ion transport protein. The primary purpose of this project will be to identify and purify the polypeptide components of this Cl channel, using monospecific polyclonal antibodies generated against a synthetic peptide comprising the DIDS-binding site region of an anion exchange protein. Our hypothesis is based on the assumption of a conserved homology of this region in the secretory Cl channel, supported by the observation that disulfonic stilbenes can, with relatively high affinity, inhibit conductive Cl transport. By immunoaffinity chromatography and Western blot analysis, we have identified a 60 kDa protein as a candidate component of this Cl channel. Once this protein is purified, we will raise both monoclonal and polyclonal antibodies against it and determine partial amino acid sequence for the arrangement of the channel protein in the membrane, and will be used as probes for the cloning and cytochemical localization of the channel protein in collaboration with projects 2 and 3 of this application. Investigations of whether the putative channel protein can be phosphorylated both in vitro and in vivo will also be undertaken. In collaboration with Project 4, studies will be done to analyze the kinetic characteristics of this Cl channel, utilizing reconstitution procedures to measure single channel properties in planar bilayer or in patches of proteoliposomes. These experiments will verify the identification of any putative Cl channel proteins, establish their functionality, and permit study of the functional consequences of phosphorylation. These studies will ultimately lead to a better understanding of the protein defect in CF, and hopefully point to possible molecular interventions that overcome the devastating consequences of the disease.
Showing the most recent 10 out of 32 publications
