Chloride channels are present in intracellular organelles of all cells and in the plasma membrane of epithelia and muscle. These proteins are coded for by diverse types of genes. Two important diseases are associated with Cl channel dysfunction; myotonia is due to disruption of a muscle plasma membrane Cl channel. Cystic fibrosis is produced by a dysfunction of an epithelial secretory Cl channel. We have proposed that the defect in CF is produced by Cl channel mediated abnormalities in the acidification of golgi with consequent defects in sialylation and sulfation of mucus glycoproteins. We plan to continue our studies on the role of vacuolar Cl channels in CF by measuring the intravesicular pH of organelles identified by specific markers such as the sialyl transferase, mannose 6P receptor, transferrin receptor and lysosomal glycoproteins. These studies will be performed in CF epithelial cells before and after rescue with transfection with the wild type CFTR. We will also introduce known disease causing mutations into these cells and examine the effect they have on sialylation and sulfation as well as on Cl secretion. These mutations are known to reach the plasma membrane but the phenotype of the disease is either severe or mild. These studies should determine whether the severity of the clinical illness correlates more with Cl secretion or with the severity of glycoprotein abnormalities. We had purified, reconstituted and cloned one vacuolar Cl channel protein, p64. We found this protein to be necessary for channel function; to demonstrate if it is sufficient also, we will overexpress it, repurify it and study its electrophysiological characteristics in planar lipid bilayers. We found that this protein is targeted to the plasma membrane in epithelia but is retained intracellularly in non-polarized cells. Identification of the structural differences between the two forms and mutational analysis should allow us to determine the targeting sequences in the protein. Biochemical studies will also be performed to examine the mechanism of retention in the plasma membrane in epithelia especially interaction with other proteins.
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