MOLECULAR-CLONING AND CHARACTERIZATION OF P64, A CHLORIDE CHANNEL PROTEIN FROM KIDNEY MICROSOMES

Chloride channels were previously purified from bovine kidney cortex m embranes using a drug affinity column. Reconstitution of the purified proteins into artificial liposomes and planar bilayers yielded chlorid e channels. A 64-kDa protein, p64, identified as a component of this c hloride channel was used to generate antibodies which depleted solubil ized kidney membranes of all chloride channel activity. This antibody has now been used to identify a clone, H2B, from a kidney cDNA library . Antibodies, affinity-purified against the fusion protein of H2B also depleted solubilized kidney cortex from all chloride channel activity . The predicted amino acid sequence of p64 shows that it contains two and possibly four putative transmembrane domains and potential phospho rylation sites by protein kinase A, protein kinase C, and casein kinas e II. There was no significant homology to other protein (or DNA) sequ ences in the data base. The protein is expressed in all cells tested. Expression of its mRNA in Xenopus laevis oocytes led to the insertion of a protein with the appropriate molecular mass in microsomes but not in the plasma membrane. It is likely that p64 represents the chloride channel of intracellular organelles.