FUNCTIONAL EXPRESSION OF A TRUNCATED CA2-ACTIVATED CL- CHANNEL AND ACTIVATION BY PHORBOL ESTER()

We have isolated a niflumic acid-insensitive, Ca2+-activated Cl- chann el (CaCC) from bovine trachea that migrates at 38 kDa (p38) on reducin g sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, a cloned CaCC isolated from a tracheal cDNA expression library by scre ening with an antibody raised against p38 has a primary cDNA transcrip t of 2712 base pairs that codes for a 100-kDa protein and is not susce ptible to dithiothreitol reduction. To test the hypothesis that the fu nctional channel may be a much smaller posttranslationally processed f orm of the 100-kDa protein, we generated a mutant construct (CaCCX, 42 .5-kDa protein) truncated at the NH2 and COOH termini. The whole cell currents of wild-type (wt) CaCC and CaCCX expressed in Xenopus oocytes were 10-fold higher than those of water-injected oocytes and were fur ther increased by ionomycin or A-23187 and inhibited by 4,4'-diisothio cyanostilbene-2,2'-disulfonic acid and dithiothreitol. Whole cell curr ents in wtCaCC- and CaCCX-expressing oocytes could also be activated b y phorbol 12-myristate 13-acetate and could be inhibited by chelery-th rine chloride, suggesting that the cloned CaCC is regulated by protein kinase C. These results suggest that a smaller form of the full-lengt h CaCC can form a functional channel.