Functional expression of a truncated Ca(2+)-activated Cl- channel and activation by phorbol ester.

We have isolated a niflumic acid-insensitive, Ca(2+)-activated Cl- channel (CaCC) from bovine trachea that migrates at 38 kDa (p38) on reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, a cloned CaCC isolated from a tracheal cDNA expression library by screening with an antibody raised against p38 has a primary cDNA transcript of 2712 base pairs that codes for a 100-kDa protein and is not susceptible to dithiothreitol reduction. To test the hypothesis that the functional channel may be a much smaller posttranslationally processed form 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 further increased by ionomycin or A-23187 and inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and dithiothreitol. Whole cell currents in wtCaCC- and CaCCX-expressing oocytes could also be activated by phorbol 12-myristate 13-acetate and could be inhibited by chelerythrine chloride, suggesting that the cloned CaCC is regulated by protein kinase C. These results suggest that a smaller form of the full-length CaCC can form a functional channel.