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.
Ji
H L
HL
Department of Physiology and Biophysics, University of Alabama at Birmingham 35294, USA.
DuVall
M D
MD
Patton
H K
HK
Satterfield
C L
CL
Fuller
C M
CM
Benos
D J
DJ
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D002712:Chlorides / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004594:Electrophysiology; D005439:Flufenamic Acid / Q000494:pharmacology; D066298:In Vitro Techniques; D015640:Ion Channel Gating / Q000187:drug effects; D007476:Ionophores / Q000494:pharmacology; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009544:Niflumic Acid / Q000494:pharmacology; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D016366:Open Reading Frames; D014176:Protein Biosynthesis; D013329:Structure-Activity Relationship; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014982:Xenopus laevis
D015220:Calcium Channels; D002712:Chlorides; D007476:Ionophores; D009544:Niflumic Acid; D005439:Flufenamic Acid; D013755:Tetradecanoylphorbol Acetate; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D002118:Calcium; D004229:Dithiothreitol