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Functional expression of a truncated Ca(2+)-activated Cl- channel and activation by phorbol ester
H L Ji1, M D DuVall, H K Patton
1Department of Physiology and Biophysics, University of Alabama at Birmingham 35294, USA.
The American Journal of Physiology
|March 5, 1998
Summary
Researchers identified a smaller, functional form of calcium-activated chloride channels (CaCC) from bovine trachea. This suggests post-translational modification of the full-length protein creates the active channel, impacting chloride transport research.
Area of Science:
- Molecular Biology
- Ion Channel Physiology
Background:
- Calcium-activated chloride channels (CaCC) play crucial roles in various physiological processes.
- Previous studies identified a 38 kDa CaCC (p38) in bovine trachea, but a cloned counterpart yielded a 100-kDa protein.
Purpose of the Study:
- To investigate if the functional CaCC is a smaller, post-translationally modified form of the cloned 100-kDa protein.
- To characterize the functional properties of truncated CaCC constructs.
Main Methods:
- Cloning and expression of wild-type (wt) CaCC and a truncated mutant (CaCCX) in Xenopus oocytes.
- Electrophysiological recordings (whole-cell currents) to assess channel activity.
- Pharmacological manipulation using ionomycin, A-23187, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, dithiothreitol, phorbol 12-myristate 13-acetate, and chelerythrine chloride.
Main Results:
- Expression of wtCaCC and CaCCX in oocytes significantly increased whole-cell currents compared to controls.
- Ionomycin and A-23187 enhanced currents, while 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and dithiothreitol inhibited them.
- Phorbol 12-myristate 13-acetate activated currents, and chelerythrine chloride inhibited them, indicating protein kinase C regulation.
Conclusions:
- A smaller, functional CaCC can be formed from the full-length 100-kDa protein, likely through post-translational processing.
- The cloned CaCC is regulated by protein kinase C.
- These findings provide insights into the molecular mechanisms of CaCC function and regulation.