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Expression and regulation of ClC-5 chloride channels: effects of antisense and oxidants
1Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, TX 77555, USA.
Abstract:
Genetic mutations of the Cl(-) channel ClC-5 cause Dent's disease in humans. We recently cloned an amphibian ortholog of Xenopus ClC-5 (xClC-5) from the A6 cell line. We now compare the properties and regulation of ClC-5 currents expressed in mammalian (COS-7) cells and Xenopus oocytes. Whole cell currents in COS-7 cells transfected with xClC-5 cDNA had strong outward rectification, Cl(-) > I(-) anion sensitivity, and were inhibited at low pH, similar to previous results in oocytes. In oocytes, antisense xClC-5 cRNA injection had no effect on endogenous membrane currents or the heterologous expression of human ClC-5. Activators of cAMP and protein kinase C inhibitors had no significant effects on ClC-5 currents expressed in either COS-7 cells or oocytes, whereas H-89, a cAMP-dependent protein kinase (PKA) inhibitor, and hydrogen peroxide decreased the currents. We conclude that the basic properties of ClC-5 currents were independent of the host cell type used for expression. In addition, ClC-5 channels may be modulated by PKA and reactive oxygen species.
Insights
Genetic mutations in ClC-5 channels cause Dent's disease. This study found ClC-5 channel properties are consistent across cell types and may be regulated by protein kinase A and reactive oxygen species.
Area of Science:
- Molecular Biology
- Cell Biology
- Physiology
Background:
- Genetic mutations in the chloride channel ClC-5 are linked to Dent's disease in humans.
- An amphibian ortholog, Xenopus ClC-5 (xClC-5), was previously cloned from the A6 cell line.
Purpose of the Study:
- To compare the properties and regulation of ClC-5 currents expressed in mammalian (COS-7) cells and Xenopus oocytes.
- To investigate the functional conservation of ClC-5 across different expression systems.
Main Methods:
- Whole-cell patch-clamp electrophysiology was used to record ClC-5 currents in COS-7 cells and Xenopus oocytes.
- Transfection of COS-7 cells with xClC-5 cDNA and injection of antisense xClC-5 cRNA into oocytes were performed.
- The effects of pH, anion selectivity, and various signaling molecules (cAMP activators, protein kinase C inhibitors, H-89, hydrogen peroxide) on ClC-5 currents were assessed.
Main Results:
- ClC-5 currents expressed in COS-7 cells exhibited strong outward rectification, chloride over iodide anion sensitivity, and inhibition at low pH, mirroring observations in oocytes.
- Antisense xClC-5 cRNA injection in oocytes did not affect endogenous currents or human ClC-5 expression.
- While cAMP and protein kinase C modulators had no significant effect, H-89 (a protein kinase A inhibitor) and hydrogen peroxide reduced ClC-5 currents.
Conclusions:
- The fundamental properties of ClC-5 currents are conserved and independent of the host cell type (mammalian vs. amphibian).
- ClC-5 channels are potentially modulated by protein kinase A (PKA) signaling and reactive oxygen species (ROS).
- These findings contribute to understanding the molecular basis of Dent's disease and ClC-5 channel function.