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Endogenous and exogenous Na-K-Cl cotransporter expression in a low K-resistant mutant MDCK cell line
J A Payne1, C Ferrell, C Y Chung
1Department of Human Physiology, School of Medicine, University of California, One Shields Ave, Davis, CA 95616, USA. japayne@ucdavis.edu
Abstract:
A low K-resistant mutant Madin-Darby canine kidney (MDCK) cell line, LK-C1, has been shown previously to lack functional Na-K-Cl cotransporter (NKCC) activity, indicating that it may be a useful NKCC "knockout" cell line for structure-function studies. Using immunological probes, we first characterized the defect in the endogenous NKCC protein of the LK-C1 cells and then fully restored NKCC activity in these cells by stably expressing the human secretory NKCC1 protein (hNKCC1). The endogenous NKCC protein of the LK-C1 cells was expressed at significantly lower levels than in wild-type MDCK cells and was not properly glycosylated. This latter finding indicated that the lack of functional NKCC activity in the LK-C1 cells may be due to the inability to process the protein to the plasma membrane. In contrast, exogenously expressed hNKCC1 protein was properly processed and fully functional at the plasma membrane. Significantly, the exogenous hNKCC1 protein was regulated in a manner similar to the protein in native secretory cells as it was robustly activated by cell shrinkage, calyculin A, and low-Cl incubation. Furthermore, when the LK-C1 cells formed an epithelium on permeable supports, the exogenous hNKCC1 protein was properly polarized and functional at the basolateral membrane. The low levels of endogenous NKCC protein expression, the absence of any endogenous NKCC transport activity, and the ability to form a polarized epithelium indicate that the LK-C1 cells offer an excellent expression system with which to study the molecular physiology of the cation Cl cotransporters.
Insights
A mutant cell line lacking functional sodium-potassium-chloride cotransporter (NKCC) activity was restored by expressing human NKCC1. This provides a new system for studying NKCC molecular physiology.
Area of Science:
- Cell Biology
- Molecular Physiology
- Membrane Transport
Background:
- A low potassium-resistant Madin-Darby canine kidney (MDCK) cell line, LK-C1, exhibits no functional Na-K-Cl cotransporter (NKCC) activity.
- This cell line presents a potential NKCC "knockout" model for structure-function investigations.
Purpose of the Study:
- To characterize the defect in endogenous NKCC protein in LK-C1 cells.
- To restore NKCC activity in LK-C1 cells by expressing human secretory NKCC1 (hNKCC1).
- To evaluate LK-C1 cells as an expression system for studying cation-chloride cotransporters.
Main Methods:
- Immunological probes were used to characterize the endogenous NKCC protein defect.
- Stable expression of human NKCC1 (hNKCC1) was achieved in LK-C1 cells.
- Functional and localization studies of exogenous hNKCC1 were performed in LK-C1 cells and epithelia.
Main Results:
- Endogenous NKCC protein in LK-C1 cells showed low expression and improper glycosylation, hindering plasma membrane localization.
- Exogenously expressed hNKCC1 was correctly processed, functional at the plasma membrane, and regulated similarly to native secretory cells.
- Exogenous hNKCC1 exhibited proper basolateral polarization when LK-C1 cells formed an epithelium.
Conclusions:
- LK-C1 cells possess defective endogenous NKCC processing, making them suitable for studying NKCC function.
- The successful restoration and regulation of hNKCC1 demonstrate the utility of LK-C1 cells as an expression system.
- These findings establish LK-C1 cells as a valuable model for investigating the molecular physiology of cation-chloride cotransporters.