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Mistargeting of a truncated Na-K-2Cl cotransporter in epithelial cells
Rainelli Koumangoye1, Salma Omer1, Eric Delpire1
1Department of Anesthesiology, Vanderbilt University School of Medicine , Nashville, Tennessee.
Abstract:
We recently reported the case of a young patient with multisystem failure carrying a de novo mutation in SLC12A2, the gene encoding the Na-K-2Cl cotransporter-1 (NKCC1). Heterologous expression studies in nonepithelial cells failed to demonstrate dominant-negative effects. In this study, we examined expression of the mutant cotransporter in epithelial cells. Using Madin-Darby canine kidney (MDCK) cells grown on glass coverslips, permeabilized support, and Matrigel, we show that the fluorescently tagged mutant cotransporter is expressed in cytoplasm and at the apical membrane and affects epithelium integrity. Expression of the mutant transporter at the apical membrane also results in the mislocalization of some of the wild-type transporter to the apical membrane. This mistargeting is specific to NKCC1 as the Na+-K+-ATPase remains localized on the basolateral membrane. To assess transporter localization in vivo, we created a mouse model using CRISPR/cas9 that reproduces the 11 bp deletion in exon 22 of Slc12a2. Although the mice do not display an overt phenotype, we show that the colon and salivary gland expresses wild-type NKCC1 abundantly at the apical pole, confirming the data obtained in cultured epithelial cells. Enough cotransporter must remain, however, on the basolateral membrane to participate in saliva secretion, as no significant decrease in saliva production was observed in the mutant mice.
Insights
A de novo mutation in the Na-K-2Cl cotransporter-1 (NKCC1) gene causes cellular defects in epithelial cells. This study investigates the mutant transporter
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- A de novo mutation in SLC12A2, encoding NKCC1, was identified in a patient with multisystem failure.
- Previous studies in nonepithelial cells did not reveal dominant-negative effects of the mutant NKCC1.
Purpose of the Study:
- To investigate the expression and localization of the mutant NKCC1 cotransporter in epithelial cells.
- To determine the in vivo effects of the mutation using a mouse model.
Main Methods:
- Heterologous expression of fluorescently tagged mutant NKCC1 in Madin-Darby canine kidney (MDCK) cells.
- Analysis of transporter localization in cultured cells and in vivo using a CRISPR/cas9-generated mouse model.
- Assessment of epithelium integrity and saliva production in mutant mice.
Main Results:
- The mutant NKCC1 is expressed in the cytoplasm and apical membrane of epithelial cells, affecting epithelium integrity.
- Apical expression of mutant NKCC1 leads to mislocalization of wild-type NKCC1 to the apical membrane, while Na+-K+-ATPase remains basolateral.
- Mutant mice show abundant wild-type NKCC1 at the apical pole in colon and salivary glands, with preserved saliva secretion.
Conclusions:
- The mutant NKCC1 cotransporter causes cellular defects in epithelial cells, including mislocalization of the wild-type transporter.
- The mouse model replicates some cellular findings but does not show an overt phenotype, suggesting compensatory mechanisms.
- NKCC1 localization and function are critical for epithelial integrity and secretion, with implications for understanding related human diseases.
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