Exploring the Cell Biological and Functional Effects of the First Disease Associated KCC1 Genetic Variant

The potassium chloride cotransporter 1 (KCC1) is ubiquitously expressed and essential for regulating cellular fluid balance. We identified a patient carrying a genetic variant (E1065K) in the KCC1 coding gene SLC12A4. This study explored the impact of the variant in ectopic cell systems and enhanced the understanding of cell biological properties of the KCC1 protein. KCC1 WT and E1065K DNA expression constructs were transfected in HEK293T, EPI7 or COS7 cells. KCC1 protein expression levels, glycosylation, intracellular trafficking, half-life and protein localization were determined with western blot and immunofluorescence microscopy. Molecular docking investigated interactions within the cotransporter. Cotransporter activity was tested with NH 4 + flux measurements. The variant reduces interactions within the cotransporter and functional activation decreases in hypotonic conditions. Other cell biology characteristics with respect to protein expression level, half-life or subcellular localization did not show any statistical difference between KCC1 WT and E1065K. However, this data provided new characteristics of KCC1 protein. Altogether, these findings are the first description of a potential pathogenic human variant in the KCC1 protein.