Novel Mouse Model of a Human Mutation in NKCC1 Confirms its Mistargeting in Epithelia

A 15‐year old patient with complete gastrointestinal and bladder failure, endocrine insufficiencies, and orthostatic intolerance carries a de novo mutation in SLC12A2, the gene encoding the Na‐K‐2Cl cotransporter‐1 or NKCC1. The 11 bp deletion leads to truncation of the cytosolic COOH‐terminal tail of the cotransporter making it non‐functional . Expression of the mutant transporter increases the amount of dimerization, suggesting the possibility that the mutant transporter might exert dominant‐negative effects on the wild‐type transporter. In polarized MDCK cells grown on permeable filters or in 3‐D culture cysts, most of the mutant transporter signal is observed on the apical membrane, in contrast to the basolateral expression of wild‐type cotransporter. The mutant transporter affects the formation of MDCK cysts, as we observed the presence of multiple lumens, as opposed to single lumen formation with wild‐type cells. The mutant transporter also causes trafficking of the wild‐type cotransporter to the apical membrane. To assess the relevance of this observation in vivo , we created a mouse model that recapitulates the human mutation. Using CRISPR/cas9, a guide RNA that targets cas9 to exon 22 of the Slc12a2 gene, and a repair oligonucleotide that deletes 11 bp from the exon, we successfully generated a female founder animal that carried the designed mutant allele in addition to a non‐homologous end joining mutation in the second allele. After one backcross that segregated the two alleles, the line was further established by backcrossing for 2 additional generations in C57BL/6J. This mouse now carries the 11 bp deletion mutant allele and a wild‐type allele. We examined the expression of NKCC1 in the salivary gland and the intestine, two Cl − secreting epithelia where the wild‐type cotransporter is typically expressed on the basolateral membrane. In both tissues, intense apical staining was observed, confirming the data obtained with MDCK cells. Preliminary studies indicate that the mouse has no overt adverse phenotype, although the mouse lives in a pathogen‐free barrier facility and has not been challenged. In conclusion, our data demonstrate that expression of a truncated Na‐K‐2Cl cotransporter causes mistargeting of the wild‐type cotransporter to the apical membrane of Cl − secreting epithelia and affects proper lumen formation in MDCK cysts. These observations may explain the multisystem dysfunction that is observed in the patient. Support or Funding Information Supported by NIH grants R21GM118944 and 1RO1DK093501. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .