Functional Characterization of the First Known Mutation of the Human SLC12A2 (NKCC1) Gene

An 8 year old girl with multiple health problems that began at the age of 6 months visited the NIH Undiagnosed Disease Program in 2010. At the time of her visit, she suffered from obstructive apnea, episodes of vomiting and dehydration; decrease energy and fatigue, exercise intolerance, dilated cardiomyopathy (left ventricle dilation) and seizure‐like episodes. In early 2015, she was found to have a de novo mutation in SLC12A2, the gene encoding the Na‐K‐2Cl cotransporter, NKCC1. Using genomic DNA isolated from the patient, we confirmed an 11‐bp deletion in exon 22 leading to protein truncation. We created the mutant cDNA for expression in Xenopus laevis oocytes. In contrast to wild‐type cotransporter which demonstrated significant bumetanide‐sensitive and hypertonic‐stimulated K + uptake, the mutant cotransporter was non‐functional. Western blot analysis of oocytes expressing of a c‐myc tagged mutant cotransporter revealed a molecular size consistent with a dimer, in contrast to oocytes expressing wild‐type cotransporters that showed all three forms of the cotransporter: core, glycosylated, and dimer. Co‐expression of mutant NKCC1 with wild‐type NKCC1 did not result in dominant‐negative effect of mutant on wild‐type transporter. K + influx measurements in fibroblasts isolated from the patient revealed that 50% of the K + influx was ouabain‐sensitive or mediated by the Na + /K + pump, whereas 41% of the flux was bumetanide‐sensitive or mediated by NKCC1. Addition of ouabain and bumetanide resulted in 87% reduction in K + influx. These data indicate that the mutation leads to a non‐functional allele without deleterious effect on the function of the cotransporter derived from the wild‐type allele. Whether the mutant protein exerts deleterious effects on other cellular functions still need to be explored. Support or Funding Information Supported by NIH grant DK093501