A Novel Role for the Na‐K‐2Cl Cotransporter in Mitochondrial Respiration

A patient recruited by the undiagnosed disease program (UDP) at the NIH suffers from multi organ failure and deficits in energy homeostasis. The multi organ failure consists of complete gastrointestinal and bladder dysfunction, as well as thyroid, parathyroid, adrenal, and pancreatic deficiencies. The deficits in energy homeostasis were evidenced in lab tests conducted on the patient's muscle and liver biopsies, where there was an abnormal increase in glycogen content and mitochondrial DNA copy number. Whole exome sequencing showed that the patient expresses a de novo mutation in an allele of the Slc12a2 gene encoding the Na‐K‐2Cl cotransporter‐1 (NKCC1). Previous studies in the laboratory demonstrated that the 11 base pair deletion causing a truncation of the carboxyl‐terminus, leads to a non‐functional transporter. We have also demonstrated that the UDP patient's fibroblasts displayed reduced total NKCC1‐mediated K + influx. Additionally, trafficking studies in transfected HEK293 cells expressing the NKCC1 truncation mutation have revealed that this mutated protein dimerizes with wild‐type (WT) NKCC1 and is trafficked to the plasma membrane along with WT NKCC1. To address a possible role of the cotransporter in metabolism, I utilized fibroblasts isolated from the patient and from healthy controls and performed mitochondrial respiration stress tests. I showed that mitochondrial respiration and ATP production was significantly higher in the patient's fibroblasts. Because the cells from the patient might carry additional mutations, I transfected MDCK cells with EGFP‐tagged wild‐type or mutant transporter, sorted the cells, and tested them. Cells expressing the mutant cotransporter also demonstrated higher mitochondrial respiration, compared to cells expressing wild‐type EGFP‐NKCC1. To further confirm the role of NKCC1 in mitochondrial respiration, I subjected control MDCK cells to bumetanide, a NKCC1 specific inhibitor. MDCK cells treated with 20 mM bumetanide for 20 min also demonstrated an increase in mitochondrial respiration. To further assess whether this effect in mitochondrial respiration is NKCC1 specific, I subjected bumetanide to two stable lines of MDCK cells (LK‐A3 and LK‐C1) that carry a distinct mutation in NKCC1, resulting in a non‐functional transporter. There was no change in mitochondrial respiration levels in these cells, which indicated that the rise in mitochondrial respiration levels in wild‐type cell is NKCC1 specific. These data reveal a novel role for NKCC1 in mitochondrial respiration. Future work will address the mechanism by which NKCC1 activity affects mitochondrial function. Support or Funding Information Supported by NIH grants R21GM118944 and RO1DK093501 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .