Functional regulation of epithelial NKCC1 by COMMD1

Disruption of the C terminus (CT) of NKCC1 in mice results in nonfunctional protein and in neurological, epithelial, and metabolic defects; however, the underlying cause for this effect is not known. We hypothesize that the defects are due to loss of proteins interacting with CT‐NKCC1. Using a yeast two hybrid approach, adaptor protein COMMD1 was found to bind to the CT‐NKCC1 (aa 1040‐1212) from airway epithelial cells. Downregulation of COMMD1 using silencing RNA led to a 97.5% loss of endogenous COMMD1, but did not affect activation of NKCC1, measured as bumetanide (BMT)‐sensitive 86 Rb uptake, by hyperosmotic sucrose. Biotinylation of the basolateral membrane of Calu‐3 cells grown on Transwell filters was applied to quantitate surface membrane expression of NKCC1. Hyperosmotic stress caused a transient increase in NKCC1 membrane expression; transient downregulation of COMMD1 reduced the baseline (unstimulated) NKCC1 expression and prevented a transient elevation in NKCC1 membrane expression. Slot blot and solution binding assays demonstrate direct binding of COMMD1 (aa 1‐191) to CT‐NKCC1 (aa 1040‐1212). Endogenous COMMD1 was detected in pulldowns using recombinant FLAG‐CT‐NKCC1. To define the site of interaction, truncations of COMD1 and CT‐NKCC1 were constructed, expressed, and purified then tested for direct binding in slot blot assays. Concentration‐dependent binding was observed between COMMD1 (aa 1‐123) and CT‐NKCC1 (aa 1040‐1134). The results indicate that COMMD1 has a role in membrane expression of airway epithelial NKCC1. Supported by a NIH grant HL‐58598 and a Research Development Grant from the Cystic Fibrosis Foundation.