Rb influx in K‐Cl cotransporter 3 (KCC3)‐transfected human embryonic kidney 293 (HEK293) cells and effect of external Na

Abstract

K‐Cl cotransport (KCC) is encoded by 4 genes (SLC12A4–7) with at least four protein isoforms (KCC1‐KCC4) and electroneutrally extrudes K and Cl under physiological conditions. Major functions of KCC involve cell volume and Cl regulation and thus abnormalities in its activity are associated with numerous pathological conditions. The purpose of this study is to characterize the functional properties of KCC3 in a human cell line. Rb, a K congener, was used to study KCC ± ouabain or bumetanide, inhibitors of other K transport mechanisms i.e. Na/K pump and Na‐K‐2Cl cotransport (NKCC), respectively, ± Cl (sulfamate replacement) ± Na (N‐methyl‐D‐glucamine replacement) in KCC3 transfected HEK293 cells. Rb uptake through KCC was linear up to 10 min whereas Rb uptake through NKCC was not linear even at the earliest time point tested (2.5 min). NKCC was 3 fold >; KCC in Na whereas KCC was about 6 fold >; NKCC in Na‐free medium. NKCC was the largest component of the total Rb flux in Na (Na/K pump 25 %, NKCC 63 %, and KCC 11 %). These findings indicate that KCC3‐transfected HEK293 cells behave like non‐transfected cells from various tissues for KCC, NKCC and Na/K pump activity both in Na or Nafree medium under baseline conditions, thus constituting a promising mammalian experimental model to study the specific role of KCC3 in cell volume regulation and in different signaling pathways. Supported by WSU Foundation and NIH R25