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Ouabain increases association between Na‐K ATPase (Na‐K) and NHE1 through N‐terminal domain of Na‐K

Low dose ouabain increases Na‐K activity in kidney basolateral membranes in an NHE1 dependent manner. TIRF and FRET microscopy suggested that 10 pM ouabain increases association between Na‐K and NHE1. The aim of the study was to identify the molecular domains on Na‐K and NHE1 required for association. Using mouse (MRPT) and human (HK11) kidney proximal tubule cells co‐transfected with full‐length GFP or RFP‐NHE1, and full length mCherry‐Na‐K a‐subunit or N‐terminal deletion mutant of YFP‐Na‐K, FRET and TIRFM were performed before and after ouabain treatment. We also measured the effect of ouabain on Na‐K activity in HK2 cells transfected with WT or scaffold domain mutated NHE1. Ouabain increased association between Na‐K and NHE1. NHE1 and Na‐K association was abolished in cells expressing N‐terminal deletion mutant Na‐K. Ouabain increased Na‐K activity in HK2 cells expressing full length NHE1 but not in cells expressing scaffold domain mutated NHE1. These data suggest that low concentrations of ouabain increase Na‐K association with NHE1 and this association involves N‐terminal domain of Na‐K and the scaffolding domain of NHE1.