Down‐regulation of Kir4.1 eliminates the basolateral K conductance in the distal convoluted tubule (DCT) and inhibits NCC activity

Loss‐of‐function mutations of the inwardly rectifying potassium channel (Kir), KCNJ10 (Kir4.1), caused EAST or SeSAME syndrome. The renal phenotype includes salt wasting, hypomagnesemia, metabolic alkalosis and hypokalemia. However, the mechanism by which KCNJ10 mutations cause the tubulopathy is not completely understood. Our previous studies demonstrated that Kir4.1 was a main contributor to the basolateral K conductance in the early distal convoluted tubule (DCT1) and the disruption of Kir.4.1 inhibited NCC expression. However, global deletion of Kir.4.1 impaired the animal growth and those mice could not survive more than two weeks after birth. Thus we have developed inducible kidney specific KCNJ10 knockout mice (Ks‐Kcnj10 KO) using a Pax8+/Cre+/Kcnj10f/f system. We fed 8 week old mice with doxycycline or vehicle for 10 days. Immunofluorescence and western analysis confirmed that more than 90% Kir4.1 protein was knocked down by this method. The patch‐clamp study detected a 40 pS K channel (a Kir4.1/5.1 heterotetramer) in the basolateral membrane of the DCT1 of wildtype (WT) mice, but the probability of finding the 40 pS K channel in doxycycline‐treated mice (KO) is lower (10% of the control) than that of WT mice. Moreover, the whole‐cell Ba2+‐sensitive K currents are largely abolished and the membrane potential is less negative in the DCT1 of KS‐Kcnj10 KO mice in comparison to that of WT mice. Thus, we have successfully generated KS‐Kcnj10 KO mice. Finally, we used western blot analysis to examine the expression of NCC in WT and KS‐Kcnj10 KO mice. The down regulation of Kir4.1 significantly inhibited the expression of total NCC protein or phosphor‐NCC. We conclude that Kir4.1 is a major type of K channel in determining the basolateral K conductance and membrane potential of DCT and determines the apical NCC expression.