H+-ATPase activity in selective disruption of H+-K+-ATPase alpha 1 gene of mice under normal and K-depleted conditions

Abstract

The outer medullary collecting duct (OMCD) plays an important role in acid-base homeostasis by two luminal proton ATPases, H⁺-ATPase and H⁺–K⁺-ATPase (HKA), both of which are in the intercalated cells (ICs) of OMCD. We showed previously that HKAα1 (gastric H⁺-K⁺-ATPase) activity is the essential H⁺-K⁺-ATPase activity under normal conditions, and that HKAα2 (colonic H⁺-K⁺-ATPase) is induced and mediates increased proton-secretion under K-depleted conditions.1, 2, 3 To better understand the role of H⁺-ATPase (potassium-independent) in acid secretion and the relationship between H⁺-ATPase and a specific HKA isoform, we examined H⁺-ATPase activity in the H⁺–K⁺-ATPaseα1 knockout (KO) mice under normal and K-depleted conditions. Mice were fed a potassium-free diet and studied after 7 days. Segments of the OMCD were perfused in vitro, and intracellular pH (pH_i) was measured by ratiometric fluorescence microscopy using the pH-sensitive indicator BCECF-AM. The isolated OMCD tubules obtained from mice fed a potassium-free diet were examined by fluorescent immunocytochemistry with an antibody to the 31-kDa subunit of H⁺-ATPase (E-11) and were compared with those obtained from a normal diet. In the absence of Na⁺ and K⁺, the H⁺-ATPase-mediate pH_i recovery rates were 6.7 ± 1.1 × 10⁻⁴ units/s (n = 7 ICs) in wild-type (WT) mice and increased to 8.7 ± 1.8 × 10⁻⁴ (P < 0.05; n = 6) in HKAα1 KO mice. K-independent proton transport activity was significantly inhibited by the H⁺-ATPase inhibitor bafilomycin A₁ (BAF, 10 nM) with luminal applied in both WT and KO mice. Comparison of the results indicated upregulation of BAF-sensitive H⁺-ATPase activity in KO mice. To determine the intracellular localization of H⁺-ATPase in the intercalated cells of OMCD, we dissected the OMCD and performed fluorescent immunocytochemistry with the H⁺-ATPase antibody in the WT and KO mice. In the WT mice, on normal diet, H⁺-ATPase staining distributed diffusely throughout the intercalated cells and was slightly polarized to the apical plasma membrane in the KO mice, consistent with increase in the H⁺-ATPase-mediate pH_i recovery in the KO mice. One week of a potassium-free diet resulted in a significant increase in the degree of H⁺-ATPase polarization at the apical plasma membrane in both WT and KO mice. Hypokalemia stimulates H⁺-ATPase in the intercalated cells of OMCD of both WT and KO mice. The enhanced activity of H⁺-ATPase plays an important role in compensatory proton secretion in the HKAα1 KO mice under normal conditions.