Gastric H+/K+ATPase beta subunit in the colonic H+/K+ATPase knockout mice

Three H+K+ATPase subunits (HKAalpha1; gastric, HKAalpha2; colonic, HKAbeta) are expressed in the renal collecting duct. It is known that the HKAalpha1 (gastric) plays an important role under normal conditions, whereas the HKAalpha2 (colonic) is induced in potassium depletion and mediates increased HCO3− in the maintenance of metabolic alkalosis. The beta‐subunit is essential for the functional expression of both the HKAalpha1 and HKAalpha2. The purpose of the current experiments was to examine the role of the beta‐subunit of HKA in wild type (WT) and in selective disruption of the HKAalpha 2 gene (KO) mice under normal and K+ depletion conditions. Mice were placed on a potassium‐deficient diet for 2 weeks. The mouse kidney medulla was dissected and lysed with a lysis buffer (pH 8). Cell lysate was incubated with monoclonal antibody (HKA beta subunit, 34 kDa) overnight under agitation at 4°C. The amount of HKA beta subunit was quantified by a modified Bradford protein assay. At a wavelength of 540 nm, the HKA beta subunit protein absorbance was 0.052 ± 0.018 (n=20) in WT mice with a potassium replete diet. In WT mice with a potassium‐deficient diet, the HKA beta subunit absorbance was 0.065 ± 0.028 (n=20, p<0.01). A similar pattern of the HKA beta subunit absorbance has been observed in KO mice with a potassium replete diet vs. a potassium‐deficient diet. Results suggested that the HKA beta‐subunit is stimulated by hypokalemia in the renal collecting ducts. The HKA beta‐subunit is likely to associate with the HKAalpha1 in the HKAalpha2 KO mice under normal and K+ depletion conditions..