Abstract
BACKGROUND: The H,K-ATPase, consisting of α and ß subunits, belongs to the P-type ATPase family. There are two isoforms of the α subunit, HKα₁ and HKα₂ encoded by different genes. The ouabain-resistant gastric HKα₁-H,K-ATPase is Sch28080-sensitive. However, the colonic HKα₂-H,K-ATPase from different species shows poor primary structure conservation of the HKα₂ subunit between species and diverse pharmacological sensitivity to ouabain and Sch28080. This study sought to determine the contribution of each gene to functional activity and its pharmacological profile using mouse models with targeted disruption of HKα₁, HKα₂, or HKbeta genes. METHODS: Membrane vesicles from gastric mucosa and distal colon in wild-type (WT), HKα₁, HKα₂, or HKß knockout (KO) mice were extracted. K-ATPase activity and pharmacological profiles were examined. RESULTS: The colonic H,K-ATPase demonstrated slightly greater affinity for K(+) than the gastric H,K-ATPase. This K-ATPase activity was not detected in the colon of HKα₂ KO but was observed in HKß KO with properties indistinguishable from WT. Neither ouabain nor Sch28080 had a significant effect on the WT colonic K-ATPase activity, but orthovanadate abolished this activity. Amiloride and its analogs benzamil and 5-N-ethyl-N-isopropylamiloride inhibited K-ATPase activity of HKα₁-containing H,K-ATPase; the dose dependence of inhibition was similar for all three inhibitors. In contrast, the colonic HKα₂-H,K-ATPase was not inhibited by these compounds. CONCLUSIONS: These data demonstrate that the mouse colonic H,K-ATPase exhibits a ouabain- and Sch28080-insensitive, orthovanadate-sensitive K-ATPase activity. Interestingly, pharmacological studies suggested that the mouse gastric H,K-ATPase is sensitive to amiloride. GENERAL SIGNIFICANCE: Characterization of the pharmacological profiles of the H,K-ATPases is important for understanding the relevant knockout animals and for considering the specificity of the inhibitors.