Colonic H-K-ATPase α- and β-subunits express ouabain-insensitive H-K-ATPase

Abstract

Active K absorption in the rat distal colon is energized by an apical H-K-ATPase, a member of the gene family of P-type ATPases. The H-K-ATPase α-subunit (HKcα) has been cloned and characterized (together with the β-subunit of either Na-K-ATPase or gastric H-K-ATPase) in Xenopus oocytes as ouabain-sensitive86Rb uptake. In contrast, HKcα, when expressed in Sf9 cells without a β-subunit, yielded evidence of ouabain-insensitive H-K-ATPase. Because a β-subunit (HKcβ) has recently been cloned from rat colon, this present study was initiated to determine whether H-K-ATPase and its sensitivity to ouabain are expressed when these two subunits (HKcα and HKcβ) are transfected into a mammalian cell expression system. Transfection of HEK-293 cells with HKcα and HKcβ cDNAs resulted in the expression of HKcα and HKcβ proteins and their delivery to plasma membranes. H-K-ATPase activity was identified in crude plasma membranes prepared from transfected cells and was 1) saturable as a function of increasing K concentration with a K m for K of 0.63 mM; 2) inhibited by orthovanadate; and 3) insensitive to both ouabain and Sch-28080. In parallel transfection studies with HKcα and Na-K-ATPase β1 cDNAs and with HKcα cDNA alone, there was expression of ouabain-insensitive H-K-ATPase activity that was 60% and 21% of that in HKcα/HKcβ cDNA transfected cells, respectively. Ouabain-insensitive 86Rb uptake was also identified in cells transfected with HKcα and HKcβ cDNAs. These studies establish that HKcα cDNA with HKcβ cDNA express ouabain-insensitive H-K-ATPase similar to that identified in rat distal colon.