Colonic H-K-ATPase alpha- and beta-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 alpha-subunit (HKcalpha) has been cloned and characterized (together with the beta-subunit of either Na-K-ATPase or gastric H-K-ATPase) in Xenopus oocytes as ouabain-sensitive (86)Rb uptake. In contrast, HKcalpha, when expressed in Sf9 cells without a beta-subunit, yielded evidence of ouabain-insensitive H-K-ATPase. Because a beta-subunit (HKcbeta) 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 (HKcalpha and HKcbeta) are transfected into a mammalian cell expression system. Transfection of HEK-293 cells with HKcalpha and HKcbeta cDNAs resulted in the expression of HKcalpha and HKcbeta 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 HKcalpha and Na-K-ATPase beta1 cDNAs and with HKcalpha cDNA alone, there was expression of ouabain-insensitive H-K-ATPase activity that was 60% and 21% of that in HKcalpha/HKcbeta cDNA transfected cells, respectively. Ouabain-insensitive (86)Rb uptake was also identified in cells transfected with HKcalpha and HKcbeta cDNAs. These studies establish that HKcalpha cDNA with HKcbeta cDNA express ouabain-insensitive H-K-ATPase similar to that identified in rat distal colon.