Colonic H-K-ATPase alpha- and beta-subunits express ouabain-insensitive H-K-ATPase

Active K absorption in the rat distal colon is energized by an apical H-K-A TPase, a member of the gene family of P-type ATPases. The H-K-ATPase alpha- subunit (HKc alpha) has been cloned and characterized (together with the P- subunit of either Na-K-ATPase or gastric H-K-ATPase) in Xenopus oocytes as ouabain-sensitive Rb-86 uptake. In contrast, HKca, when expressed in Sf9 ce lls without a beta-subunit, yielded evidence of ouabain-insensitive H-K-ATP ase. Because a beta-subunit (HKc beta) has recently been cloned from rat co lon, this present study was initiated to determine whether H-K-ATPase and i ts sensitivity to ouabain are expressed when these two subunits (HKc alpha and HKc beta) are transfected into a mammalian cell expression system. Tran sfection of HEK-293 cells with HKc alpha and HKc beta cDNAs resulted in the expression of HKc alpha and HKc beta proteins and their delivery to plasma membranes. H-K-ATPase activity was identified in crude plasma membranes pr epared from transfected cells and was 1) saturable as a function of increas ing K concentration with a K-m for K of 0.63 mM; 2) inhibited by orthovanad ate; and 3) insensitive to both ouabain and Sch-28080. In parallel transfec tion studies with KKc alpha and Na-K-ATPase beta 1 cDNAs and with HKc alpha cDNA alone, there was expression of ouabain-insensitive H-K-ATPase activit y that was 60% and 21% of that in HKc alpha/HKc beta cDNA transfected cells , respectively. Ouabain-insensitive Rb-86 uptake was also identified in cel ls transfected with HKca and HKc beta cDNAs. These studies establish that H Kc alpha cDNA with HKc beta cDNA express ouabain-insensitive H-K-ATPase sim ilar to that identified in rat distal colon.