Colonic H-K-ATPase a- and b-subunits express ouabain-insensitive H-K-ATPase

Sangan, Pitchai, Sundararajah Thevananther, Sheela Sangan, Vazhaikkurichi M. Rajendran, and Henry J. Binder. Colonic H-K-ATPase aand b-subunits express ouabain-insensitive H-K-ATPase. Am. J. Physiol. Cell Physiol. 278: C182–C189, 2000.—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 a-subunit (HKca) has been cloned and characterized (together with the b-subunit of either Na-K-ATPase or gastric H-K-ATPase) in Xenopus oocytes as ouabain-sensitive 86Rb uptake. In contrast, HKca, when expressed in Sf9 cells without a b-subunit, yielded evidence of ouabain-insensitive H-K-ATPase. Because a b-subunit (HKcb) 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 (HKca and HKcb) are transfected into a mammalian cell expression system. Transfection of HEK-293 cells with HKca and HKcb cDNAs resulted in the expression of HKca and HKcb 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 Km for K of 0.63 mM; 2) inhibited by orthovanadate; and 3) insensitive to both ouabain and Sch-28080. In parallel transfection studies with HKca and Na-K-ATPase b1 cDNAs and with HKca cDNA alone, there was expression of ouabain-insensitive H-K-ATPase activity that was 60% and 21% of that in HKca/HKcb cDNA transfected cells, respectively. Ouabain-insensitive 86Rb uptake was also identified in cells transfected with HKca and HKcb cDNAs. These studies establish that HKca cDNA with HKcb cDNA express ouabaininsensitive H-K-ATPase similar to that identified in rat distal colon.