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-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.
Sangan
P
P
Departments of Internal Medicine and Pediatrics, Yale University, New Haven, Connecticut 06520-8019, USA.
Thevananther
S
S
Sangan
S
S
Rajendran
V M
VM
Binder
H J
HJ
D000818:Animals; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D019556:COS Cells; D003106:Colon / Q000166:cytology / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004847:Epithelial Cells / Q000737:chemistry / Q000201:enzymology; D005455:Fluorescent Antibody Technique; D015971:Gene Expression Regulation, Enzymologic; D017506:H(+)-K(+)-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000166:cytology; D010042:Ouabain / Q000494:pharmacology*; D010957:Plasmids; D011188:Potassium / Q000493:pharmacokinetics; D054328:Proton Pump Inhibitors; D051381:Rats; D012414:Rubidium Radioisotopes; D014162:Transfection; D014638:Vanadates / Q000494:pharmacology
D004791:Enzyme Inhibitors; D054328:Proton Pump Inhibitors; D012414:Rubidium Radioisotopes; D014638:Vanadates; D010042:Ouabain; D017506:H(+)-K(+)-Exchanging ATPase; D011188:Potassium