Functional expression of putative H+-K+-ATPase from guinea pig distal colon.
A guinea pig cDNA encoding the putative colonic H+-K+-ATPase alpha-subunit (T. Watanabe, M. Sato, K. Kaneko, T. Suzuki, T. Yoshida, and Y. Suzuki; GenBank accession no. D21854) was functionally expressed in HEK-293, a human kidney cell line. The cDNA for the putative colonic H+-K+-ATPase was cotransfected with cDNA for either rabbit gastric H+-K+-ATPase or Torpedo Na+-K+-ATPase beta-subunit. In both expressions, Na+-independent, K+-dependent ATPase (K+-ATPase) activity was detected in the membrane fraction of the cells, with a Michaelis-Menten constant for K+ of 0.68 mM. The expressed K+-ATPase activity was inhibited by ouabain, with its IC50 value being 52 microM. However, the activity was resistant to Sch-28080, an inhibitor specific for gastric H+-K+-ATPase. The ATPase was not functionally expressed in the absence of the beta-subunits. Therefore, it is concluded that the cDNA encodes the catalytic subunit (alpha-subunit) of the colonic H+-K+-ATPase. Although the beta-subunit of the colonic H+-K+-ATPase has not been identified yet, both gastric H+-K+-ATPase and Na+-K+-ATPase beta-subunits were found to act as a surrogate for the colonic beta-subunit for the functional expression of the ATPase. The present colonic H+-K+-ATPase first expressed in mammalian cells showed the highest ouabain sensitivity in expressed colonic H+-K+-ATPases so far reported (rat colonic in Xenopus oocytes had an IC50 = 0.4-1 mM; rat colonic in Sf9 cells had no ouabain sensitivity).
Asano
S
S
Molecular Genetics Research Center, Toyama Medical and Pharmaceutical University, Toyama 930-01, Japan.
Hoshina
S
S
Nakaie
Y
Y
Watanabe
T
T
Sato
M
M
Suzuki
Y
Y
Takeguchi
N
N
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D003106:Colon / Q000201:enzymology*; D005753:Gastric Mucosa / Q000201:enzymology; D006168:Guinea Pigs; D017506:H(+)-K(+)-Exchanging ATPase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007413:Intestinal Mucosa / Q000201:enzymology*; D007668:Kidney; D007700:Kinetics; D008969:Molecular Sequence Data; D010042:Ouabain / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D014101:Torpedo; D014162:Transfection
D011994:Recombinant Proteins; D010042:Ouabain; D011189:Potassium Chloride; D017506:H(+)-K(+)-Exchanging ATPase