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Am J Physiol Cell Physiol 275: C669-C674, 1998;
0363-6143/98 $5.00
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Vol. 275, Issue 3, C669-C674, September 1998

Functional expression of putative H+-K+-ATPase from guinea pig distal colon

Shinji Asano1, Satomi Hoshina2, Yumi Nakaie2, Toshiyuki Watanabe3, Michihiko Sato4, Yuichi Suzuki5, and Noriaki Takeguchi2

1 Molecular Genetics Research Center and 2 Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Toyama 930-01; 3 Department of Physiology and 4 Central Laboratory for Research and Education, Yamagata University School of Medicine, Yamagata 990-23; and 5 Laboratory of Physiology, School of Food and Nutritional Sciences, University of Shizuoka, Shizuoka 422, Japan

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 µM. 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).

colonic proton-potassium-adenosinetriphosphatase; ouabain; proton pump inhibitor


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