FUNCTIONAL EXPRESSION OF PUTATIVE H-K+-ATPASE FROM GUINEA-PIG DISTAL COLON()

A guinea pig cDNA encoding the putative colonic H+-K+-ATPase alpha-sub unit (T. Watanabe, M. Sate, K. Kaneko, T. Suzuki, T. Yoshida, and Y. S uzuki; 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 o uabain, with its IC50 value being 52 mu M. However, the activity was r esistant 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 catalyt ic subunit (alpha-subunit) of the colonic H+-K+-ATPase. Although the b eta-subunit of the colonic H+-K+-ATPase has not been identified yet, b oth 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 ex pression of the ATPase. The present colonic H+-K+-ATPase first express ed in mammalian cells showed the highest ouabain sensitivity in expres sed colonic H+-K+-ATPases so far reported (rat colonic in Xenopus oocy tes had an IC50 = 0.4-1 mM; rat colonic in Sf9 cells had no ouabain se nsitivity).