A guinea pig cDNA encoding the putative colonic H+-K+-ATPase α-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 TorpedoNa+-K+-ATPase β-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 β-subunits. Therefore, it is concluded that the cDNA encodes the catalytic subunit (α-subunit) of the colonic H+-K+-ATPase. Although the β-subunit of the colonic H+-K+-ATPase has not been identified yet, both gastric H+-K+-ATPase and Na+-K+-ATPase β-subunits were found to act as a surrogate for the colonic β-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 IC 50 = 0.4–1 mM; rat colonic in Sf9 cells had no ouabain sensitivity).

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Cibenzoline, (±)‐2‐(2,2‐diphenylcyclopropyl‐2‐imidazoline succinate, has been clinically used as one of the Class I type antiarrhythmic agents and also reported to block ATP‐sensitive K+ channels in excised membranes from heart and pancreatic β cells. In the present study, we investigated if this drug inhibited gastric H+,K+‐ATPase activity in vitro. Cibenzoline inhibited H+,K+‐ATPase activity of permeabilized leaky hog gastric vesicles in a concentration‐dependent manner (IC50: 201 μM), whereas no effect was shown on Na+,K+‐ATPase activity of dog kidney (IC50: >1000 μM). Similarly, cibenzoline inhibited H+,K+‐ATPase activity of HEK‐293 cells (human embryonic kidney cell line) co‐transfected with rabbit gastric H+,K+‐ATPase α‐ and β‐subunit cDNAs (IC50: 183 μM). In leaky gastric vesicles, inhibition of H+,K+‐ATPase activity by cibenzoline was attenuated by the addition of K+ (0.5 – 5 mM) in a concentration‐dependent manner. The Lineweaver‐Burk plot of the H+,K+‐ATPase activity shows that cibenzoline increases Km value for K+ without affecting Vmax, indicating that this drug inhibits H+,K+‐ATPase activity competitively with respect to K+. The inhibitory effect of H+,K+‐ATPase activity by cibenzoline with normal tight gastric vesicles did not significantly differ from that with permeabilized leaky gastric vesicles, indicating that this drug reacted to the ATPase from the cytoplasmic side of the membrane. These findings suggest that cibenzoline is an inhibitor of gastric H+,K+‐ATPase with a novel inhibition mechanism, which inhibits gastric H+,K+‐ATPase by binding its K+‐recognition site from the cytoplasmic side. British Journal of Pharmacology (2001) 134, 1655–1662; doi: