The gastric H+,K+-ATPase is the proton pump responsible for the acidification of the stomach. This protein is structurally defined as a heterodimer composed of a large catalytic subunit (alpha) and a smaller type-two glycoprotein (beta) and is a member of the P2-type ATPase subfamily. Three separate studies were done to investigate the involvement of individual amino acids in K+ dependent function or association between alpha and beta subunits Membrane preparations from HEK 293T cells transiently transfected with the rabbit gastric H+,K+-ATPase show a ouabain insensitive, SCH 28080 sensitive K+-ATPase activity consistent with the pharmacological profile of the gastric H+,K +-ATPase. A recent study using DCCD suggested that E857 in the alpha subunit was important for K+-dependent function. In work presented here, site directed mutagenesis was used to change the charge or size of this amino acid sidechain. All mutants were expressed in the HEK 293T cell line and exhibited gastric H+,K+-ATPase activity indicating that the sidechain of E857 is not essential. However, the apparent affinity for SCH 28080 and K+ was altered in a way that is inconsistent with a change in the global E1 or E2 conformational equilibrium. These results, when reconciled with other studies on homologous P2-type ATPases, suggests that this region of the alpha subunit is important for cation-dependent conformation changes and may be near the entrance/exit domain of the cation transport pathway Several amino acids are essential for coordinating Ca2+ ions in the sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) are conserved in the gastric H+,K+-ATPase. In the second study, conserved amino acids to SERCA Ca2+ site I (E797, T825 and E938) as well as E774 and K793 were mutated to determine if these amino acids are essential for K+-ATPase specific activity in the gastric H+,K+-ATPase. The gastric H +,K+-ATPase specific activity, the apparent affinity for K+ activation and SCH 28080 inhibition of K+-ATPase activity were measured in mutants E774A, E774Q, K793A, K793L, E797G, E797Q, T825A, T825L, E938A, E938Q. Of the mutations in amino acids E774, E797, T825 and E938, only mutant E797G had less than 20% wildtype gastric H+,K + ATPase specific activity. Mutants T825A, T825L, E938A and E938Q each reduced the apparent affinity for K+ (3 to 6-fold), while mutants T825L and E938A also displayed a 4 and 8-fold increase in the apparent affinity for SCH 28080 (in 10 mM KCl). In contrast, E797Q showed a 4.5 fold decrease in the apparent affinity for SCH 28080 with no effect on the apparent affinity for K+. These results suggest that substitution at T825 and E938, influence, but do not destroy, the K+ binding domain Lysine 793 (K793) is unique to the gastric H+,K +-ATPase family. The homologous amino acid at this position in the Na+,K+-ATPase and SERCA families is a serine. Mutants K793A and K793L result in enzymes having less that 10% wildtype gastric H+,K+ ATPase specific activity, suggesting that this amino acid is essential for gastric H+,K + ATPase function. (Abstract shortened by UMI.)
