Conformation-dependent inhibition of gastric H+,K+-ATPase by SCH 28080 demonstrated by mutagenesis of glutamic acid 820

Gastric H+,K+-ATPase can be inhibited by imidazo pyridines like 2-methyl-8- [phenylmethoxy] imidazo-(1,2a) pyridine 3-acetonitrile (SCH 28080). The dru g shows a high affinity for inhibition of K+-activated ATPase and for preve ntion of ATP phosphorylation. The inhibition by SCH 28080 can be explained by assuming that SCH 28080 binds to both the E-2 and the phosphorylated int ermediate (E-2-P) forms of the enzyme. We observed recently that some mutan ts, in which glutamic acid 820 present in transmembrane domain six of the c atalytic subunit had been replaced (E820Q, E820N, E820A), lost their K+-sen sitivity and showed constitutive ATPase activity. This ATPase activity coul d be inhibited by similar SCH 28080 concentrations as the K+-activated ATPa se of the wild-type enzyme. SCH 28080 also inhibited ATP phosphorylation at 21 degrees C of the mutants E820D, E820N, acid E820A, although with varyin g efficacy and affinity. ATP-phosphorylation of mutant E820Q was not inhibi ted by SCH 28080; in contrast, the phosphorylation level at 21 degrees C wa s nearly doubled. These findings can be explained by assuming that mutation of Glu(820) favors the E-1 conformation in the order E820Q > E820A > E820N > wildtype = E820D. The increase in the phosphorylation level of the E820Q mutant can be explained by assuming that during the catalytic cycle the E- 2-P intermediate forms a complex with SCH 28080. This intermediate hydrolyz es considerably slower than E-2-P and thus accumulates. The high tendency o f the E820Q mutant for the E-1 form is further supported by experiments sho wing that ATP phosphorylation of this mutant is rather insensitive towards vanadate, inorganic phosphate, and K+.