CONSTITUTIVE ACTIVATION OF GASTRIC H-ATPASE BY A SINGLE MUTATION(,KAPPA(+))

In the reaction cycle of P-type ATPases, an acid-stable phosphorylated intermediate is formed which is present in an intracellularly located domain of the membrane bound enzymes. In some of these ATPases, such as Na+,K+-ATPase and gastric H+,K+-ATPase, extracellular K+ ions stimu late the rate of dephosphorylation of this phosphorylated intermediate and so stimulate the ATPase activity, The mechanism by which extracel lular K+ ions stimulate the dephosphorylation process is unresolved. H ere we show that three mutants of gastric H+,K+-ATPase lacking a negat ive charge on residue 820, located in transmembrane segment six of the alpha-subunit, have a high SCH 28080-sensitive, but K+-insensitive AT Pase activity. This high activity is caused by an increased 'spontaneo us' rate of dephosphorylation of the phosphorylated intermediate. A mu tant with an aspartic acid instead of a glutamic acid residue in posit ion 820 showed hardly any ATPase activity in the absence of K+, but K ions stimulated ATPase activity and the dephosphorylation process. Th ese findings indicate that the negative charge normally present on res idue 820 inhibits the dephosphorylation process. K+ ions do not stimul ate dephosphorylation of the phosphorylated intermediate directly, but act by neutralizing the inhibitory effect of a negative charge in the membrane.