Abstract

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 stimulate the rate of dephosphorylation of this phosphorylated intermediate and so stimulate the ATPase activity. The mechanism by which extracellular K⁺ ions stimulate the dephosphorylation process is unresolved. Here we show that three mutants of gastric H⁺,K⁺-ATPase lacking a negative charge on residue 820, located in transmembrane segment six of the -subunit, have a high SCH 28080-sensitive, but K⁺-insensitive ATPase activity. This high activity is caused by an increased 'spontaneous' rate of dephosphorylation of the phosphorylated intermediate. A mutant with an aspartic acid instead of a glutamic acid residue in position 820 showed hardly any ATPase activity in the absence of K⁺, but K⁺ ions stimulated ATPase activity and the dephosphorylation process. These findings indicate that the negative charge normally present on residue 820 inhibits the dephosphorylation process. K⁺ ions do not stimulate dephosphorylation of the phosphorylated intermediate directly, but act by neutralizing the inhibitory effect of a negative charge in the membrane.