Mutation to the glutamate in the fourth membrane segment of Na+,K+-ATPase and Ca2+-ATPase affects cation binding from both sides of the membrane and destabilizes the occluded enzyme forms.

The functional consequences of mutations Glu329 --> Gln in the Na+,K+-ATPase and Glu309 --> Asp in the sarco(endo)plasmic reticulum Ca2+-ATPase were analyzed and compared. Relative to the wild-type Na+,K+-ATPase, the Glu329 --> Gln mutant exhibited a 20-fold reduction in the apparent K+ affinity determined by titration of the rate of ATP hydrolysis at 50 microM ATP, and the rate of release of occluded K+ or Rb+ to the cytoplasmic side of the membrane was up to 30-fold enhanced by the mutation, as measured in kinetic studies of the phosphorylation by ATP of enzyme equilibrated with K+ or Rb+. The apparent affinity for extracellular K+ was 12-fold reduced by the Glu329 --> Gln mutation, as determined by K+ titration of the dephosphorylation. The maximum rate of phosphorylation by ATP of the Na+ form of the enzyme was reduced more than 2-fold by the mutation, but this effect could be counteracted by stabilizing Na+ occlusion with oligomycin. Similar studies on the Glu309 --> Asp mutant of the Ca2+-ATPase showed that the maximum rate of phosphorylation of the Ca2+ form was 8-9-fold reduced relative to that of the wild-type Ca2+-ATPase, and no Ca2+ occlusion could be detected in the mutant. Dephosphorylation of the phosphoenzyme intermediate formed with Pi was blocked in the Ca2+-ATPase mutant. The sensitivity to inhibition by thapsigargin, which binds selectively to the putative proton-occluded form of the Ca2+-ATPase, was reduced almost 300-fold in the mutant at neutral pH, but only 3-4-fold at pH 6.0. These data indicate that the mutations destabilize the occluded enzyme forms and interfere with cation binding from the extracytoplasmic side as well as with the gating process at the cytoplasmic entrance to the cation occlusion pocket.