MUTATION TO THE GLUTAMATE IN THE 4TH MEMBRANE SEGMENT OF NA-ATPASE AND CA2+-ATPASE AFFECTS CATION-BINDING FROM BOTH SIDES OF THE MEMBRANE AND DESTABILIZES THE OCCLUDED ENZYME FORMS(,K+)

The functional consequences of mutations Glu329 --> Gln in the Na+, K-ATPase and Glu309 --> Asp in the sarco(endo)plasmic reticulum Ca2+-AT Pase were analyzed and compared. Relative to the wild-type Na+, K+-ATP ase, the Glu329 --> Gln mutant exhibited a 20-fold reduction in the ap parent K+ affinity determined by titration of the rate of ATP hydrolys is at 50 mu M ATP, and the rate of release of occluded K+ or Rb+ to th e cytoplasmic side of the membrane was up to 30-fold enhanced by the m utation, as measured in kinetic studies of the phosphorylation by ATP of enzyme equilibrated with K+ or Rb+. The apparent affinity for extra cellular K+ was 12-fold reduced by the Glu329 --> Gln mutation, as det ermined 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 Glu3 09 --> 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 fo rmed with Pi was blocked in the Ca2+-ATPase mutant. The sensitivity to inhibition by thapsigargin, which binds selectively to the putative p roton-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 ind icate that the mutations destabilize the occluded enzyme forms and int erfere with cation binding from the extracytoplasmic side as well as w ith the gating process at the cytoplasmic entrance to the cation occlu sion pocket.