Identification of Asp(804) and Asp(808) as Na+ and K+ coordinating residues in alpha-subunit of renal Na,K-ATPase

Mutations to Asp(804) and Asp(808) in the alpha-subunit almost abolish Na,K-ATPase activity, but high-affinity binding of [H-3]ATP or [H-3]ouabain at equilibrium and E(1)-E(2) transitions are preserved. Titration of K+-ion displacement of [H-3]ATP or [H-3]ouabain shows that the mutations interfere with occlusion of K+ in the E(2)[2K] conformation. Reduced phosphorylation levels or affinities for Na+ in presence of oligomycin indicate that Asp(804) and Asp(808) also contribute to coordination of Na+ in the E(1)P[3Na] form. Demonstration of alternate interactions of Na+ or K+ with Asp(804) and Asp(808) support the notion of cation binding in a ping-pong sequence in catalytic models of Na,K-pumping.