Importance of intramembrane carboxylic acids for occlusion of K+ ions at equilibrium in renal Na,K-ATPase

Site-directed mutagenesis and assay of Rb+ and Tl+ occlusion in recombinant Na,K-ATPase from yeast were combined to establish structure-function relationships of amino acid side chains involved in high-affinity occlusion of K+ in the E-2[2K] form. The wild-type yeast enzyme was capable of occluding 2 Rb+ or Tl+ ions/ouabain binding site or alpha 1 beta 1 unit with high apparent affinity (Kd(Tl+) = 7 +/- 2 mu M), like the purified Na,K-ATPase from pig kidney. Mutations of Glu(327)(Gln,Asp), Asp(804)(Asn, Glu), Asp(808)(Asn, Glu) and Glu(779)(Asp) abolished high-affinity occlusion of Rb+ or Tl+ ions. The substitution of Glu(779) for Gln reduced the occlusion capacity to 1 Tl+ ion/alpha 1 beta 1-unit with a 3-fold decrease of the apparent affinity for the ion (Kd(Tl+) = 24 +/- 8 mu M). These effects on occlusion were closely correlated to effects of the mutations on K0.5(K+) for K+ displacement of ATP binding. Each of the four carboxylate residues Glu(327), Glu(779), and Asp(804) or Asp(808) in transmembrane segments 4, 5, and 6 is therefore essential for high-affinity occlusion of K+ in the E-2[2K] form. These residues either may engage directly in cation coordination or they may be important for formation or stability of the occlusion cavity.