Importance of Intramembrane Carboxylic Acids for Occlusion of K+ Ions at Equilibrium in Renal Na,K-ATPase

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Abstract
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 E2[2K] form. The wild-type yeast enzyme was capable of occluding 2 Rb+ or Tl+ ions/ouabain binding site or α1β1 unit with high apparent affinity (Kd(Tl+) = 7 ± 2 μM), like the purified Na,K-ATPase from pig kidney. Mutations of Glu327(Gln,Asp), Asp804(Asn, Glu), Asp808(Asn, Glu) and Glu779(Asp) abolished high-affinity occlusion of Rb+ or Tl+ ions. The substitution of Glu779 for Gln reduced the occlusion capacity to 1 Tl+ ion/α1β1-unit with a 3-fold decrease of the apparent affinity for the ion (Kd(Tl+) = 24 ± 8 μM). 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 Glu327, Glu779, and Asp804 or Asp808 in transmembrane segments 4, 5, and 6 is therefore essential for high-affinity occlusion of K+ in the E2[2K] form. These residues either may engage directly in cation coordination or they may be important for formation or stability of the occlusion cavity.

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