IMPORTANCE OF INTRAMEMBRANE CARBOXYLIC-ACIDS FOR OCCLUSION OF K-ATPASE( IONS AT EQUILIBRIUM IN RENAL NA,K)

Site-directed mutagenesis and assay of Rb+ and Tl+ occlusion in recomb inant Na,K-ATPase from yeast were combined to establish structure-func tion 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 c apable 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,A sp), Asp(804)(Asn, Glu), Asp(808)(Asn, Glu) and Glu(779)(Asp) abolishe d 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 correl ated to effects of the mutations on K0.5(K+) for K+ displacement of AT P binding. Each of the four carboxylate residues Glu(327), Glu(779), a nd Asp(804) or Asp(808) in transmembrane segments 4, 5, and 6 is there fore essential for high-affinity occlusion of K+ in the E-2[2K] form. These residues either may engage directly in cation coordination or th ey may be important for formation or stability of the occlusion cavity .