Evidence for Tryptophan Residues in the Cation Transport Path of the Na+,K+-ATPase.

YUDOWSKI, GUILLERMO A.; BAR SHIMON, M.; RODOLFO MARTIN GONZALEZ LEBRERO; ROLANDO CARLOS, ROSSI; PATRICIO J, GARRAHAN; BEAUGÉ, L; KARLISH, S.J.D.

BIOCHEMISTRY

AMER CHEMICAL SOC

Lugar: Washington; Año: 2003 vol. 42 p. 10212 - 10222

0006-2960

A family of aryl isothiouronium derivatives was designed as probes for cation binding sites of Na+,K+-ATPase. Previous work showed that 1-bromo-2,4,6-tris(methylisothiouronium)benzene (Br-TITU) acts as a competitive blocker of Na+ or K+ occlusion. In addition to a high-affinity cytoplasmic site (KD< 1 μM), a low-affinity site (KD ∼ 10 μM) was detected, presumably extracellular. Here we describe properties of Br-TITU as a blocker at the extracellular surface. In human red blood cells Br-TITU inhibits ouabain-sensitive Na+ transport (KD ∼ 30 μM) in a manner antagonistic with respect to extracellularNa+. In addition, Br-TITU impairs K+-stimulated dephosphorylation and Rb+ occlusion from phosphorylated enzyme of renal Na+,K+-ATPase, consistent with binding to an extracellular site. Incubation of renal Na+,K+-ATPase with Br-TITU at pH 9 irreversibly inactivates Na+,K+-ATPase activity and Rb+occlusion. Rb+ or Na+ ions protect. Preincubation of Br-TITU with red cells in a K+-free medium at pH 9 irreversibly inactivates ouabain-sensitive 22Na+ efflux, showing that inactivation occurs at an extracellular site. K+, Cs+, and Li+ ions protect against this effect, but the apparent affinity for K+, Cs+, or Li+ is similar (KD ∼ 5 mM) despite their different affinities for external activation of the Na+ pump. Br-TITU quenches tryptophan fluorescence of renal Na+,K+-ATPase or of digested ?19 kDa membranes?. After incubation at pH 9 irreversible loss of tryptophan fluorescence is observed and Rb+ or Na+ ions protect.The Br-TITU appears to interact strongly with tryptophan residue(s) within the lipid or at the extracellular membrane-water interface and interfere with cation occlusion and Na+,K+-ATPase activity.