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

• Published in: Biochemistry (Easton) 2003-09, Vol.42 (34), p.10212-10222
• Main Authors: Yudowski, Guillermo A, Bar Shimon, Meirav, Tal, Daniel M, González-Lebrero, Rodolfo M, Rossi, Rolando C, Garrahan, Patricio J, Beaugé, Luis A, Karlish, Steven J. D
• Format: Article
• Language: English
• Subjects: Animals; Binding Sites; Biological Transport, Active; Cations - metabolism; Cell Membrane - metabolism; Enzyme Inhibitors - pharmacology; Erythrocytes - metabolism; Humans; Isothiuronium - analogs & derivatives; Isothiuronium - pharmacology; Models, Molecular; Ouabain - pharmacology; Phosphorylation; Rubidium - metabolism; Sodium - pharmacokinetics; Sodium Radioisotopes; Sodium-Potassium-Exchanging ATPase - antagonists & inhibitors; Sodium-Potassium-Exchanging ATPase - chemistry; Sodium-Potassium-Exchanging ATPase - metabolism; Spectrometry, Fluorescence - methods; Swine; Tryptophan - chemistry; Tryptophan - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0342721

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 (K D < 1 μM), a low-affinity site (K D ∼ 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 (K D ∼ 30 μM) in a manner antagonistic with respect to extracellular Na+. 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 (K D ∼ 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.