title: Evidence for tryptophan residues in the cation transport path of the Na(+),K(+)-ATPase.
abstract: 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 microM), a low-affinity site (K(D) approximately 10 microM) 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) approximately 30 microM) 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 (22)Na(+) 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) approximately 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.
authors.affiliation: Laboratorio de Biofísica, Instituto M. y M. Ferreyra, INIMEC-CONICET, 5000 Córdoba, Argentina.
authors.forename: Guillermo A
authors.initials: GA
authors.lastname: Yudowski
authors.forename: Meirav
authors.initials: M
authors.lastname: Bar Shimon
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Tal
authors.forename: Rodolfo M
authors.initials: RM
authors.lastname: González-Lebrero
authors.forename: Rolando C
authors.initials: RC
authors.lastname: Rossi
authors.forename: Patricio J
authors.initials: PJ
authors.lastname: Garrahan
authors.forename: Luis A
authors.initials: LA
authors.lastname: Beaugé
authors.forename: Steven J D
authors.initials: SJ
authors.lastname: Karlish
mesh_terms: D000818:Animals; D001665:Binding Sites; D001693:Biological Transport, Active; D002412:Cations / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004912:Erythrocytes / Q000378:metabolism; D006801:Humans; D007550:Isothiuronium / Q000031:analogs & derivatives / Q000494:pharmacology; D008958:Models, Molecular; D010042:Ouabain / Q000494:pharmacology; D010766:Phosphorylation; D012413:Rubidium / Q000378:metabolism; D012964:Sodium / Q000493:pharmacokinetics; D012979:Sodium Radioisotopes; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013552:Swine; D014364:Tryptophan / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D002412:Cations; D004791:Enzyme Inhibitors; D012979:Sodium Radioisotopes; D007550:Isothiuronium; D010042:Ouabain; D014364:Tryptophan; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium