Novel aromatic isothiouronium derivatives which act as high affinity competitive antagonists of alkali metal cations...

This paper describes properties of a novel family of aromatic isothiouronium derivatives, which act as Na(+)-like competitive antagonists on renal Na/K-ATPase. The derivatives are reversible competitors of Rb+ and Na+ occlusion. Ki values of the most potent compounds, 1-bromo-2,4,6-tris(methylisothiouronium)benzene (Br-TITU) and 1,3-dibromo-2,4,6-tris(methylisothiouronium)benzene(Br2-TITU ), 0.65 and 0.32 microM, respectively, are 15-30-fold lower than Ki values of the bis-guanidinium derivatives described previously (David, P., Mayan, H., Cohen, H., Tal, D. M., and Karlish, S. J. D. (1992) J. Biol. Chem. 267, 1141-1149), and represent the lowest reported values for cation antagonists. Using fluorescein-labeled Na/K-ATPase, all derivatives have been shown to stabilize the E1 conformation when bound at high affinity sites (i.e. they are sodium-like). In addition, in one condition (10 mM Tris-HCl, pH 8.1), high concentrations of Br-TITU (KD approximately 10 microM) appear to stabilize an E2 conformation. We propose a model which allows for simultaneous binding of the antagonists to high affinity cytoplasmic sites and low affinity sites, which may be at the extracellular surface. Blockage of cation occlusion by the isothiouronium derivatives at the cytoplasmic surface probably occurs at the entrance to the occlusion sites, which is recognized both by Na+ antagonists and by Na+ or K+ ions. Unlike the alkali metal cations, the Na+ antagonists are not occluded or transported (see also Or, E., David, P., Shainskaya, A., Tal, D. M., and Karlish, S. J. D. (1993) J. Biol. Chem. 268, 16929-16937). The isothiouronium derivatives appear to be promising candidates for further development as affinity labels of cation binding domains, for kinetic analysis of isoforms or mutated Na/K pumps, or as probes of other cation transport proteins.