NOVEL AROMATIC ISOTHIOURONIUM DERIVATIVES WHICH ACT AS HIGH-AFFINITY COMPETITIVE ANTAGONISTS OF ALKALI-METAL CATIONS ON NA K-ATPASE/

This paper describes properties of a novel family of aromatic isothiou ronium derivatives, which act as Na+-like competitive antagonists on r enal Na/K-ATPase. The derivatives are reversible competitors of Rb+ an d Na+ occlusion. K-i values of the most potent compounds, 1-bromo-2,4, 6-tris(methylisothiouronium)benzene (Br-TITU) and 1,3-dibromo-2,4,6-tr is(methylisothiouronium) benzene(Br-2-TITU), 0.65 and 0.32 mu M, respe ctively, are 15-30-fold lower than K-i values of the bis-guanidinium d erivatives described previously (David, P., Mayan, H., Cohen, H., Tal, D. M., and Karlish, S. J. D. (1992) J. Biol. Chem. 267, 1141-1149), a nd represent the lowest reported values for cation antagonists. Using fluorescein-labeled Na/K-ATPase, all derivatives have been shown to st abilize the E(1) conformation when bound at high affinity sites (i.e. they are sodium-like). In addition, in one condition (10 mns Tris-HCl, pH 8.1), high concentrations of Br-TITU (K-D approximate to 10 mu M) appear to stabilize an E(1) conformation. We propose a model which all ows for simultaneous binding of the antagonists to high affinity cytop lasmic sites and low affinity sites, which may be at the extracellular surface. Blockage of cation occlusion by the isothiouronium derivativ es at the cytoplasmic surface probably occurs at the entrance to the o cclusion 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, 16929-1 6937). The isothiouronium derivatives appear to be promising candidate s 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.