INHIBITION OF AMILORIDE-SENSITIVE NA+ CHANNEL BY ISOTHIOURONIUM DERIVATIVES

The effects on the amiloride-blockable Na+ channel of a family of rece ntly synthesized isothiouronium derivatives were measured in plasma me mbrane vesicles from rat distal colon. Some of these derivatives act a s high-affinity Na+-like antagonists on the Na+-K+-adenosinetriphospha tase. One of the reagents tested, 1-bromo-2,4,6-tris(isothiouronium me thyl)benzene tribromide (Br-TITU), was found to be a potent blocker of the Nat channel. At neutral pH, Br-TITU rapidly inhibits the channel mediated Na-22(+) uptake, with an inhibition constant of 94 +/- 39 nM. The inhibition observed is specific and reversible. 1,3-Dibromo-2,4,6 -tris(isothiouronium methyl)benzene tribromide and Br-TITU derivatives with methyl and phenyl substitutions on the isothiouronium moiety wer e much less effective blockers. Incubation of cells with Br-TITU at al kaline (but not neutral) pH produces irreversible inactivation of chan nels, possibly due to covalent modification of a lysine residue. This inactivation can be attenuated by amiloride but not by Na+. Thus Br-TI TU may be a useful reagent in identifying essential residues of the ch annel protein.