Avigdor A, Asher C, Tal D M, Karlish S J, Garty H
Department of Membrane Research, Weizmann Institute of Science, Rehovot, Israel.
Am J Physiol. 1996 Nov;271(5 Pt 1):C1457-62. doi: 10.1152/ajpcell.1996.271.5.C1457.
The effects on the amiloride-blockable Na+ channel of a family of recently synthesized isothiouronium derivatives were measured in plasma membrane vesicles from rat distal colon. Some of these derivatives act as high-affinity Na(+)-like antagonists on the Na(+)-K(+)-adenosinetriphosphatase. One of the reagents tested, 1-bromo-2,4,6-tris(isothiouronium methyl)-benzene tribromide (Br-TITU), was found to be a potent blocker of the Na+ channel. At neutral pH, Br-TITU rapidly inhibits the channel mediated 22Na+ 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 were much less effective blockers. Incubation of cells with Br-TITU at alkaline (but not neutral) pH produces irreversible inactivation of channels, possibly due ot covalent modification of a lysine residue. This inactivation can be attenuated by amiloride but not by Na+. Thus Br-TITU may be a useful reagent in identifying essential residues of the channel protein.
在来自大鼠远端结肠的质膜囊泡中测定了一系列最近合成的异硫脲鎓衍生物对氨氯吡脒可阻断的钠离子通道的影响。其中一些衍生物对钠钾腺苷三磷酸酶起高亲和力类钠拮抗剂的作用。所测试的一种试剂，1-溴-2,4,6-三（异硫脲鎓甲基）苯三溴化物（Br-TITU），被发现是一种有效的钠离子通道阻滞剂。在中性pH值下，Br-TITU能迅速抑制通道介导的22Na+摄取，抑制常数为94±39 nM。观察到的抑制作用具有特异性且是可逆的。1,3-二溴-2,4,6-三（异硫脲鎓甲基）苯三溴化物以及在异硫脲鎓部分带有甲基和苯基取代基的Br-TITU衍生物作为阻滞剂的效果要差得多。在碱性（而非中性）pH值下用Br-TITU孵育细胞会导致通道不可逆失活，这可能是由于赖氨酸残基的共价修饰所致。这种失活可被氨氯吡脒减弱，但不能被钠离子减弱。因此，Br-TITU可能是鉴定通道蛋白必需残基的一种有用试剂。
