Inhibition of the amiloride-sensitive na+ channel by isothiouronium
derivatives.
Avigdor, Abraham, Carol Asher, Daniel M. Tal, Steven J. D. Karlish,
and Haim Garty.
Department of Membrane Research and Biophysics and the Department
of Biochemistry, The Weizmann Institute of Science, Rehovot 76100,
Israel
APStracts 3:0181C, 1996.
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+) ATPase. One
of the reagents tested, 1-Bromo-2,4,6-tris (isothiouroniummethyl)
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 a Ki of 94 39 nM. The inhibition observed
is specific and reversible. Br2-TITU (1,3-Dibromo-2,4,6
-tris(isothiouroniummethyl)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 to covalent modification of a lysine
residue. This inactivation can be attenuated by amiloride but not by
Na+ ions. Thus, Br-TITU may be a useful reagent in identifying
essential residues of the channel protein.
Received 26 February 1996; accepted in final form 22 May 1996.
APS Manuscript Number C105-6.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 17 June 96