Tertiary amines as antagonists of both the luminal and cytosolic K+-site of gastric H,K-ATPase

Herman G.P. Swarts, Corné H.W. Klaassen, Feico M.A.H. Schuurmans Stekhoven, Jan Joep H.H.M. De Pont*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

8 Citations (Scopus)

Abstract

Tertiary amines like imidazole and triallylamine lower the apparent affinity of K+ in the ATP hydrolysis reaction of pig gastric H,K-ATPase in a pH and amine concentration dependent way. The mechanism and sidedness of this effect was studied by analyzing the partial reactions of the enzyme in both leaky and ion-tight vesicles. In leaky vesicles Tris and Hepes had nearly no effect on the apparent Km for K+ in the ATPase reaction, but imidazole (Ki = 13 mM) and triallylamine (Ki = 1.6 mM) markedly decreased the K+ affinity. The steady-state ATP-phosphorylation level in the absence of K+ was not or only slightly affected by these compounds. The reduction of the ATP-phosphorylation level by K+, however, again depended on both the type and concentration of tertiary amine used. A comparable K+-amine antagonism was observed in the dephosphorylation reaction. In tightly sealed vesicles, where no activation of K+ at the luminal side could occur, K+ reduced the affinity for ATP in the phosphorylation reaction. Triallylamine counteracted this effect. The K+-activated p-nitrophenylphosphatase activity in these ion-tight vesicles also showed a K+-triallylamine antagonism. Inhibition of H,K-ATPase activity in these vesicles by triallylamine was immediate (with nigericin present in order to allow intravesicular K+ activation), suggesting the transmembrane feature of this inhibition. These results indicate that tertiary amines decrease the affinity for K+ at both luminal and cytosolic binding sites by interaction at the cytosolic side of the membrane. This results in shifts in the equilibrium of both the E1·H ↔ E1· K transition and in the dephosphorylation reaction, E2-P → E2· K.

Original languageEnglish
Pages (from-to)143-151
Number of pages9
JournalBBA - Biomembranes
Volume1189
Issue number2
DOIs
Publication statusPublished - 19 Jan 1994
Externally publishedYes

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