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

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 K_m for K⁺ in the ATPase reaction, but imidazole (K_i = 13 mM) and triallylamine (K_i = 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.