C.H.W. Klaassen

Role of negatively charged residues in the fifth and sixth transmembrane domains of the catalytic subunit of gastric H+,K+-ATPase

The role of six negatively charged residues located in or around the fifth and sixth transmembrane domain of the catalytic subunit of gastric H+,K+-ATPase, which are conserved in P-type ATPases, was investigated by site-directed mutagenesis of each of these residues. The acid residues were converted into their corresponding acid amides. Sf9 cells were used as the expression system using a baculovirus with coding sequences for the α- and β-subunits of H+,K+-ATPase behind two different promoters. Both subunits of all mutants were expressed like the wild type enzyme in intracellular membranes of Sf9 cells as indicated by Western blotting experiments, an enzyme-linked immunosorbent assay, and confocal laser scan microscopy studies. The mutants D824N, E834Q, E837Q, and D839N showed no 3-(cyanomethyl)-2-methyl-8(phenylmethoxy)-imidazo[1,2a]pyridine (SCH 28080)-sensitive ATP dependent phosphorylation capacity. Mutants E795Q and E820Q formed a phosphorylated intermediate, which, like the wild type enzyme, was hydroxylamine-sensitive, indicating that an acylphosphate was formed. Formation of the phosphorylated intermediate from the E795Q mutant was similarly inhibited by K+ (I50 = 0.4 mM) and SCH 28080 (I50 = 10 nM) as the wild type enzyme, when the membranes were preincubated with these ligands before phosphorylation. The dephosphorylation reaction was K+-sensitive, whereas ADP had hardly any effect. Formation of the phosphorylated intermediate of mutant E820Q was much less sensitive toward K+ (I50 = 4.5 mM) and SCH 28080 (I50 = 1.7 μM) than the wild type enzyme. The dephosphorylation reaction of this intermediate was not stimulated by either K+ or ADP. In contrast to the wild type enzyme and mutant E795Q, mutant E820Q did not show any K+-stimulated ATPase activity. These findings indicate that residue Glu820 might be involved in K+ binding and transition to the E2 form of gastric H+,K+-ATPase.

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 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.

Role of sugar residues for recombinant gastric H+,K(+)-ATPase.

A major feature of the gastric H+,K+-ATPase p subunit is the presence of six or seven consensus sequences for N-linked glycosylation which are all cotranslationally glycosylated. In several reports of Na+,K+-ATPase, it was demonstrated that N-glycosylation is not essential for enzymatic The present study investigates whether N-glycosylation is essential for H+,K+-ATPase activity. H+,K+-ATPase can be synthesized in vitro as an active enzyme using the baculovirus s y ~ t e m . ~ ~ ~ In contrast to the mammalian enzyme, the p subunit is synthesized in both a nonglycosylated and a core-glycosylated form. Complex glycosylated p subunit is either present or absent in minor amount^.^ The presence of increasing concentrations of tunicamycin, an inhibitor of N-glycosylation, in the culture medium of Sf-9 cells resulted in a highly reproducible dose-dependent decrease in the amount of functional H+,K+-ATPase synthesized (FIG. 1 ) . This decrease in H+,K+-ATPase activity is correlated with a simultaneous decrease in the amount of glycosylated p subunits. Tunicamycin treatment had no visible effect on the H+,K+-ATPase a subunit. These results strongly suggest that N-glycosylation somehow is essential for H+,K+-ATPase activity. By using deoxymannojirimycin, a specific inhibitor of a-mannosidase I, trimming of the high-mannose oligosaccharide precursor can be blocked, preventing formation of complex glycosylated forms. Analysis of glycosylated forms of the p subunit indicated that the compound was active. However, no effect on the activity of the recombinant expressed H+,K+-ATPase was measured. Thus, only the presence and not the exact structure of the oligosaccharide moieties is essential for H+,K+-ATPase activity. Functional H+,K+-ATPase subunits in the standard crude membrane preparation can be separated from nonfunctional H+,K+-ATPase subunits using a discontinuous sucrose density gradient. FIGURE 2 shows that the purified H+,K+-ATPase fraction contained more glycosylated and almost no nonglycosylated p subunits. The nonglycosylated p subunits were more abundant in the pellet fraction, in which only little H+,K+-ATPase activity was found. This supports our conclusion that glycosylation is essential for H+,K+-ATPase activity.

Tertiary Amines as Cytosolic K+-Antagonists of Gastric H+/K+-ATPase

The reported properties of the overall H+/K+-ATPase activity vary with the conditions in which they are analyzed. The affinity of K+ for H+/K+-ATPase ranges from 0.3 to 5.0 mM. Such variations in K+-affinity can be due to differences in experimental conditions lite the ATP and Mg2+ concentration, temperature and pH. Another prominent variable is the type of buffer used.

In Vitro Synthesis of H+/K+-ATPase Polypeptides in Insect Cells Using the Baculovirus Expression System

H+/K+-ATPase is the enzyme responsible for gastric acid secretion catalysing the active exchange of intracellular H+ for luminal K+ ions and generating a proton gradient of more than 6 units across the apical membrane of gastric parietal cells. The enzyme consists of two subunits, a catalytic α-subunit of approximately 114 kD and a heavily glycosylated s-subunit of 34 kD which due to its extensive glycosylation has on SDS-PAGE gels an apparent molecular mass of 60–80 kD [1].