Constitutive activation of gastric H+,K+-ATPase by a single mutation

Abstract

In the reaction cycle of P-type ATPases, an acid-stable phosphorylated intermediate is formed which is present in an intracellularly located domain of the membranebound enzymes. In some of these ATPases, such as Na ϩ ,K ϩ -ATPase and gastric H ϩ ,K ϩ -ATPase, extracellular K ϩ ions stimulate the rate of dephosphorylation of this phosphorylated intermediate and so stimulate the ATPase activity. The mechanism by which extracellular K ϩ ions stimulate the dephosphorylation process is unresolved. Here we show that three mutants of gastric H ϩ ,K ϩ -ATPase lacking a negative charge on residue 820, located in transmembrane segment six of the α-subunit, have a high SCH 28080-sensitive, but K ϩ -insensitive ATPase activity. This high activity is caused by an increased 'spontaneous' rate of dephosphorylation of the phosphorylated intermediate. A mutant with an aspartic acid instead of a glutamic acid residue in position 820 showed hardly any ATPase activity in the absence of K ϩ , but K ϩ ions stimulated ATPase activity and the dephosphorylation process. These findings indicate that the negative charge normally present on residue 820 inhibits the dephosphorylation process. K ϩ ions do not stimulate dephosphorylation of the phosphorylated intermediate directly, but act by neutralizing the inhibitory effect of a negative charge in the membrane.

Cryo-EM structure of gastric H + ,K + -ATPase with a single occupied cation-binding site

“…The density is likely to be surrounded by several amino acids involved in cation coordination (SI Appendix, Fig. S11), as determined by mutagenesis (17)(18)(19)(20)(21)(22), and most of the homologous residues in Na + ,K + -ATPase or SERCA are also involved in cation coordination (21,23). Among them, E343 is in close proximity to the strong density located at site II in our homology model (SI Appendix, Fig.…”

K+-independent Gastric H+,K+-ATPase Activity

“…This was performed by combining mutations of residue Glu 343 , which block the dephosphorylation step (4,5), with the E820Q mutation, which gives constitutive ATPase activity (9). The present study shows that the preference for the E 1 conformation in the E820Q mutant also occurs when the dephosphorylation process is blocked, indicating that these two phenomena can be uncoupled.…”

“…In previous studies, we observed that some mutants of Glu 820 (E820Q, E820A, E820N) (9) and Glu 795 (E795L, E795A) (7) possessed a relatively high ATPase activity in the absence of K ϩ . This K ϩ -independent ATPase activity (also named constitutive ATPase activity) was caused by an enhanced spontaneous dephosphorylation rate of the phosphorylated intermediate ( Fig.…”

The Non-gastric H,K-ATPase Is Oligomycin-sensitive and Can Function as an H+,NH4+-ATPase

“…For production of H,K-ATPase, 1.5⅐10 6 cells⅐ml Ϫ1 were infected at a multiplicity of infection of 1-3 in the presence of 1% (v/v) ethanol (37) and 0.1% (w/v) pluronic F-68 (ICN, Aurora, OH) in Xpress medium (BioWhittaker, Walkersville, MD) as described previously (38). After 3 days, Sf9 cells were harvested by centrifugation at 2,000 ϫ g for 5 min.…”

“…ATP-dependent Phosphorylation-ATP-dependent phosphorylation was determined as described previously (37,38,42). Sf9 membranes (1-6 g) were incubated at 0 or 21°C in 50 mM Hepes-Tris (pH 6.0) containing 1.3 mM MgCl 2 and other ions and drugs as indicated in a volume of 80 l. After 30 -60 min, 20 l of 0.5 M [␥-32 P]ATP was added, and the mixture was incubated for 10 s at the same temperature as before.…”