The negative charge of glutamic acid-820 in the gastric H+,K+-ATPase α-subunit is essential for K+ activation of the enzyme activity

The sixth transmembrane (M6) segment of the catalytic subunit plays an important role in the ion recognition and transport in the type II P-type ATPase families. In this study, we singly mutated all amino acid residues in the M6 segment of gastric H ؉ ,K ؉ -ATPase ␣-subunit with alanine, expressed the mutants in HEK-293 cells, and studied the effects of the mutation on the functions of H ؉ ,K ؉ -ATPase; overall K ؉ -stimulated ATPase, phosphorylation, and dephosphorylation. Four mutants, L819A, D826A, I827A, and L833A, completely lost the K ؉ -ATPase activity. Mutant L819A was phosphorylated but hardly dephosphorylated in the presence of K ؉ , whereas mutants D826A, I827A, and L833A were not phosphorylated from ATP. We found that almost all of these amino acid residues, which are important for the function, are located on the same side of the ␣-helix of the M6 segment. In addition, we found that amino acids involved in the phosphorylation are located exclusively in the cytoplasmic half of the M6 segment and those involved in the K ؉ -dependent dephosphorylation are in the luminal half. Several mutants such as I821A, L823A, T825A, and P829A partly retained the K ؉ -ATPase activity accompanying the decrease in the rate of phosphorylation.

Section: Discussionmentioning

confidence: 87%

Alanine-scanning Mutagenesis of the Sixth Transmembrane Segment of Gastric H+,K+-ATPase α-Subunit

Asano¹,

Io²,

Kimura³

et al. 2001

“…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.…”

Section: Methodsmentioning

confidence: 99%

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

Swarts

Koenderink

Willems

et al. 2005

Self Cite

“…This difference is due to the much faster rate of the E 2 K 3 E 1 K conversion in the latter enzyme (6,38). It has been shown that four negatively charged residues present in transmembrane segments 4 -6 play a role in cation-activated reactions of both ATPases (7)(8)(9)(10)(11)(12)(13). With the exception of a single residue in M6…”

mentioning

confidence: 99%

Chimeras of X+,K+-ATPases

Koenderink

Swarts

Stronks

et al. 2001

Self Cite

In this study we reveal regions of Na+,K+-ATPase and H+,K+-ATPase that are involved in cation selectivity. A chimeric enzyme in which transmembrane hairpin M5-M6 of H+,K+-ATPase was replaced by that of Na+,K+-ATPase was phosphorylated in the absence of Na+and showed no K+-dependent reactions. Next, the part originating from Na+,K+-ATPase was gradually increased in the N-terminal direction. We demonstrate that chimera HN16, containing the transmembrane segments one to six and intermediate loops of Na+,K+-ATPase, harbors the amino acids responsible for Na+specificity. Compared with Na+,K+-ATPase, this chimera displayed a similar apparent Na+affinity, a lower apparent K+affinity, a higher apparent ATP affinity, and a lower apparent vanadate affinity in the ATPase reaction. This indicates that theE2K form of this chimera is less stable than that of Na+,K+-ATPase, suggesting that it, like H+,K+-ATPase, de-occludes K+ions very rapidly. Comparison of the structures of these chimeras with those of the parent enzymes suggests that the C-terminal 187 amino acids and the β-subunit are involved in K+occlusion. Accordingly, chimera HN16 is not only a chimeric enzyme in structure, but also in function. On one hand it possesses the Na+-stimulated ATPase reaction of Na+,K+-ATPase, while on the other hand it has the K+occlusion properties of H+,K+-ATPase.