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

Abstract

Several mutations of residues Glu795 and Glu 820 present in M5 and M6 of the catalytic subunit of gastric H ؉ ,K ؉ -ATPase have resulted in a K ؉ -independent, SCH 28080-sensitive ATPase activity, caused by a high spontaneous dephosphorylation rate. The mutants with this property also have a preference for the E 1 conformation. This paper investigates the question of whether these two phenomena are coupled. This possibility was studied by combining mutations in residue Glu 343 , present in M4, with those in residues 795 and 820. When in combined mutants Glu and/or Gln residues were present at positions 343, 795, and 820, the residue at position 820 dominated the behavior: a Glu giving K ؉ -activated ATPase activity and an E 2 preference and a Gln giving K ؉ -independent ATPase activity and an E 1 preference. With an Asp at position 343, the enzyme could be phosphorylated, but the dephosphorylation was blocked, independent of the presence of either a Glu or a Gln at positions 795 and 820. However, in these mutants, the direction of the E 2 7 E 1 equilibrium was still dominated by the 820 residue: a Glu giving E 2 and a Gln giving E 1 . This indicates that the preference for the E 1 conformation of the E820Q mutation is independent of an active dephosphorylation process.

Involvement of the H3O+-Lys-164 -Gln-161-Glu-345 Charge Transfer Pathway in Proton Transport of Gastric H+,K+-ATPase

“…Mutant E345A, E345I, E345L, E345V, and E345K lost the K ϩ -ATPase activity, indicating that the presence of an acidic residue is necessary in site 2. However, mutant E345D also lost the K ϩ -ATPase activity, and mutant E345Q retained about half of the K ϩ -ATPase activity (7,34), which were in agreement with those reported by others (12).…”

“…In fact, mutant Q161L lost both the K ϩ -ATPase and Rb ϩ transport activities. Mutations at the position of Glu-345 of the H ϩ ,K ϩ -ATPase ␣-subunit have shown interesting results (7,12,34). Mutant E345A, E345I, E345L, E345V, and E345K lost the K ϩ -ATPase activity reflecting the absence of charge acceptor in site 2.…”

Electrogenicity of Na,K- and H,K-ATPase Activity and Presence of a Positively Charged Amino Acid in the Fifth Transmembrane Segment

“…DISCUSSION We have taken advantage of the functional differences and the high degree of sequence homology between the H,K-and Na,K-ATPases to attempt to identify the determinant of the electrogenicity of cation transport by the group IIc P-ATPases. In the fifth transmembrane segment, several amino acid residues have been shown to play a role in cation binding in both the H,K-and Na,K-ATPases (22,23,(25)(26)(27)(28)(29)(30)(31) and also in SERCA (32). The middle of the fifth transmembrane segment region is highly similar between the H,K-and Na,K-ATPases (Fig.…”

A Conformation-specific Interhelical Salt Bridge in the K+ Binding Site of Gastric H,K-ATPase

“…This can be concluded from the low concentrations of either vanadate or the specific inhibitor SCH 28080, needed to inhibit the ATPase activity (11). All mutants with K ϩ -independent ATPase activity, however, had a preference for the E1 conformation (9,11), as shown by the high vanadate concentration needed to inhibit the ATPase activity. This preference for the E 1 form also explained why pre-incubation with the specific inhibitor SCH 28080 did not prevent ATP-phosphorylation or, in some cases, even enhanced the phosphorylation level of these mutants (12).…”

“…The central theme in studies with this enzyme was the binding site for K ϩ , which ion stimulates the hydrolysis of the phosphorylated intermediate. Studies up to now suggest roles in K ϩ activation for at least Glu 820 in M6 (2-4), Glu 795 in M5 (4 -6), and Glu 343 in M4 (7)(8)(9). These glutamate residues play different roles.…”