Constitutive activation of gastric H+,K+-ATPase by a single mutation.
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 membrane-bound enzymes. In some of these ATPases, such as Na+,K+-ATPase and gastric H+, K+-ATPase, extracellular K+ ions stimulate the rate of dephosphorylation of this phosphorylated intermediate and so stimulate the ATPase activity. The mechanism by which extracellular K+ ions stimulate the dephosphorylation process is unresolved. Here we show that three mutants of gastric H+,K+-ATPase lacking a negative charge on residue 820, located in transmembrane segment six of the alpha-subunit, have a high SCH 28080-sensitive, but K+-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+, but K+ 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+ ions do not stimulate dephosphorylation of the phosphorylated intermediate directly, but act by neutralizing the inhibitory effect of a negative charge in the membrane.
Swarts
H G
HG
Department of Biochemistry, Institute of Cellular Signalling, 6500 HB Nijmegen, The Netherlands.
Hermsen
H P
HP
Koenderink
J B
JB
Schuurmans Stekhoven
F M
FM
De Pont
J J
JJ
D000818:Animals; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D005753:Gastric Mucosa / Q000201:enzymology; D017506:H(+)-K(+)-Exchanging ATPase / Q000235:genetics* / Q000378:metabolism*; D007093:Imidazoles / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation / Q000187:drug effects; D054328:Proton Pump Inhibitors; D051381:Rats
D007093:Imidazoles; D054328:Proton Pump Inhibitors; C035235:Sch 28080; D017506:H(+)-K(+)-Exchanging ATPase