Primary structure and functional expression of the mouse and frog alpha-subunit of the gastric H(+)-K(+)-ATPase.
The H(+)-K(+)-ATPase of the gastric parietal cells is responsible for the acidification of the stomach lumen. This heterodimeric protein belongs to the family of cation-translocating P-type ATPases, which includes the closely related Na(+)-ATPase. We have cloned the alpha-subunit cDNA of the Xenopus and murine gastric H(+)-K(+)-ATPase (alpha H-K). We have expressed Xenopus and murine alpha H-K along with the previously cloned gastric H(+)-K(+)-ATPase beta-subunit of rabbit (beta H-K) in Xenopus oocytes by cRNA injection. An antibody directed against the beta H-K coimmunoprecipitates under nondenaturing conditions the alpha H-K of both species, demonstrating assembly of the alpha/beta complex. Additionally, we demonstrate the presence of K(+)-transporting H(+)-K(+)-ATPase in the plasma membrane of oocytes by 86Rb- uptake. The H(+)-K(+)-ATPase-mediated K+ uptake was inhibited by the gastric H(+)-K(+)-ATPase inhibitor Sch-28080, but not by ouabain, and shows K(+)-dependent activation (K1/2 approximately 2 mM). Furthermore, H(+)-K(+)-ATPase-expressing oocytes show a Sch-28080 inhibitable proton extrusion. Our data indicate that the expressed H(+)-K(+)-ATPase behaves functionally in oocytes as in the gastric gland.
Mathews
P M
PM
Institute of Pharmacology and Toxicology, University of Lausanne, Switzerland.
Claeys
D
D
Jaisser
F
F
Geering
K
K
Horisberger
J D
JD
Kraehenbuhl
J P
JP
Rossier
B C
BC
D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D018076:DNA, Complementary / Q000235:genetics; D017506:H(+)-K(+)-Exchanging ATPase / Q000235:genetics* / Q000378:metabolism*; D051379:Mice / Q000378:metabolism*; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D009865:Oocytes / Q000378:metabolism; D011522:Protons; D012413:Rubidium / Q000378:metabolism; D013270:Stomach / Q000201:enzymology*; D014018:Tissue Distribution; D014982:Xenopus laevis / Q000378:metabolism*
D018076:DNA, Complementary; D015345:Oligonucleotide Probes; D011522:Protons; D017506:H(+)-K(+)-Exchanging ATPase; D012413:Rubidium