Chimeric domain analysis of the compatibility between H(+), K(+)-ATPase and Na(+),K(+)-ATPase beta-subunits for the functional expression of gastric H(+),K(+)-ATPase.
Gastric H(+),K(+)-ATPase consists of alpha-subunit with 10 transmembrane domains and beta-subunit with a single transmembrane domain. We constructed cDNAs encoding chimeric beta-subunits between the gastric H(+),K(+)-ATPase and Na(+),K(+)-ATPase beta-subunits and co-transfected them with the H(+),K(+)-ATPase alpha-subunit cDNA in HEK-293 cells. A chimeric beta-subunit that consists of the cytoplasmic plus transmembrane domains of Na(+),K(+)-ATPase beta-subunit and the ectodomain of H(+),K(+)-ATPase beta-subunit assembled with the H(+),K(+)-ATPase alpha-subunit and expressed the K(+)-ATPase activity. Therefore, the whole cytoplasmic and transmembrane domains of H(+),K(+)-ATPase beta-subunit were replaced by those of Na(+),K(+)-ATPase beta-subunit without losing the enzyme activity. However, most parts of the ectodomain of H(+),K(+)-ATPase beta-subunit were not replaced by the corresponding domains of Na(+), K(+)-ATPase beta-subunit. Interestingly, the extracellular segment between Cys(152) and Cys(178), which contains the second disulfide bond, was exchangeable between H(+),K(+)-ATPase and Na(+), K(+)-ATPase, preserving the K(+)-ATPase activity intact. Furthermore, the K(+)-ATPase activity was preserved when the N-terminal first 4 amino acids ((67)DPYT(70)) in the ectodomain of H(+),K(+)-ATPase beta-subunit were replaced by the corresponding amino acids ((63)SDFE(66)) of Na(+),K(+)-ATPase beta-subunit. The ATPase activity was abolished, however, when 4 amino acids ((76)QLKS(79)) in the ectodomain of H(+),K(+)-ATPase beta-subunit were replaced by the counterpart ((72)RVAP(75)) of Na(+),K(+)-ATPase beta-subunit, indicating that this region is the most N-terminal one that discriminates the H(+),K(+)-ATPase beta-subunit from that of Na(+), K(+)-ATPase.
Asano
S
S
Molecular Genetics Research Center, 2630 Sugitani Toyama 930-0194, Japan. shinji@ms.toyama-mpu.oc.jp
Kimura
T
T
Ueno
S
S
Kawamura
M
M
Takeguchi
N
N
D000595:Amino Acid Sequence; D001665:Binding Sites; D015870:Gene Expression; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008566:Membranes / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013270:Stomach / Q000201:enzymology*
D011993:Recombinant Fusion Proteins; D017506:H(+)-K(+)-Exchanging ATPase; D000254:Sodium-Potassium-Exchanging ATPase