The rat distal colon P-ATPase alpha subunit encodes a ouabain-sensitive H+, K+-ATPase.
The functional properties and the pharmacological profile of the recently cloned cDNA colonic P-ATPase alpha subunit (Crowson, M.S., and Shull, G.E. (1992) J. Biol. Chem. 267, 13740-13748) were investigated by using the Xenopus oocyte expression system. Xenopus oocytes were injected with alpha subunit cRNAs from Bufo marinus bladder or rat distal colon and/or with beta subunit cRNA from B. marinus bladder. Two days after injection, K+ uptake was measured by using 86 Rb+ as a K+ surrogate, and pH measurements were performed by means of ion-selective microelectrodes. Co-injection of alpha and beta subunit cRNAs led to a large increase in 86Rb+ uptake, an intracellular alkalinization, and an extracellular medium acidification, as compared to alpha or beta injection alone. These results indicate that the colonic P-ATPase alpha subunit, like the bladder alpha subunit, acts as a functional H+,K+-ATPase, and that co-expression of alpha and beta subunits is required for the function. External K+ activation of the 86Rb+ uptake had a K1/2 of approximately 440 microM for the bladder isoform (consistent with the previously reported value (Jaisser, F., Horisberger, J.D., Geering, K., and Rossier, B.C. (1993) J. Cell. Biol. 123, 1421-1431) and a K1/2 of approximately 730 microM for the colonic isoform. Sch28080 was ineffective to reduce 86Rb+ uptake whereas ouabain inhibited the activity expressed from rat colon alpha subunit with a Ki of 970 microM when measured at the Vmax of the enzyme. We conclude that, when expressed in Xenopus oocytes, the rat colon P-ATPase alpha subunit encodes a ouabain-sensitive H+,K+-ATPase.
Cougnon
M
M
INSERM, U323, Faculté de Médecine Necker, Université Paris V, France.
Planelles
G
G
Crowson
M S
MS
Shull
G E
GE
Rossier
B C
BC
Jaisser
F
F
D000818:Animals; D001692:Biological Transport; D002024:Bufo marinus; D003106:Colon / Q000201:enzymology*; D018076:DNA, Complementary; D005260:Female; D017506:H(+)-K(+)-Exchanging ATPase / Q000096:biosynthesis / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D009865:Oocytes / Q000378:metabolism; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012413:Rubidium / Q000378:metabolism; D001743:Urinary Bladder / Q000201:enzymology; D014981:Xenopus
D018076:DNA, Complementary; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D010042:Ouabain; D017506:H(+)-K(+)-Exchanging ATPase; D012413:Rubidium; D011188:Potassium