title: The rat distal colon P-ATPase alpha subunit encodes a ouabain-sensitive H+, K+-ATPase.
abstract: 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.
authors.affiliation: INSERM, U323, Faculté de Médecine Necker, Université Paris V, France.
authors.forename: M
authors.initials: M
authors.lastname: Cougnon
authors.forename: G
authors.initials: G
authors.lastname: Planelles
authors.forename: M S
authors.initials: MS
authors.lastname: Crowson
authors.forename: G E
authors.initials: GE
authors.lastname: Shull
authors.forename: B C
authors.initials: BC
authors.lastname: Rossier
authors.forename: F
authors.initials: F
authors.lastname: Jaisser
mesh_terms: 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
chemical_list: D018076:DNA, Complementary; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D010042:Ouabain; D017506:H(+)-K(+)-Exchanging ATPase; D012413:Rubidium; D011188:Potassium