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 Rb-86(+) as a K+ surrogate, and pH measurements were performed by means of ion-selective microelectrodes. Co-injection of alpha and beta subunit cRNAs lead to a large increase in Rb-86(+) 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 Rb-86(+) uptake had a K-1/2 similar to 440 mu M for the bladder isoform (consistent with the previously reported value (Jaisser, F., Horisberger, J. D., Geering, K., and Rossier, B. C. (1933) J. Cell Biol. 123, 1421-1431)) and a K-1/2 similar to 730 mu M for the colonic isoform, Sch28080 was ineffective to reduce Rb-86(+) uptake whereas ouabain inhibited the activity expressed from rat colon alpha subunit with a K-i of 970 mu M when measured at the V-max 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.