THE RAT DISTAL COLON P-ATPASE ALPHA-SUBUNIT ENCODES A OUABAIN-SENSITIVE H-ATPASE(,K+)

The functional properties and the pharmacological profile of the recen tly cloned cDNA colonic P-ATPase alpha subunit (Crowson, M. S., and Sh ull, G. E. (1992) J. Biol. Chem. 267, 13740-13748) were investigated b y using the Xenopus oocyte expression system, Xenopus oocytes were inj ected 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+ sur rogate, and pH measurements were performed by means of ion-selective m icroelectrodes. Co-injection of alpha and beta subunit cRNAs lead to a large increase in Rb-86(+) uptake, an intracellular alkalinization, a nd an extracellular medium acidification, as compared to alpha or beta injection alone, These results indicate that the colonic P-ATPase alp ha 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 th e 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 ineffect ive to reduce Rb-86(+) uptake whereas ouabain inhibited the activity e xpressed from rat colon alpha subunit with a K-i of 970 mu M when meas ured at the V-max of the enzyme, We conclude that, when expressed in X enopus oocytes, the rat colon P-ATPase alpha subunit encodes a ouabain -sensitive H+,K+-ATPase.