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.
Highlights
The functional properties and the pharmacological profile of the recently cloned cDNA colonic P-ATPase ␣ subunit
A novel P-type ATPase ␣ subunit cDNA has been cloned from a rat distal colon cDNA library [10, 13]
Co-expression in Xenopus laevis oocytes of synthetic cRNAs encoding the colonic P-ATPase ␣ subunit [10] and the toad urinary bladder  subunit [19] leads to a large increase in the uptake of 86Rbϩ, a surrogate of Kϩ used as a tracer, when compared to oocytes expressing the bladder  subunit alone as shown in Table I. 86Rbϩ uptake per oocyte is about twice that measured under the same experimental conditions in Xenopus oocytes co-expressing the toad bladder ␣ and  subunits (Table I). 86Rbϩ uptake in Xenopus oocytes injected with the colonic P-ATPase ␣ subunit alone is not different from those of oocytes injected with water
Summary
The functional properties and the pharmacological profile of the recently cloned cDNA colonic P-ATPase ␣ subunit Expression of gastric Hϩ,Kϩ-ATPase ␣ and  subunit cRNAs has been reported recently all along the renal collecting duct [8, 9], but not in distal colon [10].
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