Functional Expression of the Colonic H+,K+-ATPase α-Subunit: PHARMACOLOGIC PROPERTIES AND ASSEMBLY WITH X+,K+-ATPase β-SUBUNITS

Juan Codina,Bruce C Kone,Juan T Delmas-Mata,Thomas D Dubose

doi:10.1074/jbc.271.47.29759

Abstract

The functional and pharmacological properties of the alpha-subunit of the colonic H+,K+-ATPase (alphaC) were studied in Xenopus laevis oocytes. alphaC was injected with different rat beta-subunits, the beta-subunit of the gastric H+,K+-ATPase (betaG, the only H+, K+-ATPase beta-subunit identified in rat), or the beta1-subunit of the Na+,K+-ATPase (beta1) (associated with the basolateral Na+, K+-ATPase, but also expressed in the epithelial apical membranes of rat distal colon) (Marxer, A., Stieger, B., Quarini, A., Kashgarian, M., and Hauri, H. P. (1989) J. Cell Biol. 109, 1057-1069). The effect of the different beta-subunits was studied by measuring 86Rb+ uptake (a K+ congener) in the presence or absence of Sch-28080 and ouabain. Significant Na+-independent 86Rb+ uptake was observed only when alphaC was coexpressed with one of the beta-subunits. The expressed alphaCbeta1 and alphaCbetaG complexes were not inhibited by Sch-28080, were only partially sensitive to ouabain (IC50 = 400-600 microM, in the presence of external 1 mM KCl), and exhibited comparable K+ activation kinetics. Coexpression of alphaC with epitope-tagged betaG or beta1, followed by immunopurification of the alphabeta complexes, confirmed stable assembly of alphaCbetaG and alphaCbeta1 complexes. Since the beta1-subunit, but not the alpha1-subunit, of Na+,K+-ATPase is expressed in the apical membrane of rat colonocytes, our data support the view that, in rat distal colon, the beta1-subunit may play a surrogate role as the beta-subunit for the colonic H+,K+-ATPase.

Highlights

The functional and pharmacological properties of the ␣-subunit of the colonic H؉,K؉-ATPase (␣C) were studied in Xenopus laevis oocytes. ␣C was injected with different rat ␤-subunits, the ␤-subunit of the gastric H؉,K؉ATPase (␤G, the only H؉,K؉-ATPase ␤-subunit identified in rat), or the ␤1-subunit of the Na؉,K؉-ATPase (␤1)
Since the ␤1-subunit, but not the ␣1-subunit, of Na؉,K؉-ATPase is expressed in the apical membrane of rat colonocytes, our data support the view that, in rat distal colon, the ␤1-subunit may play a surrogate role as the ␤-subunit for the colonic H؉,K؉-ATPase
The results indicate that ␣C can interact with both ␤-subunits, that oligomerization is required for functional activity of the expressed enzymes in this system, and that the inhibitor sensitivities and Kϩ activation kinetics of

Summary

Introduction

The functional and pharmacological properties of the ␣-subunit of the colonic H؉,K؉-ATPase (␣C) were studied in Xenopus laevis oocytes. ␣C was injected with different rat ␤-subunits, the ␤-subunit of the gastric H؉,K؉ATPase (␤G, the only H؉,K؉-ATPase ␤-subunit identified in rat), or the ␤1-subunit of the Na؉,K؉-ATPase (␤1) (associated with the basolateral Na؉,K؉-ATPase, and expressed in the epithelial apical membranes of rat distal colon) The functional and pharmacological properties of the ␣-subunit of the colonic H؉,K؉-ATPase (␣C) were studied in Xenopus laevis oocytes.

Results

Conclusion