HIGH-AFFINITY OUABAIN BINDING BY YEAST-CELLS EXPRESSING NA+,K+-ATPASE ALPHA-SUBUNITS AND THE GASTRIC H+,K+-ATPASE BETA-SUBUNIT

Recently, a beta-subunit for the rat gastric H+,K+-ATPase (HK-beta), which is structurally similar to the beta-subunit of Na+,K+-ATPase, has been cloned and characterized. Using heterologous expression in yeast, we have tested the specificity of beta-subunit assembly with different isoforms of the alpha-subunit of Na+,K+-ATPase. Coexpression in yeast cells of the HK-beta with both the sheep alpha-1 subunit and the rat alpha-3 subunit isoforms of Na+,K+-ATPase (alpha-1 and alpha-3, respectively) leads to the appearance of high-affinity ouabain-binding sites in yeast membranes. These ouabain-binding sites (alpha-1 plus HK-beta, alpha-3 plus HK-beta) have a high affinity for ouabain (K(d), 5-10 nM) and are expressed at levels similar to those formed with the rat beta-1 subunit of Na+,K+-ATPase (beta-1) (alpha-1 plus beta-1 or alpha-3 plus beta-1). Potassium acts as a specific antagonist of ouabain binding by alpha-1 plus HK-beta and alpha-3 plus HK-beta just like sodium pumps formed with beta-1. Sodium pumps formed with the HK-beta, however, show quantitative differences in their affinity for ouabain and in the antagonism of K+ for ouabain binding. These data suggest that the structure of the beta-subunit may play a role in sodium pump function.