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 (Kd, 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.
Eakle
K A
KA
Department of Physiology and Biophysics, University of Southern California School of Medicine, Los Angeles 90033.
Kim
K S
KS
Kabalin
M A
MA
Farley
R A
RA
D000818:Animals; D015153:Blotting, Western; D046911:Macromolecular Substances; D008566:Membranes / Q000378:metabolism; D010042:Ouabain / Q000378:metabolism*; D011189:Potassium Chloride / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D012756:Sheep; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013270:Stomach / Q000201:enzymology
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D010042:Ouabain; D011189:Potassium Chloride; D000254:Sodium-Potassium-Exchanging ATPase