To study the role of the Na,K-ATPase beta subunit in the ion transport
. activity, we have coexpressed the Bufo alpha 1 subunit (alpha 1) wit
h three different isotypes of beta subunits, the Bufo Na,K-ATPase beta
1 (beta 1NaK) or beta 3 (beta 3NaK) subunit or the beta subunit of th
e rabbit gastric H,K-ATPase (beta HK), by cRNA injection in Xenopus oo
cyte. We studied the K+ activation kinetics by measuring the Na,K-pump
current induced by external K+ under voltage clamp conditions. The en
dogenous oocyte Na,K-ATPase was selectively inhibited, taking advantag
e of the large difference in ouabain sensitivity between Xenopus and B
ufo Na,K pumps. The K+ half-activation constant (K-1/2) was higher in
the alpha 1 beta 3NaK than in the alpha 1 beta 1NaK groups in the pres
ence of external Na+, but there was no significant difference in the a
bsence of external Na+. Association of alpha 1 and beta HK subunits pr
oduced active Na,K pumps with a much lower apparent affinity for K+ bo
th in the presence and in the absence of external Na+. The voltage dep
endence of the K-1/2 for external K+ was similar with the three beta s
ubunits. Our results indicate that the beta subunit has a significant
influence on the ion transport activity of the Na,K pump. The small st
ructural differences between the beta 1NaK and beta 3NaK subunits resu
lts in a difference of the apparent affinity for K+ that is measurable
only in the presence of external Na+, and thus appears not to be dire
ctly related to the K+ binding site. In contrast, association of an al
pha 1 subunit with a beta HK subunit results in a Na,K pump in which t
he K+ binding or translocating mechanisms are altered since the appare
nt affinity for external K+ is affected even in the absence of externa
l Na+.