THE INFLUENCE OF BETA-SUBUNIT STRUCTURE ON THE STABILITY OF NA+ K+-ATPASE COMPLEXES AND INTERACTION WITH K+/

Heterologous expression of the beta subunit of H+/K+-ATPase (HK beta) with alpha subunits of Na+/K+-ATPase (NK alpha) in yeast leads to the formation of ouabain binding complexes, indicating assembly of the two subunits into active ion pumps (Eakle, K. A., Kim, K. S., Kabalin, M. A., and Farley, R. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 2834 -2838). Complexes of NK alpha and HK beta are less sensitive to inhibi tion of ouabain binding by K+, suggesting that HK beta lowers the affi nity of K+ binding sites. This effect is particularly pronounced when HK beta is combined with the alpha 3 isoform of NK alpha. In this case , titration with K+ yields a biphasic curve, suggesting that there are two nonequivalent sites for K+ binding. Attempts at purifying complex es formed with either alpha 1 + HK beta or alpha 3 + HK beta using SDS extraction of microsomal membranes resulted in the loss of ouabain bi nding. Controls show that alpha 1 + beta 1 and alpha 3 + beta 1 comple xes still retain ouabain binding after SDS extraction under the same c onditions. This suggests that the HK beta subunit forms a less stable complex with NK alpha subunits. We have created chimeric beta subunits comprised of the amino-terminal cytoplasmic and transmembrane regions of HK beta combined with the carboxyl-terminal extracellular region o f Na+/K+-ATPase beta 1 (HN beta 1) and the complementary chimera with amino-terminal cytoplasmic and transmembrane regions of beta 1 combine d with the carboxyl-terminal extracellular region of HK beta (NH beta 1). When NH beta 1 is combined with either alpha 1 or alpha 3, the com plexes show profiles of K+ inhibition of ouabain binding that are very similar to HK beta combined with either alpha 1 or alpha 3. The data suggest that the extracellular region of HK beta is primarily responsi ble for the effect on apparent K+ affinity. When the HN beta 1 subunit is expressed with the alpha 3 subunit, less than 5% of the amount of ouabain binding complexes are formed compared with HN beta 1 + alpha 1 . This observation suggests that the HN beta 1 subunit either assemble s poorly or forms an unstable complex with alpha 3. After SDS extracti on, complexes of alpha 1 + NH beta 1 and alpha 3 + NH beta 1 retain ou abain binding, while alpha 1 + HN beta 1 complexes are sensitive to SD S extraction. Together, these data indicate that the cytoplasmic/trans membrane region of beta 1 is important for the assembly and stability of alpha + beta Na+/K+-ATPase complexes.