GLUTAMIC-ACID-327 IN THE SHEEP ALPHA-1 ISOFORM OF NA-ATPASE STABILIZES A K+-INDUCED CONFORMATIONAL CHANGE(,K+)

By combining the tools of site-directed mutagenesis and [H-3]ouabain b inding, the functional role of glutamic acid 327 in the fourth transme mbrane domain of the sheep al isoform of Na(+)k(+)-ATPase was examined with respect to its interactions with ouabain, Na+, K+, Mg2+, and ino rganic phosphate. Using site-directed mutagenesis, this glutamic acid was substituted with alanine, aspartic acid, glutamine, and leucine. T he mutant proteins were constructed in a sheep al protein background s uch that [H-3]ouabain binding could be utilized as a highly specific p robe of the exogenous protein expressed in NIH 3T3 cells. Na+ competit ion of [H-3]ouabain binding to the mutant forms of Na+,K+-ATPase revea led only slight alterations in their affinities for Na+ and in their a bilities to undergo Na+-induced conformational changes which inhibit o uabain binding: In contrast, K+ competition of [H-3]ouabain binding to all four mutant forms of Na+,K+-ATPase displayed severely altered int eractions between these proteins and K+. Interestingly, [H-3]ouabain b inding to the mutant E327Q was not inhibited by the presence of K+. Th is mutant was previously reported to be functionally able to support c ation transport with a 5-fold reduced K-0.5 for K+-dependent ATPase ac tivity (Jewell-Motz, E. A., and Lingrel, J. E. (1993) Biochemistry 32, 13523-13530; Vilsen, B. (1993) Biochemistry 32, 13340-13349). Thus, i t appears that this glutamic acid in the fourth transmembrane domain m ay be important for stabilizing a K+ induced conformation within the c atalytic cycle of Na+,K+-ATPase that is not rate-limiting in the overa ll ATPase cycle but that displays a greatly reduced affinity for ouaba in.