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

By combining the tools of site-directed mutagenesis and [H-3]ouabain binding, the functional role of glutamic acid 327 in the fourth transmembrane domain of the sheep al isoform of Na(+)k(+)-ATPase was examined with respect to its interactions with ouabain, Na+, K+, Mg2+, and inorganic phosphate. Using site-directed mutagenesis, this glutamic acid was substituted with alanine, aspartic acid, glutamine, and leucine. The mutant proteins were constructed in a sheep al protein background such that [H-3]ouabain binding could be utilized as a highly specific probe of the exogenous protein expressed in NIH 3T3 cells. Na+ competition of [H-3]ouabain binding to the mutant forms of Na+,K+-ATPase revealed only slight alterations in their affinities for Na+ and in their abilities to undergo Na+-induced conformational changes which inhibit ouabain binding: In contrast, K+ competition of [H-3]ouabain binding to all four mutant forms of Na+,K+-ATPase displayed severely altered interactions between these proteins and K+. Interestingly, [H-3]ouabain binding to the mutant E327Q was not inhibited by the presence of K+. This mutant was previously reported to be functionally able to support cation transport with a 5-fold reduced K-0.5 for K+-dependent ATPase activity (Jewell-Motz, E. A., and Lingrel, J. E. (1993) Biochemistry 32, 13523-13530; Vilsen, B. (1993) Biochemistry 32, 13340-13349). Thus, it appears that this glutamic acid in the fourth transmembrane domain may be important for stabilizing a K+ induced conformation within the catalytic cycle of Na+,K+-ATPase that is not rate-limiting in the overall ATPase cycle but that displays a greatly reduced affinity for ouabain.