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 [3H]ouabain binding, the functional role of glutamic acid 327 in the fourth transmembrane domain of the sheep alpha 1 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 alpha 1 protein background such that [3H]ouabain binding could be utilized as a highly specific probe of the exogenous protein expressed in NIH 3T3 cells. Na+ competition of [3H]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 [3H]ouabain binding to all four mutant forms of Na+,K(+)-ATPase displayed severely altered interactions between these proteins and K+. Interestingly, [3H]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 K0.5 for K(+)-dependent ATPase activity (Jewell-Motz, E. A., and Lingrel, J.B. (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.
Kuntzweiler
T A
TA
Department of Molecular Genetics, University of Cincinnati, College of Medicine, Ohio 45267-0524.
Wallick
E T
ET
Johnson
C L
CL
Lingrel
J B
JB
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000235:genetics; D015698:Genomic Library; D018698:Glutamic Acid; D007527:Isoenzymes / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D010042:Ouabain / Q000378:metabolism*; D017354:Point Mutation; D011188:Potassium / Q000494:pharmacology*; D011487:Protein Conformation / Q000187:drug effects*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012756:Sheep; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism*; D014162:Transfection
D007527:Isoenzymes; D011994:Recombinant Proteins; D018698:Glutamic Acid; D010042:Ouabain; D004247:DNA; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium