Glutamic Acid 327 in the Sheep α1 Isoform of Na+,K+-ATPase Stabilizes a K+-induced Conformational Change

Abstract

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.

Critical effects on catalytic function produced by amino acid substitutions at Asp804 and Asp808 of the al isoform of Na,K‐ATPase

“…In addition, Vilsen [12] reported a normal turnover for a Glu327Gln mutant, and even a radical leucine substitution also produced a functional enzyme [ 111. Finally, studies of the non-functional mutants with substitutions at position 327 are consistent with the importance of this site in conformational transitions, but not necessarily in cation binding per se [14].…”

“…Likewise, transfections with cDNAs carrying the substitutions Aspso8Ala, AspsosAsn and Binding of t3H]ouabain to intact 3T3 cells that were untransfected (3T3) or transfected with a cDNA encoding the wild-type sheep al Na,K-ATPase (WT) or the sheep a-subunit carrying the amino acid substitutions Aspso4Ala (D804A) and AspxflsAla (D808A). Conditions for binding were the same as those described in [14] except that the concentration of [3H]ouabain was 70.6 nM. Data shown are the mean f S.E.M.…”

“…With this system, [3H]ouabain can bind to the exogenous enzyme without interference from the endogenous 3T3 Na,K-ATPase [14], and high-affinity binding to the extracellular surface of intact cells would suggest that the protein is synthesized and folded properly in the plasma membrane. When ouabain binding was measured in preparations of whole cells (Fig.…”

A pump-independent function of the Na,K-ATPase is required for epithelial junction function and tracheal tube-size control

“…Across phylogeny, the Na,K-ATPase ion pump function is contained within the ␣-subunit and depends on an essential aspartic acid that interacts with the terminal phosphate group of ATP to allow hydrolysis of the high-energy bond (reviewed by Kaplan, 2002). Mutation of this invariant aspartic acid to asparagine (D369N in vertebrates) has been shown in several systems to completely abolish ATP hydrolysis and ion pumping (Ohtsubo et al, 1990;Kuntzweiler et al, 1995). Because regions of ␣-subunits containing catalytic function are nearly 100% identical between flies and vertebrates (for example, the 60 residues flanking asparatic acid 369 are 98% identical between fly ATP␣ and the vertebrate ␣-isoforms), it is highly likely that mutation of the essential aspartic acid in fly ␣-subunits completely eliminates ion pump function.…”

Substitution of Glutamic 779 with Alanine in the Na,K-ATPase α Subunit Removes Voltage Dependence of Ion Transport

“…Logical targets of these studies have been carboxyl residues (Glu-327, Glu-779, Asp-804, Asp-808, Asp-926, Glu-953, and Glu-954) 1 that may be located in the transmembrane segments of the protein (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24). Site-directed mutagenesis studies have shown that substitutions of amino acids Glu-327, Asp-926, Glu-953, or Glu-954 produce no major changes in cation affinity (16 -18, 20) or in the enzyme electrical properties (19).…”