Functional consequences of substitutions of the carboxyl residue glutamate 779 of the Na,K-ATPase.

Carboxyl-containing amino acids in the transmembrane segments appear to be important for sodium- and potassium-activated adenosinetriphosphatase (Na,K-ATPase) activity. Substitution of Glu779 with Leu in a ouabain-resistant isoform inactivates the overall enzyme activity (Jewell-Motz & Lingrel, 1993). Chemical modification of this residue results in inactivation of Na,K-ATPase in a Na+ and K+ protectable manner (Arguello and Kaplan, 1991, 1994). These experiments suggest that this residue is important in cation binding. To further understand the role of Glu779 in Na,K-ATPase function, we have substituted this with four amino acids (Gln, Asp, Ala, and Leu) using site-directed mutagenesis coupled with expression and characterized the expressed enzyme. The amino acid substitutions were introduced into a modified sheep RD alpha 1 isoform that is relatively resistant to this drug. Enzyme carrying the E779Q and E779A replacements conferred ouabain resistance to the sensitive HeLa cells, while expression of enzyme carrying the E779D and E779L substitutions did not. Further analysis of isolated plasma membranes containing altered enzymes E779Q and E779A confirmed that they retain Na,K-ATPase activity. Analysis of cation stimulation of Na,K-ATPase activity revealed that the E779Q substituted enzyme exhibited a similar apparent affinity for K+ and a 2.6-fold decrease in affinity for Na+ compared with control enzyme. The E779A replacement caused a 6.6-fold and 5-fold decrease in apparent affinity for K+ and Na+, respectively. There is no difference in apparent affinity for ATP at the low affinity site for either E779Q or E779A.(ABSTRACT TRUNCATED AT 250 WORDS)