RESUMEN
Previous chemical modification studies have suggested that Cys-369, Cys-658, Lys-482, Asp-712, and Asp-716 are essential for Na,K-ATPase function. To determine if the side chains of these amino acid residues are required for enzyme activity, rat alpha 1 cDNAs containing the mutations Cys-369-->Ser, Cys-658-->Ala, Lys-482-->Ala, Asp-712-->Asn, and Asp-716-->Asn were prepared and stably expressed in HeLa cells, which normally cannot survive in medium containing microM concentrations of ouabain. Expression of rat alpha 1 wild-type and the mutants Cys-369-->Ser, Cys-658-->Ala, and Lys-482-->Ala causes the appearance of HeLa cells that survive in medium containing 0.5 microM ouabain. Membranes isolated from the rat alpha 1 mutant clones exhibit Na,K-ATPase activities, ratios of Na,K-ATPase:phosphoenzyme, and apparent affinities for ouabain and ATP which are comparable to those of the rat alpha 1 wild-type enzyme. HeLa cells expressing mRNA and protein for Asp-712-->Asn and Asp-716-->Asn rat alpha 1 mutants cannot survive in 0.1 microM ouabain, and membranes isolated from these clones exhibit very little or no ouabain-insensitive rat alpha 1 Na,K-ATPase activity. The Asp-712-->Asn, but not Asp-716-->Asn, mutant rat alpha 1 can be phosphorylated by ATP. We conclude that Cys-369, Cys-658, and Lys-482 are not essential for Na,K-ATPase function but that the Asp residues at 712 and 716 are both essential.