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Abstract

Glutamic acid residues in transmembrane segments of the alpha subunit of the Na⁺,K⁺-ATPase have been discussed as possible candidates for the binding sites of the transported cations. Here we report on effects of mutations of Glu₃₃₄, Glu₉₅₉, and Glu₉₆₀ to alanine in ouabain-sensitive (OS) as well as ouabain-resistant (OR) ATPases of Torpedo electroplax expressed in Xenopus oocytes. All mutants are incorporated to about the same extend as the wild-type ATPases into the plasma membrane. None of the mutations produces complete inhibition of transport activity as judged from measurements of ⁸⁶Rb⁺ uptake, membrane current, and ATPase activity. After conversion of OS to OR by mutation of the bordering residues of the first extracellular loop Gln₁₁₈ to Arg and Asp₁₂₉ to Asn, the K_m value for inhibition by ouabain increases to 59 microM. Substitution of Glu₃₃₄ to Ala in the OR pump variant restores ouabain sensitivity with a K_m value of 0.12 microM, which is similar to that of the endogenous Xenopus pump. After substitution of Glu₉₆₀ by Ala in the OR pump, ouabain sensitivity is partially restored. The K_m values for pump stimulation by external K⁺ appear to be reduced in the OR compared to the OS pump. Mutation of Glu₉₅₉ and Glu₉₆₀ to Ala has no pronounced effects on the potential-dependent K_m values at external pH 7.8; only in the Glu₉₅₉-mutated OR pump, the apparent K_m at 0 mV is raised. We conclude that none of the mutated glutamic acid residues is essential for cation coordination, but that GIu₃₃₄, and in part also Glu₉₆₀, seems to be involved in preserving the ouabain-resistant conformation of the enzyme.