Significance of the glutamic acid residues Glu334, Glu959, and Glu960 of the α subunits of Torpedo Na+,K+ pumps for transport activity and ouabain binding

Abstract Glutamic acid residues in transmembrane segments of the α 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 Glu 334 , Glu 959 , and Glu 960 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 86 Rb + uptake, membrane current, and ATPase activity. After conversion of OS to OR by mutation of the bordering residues of the first extracellular loop Gln 118 to Arg and Asp 129 to Asn, the K m value for inhibition by ouabain increases to 59 μM. Substitution of Glu 334 to Ala in the OR pump variant restores ouabain sensitivity with a K m value of 0.12 μM, which is similar to that of the endogenous Xenopus pump. After substitution of Glu 960 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 Glu 959 and Glu 960 to Ala has no pronounced effects on the potential-dependent K m values at external pH 7.8; only in the Glu 959 -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 Glu 334 , and in part also Glu 960 , seems to be involved in preserving the ouabain-resistant conformation of the enzyme.