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 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 Km value for inhibition by ouabain increases to 59 microM. Substitution of Glu334 to Ala in the OR pump variant restores ouabain sensitivity with a Km 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 Km 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 Km values at external pH 7.8; only in the Glu959-mutated OR pump, the apparent Km 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.

Comparative effects of aluminum and ouabain on synaptosomal choline uptake, acetylcholine release and (Na+/K+)ATPase

“…Under our experimental conditions, 10 −3 M ouabain was sufficient to completely inhibit (Na + /K + )ATPase activity of all synaptosomal preparations. This observation is consistent with previously reported ouabain sensitivity of (Na + /K + )ATPase from rat brain (Berrebi-Bertrand et al, 1990;Gerbi and Maixent, 1999;Lopez et al, 2002), optic ganglion of the squid (Breitwieser, 1982) and torpedo electric organ (Zubler-Faivre and Dunant, 1976;Richardson and Whittaker, 1981;Vasilets et al, 1998). However, these synaptosomal fractions differ on the enzyme activity to a great extent.…”

Electrophysiological Analysis of the Mutated Na,K-ATPase Cation Binding Pocket

“…Therefore, 10 ng of cRNA ␣-subunit and 2 ng of cRNA of the ␤-subunit were injected into X. laevis oocytes. These oocytes express an endogenous Na,K-ATPase (ϳ25-nA stationary current; data not shown) that can be inhibited by 10 M ouabain (28). The current generated by the heterologously expressed rat Na,KATPase can be readily dissociated from the endogenous pump, since the latter is rather insensitive for ouabain (33).…”

Endogenous Cardiotonic Steroids: Physiology, Pharmacology, and Novel Therapeutic Targets

“…Some amino acids from the transmembrane regions (M4, M6, and M10) also interact with ouabain; this suggests that the hydrophobic regions of CTS may actually be inserted in the membrane inside which they interact with the α subunit of sodium pump (Burns et al, 1996; Croyle et al, 1997; Vasilets et al, 1998). Amino acids from 111 to 122 sequences (extracellular TM1-TM2 loop) form the most important part of the putative CTS binding site (Fig.…”