SIGNIFICANCE OF THE GLUTAMIC-ACID RESIDUES GLU(334), GLU(959), AND GLU(960) OF THE ALPHA-SUBUNITS OF TORPEDO NA+,K+ PUMPS FOR TRANSPORT ACTIVITY AND OUABAIN BINDING

Glutamic acid residues in transmembrane segments of the alpha subunit of the Na+,K+-ATPase have been discussed as possible candidates for th e 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 e lectroplax expressed in Xenopus oocytes. All mutants are incorporated to about the same extend as the wild-type ATPases into the plasma memb rane. None of the mutations produces complete inhibition of transport activity as judged from measurements of Rb-86(+) uptake, membrane curr ent, 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 increase s to 59 mu M. Substitution of Glu(334) to Ala in the OR pump variant r estores ouabain sensitivity with a K-m value of 0.12 mu M, which is si milar to that of the endogenous Xenopus pump. After substitution of Gl u(960) by Ala in the OR pump, ouabain sensitivity is partially restore d. The K-m values for pump stimulation by external K+ appear to be red uced in the OR compared to the OS pump. Mutation of Glu(959) and Glu(9 60) to Ala has no pronounced effects on the potential-dependent K, val ues at external pH 7.8; only in the Glu(959)-mutated OR pump, the appa rent K-m at 0 mV is raised. We conclude that none of the mutated gluta mic acid residues is essential for cation coordination, but that Glu(3 34), and in part also Glu(960), seems to be involved in preserving the ouabain-resistant conformation of the enzyme. (C) 1998 Elsevier Scien ce B.V.