GLUTAMIC-ACID-327 IN THE SHEEP ALPHA-1 ISOFORM OF NA+,K+-ATPASE IS A PIVOTAL RESIDUE FOR CATION-INDUCED CONFORMATIONAL-CHANGES

The cation binding characteristics of the mutant E327A formed in the sheep al isoform of the Na+,K+-ATPase were examined using [H-3]ouabain binding as a function of monovalent cation concentrations. Equilibrium competition binding assays in the presence of Mg2+, inorganic phosphate and various amounts of unlabelled ouabain indicated that both wild-type sheep al protein and the E327A mutant expressed in 3T3 cells had similar affinities for ouabain (K-D = 1.53 and 1.31 nM respectively). Sodium inhibition of ouabain binding appeared competitive in both enzymes. However, binding of three Na+ ions was required to explain the steep character of the Na+ inhibition curve for the wild-type Na+,K+-ATPase (K-i = 12.8 +/- 1.6 mM), whereas the binding of two Na+ ions was detected for the mutant E327A (K-i = 19.2 +/- 2.5 mM). Potassium inhibition of [H-3]ouabain binding displayed a partially competitive nature with Hill coefficients of 2 for both wild-type sheep alpha 1 (K-i = 0.743 +/- 0.044 mM) and E327A (K-i = 0.875 +/- 0.067 mM). At concentrations of K+ above 10 mM, the sheep alpha 1 competition curve levelled off whereas the inhibition curve for E327A displayed a stimulation in ouabain binding. This stimulation in [H-3]ouabain binding also occurred with Rb+, Cs+ and Li+, but was never observed with choline or Na+, suggesting that this effect was not due to ionic strength. From these [H-3]ouabain-binding studies, it is obvious that the mutant enzyme E327A in the presence of Mg2+, P-i and ouabain, interacts with monovalent cations in a unique fashion. One interpretation of these data is that the glutamic acid residue at position 327 is involved in a conformational transition induced by the binding of monovalent cations to the Na+,K+-ATPase and that this transition is inhibited by the mutation of E327A.