Mutation of Gly-94 in transmembrane segment M1 of Na(+),K(+)-ATPase interferes with Na(+) and K(+) binding in E(2)P conformation

The importance of Gly-93 and Gly-94 in transmembrane segment M1 of the Na(+),K(+)-ATPase for interaction with Na(+) and K(+) was demonstrated by functional analysis of mutants Gly-93-Ala and Gly-94-Ala. In the crystal structures of the Ca(2+)-ATPase, the corresponding residues, Asp-59 and Leu-60, are located exactly where M1 bends. Rapid kinetic measurements of K(+)-induced dephosphorylation allowed determination of the affinity of the E(2)P phosphoenzyme intermediate for K(+). In Gly-94-Ala, the K(+) affinity was reduced 9-fold, i.e., to the same extent as seen for mutation of the cation-binding residue Glu-329. Furthermore, Gly-94-Ala showed strongly reduced sensitivity of the E(1)P-E(2)P equilibrium to Na(+), with accumulation of E(2)P even at 600 mM Na(+), indicating that interaction of E(2)P with extracellular Na(+) is impaired. On the contrary, in Gly-93-Ala, the affinity for K(+) was slightly increased, and the E(1)P-E(2)P equilibrium was displaced in favor of E(1)P. In both mutants, the affinity of the cytoplasmically facing sites of E(1) for Na(+) was reduced, but this effect was relatively small compared with the effects seen for E(2)P in Gly-94-Ala. Comparison with Ca(2+)-ATPase mutagenesis data suggests that the role of M1 in binding of the transported ions is universal among P-type ATPases, despite the low sequence homology in this region. Structural modeling of Na(+),K(+)-ATPase mutant Gly-94-Ala on the basis of the Ca(2+)-ATPase crystal structures indicates that the alanine side chain comes close to Ile-287 of M3, particularly in E(2)P, thus resulting in a steric clash that may explain the present observations