Mutation of Gly-94 in transmembrane segment M1 of Na~+,K~+-ATPase interferes with Na~+ and K~+ binding in E_2P conformation

摘要

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 dephosphor-ylation allowed determination of the affinity of the E_2P phos-phoenzyme 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_1P-E_2P equilibrium to Na~+, with accumulation of E_2P even at 600 mM Na~+, indicating that interaction of E_2P with extracellular Na~+ is impaired. On the contrary, in Gly-93-Ala, the affinity for K~+ was slightly increased, and the E_1P-E_2P equilibrium was displaced in favor of E_1P. 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_2P 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_2P, thus resulting in a steric clash that may explain the present observations.