Mutation of Gly-94 in transmembrane segment M1 of Na+K+-ATPase interferes with Na+ and K+ binding in E2P 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 Ca²⁺-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 E2P 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 E1P-E2P equilibrium to Na⁺, with accumulation of E2P even at 600 mM Na⁺, indicating that interaction of E2P with extracellular Na⁺ is impaired. On the contrary, in Gly-93-Ala, the affinity for K⁺ was slightly increased, and the E1P-E2P equilibrium was displaced in favor of E1P. In both mutants, the affinity of the cytoplasmically facing sites of E1 for Na⁺ was reduced, but this effect was relatively small compared with the effects seen for E2P in Gly-94-Ala. Comparison with Ca²⁺-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 Ca²⁺-ATPase crystal structures indicates that the alanine side chain comes close to Ile-287 of M3, particularly in E2P, thus resulting in a steric clash that may explain the present observations.