Importance of transmembrane segment M3 of Na+,K+-ATPase for control of conformational changes and the cytoplasmic entry pathway for Na.

Abstract

A series of mutations were introduced into the sequence Glu(282)-Ile-Glu-His-Phe-Ile-His(288) of the NH(2)-terminal part of M3 of the rat kidney Na(+),K(+)-ATPase, and the resulting mutant pumps were analyzed functionally. Several of the mutations affected the conformational transitions between E(1) and E(2) forms of dephospho- and phosphoenzyme. Mutations to Glu(282) and Phe(286) affected the E(1)-E(2) and E(1)P-E(2)P equilibria in parallel, indicating a role for these two residues in both conformational changes. Mutation to His(285) preferentially affected the E(1)P-E(2)P equilibrium, and mutation to Ile(283) affected only the E(1)-E(2) equilibrium of the dephosphoenzyme, demonstrating that the conformational changes of M3 in the phospho- and dephospho-forms are not identical. Several of the mutants showed a reduced apparent affinity for Na(+), pointing to an important role of the region in optimizing the Na(+) binding properties of the enzyme. It is possible that this part of M3 is closely associated with an entry pathway through which the Na(+) ions pass from the cytoplasmic surface to reach the cation-binding pocket. Some of the mutants also displayed an increased Na(+)-ATPase activity, and a good correlation was observed between the turnover rate for Na(+)-ATPase activity and the rate of dephosphorylation in the absence of K(+), indicating an increased ability of Na(+) to activate dephosphorylation of E(2)P by binding in place of K(+) at the extracellularly facing sites. Thus, M3 also seems to be a part of the signaling pathway between the external cation sites and the catalytic site.