Functional domains of Na,K-ATPase; conformational transitions in the alpha-subunit and ion occlusion

The purified membrane-bound Na,K-ATPase from the outer renal medulla crystallizes in different forms with monomeric or dimeric unit cells, but the alpha beta-unit is the minimum asymmetric unit in P1-crystals, and cation binding sites and occlusion cavities for Na+ or K+(Rb+) are formed within the structure of the alpha beta-unit. Domains have been identified that engage in structural transitions mediating energy transduction between ATP or phosphorylation sites in the cytoplasmic protrusion and cation sites in the membrane. The purpose of this study has been to examine the relationship between ion binding and occlusion, and transitions between two conformations of the alpha-subunit, the Na-form (E1) or the K-form (E2) that are defined by the kinetics of inactivation of Na,K-ATPase and K-phosphatase activities and a specific sequence of tryptic (E1: T2(Lys30)-T3(Arg262); E2: T1(Arg438)-T2(Lys30)) or chymotryptic (E1: C3(Leu266)) cleavage of the alpha-subunit. In E1-forms stabilized in an oligomycin complex, three sites for Na+ occlusion were found per alpha-subunit. In E2-forms stabilized as the phosphorylated ouabain complex, two Na+ or two Rb+ were occluded per alpha-subunit, the E2[2Rb] conformation having a higher affinity. Selective chymotryptic cleavage (Leu266) in the second cytoplasmic loop completely blocks E1-E2 transition, ion- and charge translocation without affecting ligand binding to the protein. Structural transitions of this segment reflect changes in capacity and orientation of cation sites that are part of the ion translocation process in the alpha-subunit of the Na,K-pump.