ABSTRACT

The present PhD dissertation is based on experimental work carried out at The Department of Physiology, University of Aarhus, and deals with fundamental aspects of the mechanism of the Na + ,K + -ATPase, which is a transmembrane enzyme that utilizes the energy of ATP for vectorial translocation of Na + and K + across the cell membrane and is essential to a number of physiological functions at cellular and organ levels. The transport cycle of the Na + ,K + -ATPase follows a consecutive mechanism, which includes at least two interconvertible enzyme forms, E 1 and E 2 , which cycle between phosphorylated and dephosphorylated states. The membrane-spanning part of the enzyme, which binds and translocates the ions, is well separated from the cytoplasmic part, where ATP hydrolysis takes place. The cytoplasmic part consists of a nucleotide-binding domain (domain N), a phosphorylation domain (domain P), and a third domain (domain A) with hitherto unknown function. Recent evidence suggests that rather large rearrangements of these domains (particularly of domain A) take place during the pump cycle, but the mechanical basis for the tight coupling between the various functional parts of the enzyme is far from being understood. Furthermore, the pathways for migration of the Na + and K + ions to the coordinating ligands in the membrane have not been identified, and these pathways most likely contribute to define the cation binding properties of the enzyme. In the present PhD study, these questions have been addressed using site-directed mutagenesis combined with expression in mammalian cells and enzyme kinetic measurements to study the functional roles of single amino acid residues located in the A-M3 sector, consisting of domain A, transmembrane segment M3, and the interconnecting cytoplasmic extension of M3. Our results identify residues that are involved in the transport-associated conformational changes of the enzyme, and show that the threonine in the conserved TGES sequence in domain A plays an important role in the function of the catalytic site in the E 2 state, but not in E 1 . This is consistent with a model in which the TGES sequence moves from an isolated position in E 1 into contact with domain P in the E 2 state. Furthermore, certain M3 residues were found to be important for the binding of Na + in the E 1 state, suggesting a role in the control of Na + migration to and from the binding sites. Some M3 residues were also found to be part of the intramolecular signalling pathway between the ion sites in their extracellularly facing configuration and the catalytic site in E 2 P. Hence, the A-M3 sector seems to play a central role in mediating the cross talk between the cytoplasmic (catalytic) and membranous (ion translocating) parts of the protein.