Electrogenic exchange of Na+ and K+ by the Na,K-ATPase is responsible for establishing ion concentration gradients across cell membranes that underlie resting membrane potentials, electrical excitability and solute transport in almost all mammalian cells. The rate of ion transport by the Na,K-ATPase is strongly dependent on membrane potential (VM) in a manner that suggests extracellular Na+ and K+ ions bind in an ion well(s) located within the enzyme. However, the molecular basis for such an ion well and its relationship to cation binding sites are entirely unknown. Our preliminary data show that VM-dependent properties of the Na,K-ATPase are significantly altered by amino acid substitutions at Glu779, located in the putative 5th and 6th transmembrane segments (M5-M6 hairpin) of the alpha-subunit. One interpretation of these data is that the carboxyl side chain of Glu779 constitutes a portion of the extracellular ion well. Thus, the goal of this project is to determine the molecular mechanism and structural features of the enzyme that underlie VM-dependent extracellular ion binding by the Na,K-ATPase. The resulting data will allow us to test directly the hypothesis that an ion access channel is formed by amino acids in the M5-M6 region of the Na,K- ATPase. To accomplish this goal, experiments will determine the VM dependence, apparent ion affinity, and kinetics of extracellular Na+- and K+-dependent transport steps by the Na,K-ATPase. These experiments will measure ion transport as steady state Na pump current and as presteady state transient charge movements in voltage-clamped HeLa cells transfected with cDNA coding for Na,K- ATPase variants produced after amino acid substitutions to residues located in the M5-M6 region of the enzyme's alpha-subunit. Experiments will also probe the mechanism of VM-dependent ion binding reactions and structure of the extracellular ion well by determining the ability of organic quaternary amines, previously shown to inhibit Na,K-ATPase activity, to alter the extracellular ion and VM dependence of Na pump currant in HeLa cells expressing these Na,K-ATPase variants. By combining molecular biological and electrophysiological techniques, this project will provide detailed structure/function information on VM-dependent ion transport by the Na,K-ATPase, thereby improving our understanding of how this enzyme regulates the ion gradients critical to cell function.
