The long-term objective of this project is to obtain an understanding of the mechanism of ion translocation by the electrogenic Na+/K+ pump at the molecular and physical-chemical level. The Na+/K+ pump is present in the surface membrane of nearly all animal cells, where it provides the predominant link between the cell's metabolic production of ATP and the maintenance of the Na+ ion and K+ ion electrochemical gradients. These gradients, in turn, underlie membrane potentials and electrical activity of both non-excitable and excitable cells and numerous Na+-coupled co- and counter-transport processes. The objective will be achieved by making measurements of steady-state and transient currents of radioisotopic unidirectional fluxes mediated by the Na+/K+ pump in its various models, and measurements of acid-induced, ATP- dependent, Na+/K+ pump-mediated (ouabain-sensitive) single-channel currents with six different voltage clamp techniques: 1) two- microelectrode voltage clamp of intact Xenopus oocytes, 2) cut-open oocytes, 3) patch clamp of oocytes, 4)reconstitution of oocyte vesicles in lipid bilayers, 5) simultaneous current and flux measurements in internally-dialyzed giant axons from squid, and 6) ultra-fast voltage clamp of pre-steady-state transient currents in internally dialyzed squid giant axons. The experimental plan is to exploit the strengths of each of these techniques: the intact oocyte for its stability, wide accessible voltage range, ease of external solution change, and convenience as a heterologous expression system; the cut-open oocyte for its high-speed voltage control and ease of cytoplasmic solution change; the patch clamp and bilayer techniques for their ability to resolve single channel current, and the squid axon for the ability to control both internal and external solutions, and to simultaneously measure radiotracer flux and voltage-clamp current, as well as the ability to produce ultra-fast voltage steps. The two specific aims of the project are: 1) to investigate the charge translocating steps in the reaction cycle of the Na+/K+ pump by measurements of pre-steady transient currents in Xenopus oocytes and squid giant axons to test the hypothesis that the transient currents reflect the sequential deocclusion and release of 3 Na+ ions to the extracellular side of the enzyme, and 2) to investigate the properties of acid-induced, ouabain-sensitive steady- state and single-channel current in oocyte membranes in K+-free solutions to test the hypothesis that a titrable amino acid is necessary for the maintenance of the Na+-occluded state of the Na+/K+ pump.
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