VOLTAGE CLAMP STUDIES OF SODIUM PUMP CURRENT AND FLUX

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The long-term objective of this project is to obtain a greater understanding of the transport mechanism of the Na/K pump by identifying and characterizing the charge translocating, and hence voltage dependent, steps in the Na/K pump reaction cycle. The Na/K pump is present in the surface membrane of nearly all animal cells, where it provides the link between the cell's production of ATP and the maintenance of the Na 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 Na/K pump is also the receptor for a clinically important class of drugs, cardiotonic steroids, used in the treatment of heart failure and certain cardiac arrhythmias. The objective will be achieved by making measurements of steady-state and transient currents, and of radioisotopically-labeled unidirectional fluxes mediated by the Na/K pump in its various modes, in three voltage clamped preparations: intact Xenopus oocytes, cut-open oocytes, and internally-dialyzed, giant axons from squid. The experimental plan is to exploit the strengths of each of these preparations: 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 squid axon for the ability to control both internal and external solutions, and to simultaneously measure radiotracer flux and voltage-clamp current. The four specific aims of the project are: l) to examine the external [Na] and [K] dependence of the steady-state pump current-voltage (I-V) relationship in intact oocytes and in squid axons, 2) to examine the internal [Na] and [K] dependence of the steady-state pump I-V relationship in cut-open oocytes and in squid axons, 3) to examine the internal and external ion dependence of 22Na and 42K or 86Rb efflux mediated by the Na/K pump in its various transport modes in internally-dialyzed, voltage clamped squid giant axons, and 4) to measure and analyze voltage-jump-induced, pre-steady-state, transient currents under Na/Na exchange conditions using the cut-open oocyte voltage-clamp technique.