The objective of this project is to investigate the stoichiometry and voltage dependence of the sodium pump of squid giant axons using simultaneous measurements of isotopic 22Na efflux or 42K influx and electrogenic pump current. The electrogenic pump current is measured by the change in holding current that occurs upon addition of the reversible pump toxin dihydrodigitoxigenin (H2DTG) or of the irreversible (in squid) toxin ouabain. The toxin-sensitive change in holding current is measured using a stable, low-noise voltage-clamp apparatus. The composition of the intracellular axoplasm is controlled by the technique of internal dialysis. This method also permits the use of radioactive tracers for the measurement of both influx and efflux. By steepening the normal ionic gradients for Na+ and K+, the pump can be made to run backwards. Experiments will be done to determine the effect of changes in nucleotide and ionic composition on the magnitude of the pump current and the stoichiometry of the pump cycle. Experiments have been done that demonstrate that the backward-running sodium pump produces an electrical current in the reverse direction. The magnitude of the reverse pump current is decreased by hyperpolarization corresponding to a region of negative slope conductance in the current-voltage relationship of the reverse pump. Experiments have also shown that both the forward pump current and flux are inhibited by hyperpolarization. These results suggest that membrane voltage may be an important factor in the regulation of pump activity. The validity of the pump-clamp technique has been verified by the demonstration that H2DTG and ouabain have no effect on passive membrane conductance and that measurements of Na+-channel or K+-channel mediated flux and current are in agreement under experimental conditions that allow only a unidirectional ion flow. Future experiments will focus on the determination of the current-voltage relationship of both the forward and reverse modes of pump operation and the determination of whether the stoichiometry of the pump is fixed or variable. These studies have mechanistic implications for the operation of the sodium pump which is important in the regulation of cell volume and in establishing and maintaining the electrochemical gradient for sodium-coupled transport processes in a wide variety of animal and plant cells.
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