VOLTAGE-CLAMP STUDIES OF SODIUM PUMP CURRENT AND FLUX

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The objective of this project is to investigate the stoichiometry and voltage dependence of the Na+/K+ 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 (DHDTG) or of the irreversible toxin ouabain. A specially designed, stable, low-noise voltage-clamp circuit has been designed and tested that is capable of resolving the electrogenic Na+/K+ pump current. The magnitude of this current is on the order of 1 MuA/cm.2. Using the technique of internal dialysis, the magnitude of Na+ efflux and pump current will be measured as a function of external (K+), internal (Na+), (Mg++) and (ATP). Experiments will also be done to measure K+ influx and pump current. Preliminary results have been obtained that sugget that the pump stoichiometry may be 2Na+/1K+ or variable. Experiments will be done to determine if the coupling ratio is constant or variable and if the coupling ratio or electrogenic pump current is dependent on membrane potential. Preliminary experiments have also shown that it is possible to reverse the direction of the pump current by steepening the electrochemical gradients for Na+ and K+. Experiments will be done to examine the characteristics of the pump when it is operating in the reverse mode.