VOLTAGE-CLAMP STUDIES OF SODIUM PUMP CURRENT AND FLUX

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The objective of this project is to obtain a greater understanding of the voltage dependent step(s) in the reaction cycle of the Na/K pump. Four specific aims are: 1) measure steady-state Na/K pump current-voltage relationships (I-Vs) for various electrogenic modes of pump operation, 2) test whether the stoichiometry of the Na/K pump is fixed at 3Na/2K and is voltage-independent over wide-ranging conditions, 3) investigate transient Na/K pump currents (pump charge movement), and 4) test whether pump- mediated Na/Na exchange, though electroneutral, is voltage-dependent. To achieve these aims, simultaneous measurements of changes in pump-mediated current and flux, produced by exposure to the reversible pump inhibitor dihydrodigitoxigenin (H2DTG), will be made in voltage-clamped, internally- dialyzed squid giant axons. Control experiments have already demonstrated that: 1) H2DTG has no direct effect on ionic conductances; 2) current and 22Na efflux derive from the same membrane area; 3) changes in passive K current, due to changes of extracellular [K] on stopping the pump, are largely prevented by K channel-blocking agents; 4) pump-mediated Na/Na exchange has been minimized by adding high [ATP] and the phosphagens phosphoenolpyruvate and phosphoarginine, to the dialysis fluid. Steady- state Na/K pump I-Vs for the forward and reverse modes of pump operation will be obtained under various conditions as the difference between membrane I-Vs measured before and during exposure to H2DTG. The stoichiometry of the Na/K pump can be investigated since appropriate control experiments have shown that H2DTG-sensitive flux and current can be equated with Na/K pump flux and current, and thus used to calculate the ratio of Na to K transport, provided the pump transports no other ion. Preliminary measurements of transient pump currents have been made in voltage-clamped Xenopus oocytes in the absence of external K, which presumably limits the pump to Na/Na exchange. A prediction from such measurements, that pump-mediated electroneutral Na/Na exchange is voltage- dependent, can be tested in squid axons by assessing the voltage dependence of H2DTG-sensitive Na efflux from high-[ADP] axons into K-free seawater. Further understanding of the Na/K pump of animal cells is important since it maintains the Na electrochemical gradient which is essential for excitability and for Na-coupled transport processes.