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The Journal of General Physiology, Vol 86, 457-478, Copyright © 1985 by The Rockefeller University Press
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R Dipolo, F Bezanilla, C Caputo and H Rojas
A combination of the voltage-clamp and the intracellular dialysis techniques has been used to study the membrane potential dependence of the Nao-dependent Ca efflux in squid giant axons. In order to improve axon survival, experiments were carried out using internal solutions prepared with large impermeant organic anions and cations, which did not affect the operation of the Na/Ca exchange mechanism. In axons dialyzed with solutions prepared without internal Na, the Nao-dependent Ca efflux had a small sensitivity to membrane potential changes. For a 25-mV membrane displacement in the hyperpolarizing direction, the basal Ca efflux increased by only 7.4% (n = 13). When the dialysis medium contained Na (from 20 to 55 mM), the efflux increased 32.3% (n = 25) for the same membrane potential change. The K1/2 for this effect is approximately 5 mM Na, and saturation appears to occur at a Na concentration above 20 mM. Adding ATP to the dialysis medium increased the magnitude of the Nao-dependent Ca efflux without changing its voltage sensitivity. Wide changes in the intracellular ionized Ca concentration (from 0.1 to 230 microM) did not modify the voltage sensitivity of the exchange system. Elimination of the reversal of Na/Ca exchange (Nai-dependent Ca influx) by removing Cao did not modify the voltage sensitivity of the Nao-dependent Ca efflux. When the axon membrane potential was submitted to prolonged changes, the corresponding changes in the Ca efflux were not sustained, but declined exponentially to intermediate values. This effect may indicate a slow inactivation process in the Na/Ca exchange mechanism. Voltage-clamp pulse experiments revealed: (a) the absence of a fast inactivation process in the Na/Ca exchange, and (b) that the activation of the carrier for hyperpolarizing pulses occurs as rapidly as 1 ms.
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