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Asymmetrical properties of the Na-Ca exchanger in voltage-clamped, internally dialyzed squid axons under symmetrical
1Instituto Venezolano de Investigaciones Científicas, Centro de Biofísica y Bioquímica, Caracas, Venezuela.
Abstract:
In this work we have investigated whether the asymmetrical properties of the Na/Ca exchange process found in intact preparations are intrinsic to the exchange protein(s) or the result of the asymmetric ionic environment normally prevailing in living cells. The activation of the Na/Ca exchanger by Ca2+ ions, monovalent cations, ATP gamma S and the effect of membrane potential on the different operational modes of the exchanger (Nao/Cai, Cao/Nai, Cao/Cai, and Nao/Nai) was studied in voltage-clamped squid giant axons externally perfused and internally dialyzed with symmetrical ionic solutions. Under these conditions: (a) Ca ions activate with higher affinity from the inside (K1/2 = 22 microM) than from the outside (K1/2 = 300 microM); (b) experiments measuring the Cao-dependent Ca efflux in the conditions Lio-Trisi, Lio-Lii, Triso-Trisi, and Triso-Lii, show that the activating monovalent cation site on the exchanger faces the external surface; (c) ATP gamma S activates the Cao-dependent Ca efflux (Cao/Cai exchange) only at nonsaturating [Ca2+]i. Its effect appears to be on the Ca transport site since no alteration in the apparent affinity of the activating monovalent cation site was observed. The above results show that the Na/Ca exchange process is indeed a highly asymmetric transport mechanism. Finally, the voltage dependence of the components of the different exchange modes was measured over the range of +20 to -40 mV. The voltage dependence (approximately 26% change/25 mV) was found to be similar for all modes of operation of the exchanger except Nao/Nai exchange, which was found to be voltage insensitive. The sensitivity of the Cao/Cai exchange to voltage was found to be the same in the presence and in the complete absence of monovalent cations. This finding does not support the proposition that the voltage sensitivity of the Cao/Cao exchange is induced by the binding and transport of an external monovalent cation.