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Centro de Biofísica y Bioquímica, IVIC, Caracas, Venezuela.

1. Na(+)o-dependent Ca2+ efflux (forward Na(+)-Ca2+ exchange), and in some cases the Na(+)i-dependent Ca2+ influx (reverse Na(+)-Ca2+ exchange) were measured in internally dialysed squid axons under membrane potential control. 2. We tested the effect on the Na(+)-Ca2+ exchange of the MgATP analogue bidentate chromium adenosine-5'-triphosphate (CrATP), substrate of several kinases, and cobalt tetrammine ATP (Co(NH3)4ATP), a poor substrate of most kinases. 3. CrATP completely blocked the MgATP and MgATP-gamma-S (ATP-gamma-S) stimulation of the Na(+)o-dependent Ca2+ efflux (forward exchange) and the Na+i-dependent Ca2+ influx (reverse exchange). The analogue only blocked the nucleotide-dependent fraction of the Na(+)-Ca2+ exchange without modifying any kinetic parameters of the exchange reactions. 4. The effects of CrATP were fully reversible with a very slow time constant (t 1/2 about 30 min). 5. The MgATP stimulation of the Na(+)-Ca2+ exchange was completely saturated at 1 mM. Higher MgATP concentrations (up to 15 mM) had no additional effects. Pentalysine (internal or external), the protein kinase C inhibitor H-7 (1-(5-isoquinolinylsulphonyl)-2-methylpiperazine) and several calmodulin inhibitors did not inhibit Na(+)-Ca2+ exchange either in the absence or presence of MgATP. 6. Our results do not agree with the idea of an aminophospholipid translocase being responsible for the ATP stimulation of the Na(+)-Ca2+ exchange in squid axons; they suggest that this is due to the action of a kinase system.

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