CA2+ INFLUX INTO BOVINE RETINAL ROD OUTER SEGMENTS MEDIATED BY NA+ CA2+/K+ EXCHANGE/

Ca2+-depleted rod outer segments (ROS) were purified from bovine retin al rod photoreceptors, and factors influencing Ca2+ influx into ROS vi a the plasma membrane Na+/Ca2+/K+ exchanger were analyzed. Intracellul ar alkali cation concentrations were manipulated by 1) previous loadin g via the ionophore monensin followed by removal of monensin and 2) ad dition of the channel ionophore gramicidin during Ca2+-influx measurem ents. Ca2+ influx was measured as a rise in cytosolic free Ca2+ with t he Ca2+-indicating dye flue 3. An absolute requirement for intracellul ar Na+ was observed with a Na+ dissociation constant of 30-40 mM, wher eas intracellular K+ was a potent inhibitor of Ca2+ influx, apparently by competing with Naf for a common site on the Na+/Ca2+/K+ exchanger. Half-maximal Ca2+ influx was observed at an external free Ca2+ concen tration of 0.9 mu M when no external Na+ was present and 3.5 mu M when 10 mM external Na+ was present. Our observations are discussed in the context of 1) a three-site model for the Na+/Ca2+/K+ exchanger and 2) earlier experiments on light adaptation in rods, which depended on mi nimizing Ca2+ fluxes across the ROS plasma membrane.