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

Ca2+-depleted rod outer segments (ROS) were purified from bovine retinal rod photoreceptors, and factors influencing Ca2+ influx into ROS via the plasma membrane Na+/Ca2+/K+ exchanger were analyzed. Intracellular alkali cation concentrations were manipulated by 1) previous loading via the ionophore monensin followed by removal of monensin and 2) addition of the channel ionophore gramicidin during Ca2+-influx measurements. Ca2+ influx was measured as a rise in cytosolic free Ca2+ with the Ca2+-indicating dye flue 3. An absolute requirement for intracellular Na+ was observed with a Na+ dissociation constant of 30-40 mM, whereas 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+ concentration 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 minimizing Ca2+ fluxes across the ROS plasma membrane.