Ca2+ influx into bovine retinal rod outer segments mediated by Na+/Ca2+/K+ exchange

Abstract

Ca(2+)-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 Ca(2+)-influx measurements. Ca2+ influx was measured as a rise in cytosolic free Ca2+ with the Ca(2+)-indicating dye fluo 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 Na+ 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 microM when no external Na+ was present and 3.5 microM 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.

Na+-dependent Inactivation of the Retinal Cone/Brain Na+/Ca2+-K+ Exchanger NCKX2

“…In the presence of gramicidin and high Na ϩ , NCKX2 was able to drive intracellular free Ca 2ϩ to high levels of 15-20 M, necessitating the use of the low affinity Ca 2ϩ indicator fluo-4FF to avoid saturation of the dye. The dynamic Ca 2ϩ fluxes observed here for HEK293 cells expressing NCKX2 are not a consequence of overexpression of the NCKX2 protein in this system, as they are similar to those observed in situ for Ca 2ϩ fluxes and NCKX currents in the outer segments of retinal rod (28,36) and cone (12) , and 1 mM EDTA was subsequently added at the second arrow 150 s following the addition of NaCl. Note that, although wild-type NCKX2-transfected HEK293 cells were able to rapidly clear the cytosolic load of Ca 2ϩ , both D548E-and N572C-transfected HEK293 cells had a diminished rate of Ca 2ϩ clearance, signifying inactivation of these exchangers at lower Na ϩ concentration.…”

“…Therefore, we set out to modify our assay to allow us to verify that the reverse exchange mode of NCKX is indeed dependent on internal Na ϩ . To this end, we employed the cation channelforming ionophore gramicidin to clamp monovalent cation concentrations across the plasma membrane and to allow us to control internal Na ϩ concentrations (27,28), thereby circumventing the variability in assaying reverse exchange in intact cells.…”

Electrophysiological Characterization and Ionic Stoichiometry of the Rat Brain K+-dependent Na+/Ca2+ Exchanger, NCKX2

“…Consequently, one would expect that the externally oriented Na ϩ -binding sites of NCKX2 would be saturated under physiological conditions, hence driving the Ca 2ϩ efflux, or forward, direction of the rat brain exchanger. K ϩ has been shown to compete for Na ϩ binding to the retinal rod exchanger (32,37), which would be expected to reduce the rate of Ca 2ϩ efflux. Since [K ϩ ] is thought to vary over a broad range at the extracellular surface of actively firing neurons (33), one might suggest that this competitive interaction serves an important regulatory influence on NCKX2 function.…”

Minimal Domain Requirement for Cation Transport by the Potassium-dependent Na/Ca-K Exchanger

“…Sucrose used in the above media was added from a 600 mM stock solution that was passed over a mixed bed ion exchanger to eliminate cation contamination. Experiments on isolated bovine retinal rod outer segments were carried out on calcium-depleted rod outer segments with the sodium loading protocol and media described above; other details concerning such experiments on rod outer segments were as described previously (13,14).…”