Na+-dependent inactivation of the retinal cone/brain Na+/Ca2+-K+ exchanger NCKX2

The SLC24 gene family Na+/Ca2+-K+ exchangers (NCKX) are bidirectional plasma membrane transporters whose main function is the extrusion of Ca2+ from the cytosol. In this study, we used human embryonic kidney 293 cells expressing human retinal cone/brain NCKX2 to examine its Na+ affinity and kinetic parameters of Ca2+ transport. With the use of the ionophore gramicidin to control alkali cation concentrations across the plasma membrane, application of high intracellular Na+ promoted large NCKX2-mediated increases in intracellular free Ca2+ in the 15-20 microm range; this also resulted in inactivation of NCKX2 transport, the first description of this novel kinetic state. The affinity of NCKX2 for internal Na+ was found to be sigmoidal, with a Hill coefficient of 2.6 and Kd = 50 mm. The time-dependent (t(1/2) approximately 40s) inactivation of NCKX2 required high intracellular Na+ levels (Kd > 50 mm) as well as high occupancy of the extracellular Ca2+-binding site. Also reported are two residues whose substitution resulted in an increase in internal Na+ affinity to values of approximately 19 mm; these mutants also displayed enhanced inactivation, suggesting that inactivation requires binding of Na+ to its intracellular transport sites. These findings are the first report of a regulatory kinetic state of Ca2+ transport via NCKX2 Na+/Ca2+-K+ exchangers that may play a prominent role in regulation of Ca2+ extrusion in cellular environments such as neuronal synapses that experience frequent and dynamic Ca2+ fluxes.