Na super(+)-dependent Inactivation of the Retinal Cone/Brain Na super(+)/Ca super(2+)-K super(+) Exchanger NCKX2

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