title: Na+-dependent inactivation of the retinal cone/brain Na+/Ca2+-K+ exchanger NCKX2.
abstract: 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.
authors.affiliation: Department of Physiology and Biophysics, Faculty of Medicine, Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
authors.forename: Haider F
authors.initials: HF
authors.lastname: Altimimi
authors.forename: Paul P M
authors.initials: PP
authors.lastname: Schnetkamp
mesh_terms: D001667:Binding, Competitive / Q000502:physiology; D001921:Brain / Q000378:metabolism*; D002414:Cations, Monovalent; D002460:Cell Line; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology; D011485:Protein Binding / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D017949:Retinal Cone Photoreceptor Cells / Q000378:metabolism*; D012964:Sodium / Q000502:physiology*; D019831:Sodium-Calcium Exchanger / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: D002414:Cations, Monovalent; D019831:Sodium-Calcium Exchanger; C074530:potassium-dependent sodium-calcium exchanger; D012964:Sodium