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

Abstract

The SLC24 gene family Na ؉ /Ca 2؉ -K ؉ exchangers (NCKX) are bidirectional plasma membrane transporters whose main function is the extrusion of Ca 2؉ 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 Ca 2؉ 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 Ca 2؉ in the 15-20 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 ؉ was found to be sigmoidal, with a Hill coefficient of 2.6 and K d ‫؍‬ 50 mM. The time-dependent (t1 ⁄ 2 ϳ 40s) inactivation of NCKX2 required high intracellular Na ؉ levels (K d > 50 mM) as well as high occupancy of the extracellular Ca 2؉ -binding site. Also reported are two residues whose substitution resulted in an increase in internal Na ؉ affinity to values of ϳ19 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 Ca 2؉ transport via NCKX2 Na ؉ /Ca 2؉ -K ؉ exchangers that may play a prominent role in regulation of Ca 2؉ extrusion in cellular environments such as neuronal synapses that experience frequent and dynamic Ca 2؉ fluxes.

Residues Contributing to the Na+-binding Pocket of the SLC24 Na+/Ca2+-K+ Exchanger NCKX2

“…This particular mutant was of interest to us because an Ile to Thr mutation was found in the equivalent position of the retinal rod NCKX1 gene of two unrelated retinal disease patients (33). Some substitution mutants produced strong shifts to higher Na i ϩ affinity, a phenotype we reported on previously for D548E and N572C (31). In all of these cases, the normalized rate values increased steeply in the range of [Na ϩ ] i 10 -75 mM and declined Table S1).…”

“…NCKX is especially interesting in this regard because it is characterized, alongside NCX, by the capacity to transport multiple Na ϩ ions/cycle; moreover, both NCKX and NCX are unique in their absolute selectivity for Na ϩ over any other alkali cation (28 -30). To date, however, the residues involved in Na ϩ transport in NCKX are unknown, except for our previous identification of Asp 548 as a residue whose chargeconservative substitution dramatically increased Na i ϩ affinity; alongside Asp 548 , we also identified substitution of Asn 572 with Cys to affect Na i ϩ affinity (31). Herein, we extend our mutational analysis to 88 single-residue substitutions covering the ␣ repeats and all 14 additional acidic residues of NCKX2 found in the two sets of five TMs.…”

“…This assay was subsequently modified by using gramicidin to control internal [Na ϩ ] to obtain a measure of the affinity of wt NCKX2 for [Na ϩ ] i (31). We initially used fluo-3 measurements of changes in [Ca 2ϩ ] i induced by the addition of a limited set of [Na ϩ ] in the presence of gramicidin to scan through 102 single-residue substitutions comprising the ␣ 1 and ␣ 2 regions, as well as all other acidic residues present in the two sets of five TMs and their short connecting loops ( Fig.…”

Ion Transporter NKCC1, Modulator of Neurogenesis in Murine Olfactory Neurons

“…Focusing on ion-related proteins, we recognized two Slc-transporters, the urea transporter Slc14a2 (55) and the sodium/potassium/calcium Exchanger 2 (Slc24a2) (56) that had reduced expression in NKCC1-deficient mice. The Slc24a2 transporter functions as a calcium extruder in neurons (57). Therefore, its down-regulation impairs calcium transport and could negatively influence the excitability of the NKCC1-deficient neurons.…”

Na+/Ca2+-K+Exchangers (NCKX):Functional Properties and Physiological Roles

“…Most studies on NCX and NCKX proteins employ three different methods to assess function: 45 . In addition, a few studies have used atomic absorption measurements 3,23,26 and metalochromic dyes (arsenazo III). 2,3 The external Na + dependence of forward exchange has been measured in rod outer segments (NCKX1) or in cell lines expressing NCKX1 or NCKX2 cDNAs; The data reveal a sigmoidal dependence on the external [Na + ] with reported Hill coefficients between 2 and 3 and apparent Na + dissociation constants of 30-50 mM.…”