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Originally published In Press as doi:10.1074/jbc.M609285200 on December 12, 2006

J. Biol. Chem., Vol. 282, Issue 6, 3720-3729, February 9, 2007
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Na+-dependent Inactivation of the Retinal Cone/Brain Na+/Ca2+-K+ Exchanger NCKX2*

Haider F. Altimimi1 and Paul P. M. Schnetkamp, Scientist of the Alberta Heritage Foundation for Medical Research2

From the Department of Physiology and Biophysics, Faculty of Medicine, Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta T2N 4N1, Canada

Received for publication, October 2, 2006 , and in revised form, December 8, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 µM 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 (t1/2 ~ 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 ~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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Among the multitude of proteins that handle Ca2+ fluxes across the plasma membrane of excitable tissue, Na+/Ca2+ exchangers play a prominent role in maintaining intracellular Ca2+ homeostasis. These proteins belong to one of two gene families: SLC8, the members of which mediate electrogenic exchange of three Na+ ions for one Ca2+ ion (1), and SLC24, the members of which mediate electrogenic exchange of four Na+ ions for one Ca2+ ion and one K+ ion (2). The SLC8 gene family is composed of three distinct proteins: the Na+/Ca2+ exchanger (NCX)3 NCX1, which has widespread tissue distribution in animals, with strong expression in the heart and spleen; and NCX2 and NCX3, which are restricted to the brain and skeletal muscle (3–5). On the other hand, the SLC24 gene family is composed of five members. The Na+/Ca2+-K+ exchanger (NCKX) NCKX1 was first reported in retinal rod outer segments and is the isoform on which most extensive physiological studies have been performed (6–8). NCKX2 transcripts have been reported in the brain and retina (9–12), and recent emergent evidence indicates that this isoform is important for regulation of Ca2+ levels in synaptic terminals (13–15). NCKX3 and NCKX4 transcripts are also found in the brain, but have also been localized to smooth muscle of the aorta, uterus, and intestine (16, 17). Recently, NCKX5 transcripts have been reported to be rich in melanin-containing tissues, viz. the eye and skin (18).

Most of the available studies on NCKX structure have been carried out on NCKX2. The NCKX proteins studied to date share a common predicted structure of two sets of five and six hydrophobic domains; the current topological model suggests an arrangement of five and five transmembrane segments separated by a large cytosolic loop (19). They also have a cleaved signal peptide that is essential for plasma membrane targeting, leaving a large extracellular loop at the N terminus (the C-terminal loop is also extracellular) (20). NCKX and NCX proteins share sequence homology in only two transmembrane regions forming internal repeats termed the {alpha}1 and {alpha}2 repeats. Residues within the {alpha}1 and {alpha}2 repeats of human NCKX2 have been shown to be important for the transport function of NCKX2 and for its affinity for both Ca2+ and K+ (21–23); in addition, these transmembrane segments are in close proximity in three-dimensional space (24).

NCKX-mediated exchange of Ca2+ is characterized by a unique requirement for Na+ over any other alkali cation (25, 26). However, to date, the residues responsible for Na+ binding and transport are unknown. Hence, we sought to devise an assay that will allow us to control internal Na+ to measure the affinity of the exchanger for Na+. Additionally, although all Na+/Ca2+ exchangers characterized to date are capable of mediating bidirectional transport of Ca2+ across the plasma membrane, most methods that have been devised to measure Na+/Ca2+ exchange in intact cells rely on measuring the reverse (Ca2+ influx) mode of exchange. For this study, we sought to examine the biophysical properties of the forward (Ca2+ efflux) mode of exchange of human NCKX2, which is the mode of transport that is of physiological relevance. Finally, we set out to evaluate the contributions of other cellular Ca2+-handling mechanisms in our assay system. From the results we obtained, we discovered that NCKX2 is a highly efficient Ca2+ extrusion mechanism, but displays a previously unknown inactive kinetic state that is both time- and Na+-dependent.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
The experimental procedures have been described in detail elsewhere (22, 27), and only modifications are included herein. All chemical reagents used were from Sigma unless specified otherwise.

Assaying for NCKX Function—Human embryonic kidney 293 (HEK293) cells transiently expressing the short splice variant of Myc-tagged NCKX2 (10, 21) were trypsinized, resuspended in Dulbecco's modified Eagle's medium (Invitrogen, Burlington, Ontario, Canada) supplemented with 12 µM fluo-3 acetoxymethyl ester or fluo-4FF acetoxymethyl ester (Molecular Probes, Burlington), and incubated at room temperature for 20 min. The cells were then pelleted by centrifugation at 300 x g for 1 min and washed with a medium of buffered 150 mM LiCl (150 mM LiCl, 0.1 mM EDTA, 20 mM HEPES, 0.25 mM sulfinpyrazone (to reduce dye leakage), and 6 mM D-glucose, pH 7.4). The cells were resuspended in buffered 150 mM LiCl, and 50-µl aliquots (containing ~105 cells) were added separately to cuvettes containing 1950 µl of test medium. The test medium was buffered 150 mM NaCl (150 mM NaCl, 0.1 mM EDTA, 20 mM HEPES, 0.25 mM sulfinpyrazone, and 6 mM D-glucose, pH 7.4) or buffered 150 mM KCl (150 mM KCl, 0.1 mM EDTA, 20 mM HEPES, 0.25 mM sulfinpyrazone, 6 mM D-glucose, and 0.5 mM dithiothreitol, pH 7.4). (The same stocks of test media were used to prepare mixed test media of different ionic compositions, i.e. 75 mM LiCl and 75 mM KCl was prepared by adding equal amounts of buffered 150 mM LiCl and buffered 150 mM KCl.) For the experiments carried out in buffered 150 mM KCl, pH 8.8 (see Fig. 2A), TAPS was used in place of HEPES. For each experiment, the cuvette containing Ca2+ indicator dye-loaded NCKX2-transfected HEK293 cells was placed in the 25 °C thermostatted cuvette housing of an SLM Series 2 luminometer (SLM Instruments, Urbana, IL), and fluorescence was continuously measured under constant stirring and upon the addition of various ion concentrations as described below. To clamp the alkali cation concentrations across the plasma membrane, the channel-forming alkali cation ionophore gramicidin (2 µM) was added to the cuvette 3 min prior to the start of the experiment; other drug treatments (as described below) were also added at the same time as gramicidin. The first addition thereafter was 350 µM CaCl2, which caused an instantaneous step increase in fluorescence due to Ca2+ binding to leaked Ca2+ indicator dye; this level of fluorescence remained constant throughout the duration of experimentation and was also observed in mock-transfected Ca2+ indicator dye-loaded HEK293 cells (data not shown) and hence was subtracted during analysis of fluorescence signals. On the other hand, in experiments in which EDTA was added to the cuvette to chelate external Ca2+, an instantaneous step decrease in fluorescence occurred, which signified chelation of Ca2+ from leaked Ca2+ indicator dye in the cuvette; in those cases, the value of the step increase in fluorescence at the beginning of the experimental recording period was added to the fluorescence values at the point at which EDTA was introduced in the cuvette. At the end of each experiment, 0.02% saponin was added in the presence of saturating CaCl2 to saturate the Ca2+ indicator dye and to obtain a maximal fluorescence signal, which was used to normalize the fluorescence values for the respective experiment. In the experiments carried out with 10 µM external Ca2+, the Ca2+ was buffered by the addition of 1 mM HEDTA and 8 mM Ca2+/HEDTA to the cuvette; to chelate external Ca2+ in this case, 5 mM EDTA was added to the cuvette.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Assaying for NCKX2 Reverse Exchange and Dependence on Internal Na+—Typical reverse exchange (Ca2+ influx) assays in whole cells rely on bathing the cells in a medium free of Na+ while monitoring intracellular Ca2+ increases. During the course of our experimentation, we noticed that the activity of NCKX, as assessed by the magnitude of induced Ca2+ influx, decayed with time as NCKX-transfected cells were placed in Na+-free medium. Along with the observation that the Ca2+ influx responses of NCKX-transfected HEK293 cells originating from different stocks were quite variable, this indicated that the discrepancies were likely due to variations in cytosolic Na+. 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+. To this end, we employed the cation channel-forming ionophore gramicidin to clamp monovalent cation concentrations across the plasma membrane and to allow us to control internal Na+ concentrations (27, 28), thereby circumventing the variability in assaying reverse exchange in intact cells.

NCKX2-transfected HEK293 cells were loaded with the cell-permeant form of the Ca2+ indicator fluorescent dye fluo-3 and resuspended in buffered 150 mM LiCl. The cells were then placed in a cuvette containing buffered 150 mM KCl and treated with 2 µM gramicidin, and 350 µM CaCl2 was added to the cuvette 3 min later, which caused a step increase in fluorescence that remained constant (due to leaked dye; see "Experimental Procedures"). (Also note that all cuvette media contained 0.1 mM EDTA, and hence, the addition of 350 µM CaCl2 gave a final free Ca2+ concentration in the cuvette of 250 µM.) After 30 s, 75 mM NaCl was added to the cuvette, which caused a rapid increase in fluorescence (internal free Ca2+) that reached a plateau level within 10 s (Fig. 1A). The same aforementioned procedure was repeated, but in a cuvette with buffered 150 mM NaCl in place of buffered 150 mM KCl. In this case, the addition of KCl (40 mM) was required to initiate reverse exchange and to elevate intracellular Ca2+ (Fig. 1B), consistent with the established absolute requirement of NCKX for K+. NCKX2-transfected HEK293 cells were placed in a cuvette with buffered 150 mM LiCl and treated with gramicidin; after the addition of 350 µM CaCl2, 40 mM KCl was added to the cuvette, but the fluorescence remained at the base line until the addition of 75 mM NaCl (Fig. 1C), at which point the fluorescence rose rapidly to around the same plateau level observed in Fig. 1 (A and B). Finally, to illustrate that the exchanger is absolutely dependent on Na+, NCKX2-transfected HEK293 cells were placed in a cuvette with buffered 75 mM LiCl and 75 mM KCl, and treated with 2 µM gramicidin. The addition of 350 µM CaCl2 caused only a small step increase in fluorescence (data not shown), which remained constant for >1 min, and the fluorescence was finally elevated by the addition of 75 mM NaCl (Fig. 1D). Therefore, using this modified protocol, we were able to consistently control Na+ concentrations in the medium and thereby assess the affinity of the exchanger for Na+. In all subsequent experiments, NCKX2-transfected HEK293 cells were resuspended in buffered 150 mM LiCl, placed in cuvettes with 150 mM KCl, and treated with 2 µM gramicidin (unless specified otherwise). Reverse exchange was then initiated by the addition of NaCl subsequent to the addition of 350 µM CaCl2; this concentration of Ca2+ was chosen to promote maximal Ca2+ influx through the exchanger while minimizing influx due to capacitative Ca2+ entry following internal Ca2+ store depletion (discussed below). Activation of capacitative Ca2+ entry in HEK293 cells typically requires the addition of millimolar concentrations of Ca2+ (29, 30), consistent with our observations (data not shown). Another modification we made in subsequent experiments was related to the observation that fluorescence amplitude levels attained using the described gramicidin clamp method were significantly higher than those previously obtained with intact cells (22), suggesting that internal Ca2+ was elevated to a greater extent in the presence of the gramicidin clamp than in its absence. Hence, we tested two other intracellular Ca2+ indicators from Molecular Probes: fluo-4FF, with a reported Kd of 9.7 µM, and fluo-5N, with a Kd of 90 µM. On the basis of comparison of fluorescence increases among these various Ca2+ indicator dyes and transfection efficiencies we routinely obtained (70–80%), we estimated that the fluorescence value of 0.5 obtained with fluo-4FF (normalized to the total amount of dye present in all cells) corresponded to intracellular free Ca2+ in the range of 15–20 µM. Therefore, in all subsequent experiments, we employed the cell-permeant form of the Ca2+ indicator dye fluo-4FF, which gave the best signal resolution without saturation of the fluorescence increase signals.


Figure 1
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  		FIGURE 1.
Reverse exchange (Ca2+ import) mode of human NCKX2 and absolute requirement for internal Na+. A, fluo-3-loaded HEK293 cells transfected with human NCKX2 were placed in a cuvette with buffered 150 mM KCl (see "Experimental Procedures") and treated with the channel-forming alkali cation ionophore gramicidin (2 µM) 3 min before data collection. At the arrow, 75 mM NaCl was added (preceded by the addition of 350 µM CaCl2 30 s earlier for a final concentration of 250 µM external free Ca2+), and the fluorescence was continuously monitored. B, fluo-3-loaded NCKX2-transfected HEK293 cells were placed in a cuvette with buffered 150 mM NaCl and treated with 2 µM gramicidin. At the arrow, 40 mM KCl was added (in the presence of 250 µM external free Ca2+) to induce reverse exchange. C, fluo-3-loaded NCKX2-transfected HEK293 cells were placed in a cuvette with buffered 150 mM LiCl and treated with 2 µM gramicidin. At the first arrow, 40 mM KCl was added (in the presence of 250 µM external free Ca2+); note that base-line fluorescence level was not altered significantly. After the passage of another 30 s, 75 mM NaCl was added to the cuvette, which induced the rise in fluorescence from the cells. D, fluo-3-loaded NCKX2-transfected HEK293 cells were placed in buffered 75 mM LiCl and 75 mM KCl and treated with 2 µM gramicidin. At the arrow, 75 mM NaCl was added (in the presence of 250 µM external free Ca2+) to initiate reverse exchange; note that base-line fluorescence remained unaltered prior to the addition of NaCl.

 
Isolating the Activity of NCKX2 from Other Ca2+-handling Mechanisms—The addition of CaCl2 and Na+ to mock-transfected HEK293 cells in buffered 150 mM KCl did not result in an increase in internal Ca2+ (data not shown), indicating that HEK293 cells do not harbor endogenous Na+/Ca2+ exchange activity. However, like all eukaryotic cells, they nevertheless have other mechanisms for intracellular Ca2+ regulation, viz. the plasma membrane Ca2+-ATPase (PMCA), the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), and mitochondria. The contributions of these mechanisms to regulation of intracellular free Ca2+ in our assay system were examined. The PMCA activity was decreased by carrying out the assay in KCl medium with the pH adjusted to 8.8; SERCA was inhibited with 0.4 µM thapsigargin (Tg; Santa Cruz Biotechnology, Inc., Santa Cruz, CA); and mitochondrial Ca2+ buffering was disabled with the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) at 2 µM. All drugs were added to the cells in the cuvette at the beginning of the experiment along with gramicidin, i.e. 3 min prior to initiating reverse exchange to allow the Tg-induced rise in internal Ca2+ to return to the base line.

Application of 0.4 µM Tg to gramicidin-treated NCKX2-transfected HEK293 cells resulted in a slight increase in the amplitude of fluorescence attained after inducing Ca2+ influx via reverse exchange compared with NCKX2-transfected HEK293 cells treated with gramicidin alone (Fig. 2A). Increasing the pH of the assay medium resulted in greater enhancement of the reverse exchange amplitude of the fluorescence increase as well, but the most marked enhancement of reverse exchange activity was observed with cells treated with 2 µM FCCP. The amplitude of fluorescence attained with cells treated with FCCP reached a value of ~0.5 of maximal fluorescence compared with ~0.2 for untreated cells (Fig. 2A). These results suggest that all these mechanisms contribute to some extent to regulation of cytosolic Ca2+ in HEK293 cells, and therefore, to isolate the activity of the exchanger, we must take into account the contribution of those mechanisms. We decided to closely examine the interplay of Ca2+ handling among NCKX2, SERCA, and mitochondria, but decided against carrying out our assays in pH 8.8 media (to inhibit PMCA), as that would preclude the use of gramicidin because it would lead to strong alkalinization of the cytosol. Moreover, it was unclear whether the observed enhancement of the fluorescence increase at pH 8.8 was attributable to the decreased activity of PMCA per se because increased medium pH is also known to enhance the activity of the exchanger itself (31).


Figure 2
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  		FIGURE 2.
Contribution of other cellular Ca2+-handling mechanisms. A, fluo-4FF-loaded NCKX2-transfected HEK293 cells were placed in a cuvette with buffered 150 mM KCl and treated with 2 µM gramicidin, and 75 mM NaCl was added at the arrow (in the presence of 250 µM external free Ca2+; control (Ctrl)). In a second cuvette, NCKX2-transfected HEK293 cells from the same suspension were treated as described for the control with the addition of 0.4 µM Tg to inhibit the SERCA. In a third cuvette, NCKX2-transfected HEK293 cells from the same suspension were treated as described for the control with the addition of protonophore FCCP (2 µM) to prevent mitochondrial Ca2+ accumulation. In a fourth cuvette, NCKX2-transfected HEK293 cells from the same suspension were treated as described for the control, except that they were placed in a cuvette with 150 mM KCl adjusted to pH 8.8 to decrease PMCA activity. The traces shown are representative of results obtained in three other experiments. B, shown is a comparison of the final amplitudes of fluorescence attained by the addition of 75 mM NaCl to fluo-4FF-loaded NCKX2-transfected HEK293 cells placed in buffered 150 mM KCl with 250 µM external free CaCl2 and treated with 2 µM gramicidin with cells additionally treated with 0.4 µM Tg with or without 2 µM FCCP. Error bars represent S.D. *, significantly greater than control (p < 0.05, Student's t test, n = 3).

 
Fig. 2B compares the amplitude of the fluorescence increase in untreated NCKX2-transfected HEK293 cells with fluorescence amplitudes in NCKX2-transfected HEK293 cells pretreated with 0.4 µM Tg alone or with 2 µM FCCP. Although the fluorescence increase induced by the addition of 75 mM Na+ in Tg-treated cells was somewhat higher compared with that in untreated control cells, the results were not statistically significant (p > 0.05, Student's t test, n = 3) (Fig. 2B). This suggested that SERCA alone did not contribute significantly to the magnitude of Ca2+ signals induced in HEK293 cells under our experimental conditions. On the other hand, the fluorescence amplitude more than doubled when cells were pretreated with Tg and FCCP in combination (p < 0.05, Student's t test, n = 3), suggesting that, in HEK293 cells, mitochondria are the major buffer of large NCKX2-mediated rises in cytosolic Ca2+. Hence, to ensure in subsequent experiments that we were isolating the activity of the exchanger, all NCKX2-transfected HEK293 cells were pretreated with both 0.4 µM Tg and 2 µM FCCP prior to initiating reverse exchange (unless stated otherwise).


Figure 3
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  		FIGURE 3.
Measurement of Na+ affinity of NCKX2. A, fluo-4FF-loaded NCKX2-transfected HEK293 cells were placed in cuvettes containing buffered 150 mM KCl and treated with 2 µM gramicidin, 0.4 µM Tg, and 2 µM FCCP. To initiate Ca2+ influx via reverse exchange, the indicated millimolar concentrations of NaCl were added at time 0 to separate cuvettes (in the presence of 250 µM external free Ca2+). The traces shown are representative of results obtained in six other experiments. B, the initial rate of change in fluorescence attained after the addition of NaCl was used to derive a measure of the affinity of NCKX2 activity for internal Na+ by normalizing the initial rate of change in fluorescence at each Na+ concentration tested to the rate attained upon the addition of 150 mM NaCl. Data points represent averages, and error bars indicate S.D. (n = 3) fitted with a Hill function using SigmaPlot Version 7 software (Kd for Na+ = 50 ± 1.6 mM, Hill coefficient = 2.6).

 
Measuring the Affinity of NCKX2 for Internal Na+—To measure the affinity of NCKX2 for Na+, we normalized the initial rate of change in fluo-4FF fluorescence attained upon the addition of various concentrations of Na+ to the rate of fluorescence change obtained upon the addition of 150 mM NaCl (Vmax) in the presence of 250 µM external free Ca2+ (Fig. 3A). In our experiments, Na+ concentrations were simultaneously changed in both intracellular and external solutions, and this was inevitable because of the use of gramicidin. The addition of 250 µM external free Ca2+ proved to be sufficient to suppress competition of Ca2+ influx by external Na+ under our experimental conditions (data not shown). Fig. 3B shows the normalized rate of activity of NCKX2 in relation to the concentration of the substrate Na+. The Kd of NCKX2 for internal Na+ was 50 ± 1.6 mM (n = 3), and the fitted Hill plot gave a coefficient of 2.6, indicating that NCKX moves multiple Na+ ions in each transport cycle. This is the first reported value of the Kd of NCKX for intracellular Na+; it can be compared with values previously reported for the Kd for extracellular Na+ of human and chicken NCKX2 expressed in the insect cell line High Five (38–48 mM Na+, Hill coefficient of 1.9) (32), rat NCKX2 expressed in HEK293 cells (30 mM Na+, Hill coefficient of 2.8) (33), and in situ bovine rod NCKX1 (35 mM Na+, Hill coefficient of 2) (34, 35).

Forward Exchange (Ca2+ Extrusion) Mode of NCKX2—To examine the forward exchange (Ca2+ extrusion) mode of NCKX2 operation, we loaded NCKX2-transfected HEK293 cells with Ca2+ via the reverse exchange mode of operation of NCKX2 by the addition of 75 mM NaCl in the presence of 250 µM external free Ca2+, and after the fluorescence reached the steady-state plateau level, 1 mM EDTA was added to the cuvette to chelate external Ca2+, thereby reversing the Ca2+ gradient across the plasma membrane. The fluorescence levels dropped precipitously upon the addition of EDTA presumably because of extrusion of Ca2+ from the cytosol via NCKX2 (Fig. 4A, Ctrl trace). We repeated this assay with Tg in the absence and presence of FCCP. Whereas the pattern of fluorescence change in Tg-treated cells mirrored that in untreated control NCKX2-transfected HEK293 cells, those cells treated with both Tg and FCCP had a greatly diminished rate of Ca2+ clearance. This suggested that the rapid decrease in fluorescence observed in the untreated cells was not mediated by the exchanger, at least not entirely, but in fact, mitochondria in that case effectively and very rapidly sequestered cytosolic Ca2+. Hence, we proceeded to test the ability of NCKX2-transfected HEK293 cells pretreated with Tg and FCCP to extrude Ca2+ under a range of different Na+ concentrations and found that the rate of Ca2+ extrusion diminished with increasing Na+ concentrations (Fig. 4B). This finding was surprising, as we were expecting that Ca2+ extrusion rates would accelerate as the concentration of Na+ was increased, according to kinetic considerations. In the example shown in Fig. 4B, the addition of 75 and 150 mM Na+ promoted rapid Ca2+ influx through the reverse mode of operation of NCKX2; however, when the forward mode was engaged, the fluorescence decayed very slowly and did not reach the base line by the end of the data recording period (>2 min), signifying that the exchanger had somehow inactivated and was no longer able to effectively extrude internal Ca2+.

Time Course of Inactivation of NCKX2—To exclude the possibility that elevating intracellular Na+ in our assay resulted in the apparent inactivation of the forward mode of exchange because of competitive inhibitory interactions between Na+ and Ca2+-K+ at the intracellular binding/transport site of the exchanger, we examined the time course of inactivation of NCKX2. In Fig. 5A, NCKX2-transfected HEK293 cells were loaded with Ca2+ via the exchanger by the addition of 75 mM Na+ in the presence of 250 µM external free Ca2+. At various time points thereafter, 1 mM EDTA was introduced to chelate extracellular Ca2+ and to initiate Ca2+ efflux from the cytosol via the exchanger. Fig. 5 shows the results from separate cuvettes using the same suspension of NCKX2-transfected HEK293 cells overlaid for illustrative purposes. The rate of Ca2+ clearance diminished with time, and it was clear that inactivation of the exchanger developed with a relatively slow time course; we estimated the t1/2 of inactivation at 40 s. These results argue against competitive inhibition because the apparent inactivation process required significant time to develop and was not apparent when the cytosolic compartment was loaded with high free Ca2+ for periods of up to 40 s while still exposed to high levels of internal Na+ (75 mM). If competitive inhibition by Na+ was prominent, then the apparent inactivation process would have been expected to occur instantaneously upon the addition of EDTA. As a control, we examined the time course of the Ca2+ efflux component of NCKX2-transfected HEK293 cells loaded with 20 mM Na+ in the presence of 250 µM external free Ca2+; this concentration of Na+ was not sufficient to produce inactivation, as illustrated in Fig. 4B. As shown in Fig. 5B, in the presence of 20 mM Na+, NCKX2 was competent in rapidly clearing cytosolic Ca2+ loads regardless of the incubation time with Na+. Because of the results shown in Fig. 4A, we were interested in examining the time course of development of mitochondrial Ca2+ accumulation in NCKX2-transfected HEK293 cells. To this end, gramicidin- and Tg-treated NCKX2-transfected HEK293 cells were loaded with Ca2+ by the addition of 75 mM Na+ in the presence of 250 µM external free Ca2+, and at various time points thereafter, 1 mM EDTA was added to chelate external Ca2+. After 10 s, 2 µM FCCP was applied to release accumulated Ca2+ in the mitochondria. Fig. 5C shows the results from four separate cuvettes displaying different Ca2+ loading periods and overlaid to illustrate the development of Ca2+ accumulation in mitochondria. At the earliest time point (20 s), very little Ca2+ had accumulated in the mitochondria in comparison with the Ca2+ released by application of FCCP after 300 s of Ca2+ loading via NCKX2. These experiments also suggested that, under our experimental conditions, there was a dynamic equilibrium established between NCKX2 mediating Ca2+ influx into the cytosol and mitochondria accumulating Ca2+ from the cytosol. The experiments also suggested that the reverse mode of operation of NCKX2 seemed to operate continuously, as the magnitude of the Ca2+ pool accumulated by mitochondria grew larger with time presumably because of the continued influx of external Ca2+ through the exchanger.


Figure 4
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  		FIGURE 4.
Forward exchange (Ca2+ extrusion) mode of NCKX2. A, fluo-4FF-loaded NCKX2-transfected HEK293 cells were placed in cuvettes with buffered 150 mM KCl and treated with 2 µM gramicidin. After loading the cells with Ca2+ using the reverse exchange mode of NCKX2 by the addition of 75 mM NaCl (in the presence of 250 µM external free Ca2+), 1 mM EDTA was added to chelate external Ca2+, thereby reversing the Ca2+ gradient and inducing Ca2+ extrusion via the exchanger. In separate cuvettes, cells were also treated with Tg alone to inhibit SERCA or with Tg and FCCP in combination to inhibit both SERCA and mitochondrial Ca2+ sequestration. Note that, in both untreated control (Ctrl) and Tg-treated cells, the fluorescence rapidly returned to base-line levels upon the addition of EDTA; however, when mitochondria were additionally disabled, the fluorescence decrease (internal Ca2+ clearance) rate was greatly diminished. The traces shown are representative of results obtained in six other experiments. B, fluo-4FF-loaded HEK293 cells were placed in separate cuvettes with buffered 150 mM KCl and treated with 2 µM gramicidin, 0.4 µM Tg, and 2 µM FCCP. At the first arrow, the indicated millimolar concentrations of NaCl were added to the cuvettes (in the presence of 250 µM external free Ca2+). After 150 s, 1 mM EDTA was added to each cuvette to chelate external Ca2+ and to initiate forward exchange. Note that the rate of internal Ca2+ clearance diminished with increasing Na+ concentrations. The traces shown are representative of results obtained in six other experiments.

 


Figure 5
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  		FIGURE 5.
Time course of inactivation of NCKX2 and accumulation of the mitochondrial Ca2+ pool. A, fluo-4FF-loaded NCKX2-transfected HEK293 cells were placed in cuvettes with buffered 150 mM KCl and treated with 2 µM gramicidin, 0.4 µM Tg, and 2 µM FCCP. At time 0, 75 mM NaCl was added to each cuvette in the presence of 250 µM external free Ca2+. At various time points thereafter, 1 mM EDTA was added to the cuvette as indicated by the arrows. The data shown are from separate cuvettes overlaid to illustrate the difference in kinetics of Ca2+ clearance. The traces shown are representative of results obtained in four other experiments. B, the same experimental conditions as described for A were used, except that Ca2+ influx was induced by the addition of 20 mM NaCl at time 0 (concentration not sufficient to produce strong inactivation; see Fig. 4B). The traces shown are representative of results obtained in two other experiments. C, fluo-4FF-loaded NCKX2-transfected HEK293 cells were placed in cuvettes with buffered 150 mM KCl and treated with 2 µM gramicidin and 0.4 µM Tg. In separate cuvettes, at time 0, 75 mM NaCl was added, and at the arrows, 1 mM EDTA was added to the cuvette to chelate external Ca2+ and to initiate Ca2+ efflux. Following the addition of EDTA at 10 s, 2 µM FCCP was added to release accumulated Ca2+ in mitochondria (indicated by the arrowheads). The data shown are from four separate cuvettes overlaid for illustrative purposes. The traces shown are representative of results obtained in three other experiments.

 
NCKX2 Inactivation Is Dependent on External Ca2+—To further address whether the apparent inactivation of the exchanger may have been due to application of high Na+ at the intracellular surface of the exchanger, thereby effectively competing with Ca2+ at the Ca2+-K+-binding pocket, we examined the process of inactivation of NCKX2 in HEK293 cells loaded with different starting values of Ca2+. We had predicted that, if competitive inhibition was a major contributing factor to the apparent inactivation, then lowering the starting external Ca2+ levels should enhance the apparent inactivation (because less Ca2+ would enter the cytosolic compartment via the exchanger, and hence, Na+ would more effectively compete with Ca2+ for the intracellular binding pocket). However, we observed the opposite effect; as the starting concentration of Ca2+ was decreased, inactivation did not develop. Fig. 6 shows a typical example of NCKX2-transfected HEK293 cells assayed with either 250 or 10 µM external free Ca2+. In both experiments, the cells were loaded with Ca2+ via the reverse exchange mode of NCKX2 by the addition of 75 mM Na+. The cells that had been exposed to 10 µM external free Ca2+ were able to rapidly clear the cytosolic load of Ca2+, whereas those exposed to 250 µM external free Ca2+ (the concentration used in previous experiments) displayed a diminished rate of Ca2+ clearance from the cytosol upon initiation of Ca2+ extrusion. This suggested that inactivation of NCKX2 required high intracellular Na+ and high extracellular Ca2+; the results also argued against competitive inhibitory interactions between Na+ and Ca2+.In addition, we considered the possibility of abrupt changes in pH mediating the observed inactivation process. To this end, we loaded NCKX2-transfected HEK293 cells with the proton indicator cell-permeant form of the dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (Molecular Probes) and carried out the assays described herein, but under no conditions did we detect any large or abrupt changes in intracellular pH, even upon the addition of FCCP and the various solutes (data not shown).


Figure 6
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  		FIGURE 6.
NCKX2 inactivation does not develop with low extracellular Ca2+. Fluo-4FF-loaded NCKX2-transfected HEK293 cells were placed in cuvettes with buffered 150 mM KCl and treated with 2 µM gramicidin, 0.4 µM Tg, and 2 µM FCCP. At the first arrow, 75 mM NaCl was added in the presence of 250 or 10 µM external free Ca2+, achieved by the addition of 1 mM HEDTA and 8 mM Ca2+/HEDTA (see "Experimental Procedures"). At the second arrow, 5 mM EDTA was added to chelate external Ca2+ and to initiate Ca2+ extrusion via the exchanger. Note that the rate of Ca2+ efflux was markedly slower in the cells incubated with 250 µM external free Ca2+, signifying inactivation of NCKX2. The traces shown are representative of results obtained in three other experiments.

 
Relief of Inactivation of NCKX2—Further evidence that the inactivation of the exchanger was both time- and Na+-dependent was that it could be relieved by reducing the Na+ concentration in the cuvette. This was achieved by loading the cells with Ca2+ using the reverse exchange mode of NCKX2 in 500 µl of buffered 100 mM NaCl and 50 mM KCl, after a passage of 150 s, the medium was diluted 4-fold with 150 mM buffered KCl (containing 1 mM EDTA) to decrease the Na+ concentration from 100 to 25 mM. This caused a time-dependent relief of inactivation (Fig. 7). In contrast, upon dilution with 150 mM buffered NaCl (containing 1 mM EDTA), the inactivated state of forward exchange was maintained despite the fact that the high Na+ concentration was expected to saturate the external binding sites and to maximize Ca2+ efflux. When the assay medium was diluted with buffered 150 mM KCl (containing 1 mM EDTA) 40 s following initiation of reverse exchange, the fluorescence levels dropped precipitously, consistent with results of the time dependence plots in Fig. 5A, suggesting that exposure to high intracellular Na+ alone was not sufficient to completely inactivate the exchanger and that the full inactivation required >40 s to become apparent. Note that dilution with 150 mM buffered NaCl (containing 1 mM EDTA) 40 s following initiation of reverse exchange resulted initially in rapid clearing of cytosolic Ca2+, with the rate then subsiding due to progressive inactivation. As a result, the final cytosolic Ca2+ concentration at the end of our recording in 150 mM buffered NaCl was higher than the level achieved by dilution of the assay medium with buffered 150 mM KCl.


Figure 7
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  		FIGURE 7.
Relief of inactivation of NCKX2. Fluo-4FF-loaded NCKX2-transfected HEK293 cells were placed in cuvettes with 500 µl of buffered 100 mM NaCl and 50 mM KCl, and treated with 2 µM gramicidin, 0.4 µM Tg, and 2 µM FCCP. The cells were then loaded by the addition of 250 µM external free Ca2+ to the cuvette. After either 40 or 150 s, the medium in the cuvette was diluted 4-fold by the addition of 1500 µl of buffered 150 mM KCl (supplemented with 1 mM EDTA) or 1500 µl of buffered NaCl (supplemented with 1 mM EDTA). The traces represent dilution with either buffered KCl or NaCl and, after a period of 40 or 150 s, incubation with Na+; dilution was at time 0 in each case. The traces shown are representative of results obtained in three other experiments.

 
NCKX2 Inactivation Is an Inherent Property of the Exchanger Related to Its Affinity for Internal Na+—The NCKX2 inactivation described above appeared to be related to occupancy of the internal binding sites of NCKX2 with Na+. To test this notion, it would be desirable to have NCKX2 mutants with altered Na+ affinity. We are currently in the process of screening all the NCKX2 mutants reported previously (21) for changes in Na+ affinity using the experimental protocol illustrated in Fig. 3. Two single residue substitutions were found to be useful for this study, as they resulted in a marked increase in affinity for internal Na+. Previously, we had reported that a key acidic residue, Asp548, which lies within the midplane of transmembrane-spanning domain H8 of NCKX2, is important for the binding and transport of both Ca2+ and K+ (22). Substitution of Asp548 with glutamate (D548E) caused dramatic decreases in the affinity of the exchanger for K+ and Ca2+ by 10- and 100-fold, respectively. In this study, we assayed mutant D548E for internal Na+ affinity at the same concentrations tested with wild-type NCKX2. As shown in Fig. 8A, the apparent affinity for intracellular Na+ was increased with the D548E substitution; the Kd value was 19.3 ± 1.6 mM (n = 3) as opposed to 50 mM for wild-type NCKX2 (Fig. 3B). The Hill coefficient was slightly increased to a value of 3.0 as opposed to 2.6 for wild-type NCKX2. Another residue whose substitution we also found to increase the apparent affinity of the exchanger for Na+ was Asn572; its substitution to cysteine (N572C) also resulted in increased Na+ affinity, with Kd = 18.5 ± 1.9 (n = 3) (Fig. 8B) and a Hill coefficient of 3.1. Fig. 8C compares the Vmax values of the three NCKX2 constructs as judged by the maximal amplitude of the Na+-induced changes in fluo-4FF fluorescence changes. Wild-type NCKX2 and the D548E mutant showed comparable values, whereas the Vmax of the N572C mutant was lower. Because we had observed that NCKX2 inactivated when exposed to high intracellular Na+, we predicted that those mutants with increased affinity for intracellular Na+ would display inactivation of the forward mode of exchange at lower Na+ concentrations than would wild-type NCKX2. Fig. 8D shows that this was indeed the case; although we observed inactivation of wild-type NCKX2 only when intracellular Na+ was elevated to 75 mM or higher (see Fig. 4B), both the D548E and N572C mutants displayed greatly decreased Ca2+ clearance when intracellular Na+ was elevated to 35 mM. Note that with the level of Ca2+ loading induced by the addition of 35 mM Na+, D548E-transfected HEK293 cells were loaded with the same level of Ca2+ as cells transfected with wild-type NCKX2; however, those transfected with N572C had about half the level of intracellular Ca2+ increase upon the addition of Na+. When looking at the forward exchange mode, only the mutants (with increased Na+ affinity) were inactivated and did not rapidly lower intracellular Ca2+ back to the base line upon the addition of EDTA, whereas wild-type NCKX2-transfected HEK293 cells rapidly cleared the cytosolic load of Ca2+ when exposed to 35 mM Ca2+ (see also Fig. 4B). Although in our previous work we had shown that D548E displays a decreased affinity for Ca2+ and K+ (22), this does not account for this inability of this mutant protein to clear cytosolic Ca2+ (as shown in Fig. 8) because, when forward exchange was engaged at an earlier time point, D548E rapidly cleared the rise in intracellular Ca2+ (as did N572C) (data not shown).


Figure 8
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  		FIGURE 8.
Enhanced inactivation of NCKX2 mutants with increased Na+ affinity. A, fluo-4FF-loaded HEK293 cells transfected with the NCKX2 mutant D548E were assayed for Na+ affinity as described in the legend to Fig. 3. Data points represent averages, and error bars indicate S.D. The Kd for Na+ was 19.3 ± 0.6 mM, and the fitted Hill plot gave a Hill coefficient of 3.0. B, fluo-4FF-loaded HEK293 cells transfected with the NCKX2 mutant N572C were assayed for Na+ affinity as described in the legend to Fig. 3. The Kd for Na+ was 18.5 ± 0.3 mM, and the fitted Hill plot gave a Hill coefficient of 3.1. C, shown is a comparison of the Vmax values for wild-type (wt) NCKX2, D548E, and N572C based on the maximal plateau amplitude of fluorescence attained. D, fluo-4FF-loaded HEK293 cells transfected with wild-type NCKX2, D548E, or N572C were placed in cuvettes with buffered 150 mM KCl and treated with 2 µM gramicidin, 0.4 µM Tg, and 2 µM FCCP. At the first arrow, 35 mM NaCl was added to the cuvettes in the presence of 250 µM external free Ca2+, 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 Ca2+, both D548E- and N572C-transfected HEK293 cells had a diminished rate of Ca2+ clearance, signifying inactivation of these exchangers at lower Na+ concentration.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The SLC24 gene family of NCKX Na+/Ca2+-K+ exchangers comprises bidirectional and electrogenic Ca2+ transporters. The transport of Ca2+ through these carriers is dependent on the relative gradients of Na+, Ca2+, and K+ as well as membrane potential. NCKX displays a high turnover rate and hence a large capacity to change intracellular free Ca2+ over a large range of free Ca2+ concentrations and even at low intracellular free Ca2+ in the nanomolar range (36). Here, we have described a method for measuring the affinity of the human retinal cone/brain exchanger NCKX2 for Na+ and how to examine the physiologically relevant mode of Ca2+ extrusion from the cytosol; in the process, we discovered a novel feature of Na+-dependent inactivation of Ca2+ transport. We suggest that this kinetic transition between an active and inactive mode of the exchanger serves as a regulatory mechanism to prevent lowering of intracellular Ca2+ to unfavorable levels.

Controlling Alkali Cation Concentrations Using Gramicidin— The method of using the gramicidin clamp to control alkali cation concentrations across the plasma membrane has proven very useful and indeed necessary for accurately and quantitatively assaying the affinity of the exchanger for Na+. The resting Na+ concentration of cells is typically ~10 mM, which is the concentration of Na+ at the initial sigmoidal ramp of exchanger activity (Fig. 3B); hence, relying on the low resting intracellular Na+ concentration to drive Ca2+ import can be very variable because of the sensitivity of the exchanger to small changes in Na+ concentrations in this range. In the presence of gramicidin and high Na+, NCKX2 was able to drive intracellular free Ca2+ to high levels of 15–20 µM, necessitating the use of the low affinity Ca2+ indicator fluo-4FF to avoid saturation of the dye. The dynamic Ca2+ 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 Ca2+ fluxes and NCKX currents in the outer segments of retinal rod (28, 36) and cone (12) photoreceptors.

Interplay between NCKX2 and Other Cellular Ca2+-handling Mechanisms—As our results have clearly shown, whereas NCKX can mediate large increases in intracellular Ca2+, when the Na+ gradient is reversed so as to favor Ca2+ efflux, only a portion of the Ca2+ efflux signal component is actually mediated by the exchanger (when intracellular Na+ is high). Specifically, it appears that a dynamic equilibrium of Ca2+ influx via the exchanger and Ca2+ sequestration by mitochondria is established once intracellular Ca2+ levels reach a certain threshold. On the basis of experiments we carried out with the two dyes fluo-3 (with a reported Kd for Ca2+ of ~0.4 µM; data not shown) and fluo-4FF (with Kd for Ca2+ of ~10 µM), we estimated that the level at which mitochondria began accumulating Ca2+ was ~2–3 µM. Kim et al. (37) found a similar pattern when investigating Ca2+ clearance at synaptic terminals, where Na+/Ca2+ exchange activity (including a significant K+-dependent component) dominates in Ca2+ clearance, with mitochondria participating only when Ca2+ reaches levels >2.5 µM or the Ca2+ load is prolonged. Likewise, in adrenal chromaffin neuroendocrine cells, mitochondria do not participate in clearance of Ca2+ loads (induced by depolarization) up to 0.5 µM; however, mitochondria are the major mechanism for Ca2+ clearance when intracellular Ca2+ reaches 2–3 µM (38). Coincidentally, the level of Ca2+ at which mitochondria started to accumulate Ca2+ in our cells was the same as that required to observe inactivation of the exchanger, raising the possibility of an underlying interaction between mitochondria and the exchanger at the plasma membrane. Such an interaction between the plasma membrane Na+/Ca2+ exchanger and mitochondria has been suggested previously to mediate inactivation of exchange of NCX (39). Although our results cannot conclusively rule out this intriguing possibility, we do not think that such is the case for NCKX; however, this issue deserves further investigation.

Distinguishing Inactivation from Competitive Inhibition of NCKX2 Activity—Arguably, the use of gramicidin introduced the caveat that Na+ was present on both sides of the membrane (likewise with K+), and hence, exchange activity in the reverse (or forward) mode may have been slowed because of competition of Na+ on the external (or internal) side of the membrane. We do not believe this to be the case because, by carrying out the assay in a medium of high KCl, we were able to effectively minimize inhibitory interactions between Na+ and Ca2+ by favoring the binding of Ca2+, as was shown previously in studies of Na+/Ca2+-K+ exchange in situ in rod outer segments (36). Moreover, several of our findings herein argue against competitive inhibition. First, the inactivation process was time-dependent as illustrated in Fig. 5, although if competitive inhibition was prominent, it would have been observed at all time points examined. In line with this finding, we have also shown time-dependent relief of inactivation by lowering intracellular Na+ from 100 to 25 mM (Fig. 7). Second, inactivation did not develop when extracellular Ca2+ was lowered to 10 µM (Fig. 6). This result was intriguing because it would be expected that lowering the external Ca2+ and ultimately the Ca2+ availability in the cytoplasm would promote competitive inhibition in the presence of high internal Na+. Interestingly, the process of Na+-dependent inactivation described herein for NCKX2 is similar in characteristics to Na+-dependent inactivation described for the cardiac Na+/Ca2+ exchanger (40), for which it was proposed that the inactive state occurs when the exchanger transport sites are exposed to high Na+ only on the cytoplasmic side and that it requires high extracellular Ca2+ as well. Hilgemann et al. (40) suggested that inactivation of the cardiac exchanger does not develop with low extracellular Ca2+ because, under those conditions, the exchanger binding sites would favor the exoplasmic face, which prevents them from exposure to high internal Na+ on the cytoplasmic face, the only conformation that will inactivate the exchanger. Finally, our results with the NCKX2 point mutants D548E and N572C suggest that inactivation of NCKX2 is dependent on its affinity for Na+, as both these mutants showed enhanced inactivation at lower intracellular Na+, concomitant with their increased affinity for this ion (Fig. 8).

Residues Important for Na+ Binding—No studies have reported previously on residues important for Na+ transport of NCKX transporters or on residues in the {alpha}2 repeat of NCX transporters important for Na+ transport. Asp548 and Asn572 are located and conserved in the {alpha}2 repeats of all NCX and NCKX sequences currently in the data base, and our observation of altered Na+ affinities suggests that these two residues may form part of the cation-binding pocket of all NCKX and NCX transporters. We reported previously that Asp548 is one of the two most important residues (together with Glu188) that determine the Ca2+ affinity of NCKX transporters (22).

Physiological Implications—Previous work from our laboratory on retinal rod outer segments that carry the related NCKX1 exchanger suggest that the NCKX1 exchanger operates in "bursts" of activity, which then drastically diminish (41, 42). Our results here suggest the presence of different kinetic states of the NCKX2 exchanger and reveal some of the ionic conditions necessary for the process of inactivation to occur: high extracellular Ca2+ (≥250 µM) and high intracellular Na+ (>35 mM). Little is known about the molecular mechanism of the above regulatory features of NCKX1 and NCKX2 and to what degree they are related or distinct. The experimental paradigms described in this study and the capability of HEK293 cells to express (mutant) NCKX proteins at levels comparable with those found in situ in rod and cone photoreceptors will be useful to further elucidate the regulatory mechanisms and kinetic states of NCKX exchangers.

Inactivation of NCKX2 may be a useful regulatory mechanism that operates under physiological conditions. Mounting evidence points to NCKX2 being an important Ca2+ clearance mechanism in neuronal synaptic terminals (13–15), which are specialized compartments that experience frequent and dynamic Ca2+ fluxes and depolarized membrane potential resulting from frequent invasions of action potential spikes. Persistent presynaptic activity leads to persistent elevations in presynaptic terminal Ca2+ concentrations (43, 44), associated with an increase in intraterminal Na+ levels (up to peaks of 80 mM) (45). This accumulation of intraterminal Na+ has been suggested to induce Ca2+ influx into the neuronal terminal through plasma membrane Na+/Ca2+ exchangers operating in the reverse mode as well as from delayed release of Ca2+ from mitochondria (46, 47). Although NCX operating at an exchange stoichiometry of three Na+ ions for one Ca2+ ion may indeed reverse under such conditions, NCKX with a stoichiometry of four Na+ ions for one Ca2+ ion + one K+ ion is less likely to reverse and begin Ca2+ import. However, a significant component of the persistent rise in intraterminal Ca2+ may be explained by the diminished rate of Ca2+ clearance by NCKX2 as it becomes inactivated by the prolonged high levels of intracellular Na+ until presynaptic activity subsides, at which point ionoregulatory mechanisms can restore resting ionic conditions, and the exchanger may resume in clearance of the presynaptic terminal of the high load of Ca2+.


   FOOTNOTES
 
* This work was supported in part by an operating grant from the Canadian Institutes of Health Research (to P. P. M. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Recipient of a studentship from the Foundation Fighting Blindness-Canada. Back

2 To whom correspondence should be addressed: Dept. of Physiology and Biophysics, Faculty of Medicine, University of Calgary, 3330 Hospital Dr. NW, Calgary, Alberta T2N 4N1, Canada. Tel.: 403-220-5448; Fax: 403-283-8731; E-mail: pschnetk{at}ucalgary.ca.

3 The abbreviations used are: NCX, Na+/Ca2+ exchanger; NCKX, Na+/Ca2+-K+ exchanger; HEK293, human embryonic kidney 293 cells; TAPS, 3-[tris(hydroxymethyl)methyl]aminopropanesulfonic acid; HEDTA, N-(2-hydroxyethyl)ethylenediaminetriacetic acid; PMCA, plasma membrane Ca2+-ATPase; SERCA, sarco/endoplasmic reticulum Ca2+-ATPase; Tg, thapsigargin; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Back



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