Identification and Characterization of K+-Dependent Na+-Ca2+

Apr 19, 2016 - ... of Physiology & Pharmacology, Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Calgary T2N 4N1, Canada...
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Identification and Characterization of K+‑Dependent Na+‑Ca2+ Exchange Transport in Pigmented MEB4 Cells Mediated by NCKX4 Robert T. Szerencsei,† Rebecca S. Ginger,‡ Martin R. Green,‡ and Paul P. M. Schnetkamp*,† †

Department of Physiology & Pharmacology, Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Calgary T2N 4N1, Canada ‡ Unilever R&D, Colworth Science Park, Sharnbrook, Bedfordshire MK44 1LQ, U.K. ABSTRACT: The SLC24 gene family encodes K+-dependent Na+-Ca2+ exchangers or NCKX proteins. The NCKX4 and NCKX5 isoforms have been shown to be important for pigmentation, and single nucleotide polymorphism (SNP) in both alleles of the SLC24a5 gene is the major genetic determinant for light skin in Caucasians. NCKX4 is thought to operate in the surface membrane of cells, whereas NCKX5 is thought to be located in intracellular membranes. However, no functional data have yet been reported to describe either NCKX4 or NCKX5 activity in pigmented cells. In this study, we used the B16 and MEB4 mouse pigmented cell lines to investigate NCKX-mediated Ca2+ fluxes using 45Ca uptake experiments and measurements of changes in intracellular free Ca2+ with the fluorescent Ca2+-indicating dye Fluo-4. We used siRNA-mediated knockdown to selectively reduce either NCKX4 or NCKX5 expression. The results show that both B16 and MEB4 cells contain roughly equal amounts of NCKX4 and NCKX5 transcript, but surface membrane NCKX activity is restricted to NCKX4. Intracellular NCKX4 activity was also observed, but we could not unambiguously detect any NCKX5 activity. We were able to demonstrate that NCKX5 is a functional K+-dependent Na+-Ca2+ exchanger located in internal membranes after transfection of NCKX5 cDNA in HEK293 cells. We conclude that pigment cells express robust, functional NCKX4 activity, but that the role of NCKX5 remains enigmatic ten years after the discovery of its link to pigmentation. we analyzed Ca2+ fluxes in pigmented B16 and MEB4 cells, both of which express NCKX4 and NCKX5 transcripts. We also used 45Ca fluxes and measurement of changes in cytosolic free Ca2+ using the fluorescent Ca2+-indicating dye Fluo-4 to assess NCKX-mediated Ca2+ transport across the plasma and internal membranes. Using a range of assay formats, we found conclusive evidence for and have characterized NCKX4mediated exchange activity at the plasma membrane and in internal membranous compartment(s) in these cells. Knockdown experiments support a much smaller but not conclusively proven NCKX5 ion exchange component.

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he SLC24 gene family of K + -dependent Na + -Ca 2+ exchangers encodes five distinct NCKX proteins that have been shown to be important for a wide range of biological processes.1 NCKX proteins use both the inward Na+ gradient and the outward K+ gradient to extrude Ca2+ from animal cells, in particular in excitable tissues where fast Ca2+ clearance is required. However, NCKX proteins have been reported to be important for other processes as well, including in pigmented cells of the human skin, hair, and iris. Human genome-wide association studies have shown that NCKX5 variants have a major role in the natural variation in human skin color,2,3 and knockdown of this protein causes a major loss of pigment production in human melanocytes.4 Similar genetic investigations into natural human variation have also linked NCKX4 to pigment production, particularly that of hair and eye color.5−7 The melanogenic mechanisms by which high capacity Na+-Ca2+ exchangers affect pigmentation are almost completely unknown, though Ginger et al. have proposed possible mechanisms on the basis of the demonstrated location of NCKX5 in the trans-Golgi network.4 In addition to its proposed role in hair and eye color, NCKX4 has also been shown to play an important role in olfaction8 and melanocortin-4-receptordependent satiety9 in mice and enamel maturation in humans.10,11 To begin the investigation of how ion exchange fluxes mediated by NCKX4 and NCKX5 regulate melanogenesis, © 2016 American Chemical Society

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METHODS Unless otherwise indicated, all chemicals were purchased from Sigma-Aldrich. Cell Culture. HEK293, B16, and MEB4 cells were grown on 10 cm plates (8 million cells) in complete DMEM (DMEM + 10% FBS, 2 mM L-glutamine, 50 units/mL penicillin, 50 μg/mL streptomycin, and 2.5 μg/mL Fungizone B (Invitrogen)) and supplemented with 50 μM forskolin when indicated. In preparation for the experiments, the medium was discarded; Received: January 8, 2016 Revised: March 31, 2016 Published: April 19, 2016 2704

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Figure 1. NCKX transport activity measured in pigmented B16 and MEB4 cells. Panel A: 45Ca uptake in Na+-loaded B16 cells was measured as described in Methods. Filled circles represent 45Ca uptake in a KCl medium, and the filled squares and triangles represent the uptake in LiCl and NaCl media, respectively. The data represent the average of five separate experiments, and the error bars represent the standard error of the mean. Panel B: Ca2+ influx was initiated at time zero by addition of 0.25 mM CaCl2 to Na+-loaded B16 cells diluted into a KCl, LiCl, or NaCl medium as indicated. The rise of cystosolic free Ca2+ was measured with the fluorescent Ca2+indicating dye Fluo-4 as described in Methods. NCKX activity is the difference in Ca2+ influx observed in the KCl medium versus that observed in the LiCl or NaCl medium. The gramicidin treatment depletes the internal Na+ from the cell, and consequently, we see a loss of NCKX-mediated Ca2+ influx. Panels C and D: NCKX activity was measured with either Na+-loaded (Panel C) or Li+-loaded (Panel D) MEB4 cells with the fluorescent Ca2+-indicating dye Fluo-4. As above, the NCKX activity is the difference between Ca2+ influx observed in the KCl medium versus that observed in the LiCl or NaCl medium.

Figure 2. NCKX activity in different fractions of B16 cells obtained after gradient centrifugation. 45Ca uptake of different membrane fractions of B16 cells was measured in KCl, LiCl, or NaCl medium as described in Methods. The difference between 45Ca uptake observed in the KCl medium and that observed in either the LiCl or NaCl medium represents reverse NCKX activity. Panel A represents the upper white nonpigmented band, and panel B represents the lower black pigmented bands.

Ca2+ accumulation in mitochondria and endoplasmic reticulia, respectively. The cells were spun down for 2 min at 240g; the supernatant was discarded, and the cells were washed with 1 mL of the above Na+-loading medium. The cell pellets were used for experiments as described below. Sucrose Gradient Ultracentrifugation. Six T-150 flasks of B16 cells were grown at 37 °C with 5% CO2 to 100% confluence. We harvested the cells by first removing the media and rinsing the plates with 10 mL of PBS. Next, 2 mL of 0.05% trypsin/0.53 mM EDTA was added to the plates and left for 90 s, at which time 20 mL of fresh DMEM with 10% FBS was added to inactivate the trypsin. The cells were collected and spun down for 2 min at 340g. The pelleted cells were washed with 25 mL of PBS and spun down again. The pellets were pooled after resuspension in 5 mL of 240 mM sucrose, 20 mM HEPES (pH 7.4), 50 mM NaCl, and 50 μM EDTA containing a protease inhibitor tablet (Roche, 05892791001). The cells were Dounced 60 times using the tight pestle (Wheaton, 357542) and spun down at 500g for 20 min at 4 °C. The supernatant was saved while the pellet was Dounced twice.

the cells were rinsed with 5 mL of 1× PBS, and 1 mL of 0.05% Trypsin-EDTA (Invitrogen) was added for 90 s. Complete DMEM (15 mL) was added to inactivate the trypsin, and the cells were transferred to a 50 mL centrifuge tube and spun down for 2 min at 400g. The supernatant was discarded, and the cells were washed once more with 15 mL of PBS-0.1 mM EDTA. Next, the cells were resuspended in 500 μL of Na+loading medium containing 150 mM NaCl, 20 mM HEPES (pH 7.4), 10 mM D-glucose, 250 μM sulfinpyrazone, 500 μM ouabain, 200 μM EDTA, and 10 μM Fluo-4AM (Invitrogen Life Technologies). The latter was absent in the 45Ca experiments. The cells were incubated for 30 min in the dark at room temperature, and 20 μM FCCP and 10 μM thapsigargin were added when indicated. FCCP (0.5 μM final concentration) and thapsigargin (2.5 μM final concentration) were added to prevent 2705

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Figure 3. siRNA inhibition of the endogenous NCKX-mediated transport activity in MEB4 cells. MEB4 cells were transfected with siRNA toward either scrambled control (a), NCKX4 (b), NCKX5 (c), or, as another control, NCKX2 (d). Ca2+ influx via reverse NCKX activity was initiated by the addition of 50 mM KCl at time zero in the presence of 150 μM external Ca2+. The trace labeled 0 mM KCl represents nonspecific Ca2+ influx in the absence of external K+.

Table 1. Kinetic Parameters of NCKX4-Mediated Ca2+ Transport in HEK Cells Transfected with NCKX4 cDNA or NCKX4 Endogenous to MEB4 Cellsa +

K Ca2+ Na+ Na+ competition DCB K+ Ca2+ Na+ Na+ competition DCB

cell type

Km

Ki

HEK-hNCKX4 HEK-hNCKX4 HEK-hNCKX4 HEK-hNCKX4 HEK-hNCKX4 MEB4 MEB4 MEB4 MEB4 MEB4

3.13 mM 7.97 μM 46.18 mM 32.08 mM 19.54 μM 9.87 mM 11.57 μM 32.66 mM 19.72 mM 10.68 μM

SEM 0.76 1.41 6.73 5.54 2.00 0.61 1.33 2.83 1.87 2.17

a

Data are average values with standard error of the mean (SEM) of 4−6 separate experiments. Data for the HEK cells expressing NCKX4 were taken from ref 16.

Figure 4. Effect of siRNA toward mNCKX4 or mNCKX5 on NCKXmediated transport activity in MEB4 cells. The bar diagram represents NCKX transport activity that remains following treatment with the indicated NCKX siRNA, normalized with respect to the scrambled control (number of observations listed on top of each column). Error bars represent standard error of the mean. The p-value is given on the bottom of each column. We used the two-tailed unpaired t-test, which compares the means of two unmatched groups, assuming that the values follow a Gaussian distribution.

medium, and 70 μL aliquots of the solution were placed in Eppendorf tubes. Microsomes were prepared by collection of cell pellets from four to eight 10 cm plates and stored frozen at −80 °C. Cell pellets were thawed and resuspended in 4 mL of 240 mM sucrose, 20 mM HEPES (pH 7.4), 0.1 mM EDTA, and 50 mM NaCl (one protease inhibitor tablet was included (Roche, 05892791001) per 30 mL of cocktail). On ice, the suspension was Dounced 60 times with the tight pestle, and the cell homogenate was transferred into a centrifuge tube and spun down at 4 °C for 15 min at 1000g. The supernatant was kept, and the pellet was Dounced again with 4 mL of fresh sucrose-Na cocktail and spun down. The combined supernatants were centrifuged at 4 °C for 15 min at 5500g. The supernatant was discarded; the pellet was resuspended in 260 μL of 300 mM sucrose, 20 mM HEPES (pH 7.4), and 50 μM EDTA, and 70 μL aliquots of the solution were placed in Eppendorf tubes. The Eppendorf tubes containing 70 μL of either intact cells or microsomes were used for the uptake experiments. 45Ca uptake was initiated by addition of 350 μL of 150 mM NaCl, KCl, or LiCl (as indicated), 20 mM HEPES (adjusted to pH 7.4 with arginine), 1 μCi of 45Ca (Amersham), and 30 μM CaCl2. At various time points, 60 μL samples were taken; 45Ca uptake

All three supernatants were pooled and layered on a continuous density gradient made of 1−2 M sucrose containing 20 mM HEPES (pH 7.4), 50 mM NaCl, and 50 μM EDTA. The gradients were centrifuged in a Beckman ultracentrifuge using a SW28 rotor at 4 °C for 1 h at 120000g. The bands were collected separately, washed with 10× excess of 240 mM sucrose, 20 mM HEPES (pH 7.04), 50 mM NaCl, and 50 μM EDTA, and spun down at 27000g for 15 min at 4 °C. The supernatant was discarded, and the pellets were resuspended in 280 μL of 300 mM sucrose, 20 mM HEPES (pH 7.4), and 30 μM EDTA and used for the 45Ca uptake experiments described below. 45 Ca Uptake. For uptake measured in intact cells, the cells were washed once with 1 mL of 150 mM LiCl, 20 mM HEPES (pH 7.4), and 50 μM EDTA and resuspended in 200 μL of this 2706

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Figure 5. Ca2+, Na+, and K+ dependencies of NCKX transport in MEB4 cells. Cation dependencies of NCKX transport were measured in Fluo-4loaded MEB4 cells as previously described in detail.16 NCKX-mediated Ca2+ influx was measured as a function of the indicated external K+ concentration (panel A, external Ca2+ concentration fixed at 0.15 mM) and as a function of the indicated external free Ca2+ concentration (panel B, external K+ concentration fixed at 50 mM). Panel C shows NCKX-mediated Ca2+ influx measured as a function of the indicated internal Na+ concentration in Li+-loaded cells treated with gramicidin and diluted into a KCl medium containing 1 mM CaCl2 (see Figure 1). Panel D illustrates inhibition of Ca2+ influx (external Ca2+ concentration fixed at 50 μM) in a KCl medium through an increase in the external Na+ concentration. Inhibition represents competition between Ca2+ and Na+ for a common binding site on the NCKX protein.19

was stopped by addition of 5 mL of ice-cold stop solution (140 mM KCl, 20 mM HEPES (pH 7.4), 5 mM MgCl2, and 1 mM EGTA), and the cells or microsomes were filtered through borosilicate glass fiber filters as described previously.12 All 45Ca uptake experiments were carried out in the presence of 1 μM FCCP to prevent 45Ca uptake in the mitochondria. The filters were placed into a 20 mL scintillation vial containing 10 mL of CytoScint scintillation cocktail (MP Biomedicals), and radioactivity was counted in a Beckman LS6500 scintillation counter. All 45Ca uptake experiments were carried out at 25 °C. Fluo-4 Experiments. The cell pellet obtained from one 10 cm plate was resuspended in a medium containing 150 mM NaCl, 20 mM HEPES (pH 7.4), 10 mM D-glucose, 250 μM sulfinpyrazone, 500 μM ouabain, and 200 μM EDTA. MEB4 cells from one T-150 flask were collected by removal of the medium followed by rinsing with 10 mL of PBS. The cells were lifted by addition of 2 mL of 0.05% trypsin/0.53 mM EDTA and incubation for 2 min at room temperature. Trypsin was

inactivated by addition of 20 mL of DMEM and 10% FBS. The cells were transferred to a 50 mL centrifuge tube and spun down for 2 min at 500g. The supernatant was discarded, and the cells were washed with 25 mL of PBS and 0.2 mM EDTA. The final pellet was resuspended in 500 μL of a NaCl-ouabain solution (150 mM NaCl, 20 mM HEPES (pH 7.4), 10 mM D-glucose, 200 μM EDTA, 250 μM sulfinpyrazone, and 500 μM ouabain) plus 10 μM Fluo-4AM (Invitrogen Life Technologies). The cells were incubated in this medium for 30 min at room temperature in the dark. At the end of incubation, 10 μM FCCP (final concentration) was added, and the cells were spun down for 2 min at 340g. The supernatant was discarded, and the cells were washed with 1 mL of the above NaClouabain solution. The cells were resuspended in 600 μL of the NaCl-ouabain solution at room temperature and used for the Ca2+ influx assays within 20 min (50 μL of cell suspension was used for each cuvette, containing a final volume of 2 mL in the indicated media). The increase in fluorescence observed was 2707

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Biochemistry normalized to the change in fluorescence observed when the Fluo-4 dye was saturated by addition of the membranepermeabilizing agent saponin (0.01%) in the presence of a saturating Ca2+ concentration of 1 mM. All Fluo-4 experiments were carried out at 25 °C with an SLM Series 2 luminescence spectrometer.

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RESULTS AND DISCUSSION As has become customary in Na+/Ca2+(−K+) exchange transport studies, we took advantage of the bidirectional nature of Na+/Ca2+(−K+) exchangers to carry out both Ca2+ influx (reverse exchange) and Ca2+ efflux (forward exchange), dependent on the direction of the transmembrane Na+ gradient. When either intact cells or membrane vesicles derived from cells are studied, quantitative measurements of reverse exchange-associated Ca2+ fluxes in Na+-loaded cells or vesicles have become a standard assay for detecting NCKX activity. 45 Ca Uptake in B16 Cells and Membrane Fractions from B16 Cells. Direct measurements of Ca2+ fluxes associated with K+-dependent reverse Na+/Ca2+ exchange were investigated by means of 45Ca uptake in Na+-loaded pigmented B16 cells. This assay represents a simple and quantitative assay for NCKX activity.12 Figure 1a illustrates the results of such an experiment. A significantly greater 45Ca uptake was observed in KCl medium compared to that observed in LiCl or NaCl medium; the latter was expected to specifically inhibit reverse Na+/Ca2+ exchange, as the high Na+ concentration competes with 45Ca for transport via NCKX. The lack of uptake in LiCl medium indicates the presence of NCKX in the plasma membrane rather than the K+-independent Na+/Ca2+ exchanger NCX. We repeated this experiment with the membrane fractions obtained from B16 cells after cell homogenization and sucrose gradient centrifugation. The lower density membrane fractions were typically free of melanosomes, as judged by the lack of pigmentation and the absence of the melanosomal marker TYRP1,4 while higher density membrane fractions contained large amounts of melanin pigment. Figure 2 illustrates that both fractions contained significant NCKX activity, as judged by the observation that 45Ca uptake in KCl medium was significantly greater than 45Ca uptake observed in LiCl or NaCl medium. These experiments suggest that membranes isolated from B16 cells contain NCKX activity and that this activity does not correlate with the melanin content of these fractions. The decoupling of NCKX activity from melanin pigment in the different B16 membrane fractions suggests that some of this activity is not located in pigmented melanosomes. Changes in Free Cytosolic Ca 2+ Concentration Mediated by NCKX Activity. Having established that B16 cell membranes display NCKX-mediated 45Ca fluxes, we used fluorescent Ca2+-indicating dyes located in the cytosol to obtain higher resolution recordings of NCKX activity in B16 cells. In the process, we switched to the related melanocyte MEB4 cell line13 (RIKEN Bioresource Center) and included forskolin in the culture medium to obtain more consistent NCKX-mediated Ca2+ influx signals. We observed that addition of forskolin significantly reduced run-down of NCKX activity during cell passage, and the cells also maintained better pigmentation levels under these conditions (data not illustrated). It has been previously reported that transcripts of several pigmentation genes, including Slc24a5, are maintained at significantly higher levels in the presence of forskolin.14 Finally, we included both FCCP and thapsigargin in the dye-loading medium to eliminate sequestration of Ca2+ that entered the MEB4 cells by the

Figure 6. Internal NCKX activity measured in gramicidin-treated MEB4 cells. Panel A: Na+-loaded and Fluo-4-loaded MEB4 cells were diluted 80 times in a medium containing 150 mM KCl, 20 mM HEPES (pH 7.4), and 0.1 mM EDTA. Release of Ca2+ from internal stores was initiated at time zero by addition of the indicated concentration of NaCl and measured as an increase in Fluo-4 fluorescence located in the cytosol. Changes in external Na+ concentration are transmitted to the cytosol via channel ionophore gramicidin. Note: changes in cytosolic Ca2+ must originate from internal stores as the Ca2+ concentration is buffered to very low values in the presence of EDTA in the external medium. Panel B illustrates the sigmoidal relationship between the initial rate of change in cytosolic Ca2+ concentration and the Na+ concentration. The Na+ Km for the internal NCKX signal is 106 mM (n = 6, standard deviation of 15, Hill slope of 1.73).

endoplasmic reticulum and mitochondria, respectively, thus maximizing changes in cytosolic free Ca2+ mediated by Ca2+ entry across the plasma membrane. We previously illustrated the use of tools when characterizing NCKX-mediated Ca2+ fluxes in HEK293 cells expressing NCKX2.15 In the experiment illustrated in Figure 1b, we used Fluo-4 to monitor rises in intracellular free Ca2+ concentration in Na+-loaded B16 cells under conditions that favor NCKX-mediated Ca2+ entry via reverse exchange and under conditions that inhibit such entry. NCKX-mediated Ca2+ entry was prevented when the external medium contained high NaCl and when intracellular Na+ was removed by application of the cation channel ionophore gramicidin in a Na+-free medium. A large, fast rise in intracellular free Ca2+ was observed in KCl medium but not in LiCl or NaCl medium, consistent with Ca2+ entry via NCKX. As the rise in intracellular Ca2+ observed in LiCl medium was indistinguishable from that seen in NaCl medium, we conclude 2708

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Figure 7. siRNA-mediated knockdown of internal NCKX activity measured in gramicidin-treated MEB4 cells. siRNA transfected MEB4 cells were subjected to the experimental protocol described in the caption of Figure 6. Release of Ca2+ from internal stores was initiated at time zero by addition of the indicated concentration of NaCl and measured as an increase in Fluo-4 fluorescence located in the cytosol. Release of Ca2+ from internal stores was observed only in the presence of gramicidin, which allows changes in external Na+ concentration to be transmitted to the cytosol. Results from a single experiment are illustrated, representative of 12 control experiments (A), 7 transfection experiments with C07 (B), and 8 transfection experiments with H07 (C).

the NCKX5 siRNA is not clear, as H07 was less efficacious in reducing the NCKX5 transcript level when compared to the effectiveness of H03. H05 and H07 did not appear to affect the NCKX4 transcript level (data not shown). Unfortunately, commercially available NCKX5 antibodies did not detect our myc-tagged NCKX5 clones overexpressed in HEK293 cells and did not result in clear Western blots of MEB4 cell membranes, hindering protein-based investigations (data not illustrated). Characterization of NCKX4 Activity in MEB4 Cells in Comparison with NCKX4 Expressed in HEK293 Cells. The experiments described above strongly suggest that the NCKX-mediated Ca2+ influx observed in MEB4 cells is mediated predominantly by NCKX4 expressed in these cells. To our knowledge, this is the first cell line described that expresses a native NCKX protein in an assayable format and thus presents the first opportunity to characterize endogenous NCKX4. Table 1 summarizes the Km values we observed for the external Ca2+ and K+ dependencies of Ca2+ influx, the internal Na+ dependence of Ca2+ influx, and the external Ki of the inhibition by external Na+ dependence of Ca2+ influx via reverse exchange. Exemplars of the influx traces used to calculate the Km and Ki values are illustrated in Figure 5. The cation dependencies are consistent with the presence of NCKX4, although the Km and Ki values observed for MEB4 cells differ somewhat from those observed in HEK293 cells transfected with human NCKX4 cDNA reported previously16 and summarized in Table 1. NC(K)X Activity in Internal Compartments within MEB4 Cells. The above results suggest that the NCKX activity observed in our experiments with intact cells reflects predominantly NCKX4 activity in the MEB4 cell plasma membrane. Furthermore, when we transfected myc-tagged NCKX5 DNA into either B16 or MEB4 cells, we never observed an increase in plasma membrane NCKX-mediated Ca2+ influx despite the fact that immunoblots showed abundant expression of the myc-tagged NCKX5 (data not illustrated). This suggests that, consistent with earlier published studies,2,4 NCKX5 is not found in the plasma membrane but in organellar membranes. Therefore, we developed two assays to measure intracellular NC(K)X activity. First, as before, we used the channel ionophore gramicidin to selectively permeabilize the plasma membrane to monovalent

that MEB4 cells do not express measurable amounts of the K+-independent Na+/Ca2+ exchangers NCX1−3, consistent with transcript measurements of NCX1−3 in MEB4 cells (data not shown). We repeated this protocol when we switched to MEB4 cells and obtained a very similar result (Figure 1c). To demonstrate that this K+-dependent Ca2+ influx was dependent on intracellular Na+ and represents reverse Na+/Ca2+ + K+ exchange, we carried out two controls. First, we added gramicidin to the Na+-loaded cells, which permitted release of all internal Na+ into the large volume of the external KCl medium. Addition of gramicidin led to a near complete abolition of Ca2+ influx observed in KCl medium (Figures 1b and c, trace labeled “gramicidin”). Second, we used Li+ (rather than Na+)loaded cells, and no Ca2+ influx was observed regardless of the presence of K+ in the external medium (Figure 1d). Combined, we believe the experiments illustrated in Figure 1 establish that the plasma membranes of both B16 and MEB4 cells contain an active Na+/Ca2+ + K+ exchanger but very little if any K+-independent Na+/Ca2+ exchangers. Knockdown of NCKX4 and NCKX5 Transcripts. It has been reported that B16 cells contain significant NCKX4 transcripts in addition to NCKX5 transcripts, unlike human epidermal melanocytes which contain NCKX5 transcripts but very few NCKX4 transcripts.4 As observed for B16 cells, we found that MEB4 cells contain about equal amounts of NCKX4 and NCKX5 transcripts but very few transcripts of the other three NCKX isoforms (data not illustrated). To identify the NCKX isoform responsible for the NCKX-mediated Ca2+ fluxes illustrated in Figure 1, we used siRNA to knock down either NCKX4 or NCKX5 transcripts. Figure 3 illustrates a typical experiment in which siRNA directed against NCKX4 transcripts caused a large (75−80%) reduction of NCKX-mediated Ca2+ influx, whereas siRNA directed against NCKX5 transcripts had much less of an effect, although in all cases a statistically significant reduction (25−30%) of NCKX-mediated Ca2+ influx was observed when compared to that observed with scrambled siRNA or siRNA directed against NCKX2 transcripts (used as a negative control). Figure 4 illustrates a statistical analysis of a large number of such experiments. The significance of the modest reduction in NCKX-mediated Ca2+ influx observed for 2709

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Biochemistry alkali cations. When MEB4 cells were maintained in a physiological solution with an elevated Ca2+ concentration, we reasoned that this might increase the Ca2+ content of intracellular stores. MEB4 cells were diluted in the cuvette in a KCl/EDTA medium, and gramicidin was added to equilibrate intra- and extracellular K+ and Na+ concentrations. Next, different NaCl concentrations were added to separate cuvettes, and as the Na+ has access to the cytosol via the gramicidin ionophore, the increase in cytosolic Na+ concentration may cause Ca2+ release from intracellular organelles if the organellar membrane contains NC(K)X. Figure 6 illustrates a typical result of this experiment carried out with MEB4 cells. The sigmoidal dependence of the rate of increase in intracellular free Ca2+ concentration when the intracellular Na+ concentration was increased is consistent with NC(K)X activity, which exchanges one Ca2+ ion against multiple Na+ ions. Also, no significant increases in intracellular free Ca2+ concentration were observed when LiCl replaced NaCl in this experiment (not illustrated). This result also suggests that the NC(K)X activity observed in Figure 6 is unlikely to represent the mitochondrial NCLX Na+/Ca2+ exchanger because NCLX operates equally well as a Li+/Ca2+ exchanger.17 In six separate experiments, the average Km for Na+ was 106 mM (standard error of the mean (SEM) = 15), while the average Hill coefficient was 1.7 (SEM = 0.09). When we used siRNA directed toward NCKX4 or NCKX5 transcripts, we observed a significant reduction in the Na+-induced rise in intracellular free Ca2+ concentration in the case of siRNA directed against NCKX4 but much less so in the case of siRNA directed against NCKX5 (Figure 7). The same result was obtained in five additional experiments. Second, we selectively permeabilized the plasma membrane through the addition of saponin and measured 45Ca uptake in Na+-loaded cells using protocols identical for those used for intact cells (e.g., Figure 1). The assumption was that Na+ loading would also lead to an increase in organellar Na+ content and would result in a K+-dependent Ca2+ uptake if the organellar membrane contained NCKX. As observed in intact cells, saponinpermeabilized MEB4 cells showed K+-dependent 45Ca2+ uptake consistent with NCKX activity; siRNA knockdown suggested a predominant contribution of NCKX4, although the results are less statistically significant when compared with those of the Fluo-based assays, in large part due to the rather low resolution of the 45Ca uptake data (Figure 8). NCKX5 Activity in Permeabilized HEK293 Cells or Microsomes Isolated from Transfected HEK293 Cells. MEB4 cells have slightly higher transcript levels of NCKX5 compared with those of NCKX4 (data not illustrated), although lack of a specific NCKX5 antibody does not permit an assessment of the respective protein levels. In the experiments described so far, we identified NCKX-mediated Ca2+ fluxes in MEB4 cells that can be resolved with high resolution and represent NCKX4 found in the plasma and internal membranes. It is less clear whether NCKX5 was responsible for any of the NC(K)X-mediated Ca2+ fluxes observed so far. To demonstrate that NCKX5 is a functional Na+/Ca2+ + K+ exchanger, we expressed NCKX5 (and NCKX2 for comparison) in HEK293 cells and isolated microsomal membranes from these cells in a high NaCl medium to provide for internal Na+ loading. Using these microsomal membranes, we repeated the 45Ca uptake experiment illustrated in Figure 2. Microsomes obtained from untransfected HEK293 cells showed no evidence of K+-dependent 45Ca uptake, whereas cells transfected with either NCKX2 or NCKX5 cDNA showed well-defined K+-dependent 45Ca uptake, as uptake in a KCl

Figure 8. Effect of NCXK4 and NCKX5 siRNA on 45Ca uptake into internal stores of Na+-loaded and saponin-permeabilized MEB4 cells. Panel A: Saponin (1.5 μL of a 6% solution) was added to Na+-loaded MEB4 cells (50 μL), and the solution was incubated for 1 min. The saponin-treated cells were diluted with 350 μL of KCl, NaCl, or LiCl medium as indicated, and the 45Ca uptake assay was carried out as described in Figure 1a. Panel B: The bar diagram illustrates the ratio of 45Ca uptake in a KCl medium over 45Ca uptake in a NaCl medium as an indicator of NCKX-mediated 45Ca uptake (in the absence NCKX-mediated transport this ratio is expected to be 1). The average results from six experiments are shown, and the error bars indicate the standard error of the mean.

medium significantly exceeded that observed in a NaCl or LiCl medium (Figure 9, top three panels). We also used saponinpermeabilized HEK cells and obtained similar data as described above for microsomes (Figure 9, bottom three panels). These data clearly show that heterologously expressed NCKX5 is a functional Na+/Ca2+ + K+ exchanger and demonstrate that its activity can be measured in internal membranes using the 45Ca radiotracer assay formats described here.

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CONCLUSIONS Since the first paper reporting on the critical role of SLC24A5 (encoding the NCKX5 protein) in human skin pigmentation2 was published ten years ago, very little progress has been made in elucidating the underlying molecular mechanism. Moreover, apart from the study by Ginger et al.,4 no functional or molecular studies on NCKX5 have been published, which probably reflects the fact that, unlike the NCKX1−4 proteins, NCKX5 does not appear to be present in the plasma membrane and is consequently difficult to assay and study. In addition to SLC24A5 transcripts, B16 cells contain significant Slc24a4 transcripts,4 and 2710

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Biochemistry

Figure 9. NCKX2- and NCKX5-mediated 45Ca uptake in microsomes isolated from transfected HEK293 cells. Panels A−C: Microsomes were isolated from HEK293 cells transfected with empty vector (negative control) or hNCKX2 (positive control) and hNCKX5 cDNAs as indicated. 45Ca uptake was measured in a LiCl medium (triangles) to represent background uptake not mediated by NCKX or in a KCl medium (circles) to represent additional NCKX-mediated 45Ca uptake. As a second control, 45Ca uptake was also measured in a NaCl medium (squares), which fully inhibits NCKX-mediated uptake because the high Na+ concentration competes with Ca2+ for NCKX-mediated uptake. NCKX-mediated 45Ca uptake is the difference between uptake observed in a KCl medium and that observed in a NaCl or LiCl medium. The error bars represent standard error of the mean of four experiments. Panels D and E: NCKX activity in internal organelles of transfected HEK293 cells was also measured after selective permeabilization of the plasma membrane with 0.01% saponin. Transfected HEK cells were Na+-loaded and then treated with FCCP and 0.01% saponin. Other details are the same as in panels A−C. The error bars represent standard error of the mean of six experiments.

We believe that the NCKX4 activity and assay formats described herein for MEB4 and B16 cells will aid the study of this protein and its important role in pigmentation regulation. We demonstrated microsomal Ca2+ uptake activity mediated by NCKX5 in HEK293 cells transfected with NCKX5 cDNA.

NCKX4 is expected to be, at least in part, trafficked to and functionally active in the plasma membrane, as has been shown in the case of transfected HEK293 cells.16,18 Here, we presented a detailed analysis of Na+-dependent Ca2+ fluxes in the pigmented B16 and MEB4 cell lines. We conclude that the predominant NCKX exchange activity present in B16 and MEB4 pigmented cells, both in the plasma membrane and in internal membranes, arises from NCKX4 (Figures 3, 4, 7, and 8). We previously reported that dark and light normal human epidermal melanocytes have NCKX5 but not NCKX4 transcripts,4 and we failed to detect any evidence of NCKX transport function in the surface membrane of these cells (data not shown). It is consistent with our observations on B16 and MEB4 cells herein that NCKX activity observed in these cells can be accounted for by the presence of NCKX4, and any contribution by NCKX5 remains unclear. Our 45Ca assay format is capable of detecting internal NCKX5 activity in microsomes derived from transfected HEK293 cells (Figures 9b and e), showing that NCKX5 operates as a K+-dependent Na+/Ca2+ exchanger. However, we were unable to confirm with certainty that any of the NCKX activity observed here in MEB4 cells represented NCKX5 activity, as assessed with several different assay formats, and this likely reflects the fact that NCKX5 resides in an internal compartment inaccessible to our assay formats. Given our observation that MEB4 cells contain very similar amounts of NCKX4 and NCKX5 transcripts, we do not believe that our assays lack the sensitivity to detect NCKX5 activity, as we can detect NCKX4 activity with significant resolution.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Funding

This research was funded in part by an operating grant from the Canadian Institutes for Health Research (PPMS, CIHR MOP-81327) and in part by a sponsored research agreement with Unilever R&D (PPMS). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS NCKX, K+-dependent Na+/Ca2+ exchangers; SLC24, solute carrier 24 gene family; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; DCB, 3′4′-dichlorobenzamil; EDTA, ethylenediaminetriacetic acid; HEDTA, hydroxyethyl ethylenediaminetriacetic acid; TBST, Tris-buffered saline and Tween; Km, Michaelis−Menten constant 2711

DOI: 10.1021/acs.biochem.6b00017 Biochemistry 2016, 55, 2704−2712

Article

Biochemistry

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(15) Altimimi, H. F., and Schnetkamp, P. P. M. (2006) Na+dependent inactivation of the retinal cone/brain Na+/Ca2+-K+ exchanger NCKX2. J. Biol. Chem. 282, 3720−3729. (16) Jalloul, A. H., Szerencsei, R. T., and Schnetkamp, P. P. (2016) Cation dependencies and turnover rates of the human K(+)dependent Na(+)-Ca(2+) exchangers NCKX1, NCKX2, NCKX3 and NCKX4. Cell Calcium 59, 1−11. (17) Palty, R., Silverman, W. F., Hershfinkel, M., Caporale, T., Sensi, S. L., Parnis, J., Nolte, C., Fishman, D., Shoshan-Barmatz, V., Herrmann, S., Khananshvili, D., and Sekler, I. (2010) NCLX is an essential component of mitochondrial Na+/Ca2+ exchange. Proc. Natl. Acad. Sci. U. S. A. 107, 436−441. (18) Li, X. F., Kraev, A. S., and Lytton, J. (2002) Molecular cloning of a fourth member of the potassium-dependent sodium-calcium exchanger gene family, NCKX4. J. Biol. Chem. 277, 48410−48417. (19) Schnetkamp, P. P., Jalloul, A. H., Liu, G., and Szerencsei, R. T. (2014) The SLC24 family of K(+)-dependent Na(+)-Ca(2)(+) exchangers: structure-function relationships. Curr. Top. Membr. 73, 263−287.

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DOI: 10.1021/acs.biochem.6b00017 Biochemistry 2016, 55, 2704−2712