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Modulation of Na⁺-Ca²⁺ Exchanger Expression by Immunosuppressive Drugs Is Isoform-Specific

Department of Biochemistry Hebrew University-Hadassah Medical School, Jerusalem, Israel (B.E., A.A., H.R.); Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (B.E., M.M.G., H.R.); and Center for Biologics Evaluation and Research, United States Food and Drug Administration, Bethesda, Maryland (C.K.)

	Abstract

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The Na⁺-Ca²⁺ exchanger (NCX) is a major Ca²⁺-regulating protein encoded by three genes: NCX1, NCX2, and NCX3. They share a sequence homology of approximately 65%. NCX1 protein is expressed ubiquitously, and NCX2 and NCX3 are expressed almost exclusively in the brain. We have shown previously (Kimchi-Sarfaty et al., 2002) that treatment of NCX1-transfected human embryonic kidney (HEK) 293 cells with the immunosuppressive cyclosporin A (CsA) and its nonimmunosuppressive analog PSC833 (valspodar) results in down-regulation of surface expression and transport activity of the protein without a decrease in expression of cell NCX1 protein. In this study, we show that cyclosporin A and PSC833 treatment of NCX2- and NCX3-transfected HEK 293 cells also resulted in dose-dependent down-regulation of surface expression and transport activity of the two brain NCX proteins; however, whereas CsA had no effect on total cell NCX protein expression, PSC833 reduced mRNA and cell protein expression of NCX2 and NCX3. Moreover, tacrolimus (FK506), which had no effect on NCX1 protein expression, down-regulated NCX2 and NCX3 surface expression and transport activity without any significant effect on cell protein expression. Sirolimus (rapamycin) had no effect on NCX2 and NCX3 protein expression, yet it reduced NCX2 and NCX3 transport activity. Because all of the experimental conditions in our studies were identical, presumably the different drug response is related to structural differences between NCX isoforms. Clinical studies suggested that immunosuppressive regimes of patients who have received transplants resulted in complications related to Ca²⁺. Expression of NCX genes is tissue-specific. Hence, our results can potentially provide a tool for choosing the immunosuppressive protocol to be used.

Immunophilins are also involved in protein folding, mediated by two different activities localized within separate protein domains: peptidyl prolyl cis-trans isomerase (PPIase) activity, which is rate-limiting in acquisition of configuration of X-proline peptide bonds, and chaperone activity (Galat, 2003; Barik, 2006). The role of both activities in acquisition of functional conformation of proteins was supported by many studies, and the relevant protein segments responsible for these activities were identified (Fischer et al., 1989; Pirkl et al., 2001; Galat, 2003; Barik, 2006; Mok et al., 2006). Binding of immunosuppressive drugs to their immunophilins receptors inhibits both PPIase and chaperon activity (Galat, 2003; Barik, 2006).

View larger version (19K): [in this window] [in a new window]   Fig. 1. The effect of different concentrations of CsA on Na+-dependent Ca2+ uptake in HEK 293 cells expressing the FN-NCX2 and FN-NCX3 proteins. Cells were transfected with plasmids encoding the FN-NCX2 (A) and FN-NCX3 (B). Na+-dependent Ca2+ uptake was determined 24 h after transfection, as described under Materials and Methods, without and with exposure of the expressing cells to 10, 20, and 30 µM CsA. Transport activities of FN-NCX2- and FN-NCX3-transfected cells with DMSO treatment were taken as 100%, and the transport activities measured in the drug-treated cells were calculated in relative values. The bars represent S.D. (*, P < 0.05; **, P < 0.01).

The Na⁺-Ca²⁺ exchanger is a major Ca²⁺-regulating protein expressed in all excitable and many nonexcitable cells. It transports Ca²⁺ across the plasma membrane in a bidirectional manner in response to driving Na⁺ gradient and changes in membrane potential (Blaustein and Lederer, 1999). Three separate genes, NCX1, NCX2, and NCX3, code for this activity (Philipson and Nicoll, 2000). Whereas NCX1 gene products are almost ubiquitously expressed, NCX2 and NCX3 are expressed mostly in the brain (Quednau et al., 1997; Blaustein and Lederer, 1999; Annunziato et al., 2004). NCX1, NCX2, and NCX3 share an overall sequence homology of 65% (Annunziato et al., 2004), which is highest in the transmembrane helices.

We have shown previously that treatment of human embryonic kidney (HEK) 293 cells transfected with the NCX1.1 and NCX1.5 genes with CsA and its nonimmunosuppressive analog PSC833 (valspodar; SDZ 215-833) (Boesch et al., 1991), results in a dose-dependent decrease of surface expression and Na⁺-Ca²⁺ exchange activity without a significant change in total cell NCX1 protein (Kimchi-Sarfaty et al., 2002). Neither FK506 nor rapamycin treatment of transfected HEK 293 cells had any effect on expression of the Na⁺-Ca²⁺ exchanger NCX1. Moreover, we have shown that CsA treatment of L6, H9c2, and primary cultured smooth muscle cells, all expressing the Na⁺-Ca²⁺ exchanger NCX1, led to down-regulation of transport activity and surface expression (Rahamimoff et al., 2007). This result suggests that the phenomenon was not restricted to heterologous expression systems. However, not all membrane proteins are modulated by treatment with CsA. For example, no reduction in functional expression of the human multidrug transporter (MDR) P-glycoprotein, transfected into HEK 293 cells, was obtained by treatment with CsA (Kimchi-Sarfaty et al., 2002).

Clinical studies suggested that immunosuppressive regimes of patients who have received transplants resulted in complications related to Ca²⁺ such as nephrotoxicity, hypertension, bone loss, and neurotoxicity (Cameron et al., 1995; Cardenas et al., 1995; Bechstein, 2000; Hariharan et al., 2000). CsA, FK506, rapamycin, and PSC833 were shown to cross the blood-brain barrier (Shirai et al., 1994; Lemaire et al., 1996; Kochi et al., 1999; Tai, 2000; Pong and Zaleska, 2003; Hsiao et al., 2006). On the basis of these findings, we decided to assess the effects of CsA, FK506, rapamycin, and PSC833 on the expression of NCX2 and NCX3 proteins.

In this study, we show that, with the exception of CsA, which modulated the expression of all three NCX proteins (NCX1, NCX2, and NCX3) in a similar manner, all other immunosuppressive and nonimmunosuppressive drugs tested (PSC833, FK506, and rapamycin) modulated functional expression of NCX2 and NCX3 in transfected HEK 293 cells in a different manner than they modulated the expression of NCX1.

	Materials and Methods

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Cell Line and Cell Culture. HEK 293 cells (American Type Culture Collection, Manassas, VA) were used in all of the transfection experiments, and they were grown in Dulbecco's modified Eagle's medium (Biological Industries Ltd., Kibbutz Beit Haemek, Israel or Invitrogen, Carlsbad, CA) with 1% glutamine, 1% penicillin-streptomycin, and 10% fetal bovine serum (Biological Industries Ltd. or Gibco BRL Life Technologies) at 37°C in humid air containing 5% CO₂.

View larger version (30K): [in this window] [in a new window]   Fig. 2. The effect of various concentrations of CsA on the surface and total protein expression of the FN-NCX2 and FN-NCX3 proteins expressed in HEK 293 cells. Cells expressing the FN-NCX2 and FN-NCX3 were treated with various concentrations of CsA (10-30 µM) or equivalent volume of DMSO. Twenty-four hours after transfection, the cells were analyzed for cell surface and total expression using M2 anti-FLAG antibody and Alexa-Green 488 anti-mouse secondary antibody. A, nonpermeabilized FN-NCX2-transfected cells treated with 0 to 30 µM CsA. B, permeabilized FN-NCX2-transfected cells treated with 0 to 30 µM CsA. C, nonpermeabilized FN-NCX3-transfected cells treated with 0 to 30 µM CsA. D, permeabilized FN-NCX3-transfected cells treated with 0 to 30 µM CsA. The key to the lines used in all panels is identical (see A).

Transfection Procedure. Transfection was carried out with the Lipofectamine and Plus reagents (Invitrogen) according to the manufacturer's protocol. One or 2 µg of pDNA was used to transfect cells plated into one well of a 12- or 6-well plate, respectively. In some transfection experiments, calcium phosphate (Sambrock et al., 1989) was used. No significant differences in the relative transport activities without or with drug treatment were observed between the two transfection reagents. Each type of the transfection experiments was repeated four to six times. The efficiency of the transfection was calculated from statistical values obtained from FACS analyses (see Immunostaining to Detect Total and Surface Expression of the Na⁺-Ca²⁺ Exchanger by FACS, below), by measuring only the area under the curve of the positive transfected/treated sample (M2)—not including the area under the curve of the control sample (M1). The average number of transfected cells was 68% (range, 56-84%).

Drug Treatments. Cyclosporin A (Calbiochem Corp., San Diego, CA), FK506 (LC Laboratories, Woburn MA), rapamycin (Calbiochem Corp. or LC Laboratories), and PSC833 (a gift to H.R. from Novartis Pharma AG, Basel, Switzerland) were dissolved in dimethyl sulfoxide (DMSO) and added at 3 h after transfection together with the fetal bovine serum-Dulbecco's modified Eagle's medium supplement to the transfected cells. The amount of DMSO added to each well was equal, and the total volume never exceeded 1% of the volume in the wells.

The effect of each drug on the transport assay (see Determination of Na⁺-Dependent Ca²⁺ Uptake) was examined as well. This was done by adding different concentrations of each drug to both the buffered Ca²⁺-containing NaCl and KCl solutions directly, and no drug was added to the transfected cells (see Table 1).

Determination of Na⁺-Dependent Ca²⁺ Uptake. Determination of transport activity in whole cells was carried out essentially as described previously (Kasir et al., 1999; Kimchi-Sarfaty et al., 2002). In principle, expressing cells were preloaded with 0.16 M NaCl and 0.01 M Tris-HCl, pH 7.4, using 25 µM nystatin (Sigma-Aldrich, Rehovot, Israel). Cells were washed with the same buffered NaCl solution (without MgCl₂) to remove nystatin. Transport was initiated by overlaying the cells with the same buffered Na⁺ or K⁺ containing solution, to which 25 µM ⁴⁵Ca²⁺ (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK) was added. All solutions also contained 1 mM ouabain (Sigma-Aldrich). Na⁺-dependent Ca²⁺ uptake was determined by subtracting the Ca²⁺ taken up in the absence of a Na⁺ gradient from that taken up in its presence. In some experiments, Na⁺-preloaded cells were collected by centrifugation at 1500 rpm at 4°C and suspended in a minimal volume of the buffered NaCl solution (without MgCl₂). A total of 3 µl of the Na⁺-loaded cells (approximately 40 µg of protein) were diluted into 100 µl of buffered K⁺ or Na⁺ to which 25 µM ⁴⁵Ca²⁺ was added (the same solution as described above). The transport reactions were terminated by filtration via 0.45-µm filters (Whatman Schleicher and Schuell, Keene, NH). Washes of the filters and calculation of the net Na⁺-dependent Ca²⁺ uptake was done as described above for adherent cells in situ. Transport measurements were done in triplicate, and each experiment was repeated four to six times. In each experiment, the transport activity of FN-NCX2- and FN-NCX3-transfected cells with DMSO treatment was taken as 100%, and the transport activities measured in the drug-treated transfected cells were calculated in relative values.

View larger version (24K): [in this window] [in a new window]   Fig. 3. Determination of the transport activities of HEK 293 cells expressing the FN-NCX2 and FN-NCX3 proteins exposed to different concentrations of FK506. Cells were transfected with plasmids encoding the FN-NCX2 (A) and FN-NCX3 (B). Na+-dependent Ca2+ uptake was determined 24 h after transfection (as described under Materials and Methods) without and with exposure of the expressing cells to 10, 20, and 30 µM FK506. The transport activity of FN-NCX2- and FN-NCX3-transfected cells with DMSO treatment was taken as 100%, and the transport activities measured in the drug-treated cells were calculated in relative values. The bars represent S.D. (*, P < 0.05; **, P < 0.01).

View larger version (30K): [in this window] [in a new window]   Fig. 4. The effect of FK506 on the surface and total FN-NCX2 and FN-NCX3 protein expressed in HEK 293 cells. Parallel transfections to those described in Fig. 3 were carried out. Twenty-four hours after transfection, the cells were analyzed for cell surface and total NCX protein expression using M2 anti-FLAG antibody and FITC-conjugated anti-mouse secondary antibody. A and C, nonpermeabilized FN-NCX2 (A) and FN-NCX3 (C)-transfected cells treated with FK506. B and D, permeabilized FN-NCX2 (B) and FN-NCX3 (D)-transfected cells treated with 0, 20, 30, and 50 µM FK506. The key to the lines used in all panels is identical to that in A.

View larger version (19K): [in this window] [in a new window]   Fig. 5. Determination of the transport activities of HEK 293 cells expressing the FN-NCX2 and FN-NCX3 proteins exposed to different concentrations of rapamycin. Cells were transfected with plasmids encoding FN-NCX2 (A) and FN-NCX3 (B). Na+-dependent Ca2+ uptake was determined 24 h after transfection (as described under Materials and Methods) without and with exposure of the expressing cells to 10 and 20 µM rapamycin. The transport activity of FN-NCX2- and FN-NCX3-transfected cells with DMSO treatment was taken as 100%, and the transport activities measured in the drug-treated cells were calculated in relative values. The bars represent S.D. (*, P < 0.05; **, P < 0.01).

Determination of FN-NCX2 and FN-NCX3 mRNA by Quantitative PCR. RNA was isolated (1 µg) from FN-NCX2 and from FN-NCX3-transfected HEK 293 cells, which were without or with PSC833 treatment, using TRI Reagent (Sigma-Aldrich) according to the manufacturer's instructions. cDNA synthesis was carried out with the RobusT II RT-PCR kit (Finnzymes, Espoo, Finland) according to the manufacturer's instructions.

Quantitative real-time PCR was done using the TaqMan Gene Expression assay (Applied Biosystems, Foster City, CA). The reaction was carried out in an ABI Prism 7000 spectrofluorometric thermal cycler (Applied Biosystems). The assay identification of the genes was as follows: Rn00589573_m1 (for NCX2); Rn01517854_m1 (for NCX3); and Hs99999910_m1 (for TATA box binding protein, used as endogenous control) (purchased from Applied Biosystems). Data analysis was done using ABI Prism 7000 software (Applied Biosystems). Each RNA sample was isolated from three separate transfections. Each assay was done in triplicate (for each NCX and respective TATA box binding protein).

	Results

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Cyclosporin A Treatment of HEK 293 Cells Transfected with FN-NCX2 and FN-NCX3 Resulted in Down-Regulation of the Na⁺-Dependent Ca²⁺ Uptake and Surface Expression. To examine the effects of CsA on the Na⁺-Ca²⁺ exchangers NCX2 and NCX3, we transfected HEK 293 cells with the cloned FN-NCX2 or cloned FN-NCX3. The transfected cells were exposed to 10 to 30 µM CsA in DMSO or an equal volume of DMSO, and Na⁺-dependent Ca²⁺ uptake was determined 24 h after transfection, as described under Materials and Methods. Figure 1, A and B, summarize the results of these experiments. As can be seen, exposure of the cells expressing the cloned exchangers to CsA resulted in a reduction of Na⁺-dependent Ca²⁺ uptake in a concentration-dependent manner. Exposure of the cells expressing the transporter to 10 µM CsA or above resulted in a significant decrease of the transport activity relative to that expressed in the absence of the drug. Control experiments in which the cells were not exposed to the drug during transfection and the drug was added only to the transport solutions during the assay did not result in a decrease in the Na⁺-dependent Ca²⁺ uptake (see Table 1).

To study the effects of CsA on the surface and total expression of the Na⁺-Ca²⁺ exchangers FN-NCX2 and FN-NCX3, parallel transfections to those used for transport experiments were carried out. Surface and total protein expression was determined by measuring the fluorescence intensity using M2 antibody staining via FACS analysis (for details, see Materials and Methods). Figure 2, A and B, show the surface and total FN-NCX2 protein expression, and Fig. 2, C and D, show the surface and total FN-NCX3 at 0, 10, 20, and 30 µM CsA. It can be seen that the fluorescence of M2 antibody cell surface-labeled proteins decreases in a concentration-dependent manner that parallels the decrease of their respective transport activities (Fig. 1). The mean fluorescence intensity (MFI) was calculated by compiling the MFI data from three separate transfections. The MFI of surface-expressed protein in 0 CsA (DMSO-treated cells) was taken as 100%, and all other values were normalized. For FN-NCX2-expressing cells, calculations were as follows: 0 to 100; 10 µM, 74.6 (S.D. 14.1); 20 µM, 67.1 (S.D. 14.2); and 30 µM, 25.8 (S.D. 8.9). For FN-NCX3-expressing cells, calculations were as follows: 0 to 100; 10 µM, 64.4 (S.D. 7.6); 20 µM, 46.3 (S.D. 15.9); and 30 µM, 32.6 (S.D. 11.1). The immunofluorescence detected in permeabilized cells (total cell NCX protein) was calculated in the same manner. The average MFIs did not significantly change when they were exposed to the same concentration of CsA.

View larger version (32K): [in this window] [in a new window]   Fig. 6. The effect of rapamycin on surface and total FN-NCX2 and FN-NCX3 protein expressed in HEK 293 cells. Parallel transfections to those described in Fig. 5 were carried out. Twenty-four hours after transfection, the cells were analyzed for cell surface and total NCX protein expression using M2 anti-FLAG antibody and FITC-conjugated anti-mouse secondary antibody. A and C, nonpermeabilized FN-NCX2 (A) and FN-NCX3 (C)-transfected cells. B and D, permeabilized FN-NCX2 (B) and FN-NCX3 (D)-transfected cells. The key to the lines used in all panels is identical to that shown in A.

Rapamycin Reduced Na⁺-Dependent Ca²⁺ Uptake Activity but Had No Effect on Surface Expression and Total FN-NCX2 and FN-NCX3 Protein Expression. We have also examined the effect of rapamycin treatment (5-20 µM) on the expression of FN-NCX2- and FN-NCX3-transfected HEK 293 cells. Rapamycin treatment of FN-NCX2- and FN-NCX3-transfected HEK 293 resulted in a decrease in FN-NCX2 and FN-NCX3 Na⁺-dependent Ca²⁺ uptake activity (Fig. 5, A and B). However, rapamycin treatment had no effect on surface or total FN-NCX2 and FN-NCX3 protein expression (Fig. 6, A-D).

Rapamycin could have potentially inhibited the transport assay. To rule out this possibility, we have not added the drug to the cells during the transfection procedure but added it directly to the transport assay. Addition of rapamycin to the transport assay did not inhibit Na⁺-dependent Ca²⁺ uptake activity (Table 1).

The Nonimmunosuppressive PCS833 Reduced Surface Expression, Na⁺-Ca²⁺ Exchange Activity, mRNA, and Total Immunoreactive Cell FN-NCX2 and FN-NCX3 Protein. PSC833 is a nonimmunosuppressive analog of CsA. Hence, it was interesting to examine its effect on the expression of FN-NCX2 and FN-NCX3 in transfected HEK 293 cells. Figures 7 and 8 summarize these data. In Figs. 7A (FN-NCX2-transfected cells) and 8A (FN-NCX3-transfected cells), it can be seen that treatment of HEK 293 cells with PSC833 leads to dose-dependent reduction in Na⁺-Ca²⁺ exchange activity. The sensitivity of the FN-NCX2 and FN-NCX3 proteins to PSC833 is higher than that to CsA: 20 µM PSC833 reduces approximately 20% of the relative transport activity, and treatment of the cells with 30 µM PSC833 reduces the expression of the relative transport activity to approximately 10% compared with untreated (DMSO only) transfected HEK 293 cells.

View larger version (31K): [in this window] [in a new window]   Fig. 7. The effect of different concentrations of PSC833 on the expression of FN-NCX2 in HEK 293 cells. Na+-dependent Ca2+ uptake, surface expression, mRNA expression, and total FN-NCX2 protein expression were determined 24 h after transfection (as described under Materials and Methods) without and with exposure of the transfected cells to 0 to 30 µM PSC833. A, The transport activity of FN-NCX2-transfected cells with DMSO treatment was taken as 100%, and the transport activities measured in the drug-treated cells were calculated in relative values. The bars represent S.D. (*, P < 0.05; **, P < 0.01). B, surface expression was determined by FACS analysis and by surface biotinylation (insert in B). The M2 anti-FLAG antibody and FITC anti-mouse secondary antibody were used for detection of FN-NCX2 by FACS analysis, and the HRP-conjugated anti-mouse secondary antibody was used for detection of surface-biotinylated FN-NCX2. C, FN-NCX2 mRNA levels were measured by quantitative PCR as described under Materials and Methods. All results were normalized to untreated samples and TATA box binding protein values. The S.D. represents results from three independent experiments. D, Western blot analysis was used to determine total immunoreactive FN-NCX2 protein using the M2 anti-FLAG antibody and HRP-conjugated anti-mouse secondary antibody. Twenty micrograms of transfected cell extract was loaded on each lane. Immunodetection of actin was used as an equal loading control.

View larger version (30K): [in this window] [in a new window]   Fig. 8. The effect of different concentrations of PSC833 on the expression of FN-NCX3 in HEK 293 cells. Na+-dependent Ca2+ uptake, surface expression, mRNA expression, and total FN-NCX3 protein expression were determined 24 h after transfection (as described under Materials and Methods) without and with exposure of the transfected cells to 0 to 30 µM PSC833. Experimental conditions and statistical analysis are identical to those described in detail in Fig. 7.

In a manner similar to the effect of CsA treatment of transfected FN-NCX2 and FN-NCX3 HEK 293 cells, PSC833 treatment resulted in reduced dose-dependent surface protein expression. This was shown by immunostaining and FACS analysis (Fig. 7B) and surface biotinylation (insert in Fig. 7B) for FN-NCX2-transfected cells, and the same result is shown in Fig. 8B for FN-NCX3-transfected cells. Immunostaining with M2, the anti-FLAG antibody, shows that FN-NCX2 and FN-NCX3 proteins migrate as a double band (insert in Figs. 7B and 8B). This presumably represents two slightly different conformational forms derived both from the same cloned Na⁺-Ca²⁺ exchanger, because only the cloned constructs bear the FLAG epitope, which is stained by the monoclonal antibody.

Unlike the effect of CsA on FN-NCX2 and FN-NCX3 expression, PSC833 treatment resulted in dose-dependent reduction of total cell FN-NCX2 protein (Fig. 7D) and respective FN-NCX3 and protein (Fig. 8D). To elucidate the mode of action of PSC833 on total FN-NCX2 and FN-NCX3 cell protein expression, we have measured the corresponding mRNA levels (Figs. 7C and 8C) without (DMSO-treated cells) and with PSC833 treatment by quantitative PCR. The PSC833-dependent reduction in mRNA levels suggests that the effect of PSC833 on FN-NCX2 and FN-NCX3 expression is at the transcriptional level.

	Discussion

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In this study, we have examined the effect of the commonly used immunosuppressive drugs CsA, FK506, and rapamycin and the nonimmunosuppressive PSC833 on the expression of NCX2 and NCX3 proteins in HEK 293 cells. This was done because many of the immunosuppressive drug-related clinical complications are linked to impaired homeostasis of cell Ca²⁺, and the Na⁺-Ca²⁺ exchanger is a major Ca²⁺ regulator. The exchanger is the only surface membrane Ca²⁺ handling protein that transports Ca²⁺ in a bidirectional manner. Direction of Ca²⁺ flux changes in response to changes in membrane potential and respective Na⁺ and Ca²⁺ gradients. Hence, changes in NCX expression can potentially modulate Ca²⁺ efflux, influx, and homeostasis (Blaustein and Lederer, 1999).

Na⁺-Ca²⁺ exchange activity is encoded by three genes: NCX1, NCX2, and NCX3. They share considerable sequence homology (Quednau et al., 1997). Therefore, we expected that the effects of CsA, FK506, rapamycin, and PSC833, which we described previously on the expression of NCX1 (Kimchi-Sarfaty et al., 2002; Rahamimoff et al., 2002), would be similar. We were surprised to find that only the effect of CsA treatment of NCX2- and NCX3-transfected cells is similar to that observed with NCX1-transfected cells, leading to down-regulation of the expression of each one of the NCX proteins in the surface membrane in a dose-dependent manner, parallel reduction in the Na⁺-Ca²⁺ exchange activity, and no significant change in immunoreactive total cell NCX protein. These findings can be explained by CsA binding to cyclophilin and inhibition of either the cis-trans isomerization of target X-Pro peptide bonds and/or chaperone activity. This would result in impaired post-translational NCX protein maturation, folding, and cell retention (Kopito, 1997). In addition, CsA treatment could indirectly impair trafficking between organelles, increase membrane retrieval, or modify any other post-translational process that could reduce surface expression of the protein and not change total NCX protein (Ellgaard and Helenius, 2003).

Our studies show three major differences, elucidated by the drug treatments, between NCX1-expressing cells and cells expressing NCX2 and NCX3 proteins: 1) their response to the immunosuppressive FK506 treatment, 2) their response to the immunosuppressive rapamycin treatment, and 3) their response to the nonimmunosuppressive PSC833 treatment. NCX1 expression was not modulated by FK506 or by rapamycin treatments of transfected HEK 293 cells (Kimchi-Sarfaty et al., 2002; Rahamimoff et al., 2002). However, when FN-NCX2- and FN-NCX3-transfected cells were treated with FK506, surface expression and Na⁺-dependent Ca²⁺ uptake of both FN-NCX2 and FN-NCX3 decreased in a concentration-dependent manner without any change in total cell NCX protein. This finding suggests that FK506 treatment in a similar manner to CsA treatment of transfected cells could have impaired NCX2 and NCX3 protein folding and/or maturation, in a post-translational manner, presumably by inhibition of PPIase and/or chaperon activity of FKBP or any other unknown mechanism as suggested for the mode of action of CsA.

Because total cell NCX2 and NCX3 protein was not reduced by exposing the transfected HEK 293 cells to CsA and to FK506, presumably drug-dependent inhibition of transcription via the calcineurin pathway (or another unknown pathway) was not involved. Although both FK506 and rapamycin bind to immunophilins from the FKBP family, rapamycin treatment of FN-NCX2- and FN-NCX3-transfected HEK 293 cells did not result in any decrease in either surface or total immunoreactive protein expression, yet a decrease in Na⁺-dependent Ca²⁺ uptake was consistently obtained. This suggests that rapamycin modulates FN-NCX2 and FN-NCX3 expression in a different manner than FK506. Because addition of rapamycin had no effect on the transport assay itself, it is possible that it impaired (in an unknown yet mode of action) the correct functional expression of the FN-NCX2 and FN-NCX3 protein, and the impaired protein bypassed the quality control in the endoplasmic reticulum (Kopito, 1997) and trafficked to the surface membrane. Wild-type-like surface expression of impaired protein has been described previously in several cases. For example, mutant G420H of the scavenger receptor class B type I (Parathath et al., 2007) exhibited reduced high-density lipoprotein cholesterol ester uptake but had wild-type-like surface expression and total receptor protein expression. A deletion mutant of P-glycoprotein (Loo and Clarke, 1999) that was cell retained did express in the surface membrane after transfected HEK 293 cells were treated with verapamil, vinblastine, capsaicin, or CsA. P112L mutant of NCX1 protein (Lichtenstein, 2004) exhibited no Na⁺-dependent Ca²⁺ uptake activity when expressed in HEK 293 cells, yet it had wild-type-like surface expression. Haplotype C1236T-G2677T-C3435T of MDR1 exhibited altered function but wild-type-like total protein and cell surface expression (Kimchi-Sarfaty et al., 2007). Additional studies are needed to provide an explanation of how these altered proteins bypass cellular quality control and express in the surface membrane. In addition, it is interesting to speculate that rapamycin could have potentially impaired folding of FN-NCX2 and FN-NCX3 nascent chains in the ribosomal exit tunnel (Etchells and Hartl, 2004; Amit et al., 2005) to which they bind.

PSC833 is a nonimmunosuppressive analog of CsA, mostly studied as a potent MDR modulator (Smith et al., 1998). To provide an explanation for our results, its involvement in modulating protein expression directly or by binding to immunophilins without inhibition of calcineurin needs to be studied.

Taking these data together, we suggest that the different response of NCX2 and NCX3 proteins to immunosuppressive and nonimmunosuppressive drugs studied in this research, compared with the response of NCX1 protein that we described previously (Kimchi-Sarfaty et al., 2002), is due to the structural differences among the three NCX exchangers. In every other respect, the experiments were done using identical experimental conditions: Lipofectamine and Plus reagent or calcium phosphate-mediated transfection of HEK 293 cells and pcDNA 3.1, the mammalian expression vector encoding each one of the NCX genes. The cells express a repertoire of immunophilins that can bind CsA, FK506, and rapamycin (Rahamimoff et al., 2002). Therefore, the interaction of the immunophilins-drug complex with NCX and the modulation of its expression, which involves protein-protein interaction, are specific for the appropriate NCX protein and the appropriate drug. However, these still have to be elucidated.

The Na⁺-Ca²⁺ exchanger genes and their multiple isoforms are expressed in a tissue-selective manner (Quednau et al., 1997; Annunziato et al., 2004). The drug concentrations that were used in our study are medically relevant. In this respect, it is interesting that clinical research studies comparing the action of FK506 and CsA suggest that clinical complications, patient survival, and graft survival in patients who have received kidney transplants are better when FK506 is used for immunosuppression (First, 2004) rather than CsA. It is possible that one of the contributing factors to this observation could be our finding that the surface expression of NCX1, the major and most abundant Na⁺-Ca²⁺ exchanger, is not modulated by FK506 treatment.

	Acknowledgements

 
We thank George Leiman (National Cancer Institute, National Institutes of Health, Bethesda, MD) for help with the preparation of the manuscript. The findings and conclusions in this article have not been formally disseminated by the United States and Food and Drug Administration and should not be construed to represent any agency determination or policy.

	Footnotes

 
This study was supported in part by research grants from the Israel Science Foundation, the Israel Ministry of Health (to H.R.), and the Intramural Research Program of the National Institutes of Health, National Cancer Institute (to M.M.G.).

ABBREVIATIONS: CsA, cyclosporin A; FKBP, FK506 binding protein; PPIase, peptidyl prolyl cis-trans isomerase; NCX, Na⁺-Ca²⁺ exchanger; HEK, human embryonic kidney; MDR, multidrug transporter; PCR, polymerase chain reaction; FN, FLAG epitope tagged; DMSO, dimethyl sulfoxide; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; NHS-SS-biotin, 2-(biotinamido)-ethyl-1,3-dithiopropionate; HRP, horseradish peroxidase; MFI, mean fluorescence intensity; PSC833, valspodar.

Address correspondence to: Dr. Hannah Rahamimoff, Department of Biochemistry, Hebrew University-Hadassah Medical School, P.O. Box 12272, Jerusalem 91120, Israel. E-mail: hannah.rahamimoff{at}huji.ac.il

	References

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Amit M, Berisio R, Baram D, Harms J, Bashan A, and Yonath A (2005) A crevice adjoining the ribosome tunnel: hints for cotranslational folding. FEBS Lett 579: 3207-3213.[CrossRef][Medline]

Annunziato L, Pignataro G, and Di Renzo GF (2004) Pharmacology of brain Na⁺/Ca²⁺ exchanger: from molecular biology to therapeutic perspectives. Pharmacol Rev 56: 633-654.[Abstract/Free Full Text]

Barik S (2006) Immunophilins: for the love of proteins. Cell Mol Life Sci 63: 2889-2900.[CrossRef][Medline]

Bechstein WO (2000) Neurotoxicity of calcineurin inhibitors: impact and clinical management. Transpl Int 13: 313-326.[CrossRef][Medline]

Blaustein MP and Lederer WJ (1999) Sodium/calcium exchange: its physiological implications. Physiol Rev 79: 763-854.[Abstract/Free Full Text]

Boesch D, Gaveriaux C, Jachez B, Pourtier-Manzanedo A, Bollinger P, and Loor F (1991) In vivo circumvention of P-glycoprotein-mediated multidrug resistance of tumor cells with SDZ PSC 833. Cancer Res 51: 4226-4233.[Abstract/Free Full Text]

Cameron AM, Steiner JP, Sabatini DM, Kaplin AI, Walensky LD, and Snyder SH (1995) Immunophilin FK506 binding protein associated with inositol 1,4,5-trisphosphate receptor modulates calcium flux. Proc Natl Acad Sci U S A 92: 1784-1788.[Abstract/Free Full Text]

Cardenas ME, Zhu D, and Heitman J (1995) Molecular mechanisms of immunosuppression by cyclosporine, FK506, and rapamycin. Curr Opin Nephrol Hypertens 4: 472-477.[Medline]

Chen H, Kubo Y, Hoshi T, and Heinemann SH (1998) Cyclosporin A selectively reduces the functional expression of Kir2.1 potassium channels in Xenopus oocytes. FEBS Lett 422: 307-310.[CrossRef][Medline]

Ellgaard L and Helenius A (2003) Quality control in the endoplasmic reticulum. Nat Rev Mol Cell Biol 4: 181-191.[CrossRef][Medline]

Etchells SA and Hartl FU (2004) The dynamic tunnel. Nat Struct Mol Biol 11: 391-392.[CrossRef][Medline]

First MR (2004) Tacrolimus based immunosuppression. J Nephrol 17 (Suppl 8): S25-S31.

Fischer G, Wittmann-Liebold B, Lang K, Kiefhaber T, and Schmid FX (1989) Cyclophilin and peptidyl-prolyl cis-trans isomerase are probably identical proteins. Nature 337: 476-478.[CrossRef][Medline]

Galat A (2003) Peptidylprolyl cis/trans isomerases (immunophilins): biological diversity-targets-functions. Curr Top Med Chem 3: 1315-1347.[CrossRef][Medline]

Hariharan S, Johnson CP, Bresnahan BA, Taranto SE, McIntosh MJ, and Stablein D (2000) Improved graft survival after renal transplantation in the United States, 1988 to 1996. N Engl J Med 342: 605-612.[Abstract/Free Full Text]

Helekar SA, Char D, Neff S, and Patrick J (1994) Prolyl isomerase requirement for the expression of functional homo-oligomeric ligand-gated ion channels. Neuron 12: 179-189.[CrossRef][Medline]

Helekar SA and Patrick J (1997) Peptidyl prolyl cis-trans isomerase activity of cyclophilin A in functional homo-oligomeric receptor expression. Proc Natl Acad Sci U S A 94: 5432-5437.[Abstract/Free Full Text]

Ho SN, Hunt HD, Horton RM, Pullen JK, and Pease LR (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77: 51-59.[CrossRef][Medline]

Hsiao P, Sasongko L, Link JM, Mankoff DA, Muzi M, Collier AC, and Unadkat JD (2006) Verapamil P-glycoprotein transport across the rat blood-brain barrier: cyclosporine, a concentration inhibition analysis, and comparison with human data. J Pharmacol Exp Ther 317: 704-710.[Abstract/Free Full Text]

Kasir J, Ren X, Furman I, and Rahamimoff H (1999) Truncation of the C-terminal of the rat brain Na⁺-Ca²⁺ exchanger RBE-1 (NCX1.4) impairs surface expression of the protein. J Biol Chem 274: 24873-24880.[Abstract/Free Full Text]

Kimchi-Sarfaty C, Kasir J, Ambudkar S, and Rahamimoff H (2002) Transport activity and surface expression of the Na⁺-Ca²⁺ exchanger NCX1 is inhibited by the immunosuppressive agent cyclosporin A and the non-immunosuppressive agent PSC833. J Biol Chem 277: 2505-2510.[Abstract/Free Full Text]

Kimchi-Sarfaty C, Oh JM, Kim IW, Sauna ZE, Calcagno AM, Ambudkar SV, and Gottesman MM (2007) A "silent" polymorphism in the MDR1 gene changes substrate specificity. Science 315: 525-528.[Abstract/Free Full Text]

Kochi S, Takanaga H, Matsuo H, Naito M, Tsuruo T, and Sawada Y (1999) Effect of cyclosporin A or tacrolimus on the function of blood-brain barrier cells. Eur J Pharmacol 372: 287-295.[CrossRef][Medline]

Kopito RR (1997) ER quality control: the cytoplasmic connection. Cell 88: 427-430.[CrossRef][Medline]

Lemaire M, Bruelisauer A, Guntz P, and Sato H (1996) Dose-dependent brain penetration of SDZ PSC 833, a novel multidrug resistance-reversing cyclosporin, in rats. Cancer Chemother Pharmacol 38: 481-486.[CrossRef][Medline]

Levy GA (2000) Neoral use in the liver transplant recipient. Transplant Proc 32: 2S-9S.[CrossRef][Medline]

Li Z, Matsuoka S, Hryshko LV, Nicoll DA, Bersohn MM, Burke EP, Lifton RP, and Philipson KD (1994) Cloning of the NCX2 isoform of the plasma membrane Na⁺-Ca²⁺ exchanger. J Biol Chem 269: 17434-17439.[Abstract/Free Full Text]

Lichtenstein Y (2004) The involvement of proline residues in the expression and activity of the Na⁺-Ca²⁺ exchanger. M.Sc. thesis, The Hebrew University, Jerusalem, Israel.

Liu J, Albers MW, Wandless TJ, Luan S, Alberg DG, Belshaw PJ, Cohen P, MacKintosh C, Klee CB, and Schreiber SL (1992) Inhibition of T cell signaling by immunophilin-ligand complexes correlates with loss of calcineurin phosphatase activity. Biochemistry 31: 3896-3901.[CrossRef][Medline]

Loo TW and Clarke DM (1999) The transmembrane domains of the human multidrug resistance P-glycoprotein are sufficient to mediate drug binding and trafficking to the cell surface. J Biol Chem 274: 24759-24765.[Abstract/Free Full Text]

Mok D, Allan RK, Carrello A, Wangoo K, Walkinshaw MD, and Ratajczak T (2006) The chaperone function of cyclophilin 40 maps to a cleft between the prolyl isomerase and tetratricopeptide repeat domains. FEBS Lett 580: 2761-2768.[CrossRef][Medline]

Nicoll DA, Quednau BD, Qui Z, Xia Y-R, Lusis AJ, and Philipson KD (1996) Cloning of a third mammalian Na⁺-Ca²⁺ exchanger, NCX3. J Biol Chem 271: 24914-24921.[Abstract/Free Full Text]

Parathath S, Darlington YF, de la Llera Moya M, Drazul-Schrader D, Williams DL, Phillips MC, Rothblat GH, and Connelly MA (2007) Effects of amino acid substitutions at glycine 420 on SR-BI cholesterol transport function. J Lipid Res 48: 1386-1395.[Abstract/Free Full Text]

Philipson KD and Nicoll DA (2000) Sodium-calcium exchange: a molecular perspective. Annu Rev Physiol 62: 111-133.[CrossRef][Medline]

Pirkl F, Fischer E, Modrow S, and Buchner J (2001) Localization of the chaperone domain of FKBP52. J Biol Chem 276: 37034-37041.[Abstract/Free Full Text]

Pong K and Zaleska MM (2003) Therapeutic implications for immunophilin ligands in the treatment of neurodegenerative diseases. Curr Drug Targets CNS Neurol Disord 2: 349-356.[CrossRef][Medline]

Proud CG (2007) Signalling to translation: how signal transduction pathways control the protein synthetic machinery. Biochem J 403: 217-234.[CrossRef][Medline]

Quednau BD, Nicoll DA, and Philipson KD (1997) Tissue specificity and alternative splicing of the Na⁺/Ca²⁺ exchanger isoforms NCX1, NCX2, and NCX3 in rat. Am J Physiol 272: C1250-C1261.[Medline]

Rahamimoff H, Elbaz B, Alperovich A, Kimchi-Sarfaty C, Gottesman MM, Lichtenstein Y, Eskin-Shwartz M, and Kasir J (2007) Cyclosporin A dependent down regulation of the Na⁺-Ca²⁺ exchanger expression. Ann N Y Acad Sci 1099: 204-214.[CrossRef][Medline]

Rahamimoff H, Ren X, Kimchi-Sarfaty C, Ambudkar S, and Kasir J (2002) NCX1 surface expression: a tool to identify structural elements of functional importance. Ann N Y Acad Sci 976: 176-186.[Medline]

Ren X, Kasir J, and Rahamimoff H (2001) The transport activity of the Na⁺-Ca²⁺ exchanger NCX1 expressed in HEK 293 cells is sensitive to covalent modification of intracellular cysteine residues by sulfhydryl reagents. J Biol Chem 276: 9572-9579.[Abstract/Free Full Text]

Sambrock J, Fritsch E, and Maniatis T (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Shirai A, Naito M, Tatsuta T, Dong J, Hanaoka K, Mikami K, Oh-hara T, and Tsuruo T (1994) Transport of cyclosporin A across the brain capillary endothelial cell monolayer by P-glycoprotein. Biochim Biophys Acta 1222: 400-404.[Medline]

Shiraishi S, Yamamoto R, Yanagita T, Yokoo H, Kobayashi H, Uezono Y, and Wada A (2001) Down-regulation of cell surface insulin receptors by sarco(endo)plasmic reticulum Ca²⁺-ATPase inhibitor in adrenal chromaffin cells. Brain Res 898: 152-157.[CrossRef][Medline]

Smith AJ, Mayer U, Schinkel AH, and Borst P (1998) Availability of PSC833, a substrate and inhibitor of P-glycoproteins, in various concentrations of serum. J Natl Cancer Inst 90: 1161-1166.[Abstract/Free Full Text]

Stephan MM, Chen MA, Penado KM, and Rudnick G (1997) An extracellular loop region of the serotonin transporter may be involved in the translocation mechanism. Biochemistry 36: 1322-1328.[CrossRef][Medline]

Tai HL (2000) Technology evaluation: Valspodar, Novartis AG. Curr Opin Mol Ther 2: 459-467.[Medline]

Tran TT, Dai W, and Sarkar HK (2000) Cyclosporin A inhibits creatine uptake by altering surface expression of the creatine transporter. J Biol Chem 275: 35708-35714.[Abstract/Free Full Text]

Tsang CK, Qi H, Liu LF, and Zheng XF (2007) Targeting mammalian target of rapamycin (mTOR) for health and diseases. Drug Discov Today 12: 112-124.[CrossRef][Medline]

Zhou Y, Gottesman MM, and Pastan I (1999) Domain exchangeability between the multidrug transporter (MDR1) and phosphatidylcholine flippase (MDR2). Mol Pharmacol 56: 997-1004.[Abstract/Free Full Text]

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