NCLX is an essential component of mitochondrial Na+/Ca2+ exchange Raz Paltya, William F. Silvermanb, Michal Hershfinkelb, Teresa Caporalec, Stefano L. Sensic,d, Julia Parnise, Christiane Noltee, Daniel Fishmanb, Varda Shoshan-Barmatzf, Sharon Herrmanna, Daniel Khananshvilig, and Israel Seklera,1 Departments of aPhysiology, bMorphology, and cLife Sciences, and the Zlotowski Center for Neuroscience, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel; cDepartment of Basic and Applied Medical Science, Molecular Neurology Unit, CESI–Center for Research on Aging, University G. d’Annunzio, Chieti 66013, Italy; dDepartment of Neurology, University of California, Irvine, CA, 92697; eDepartment of Cellular Neuroscience, Max Delbrueck Center for Molecular Medicine (MDC), D-13122 Berlin-Buch, Germany; Departments of fPhysiology and gPharmacology, Sackler School of Medicine, Tel Aviv University, Ramat-Aviv 69978, Israel Edited by Harald Reuter, University of Bern, Hinterkappelen, Switzerland, and approved November 10, 2009 (received for review July 22, 2009)
Mitochondrial Ca2+ efflux is linked to numerous cellular activities and pathophysiological processes. Although it is established that an Na+-dependent mechanism mediates mitochondrial Ca2+ efflux, the molecular identity of this transporter has remained elusive. Here we show that the Na+/Ca2+ exchanger NCLX is enriched in mitochondria, where it is localized to the cristae. Employing Ca2+ and Na+ fluorescent imaging, we demonstrate that mitochondrial Na+-dependent Ca2+ efflux is enhanced upon overexpression of NCLX, is reduced by silencing of NCLX expression by siRNA, and is fully rescued by the concomitant expression of heterologous NCLX. NCLX-mediated mitochondrial Ca2+ transport was inhibited, moreover, by CGP-37157 and exhibited Li+ dependence, both hallmarks of mitochondrial Na+-dependent Ca2+ efflux. Finally, NCLXmediated mitochondrial Ca2+ exchange is blocked in cells expressing a catalytically inactive NCLX mutant. Taken together, our results converge to the conclusion that NCLX is the long-sought mitochondrial Na+/Ca2+ exchanger.
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mitochondrial calcium exchanger mitochondrial calcium homeostasis sodium calcium exchanger CGP-37157
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part from their metabolic role, mitochondria are a major hub of cellular Ca2+ homeostasis (1). Powered by the steep mitochondrial membrane potential, Ca2+ enters into this organelle via a mitochondrial uniporter and is extruded by either an H+- or an Na+-coupled mitochondrial exchanger. Activity of the Na+/Ca2 + exchanger is ubiquitously found in most cell types and tissues studied so far and is particularly robust in excitable cells, whereas the activity of the H+/Ca2+ exchanger is primarily found in nonexcitable cells. Recently a mitochondrial H+/ Ca2+ exchanger termed Letm1 has been identified, but its role in Ca2+ extrusion is not clear (2). By catalyzing Na+-dependent Ca2+ efflux, the putative mitochondrial Na+/Ca2+ exchanger plays a fundamental role in regulating mitochondrial Ca2+ homeostasis (3), oxidative phosphorylation (4), and Ca2+ crosstalk among mitochondria, cytoplasm, and the endoplasmic reticulum (ER) (5). Although the activity of this transporter was documented more than 30 years ago (6), its molecular identity remained unknown. We and others previously identified and characterized NCLX, a novel Na+/Ca2+ exchanger, and the single mammalian member of a phylogenetically ancestral branch of the Na+/Ca2+ exchanger superfamily (7, 8). Intriguingly, NCLX catalyzes Na+or Li+-dependent Ca2+ transport at similar rates, a feature shared only with the unidentified mitochondrial exchanger. Although the activity of ectopically expressed human NCLX was first monitored at the plasma membrane, the unique phylogenetic and functional properties of NCLX prompted us to investigate whether NCLX is linked to the mitochondrial exchanger. Results We initially sought to determine if NCLX is found in the mitochondria. Comparison of NCLX levels in total versus mitochondrial436–441 | PNAS | January 5, 2010 | vol. 107 | no. 1
enriched fractions from mouse heart and brain showed that the 50and 70-kDa forms of NCLX (7) and an additional 100 kDa form were enriched in the mitochondrial fraction (Fig. 1A). Augmentation of NCLX expression was also observed in the mitochondrial fraction from HEK-293 cells heterologously or endogenously expressing NCLX (Fig. 1 B and D and Figs. S1A and S2 C–F). Based on the well documented ability of NCX and other membrane proteins to form SDS-resistant dimers, we reasoned that the 100-kDa form is related to such an NCLX dimer. This was confirmed by the simultaneous reduction in expression of the 50-kDa and 100-kDa forms of NCLX in cells transfected with siNCLX (Fig. 1C), and by coimmunoprecipitation of NCLX monomers (see Fig. S1C). Also, dissociation of the 100-kDa form to the 50-kDa form was observed following lengthy incubation in denaturing buffer (Fig. S1B). Intriguingly, the mitochondrial NCLX is remarkably similar in size to molecularly unidentified mitochondrial polypeptides that, when purified and reconstituted, exhibited Na+/Ca2+ exchange activity (10, 11). We next determined the subcellular distribution of NCLX by analyzing endogenous NCLX expression in plasma membrane, ER, and mitochondrial fractions from HEK-293 cells or rat heart (Fig. 1 D and E) and found that endogenous NCLX was enriched primarily in the mitochondrial fraction, but not in the ER or the plasma membrane, as previously suggested (7, 9). Altogether, these results indicate that the mitochondria are the major cellular site of NCLX localization. Additional support for the mitochondrial localization of NCLX came from immunoelectron microscopy analysis of brain sections and CHO cells. Intense labeling was observed in mitochondria, but not in nuclear, sarcoplasmic reticulum, or plasma membrane structures or in preparations reacted with preimmune serum (Fig. 2 A–C). Finally, to define the localization of NCLX within mitochondria, a postembedding cryo-EM procedure featuring protein A gold–labeled, anti-NCLX antibodies was employed (Fig. 2 D–E and Fig. S1D). Discrete gold particles were observed within mitochondria, particularly within the cristae, of control or NCLX-overexpressing SHSY-5Y cells. Individual gold particles unrelated to mitochondria were occasionally observed (see Materials and methods), showed no organelle or compartment preference, and apparently represent nonspecific background staining. Comparison of the number of gold particles observed on ultrathin mitochondrial profiles from control and
Author contributions: R.P., W.F.S., D.K., and I.S. designed research; R.P., W.F.S., T.C., S.L.S., J.P., C.N., D.F., S.H., and D.K. performed research; R.P., W.F.S., M.H., S.L.S., C.N., D.F., V.S.B., S.H., D.K., and I.S. analyzed data; and R.P., W.F.S., M.H., S.L.S., C.N., V.S.-B., and I.S. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1
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NCLX-overexpressing SHSY-5Y cells revealed that mitochondria from the latter contained more particles than did control mitochondria (35 vs. 14; P < 0.01; Fig. 2F). This result indicates that the observed labeling is related to NCLX and that both overexpressed and endogenous NCLX, like the mitochondrial exchanger, are localized to the mitochondrial cristae. If NCLX is to play a functional role in mitochondria, it should be linked to mitochondrial Ca2+ efflux. Mitochondrial Ca2+ signals were determined by using the mitochondrial-targeted calcium-sensing protein Pericam [RP-mt (12), also see Fig. S3B]. RP-mt was excited at the pH-insensitive 430-nm wavelength and not at the 485-/430-nm ratiometric mode because of the pH sensitivity of Pericam to excitation at the 485-nm wavelength and the potential interference by mitochondrial pH changes we and others have observed (see Fig. S3 E–G and ref. 13). In this set of experiments, we have expressed the murine isoform of NCLX that, consistent with previous studies (9), did not reach the plasma membrane and did not exhibit plasma membrane Na+/Ca2+ exchange activity (see Fig. S2 B and F). Plasma membrane Na+/Ca2+ exchange activity was also absent in the control nontransfected cells (see Fig. S2B). These results indicate that neither the endogenous nor the ectopically expressed murine NCLX contribute to plasma membrane Ca2+ fluxes. We then asked if overexpression of NCLX will modulate mitochondrial Ca2+ efflux. Rates of mitochondrial Ca2+ efflux in control cells were taken as the reference point (100%) and changes in activity following NCLX overPalty et al.
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Fig. 1. NCLX is localized to mitochondria. (A) Immunoblot analysis of NCLX in total tissue lysate (Total) and mitochondrial fractions (Mitochondria) purified from mouse heart and brain (15 μg). (B) Immunoblot analysis of total cellular and mitochondrial fractions purified from HEK-293-T cells overexpressing murine NCLX (10 μg). (Lower) Immunoblots of ANT or VDAC serving as markers. (C) Immunoblot analysis of NCLX in mitochondrial fractions purified from HEK-293 cells transfected with siNCLX or scrambled siRNA (siControl). Note that siNCLX diminishes expression levels of both the 50-kDa and 100-kDa forms of NCLX. (D and E) Expression of NCLX in cellular and tissue fractions of the indicated components purified from HEK-293 cells (20 μg) (D) or rat heart (10 μg) (E). (Lower) Immunoblots of ANT (mitochondrial marker), Na+/K+ ATPase, or N-cadherin (plasma membrane, PM, marker) and Calnexin or Sec-62 (ER, marker). Note that the mitochondria are the major site of NCLX localization (the slight NCLX signal in cardiac sarcoplasmic reticulum is presumably related to cross-contamination with mitochondria; see ANT staining).
expression, silencing, or inhibition were presented as percentage of control. Application of ATP (40 μM) was followed by mitochondrial Ca2+ uptake and a subsequent slower Ca2+ efflux phase (Fig. 3A), corresponding to the established activity of the mitochondrial Ca2+ uniporter and the Na+/Ca2+ exchanger, respectively. Mitochondrial Ca2+ uptake was similar in NCLX-expressing or control cells but the rate of Ca2+ efflux was higher in the NCLX-expressing cells (194 ± 23% of control; Fig. 3 A and C), indicating that NCLX expression is linked to enhanced mitochondrial Ca2+ efflux. Application of ATP triggered a similar increase in cytoplasmic Ca2+ (Fig. S4E) in the NCLX-overexpressing cells and control. Furthermore, steady-state mitochondrial Ca2+ levels and membrane potential were similar in NCLX-expressing and control cells (Fig. S3A and Fig. S4 A–C), arguing against an indirect effect of NCLX activity on the cytoplasmic Ca2+ response or mitochondrial Ca2+ uptake. However, when ATP was applied for longer intervals in the presence of Ca2+-containing Ringer solution, thereby imposing a stronger mitochondrial Ca2+ load, the cytoplasmic Ca2+ levels were elevated in cells overexpressing NCLX (Fig. S4 F and G). This is consistent with the physiological role of the mitochondrial exchanger in also modulating cytoplasmic Ca2+ responses by enhancing mitochondrial Ca2+ efflux and augmenting the store-operated calcium entry into cells (13). Because of the dimeric nature of NCLX (Fig. S1 B and C and ref. 14), we hypothesized that a catalytically inactive mutant of NCLX would exert a dominant-negative effect on mitochondrial Ca2+ efflux, thereby providing an additional and highly specific tool for analyzing the mitochondrial role of NCLX. Accordingly, we generated a mutant in which threonine 468, which is catalytically essential for the activity of NCLX and other members of NCX family, was replaced by a serine (NCLX-S468T) (14). Like the native protein, NCLX-S468T was targeted to the mitochondria (see Fig. S2E). Applying the same paradigm as described in Fig. 3A, we found that expression of NCLX-S468T was followed by profound inhibition of endogenous mitochondrial Ca2+ efflux rate in SHSY-5Y (20 ± 15% of control; Fig. 3 A and C) or HEK-293 cells (26 ± 5% of control; Fig. S5 A–B). These results indicate that NCLX is catalytically essential for mitochondrial Ca2+ efflux. The benzothiazepine compound CGP-37157 is an established inhibitor of the mitochondrial Na+/Ca2+ exchanger (15). We reasoned that, if NCLX is this exchanger, its activity should be blocked by this compound. Application of CGP-37157 (10 μM) to SHSY-5Y cells heterologously expressing NCLX or to control cells resulted in profound inhibition of mitochondrial Ca2+ efflux in both (Fig. 3 B and C). In addition, dose-dependence analysis of the effect of CGP-37157 on Ca2+ efflux in NCLX-overexpressing SHSY-5Y cells showed approximately 50% inhibition using 5 μM and complete inhibition of mitochondrial Ca2+ efflux at 7.5 μM (Fig. 3D). This analysis argues against any heterogeneous effect of CGP-37157 on distinct targeting sites and thus indicates that endogenous and overexpressed NCLX share similar sensitivity to GCP-37157. Moreover, the concentration of CGP-37157 required for inhibition of Ca2+ efflux in NCLX-overexpressing SHSY-5Y cells was similar to that reported for inhibition of the mitochondrial exchanger in intact cells (16), and is considerably lower than that reported for partial inhibition of other members of the NCX family (17, 18). Hence, the results of this set of experiments point to NCLX as being the molecular moiety catalytically linked to mitochondrial Ca2+ efflux, and its sensitivity to CGP-37157 provides additional evidence that NCLX is the mitochondrial Na+/ Ca2+ exchanger. If NCLX mediates mitochondrial exchange activity, then reducing its endogenous expression should attenuate mitochondrial Ca2+ efflux. Transfection of cells with NCLX siRNA (siNCLX) lowered NCLX expression (Fig. 1C). These cells exhibit similar resting mitochondrial Ca2+ levels as untreated cells, their mitochondria being slightly hyperpolarized (Figs. S3A and S4C). Then the same ATP-induced mitochondrial Ca2+ efflux assay described in Fig. 3A was applied. Whereas mitochondrial Ca2+
Fig. 2. NCLX is found on the inner membrane of mitochondria. (A–C) Electron micrographs of rat cortical slices (A) or CHO cells (B) stained with anti-NCLX antibodies or NCLX preimmune serum (C). N, ER, and PM denote the nucleus, endoplasmic reticulum, and plasma membrane structures, respectively. Positive immunolabeling, defined as the presence of dense DAB precipitate, is observed primarily in the mitochondria. (Scale bar, 0.5 μm.) (D and E) Immunogold labeling of SHSY-5Y cells overexpressing (D) or endogenously expressing (E) NCLX. Note that NCLX labeling is found primarily in the cristae of the mitochondria. (Scale bar, 0.2 μm.) (F) The distribution of numbers of gold particles per mitochondrion is shown. Quantitative analysis of mitochondrial gold particle distribution shows that the overall number of particles in NCLX-overexpressing cells was 35 versus 14 in the control cells (number of mitochondria examined, n = 10 for each group; **P < 0.01).
uptake was unaffected, in cells in which NCLX expression had been silenced, the Ca2+ efflux rate was reduced by approximately 75% of the rate measured in control cells (22 ± 6.5% of control; Fig. 4A). A similar decrease in mitochondrial Ca2+ efflux following silencing of NCLX expression was also observed in CHO cells (Fig. S5 D–F). We further reasoned that, if NCLX directly participates in endogenous mitochondrial exchange activity, expression of a heterologous species of NCLX should rescue the activity reduced by the silencing. To address this hypothesis, SHSY-5Y cells were transfected with a plasmid encoding NCLX shRNA (see Materials and Methods), thereby generating a sequence that specifically targets a UTR region in the human NCLX mRNA sequence (i.e., shNCLX) but not in the murine homologue. Human SHSY-5Y cells transfected with the shNCLX exhibited a 56 ± 6.7% reduction in mitochondrial Ca2+ efflux activity compared with control cells (Fig. 4B). In contrast, coexpression of murine NCLX with the shNCLX plasmid induced a fourfold increase in the rate of Ca2+ efflux versus cells transfected with shNCLX only. Hence, Ca2+ efflux activity had been fully restored (Fig. 4B). Taken together, the results from this set of experiments support the concept that NCLX is an essential mediator of endogenous mitochondrial Ca2+ efflux and that heterologous expression of NCLX is sufficient to functionally complement the loss of the endogenous activity. We next examined whether NCLX activity is linked to mitochondrial Na+/Ca2+ exchange by assessing whether it displays a strict Na+ dependence in mediating Ca2+ efflux. The Na+ dependence of mitochondrial NCLX was assayed in digitonin-permeabilized cells, thus allowing equilibration of the intracellular and extracellular compartments and control of the extramitochondrial ionic environment while eliminating any possible interference by plasma membrane transporters. To ascertain that the cells were permeabilized using this protocol, they were loaded with carboxyfluorescein diacetate (CFDA) succinimidyl ester dye that was trapped within the cells. Subsequent addition of digitonin triggered a strong decrease of CFDA fluorescence, suggesting that the entrapped dye was removed out of the cells and hence that the cells were permeabilized (Fig. S3C). Permeabilized HEK-293 cells transiently expressing RP-mt alone or together with NCLX were s\perfused with a Ca2+-containing (60 μM) Na+-free (replaced with NMDG+, see Materials and Methods) solution that triggered a similar increase in mitochondrial Ca2+ levels in control and NCLX-transfected cells (Fig. S3D). Strong Ca2+ efflux was monitored only after 20 mM Na+ (Fig. 5A) were reintroduced, 438 | www.pnas.org/cgi/doi/10.1073/pnas.0908099107
indicating that the Ca2+ efflux mediated by Na+-dependent exchange is the major mitochondrial Ca2+ extrusion pathway in these cells. A marked increase (259 ± 45%) in mitochondrial Na+-dependent Ca2+ efflux was monitored in NCLX-overexpressing versus control cells (Fig. 5A). To directly determine if Na+ was the counter ion transported into mitochondria by NCLX, the same paradigm was applied to cells overexpressing NCLX loaded with the mitochondrial Na+sensitive dye CoroNa Red. As shown in Fig. 5B, application of 20 mM Na+ following mitochondrial Ca2+ loading triggered a rise in CoroNa Red fluorescence. The rate of Na+ influx was increased (170 ± 47%) in NCLX-overexpressing cells compared with controls, but not in cells superfused with Na+-free solution. indicating that the fluorescence increase resulted from Na+ influx into the mitochondria. This apparent coupling between mitochondrial Na+ influx and Ca2+ efflux with elevated expression levels of NCLX is consistent with mitochondrial Na+/Ca2+ exchange activity being mediated by this protein. Further substantiating the link between NCLX and mitochondrial exchanger activity, the observed Na+-dependent Ca2+ efflux was profoundly inhibited (to 28 ± 8.7% of control) in HEK-293 cells transfected with the siNCLX construct (Fig. 5C). Importantly, Ca2+ efflux was virtually eliminated in NCLX-overexpressing (Fig. 5 A and B) or control (Fig. 5C) cells that were superfused with Na+-free solution. indicating that Ca2+ efflux mediated by the mitochondrial exchanger and mitochondrial NCLX share the same strict Na+ dependence. Finally, Li+/Ca2+ exchange activity is a unique property of both NCLX (7) and the mitochondrial exchanger (19). We therefore asked if NCLX conducts mitochondrial Li+-dependent Ca2+ transport. Superfusion of permeabilized HEK-293 control cells with 30 mM Li+ in a Na+-free solution facilitated robust Ca2+ efflux from the mitochondria, consistent with the unique ability of the mitochondrial exchanger to mediate Li+-dependent Ca2+ exchange (Fig. 5D). Importantly, silencing of NCLX expression was followed by a strong reduction in the rate of Li+-dependent Ca2+ efflux, (35 ± 8% of control; Fig. 5D). Similar attenuation of Li+-dependent Ca2+ efflux activity upon silencing of NCLX expression was also recorded in CHO cells (see Fig. S5 G–I). These results indicate that NCLX is the molecular determinant essential for mitochondrial Na+/Ca2+ and Li+/Ca2+ exchange activity. Discussion Numerous studies have reported that the Na+-dependent mitochondrial exchanger, similar to other members of the NCX Palty et al.
family, mediates Na+/Ca2+ exchange, albeit with unique functional properties (15, 19), including altered selectivity to monovalent cations and distinct sensitivity to inhibitors. In our studies, we have focused on NCLX because, although it is a member of the NCX family, it is phylogenetically and functionally distinct from other known NCX or NCKX family members (20). Support for NCLX being the mitochondrial Na+/Ca2+ exchanger come from the following: (i) NCLX is expressed in mitochondria, where it is localized to the inner membrane of this organelle, and has a molecular weight similar to that of the purified mitochondrial exchanger (21). (ii) NCLX expression accelerates mitochondrial Ca2+ efflux activity, whereas silencing of NCLX expression attenuates this process. Importantly, Ca2+ efflux was fully rescued in NCLX-silenced cells by concomitant overexpression of NCLX, indicating that the expression of NCLX is essential and sufficient to functionally complement mitochondrial Na+/Ca2+ exchange. Despite the profound effect of NCLX expression on mitochondrial Ca2+ efflux, it did not affect the steady-state resting mitochondrial Ca2+ level. The relatively low affinity of the mitochondrial exchanger to Ca2+ (10) or the effective phosphate-based mitochondrial Ca2+ buffering system that is capable of keeping steady-state Ca2+ concentration constant over a wide range by a reversible formation or dissociation of insoluble Ca2+-phosphate precipitates may account for this effect Palty et al.
(22). Hence, NCLX may be playing a similar role to plasma membrane members of the NCX family that primarily mediate and respond to rapid and robust Ca2+ changes rather than fine tuning of the steady-state Ca2+ level (23). (iii) Mitochondrial Ca2+ efflux mediated by NCLX is strictly Na+-dependent and coupled to accelerated mitochondrial Na+ influx. The strong endogenous Na+ dependence of Ca2+ efflux that we have assayed in permeabilized cells (Fig. 5) indicates that NCLX is the dominant Ca2+ extruding transporter in these cells. Further studies comparing the activity of NCLX and the H+/Ca2+ exchanger are required to address their specific roles in distinct cell types and tissues. (iv) Replacement of a single residue in the putative catalytic α-domain of NCLX was sufficient to fully block mitochondrial Ca2+ efflux. The role of the α-domains as the catalytic core of NCX members is firmly established and hence this finding will provide the basis and rational for further studies on the unique and common structural/functional properties of the mitochondrial Na+/Ca2+ exchanger. (v) NCLX is fully inhibited by the mitochondrial Na+/Ca2+ exchange inhibitor CGP-37157 and (vi) NCLX mediates Li+/Ca2+ exchange, a functional property that, among NCX proteins, is shared exclusively with the mitochondrial exchanger. Thus, the mitochondrial localization, the link between NCLX expression/inactivation with mitochondrial exchange activity, and its functional properties all indicate that NCLX is the mitochondrial exchanger. Our results show that endogenous expression of NCLX in HEK 293 cells, as well as in other cells studied, is most prominent in mitochondria (Fig. 1); this is also seen in cells overexpressing the murine NCLX isoform. Thus, the enhanced plasma membrane Ca2+ transport activity that was previously found in HEK-293 cells ectopically expressing the human isoform of NCLX (7) is probably linked to a “spillover” of NCLX to the plasma membrane, a phenomenon commonly linked to strong overexpression in HEK-293 and other cell types (24, 25). Importantly, this spillover of the human NCLX protein to the plasma membrane fortuitously provided us with the seminal finding that NCLX mediates Li+/Ca2+ exchange PNAS | January 5, 2010 | vol. 107 | no. 1 | 439
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Fig. 3. Expression of NCLX enhances mitochondrial Ca2+ efflux that is blocked by mutation in the catalytic site of NCLX and by the mitochondrial exchanger inhibitor CGP-37157. (A) SHSY-5Y cells transfected with plasmids encoding the mouse NCLX, NCLX-S468T mutant, or the vector (control) were cotransfected to express RP-mt. Cells were superfused with ATP-containing Ringer solution (40 μM at the indicated time) while monitoring mitochondrial Ca2+ fluorescence. Note that enhancement of NCLX levels increased mitochondrial Ca2+ efflux, and the mutant was not only inactive but also exerted a dominant-negative effect on endogenous activity. (B) Illustration of the same experimental paradigm as in A repeated in control or NCLX-expressing cells in the presence of the mitochondrial exchanger inhibitor CGP-37157 (10 μM). Similar inhibition of Ca2+ efflux is seen in both. (C) Averaged mitochondrial Ca2+ efflux rates (n = 9; **P < 0.01). (Inset) Experimental paradigm for Ca2+ efflux rate measurement based on determination of the initial rate of the mitochondrial Ca2+ efflux phase. (D) Dosedependence analysis of the effect of CGP-37157 on mitochondrial Ca2+ efflux in cells expressing NCLX. Mitochondrial Ca2+ efflux rate was measured in the presence of the indicated concentrations of CGP-37157 and presented as percentage of the rate measured in its absence. The dashed line is a fit of the data to the equation I = Io / [1+([CGP] / IC50)].
Fig. 4. Silencing the expression of endogenous NCLX decreases mitochondrial Ca2+ efflux that can be rescued by expression of recombinant murine NCLX. (A) Mitochondrial Ca2+ responses following application of ATP (40 μM, as in Fig. 3A) was measured in HEK-293 cells cotransfected with either the siNCLX or a scrambled siRNA construct (si control) and the RP-mt–expressing plasmid. Averaged rates of mitochondrial Ca2+ efflux are shown (Right) (n = 9, **P < 0.01). (B) The same experiment as in A was performed on SHSY-5Y cells transfected with NCLX shRNA plasmid alone or together with the murine NCLX-encoding plasmid (which is insensitive to the NCLX shRNA construct). Averaged rates of mitochondrial Ca2+ efflux are shown (Right) (n = 11, *P < 0.05).
Fig. 5. The mitochondrial Na+- or Li+-dependent Ca2+ exchange is mediated by NCLX. (A) Traces and rates of Na+-dependent mitochondrial Ca2+ efflux. Mitochondrial Ca2+ levels were recorded by monitoring RP-mt fluorescence in HEK-293 cells overexpressing either the human NCLX isoform (NCLX) or vector alone (control) and coexpressing RP-mt. Experiments were conducted on digitonin-permeabilized cells (see Materials and methods). Mitochondrial Ca2+ uptake was induced by superfusion with Na+-free solution (replaced by NMDG+) containing Ca2+ (60 μM). Ca2+ efflux was monitored following superfusion in Ca2+-free solution in the presence or absence of Na+. Note that the Ca2+ efflux was strictly Na+-dependent and was enhanced by the expression of NCLX (n = 8, **P < 0.01). (B) Traces and rates of Na+-dependent mitochondrial Na+ influx. Mitochondrial Na+ levels were measured in cells loaded with the Na+-sensitive dye CoroNa Red (see Materials and methods) as in A. Note the enhanced and reciprocal nature of the Na+ and Ca2+ transport rates, mediated by mitochondrial NCLX (n = 8, **P < 0.01). (C) Traces and rates of Na+-dependent mitochondrial Ca2+ efflux in cells transfected with either siNCLX or a scrambled siRNA (si control). The same experimental paradigm described in A was applied. Silencing of NCLX eliminated mitochondrial Na+-dependent Ca2+ efflux (n = 11, **P < 0.01). (D) Traces and rates of Li+-dependent mitochondrial Ca2+ efflux in cells transfected with either siNCLX or si control, using Li+-containing solution (replacing Na+; n = 11; *P < 0.05). Silencing of NCLX also diminished Li+dependent Ca2+ efflux.
and prompted us to investigate if NCLX is the mitochondrial exchanger. We do not rule out, however, the possibility that NCLX or its spliced isoforms may also be found in other cellular compartments (7, 9). Indeed, NCX members are often found at multiple cellular sites. Among them, NCX1 is found both in the plasma membrane and nucleus (26). Dual localization is also shared by other mitochondrial proteins such as several members of the cytochrome P450 family that are targeted to both the ER and mitochondrial compartments (27, 28) and the potassium channel, KCa3.1, that can be found in the mitochondria or plasma membrane (29). Our finding that NCLX is the mitochondrial Na+/Ca2+ exchanger is of particular interest in light of the intense expression of NCLX in skeletal muscle, stomach, and pancreas (7) and the critical role played by the mitochondrial Ca2+ transport in these organs. A remarkable feature of mitochondrial Ca2+ transport in skeletal muscle is the ultrafast Ca2+ efflux rate (30), which equals the rate of influx mediated by the uniporter; compared with the 2 to 3 orders of magnitude slower efflux rate found in cardiomyocytes. The dramatically higher expression of NCLX in skeletal muscle versus cardiac tissue further links the expression of NCLX to mitochondrial Ca2+ efflux and suggests that expression of NCLX is a major rate limiting determinant of the uniquely fast mitochondrial Ca2+ transport in skeletal muscle. Smooth muscle accounts for the largest mass of tissue in the stomach, and it is therefore conceivable that the intense NCLX expression in the stomach results from its ubiquitous expression in smooth muscle (7). The essential role of the mitochondrial Na+/Ca2+ exchanger in refilling smooth muscle Ca2+ stores and in facilitating the Ca2+ sparks leading to muscle contraction is documented (31, 32) and agrees well with the high ex440 | www.pnas.org/cgi/doi/10.1073/pnas.0908099107
pression of NCLX in this tissue. A role of mitochondrial Na+/Ca2+ exchange in insulin secretion is of profound physiological interest particularly because of the link between mitochondrial Ca2+ levels and the rate of ATP synthesis, but is controversial because of potential interference of CGP-37157 with the L-type Ca2+ channel activity (16). In the exocrine pancreas, mitochondria have a well documented role in limiting the apical to the basolateral propagation of Ca2+ waves and in controlling plasma membrane Ca2+ influx (33). Much has to be learned on the specific role of the mitochondrial exchanger in the function of endocrine and exocrine pancreas, and the present study will provide the molecular tools required for this goal. Finally, the profound change in mitochondrial Ca2+ exchange activity observed for example in a neuronal Parkinson model (34) underscores the pathophysiological relevance of NCLX regulation. Hence, the identification of the mitochondrial Na+/Ca2+ exchanger will provide insight on the molecular basis underlying these processes. Materials and Methods Fluorescent Measurements of Intracellular Ions. Cell culture and transfection procedures are described in SI Methods. Cytosolic Ca2+ levels were recorded from Fura-2–loaded cells, excited at wavelengths of 340 and 380 nm, and imaged with a 510 nm long-pass filter as previously described (7). Mitochondrial Ca2+ or H+ levels were monitored in cells transiently expressing the RP-mt protein at excitation wavelengths of 430 nm [i.e., Ca2+-sensitive wavelength (12), presented as F430 (1-F/F0)] and 485 nm [i.e., pH-sensitive wavelength (35, 36), presented as F485 F/F0], respectively, and the emission collected using a 535-nm band-pass filter. In experiments in which digitoninpermeabilized HEK-293 cells were employed, F430 was normalized to the value obtained following the Ca2+-loading phase (FT-200). Mitochondrial Na+
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levels were monitored in CoroNa-Red (Molecular Probes)–loaded cells excited at 545 nm and imaged using a 630-nm long-pass filter. Unless stated otherwise, all experiments in nonpermeabilized cells were conducted using Ringer solutions containing 130 mM NaCl or NMDG (Na+-free), 20 mM Hepes, 15 mM glucose, 5 mM KCl, and 0.8 mM MgCl2, with the pH adjusted to 7.4. Ringer solution was supplemented with 0.5 or 2 mM CaCl2 (for only experiments described in SI Materials and Methods; Figs. S2 and S4F) or in 0.5 mM EDTA and 40 μM ATP as indicated. Mitochondrial Ca2+ or Na+ in digitonin-permeabilized (0.007% digitonin) cells were determined as previously described (37) in a buffer containing 220 mM sucrose, 10 mM Hepes, 5 mM succinate, 2.5 mM KH2PO4, 0.4 mM EGTA, and 1 μM cyclosporine A, with the pH adjusted to 7.4 with KOH. Following the addition of 60 μM Ca2+, and Ca2+ loading of the mitochondria, 1 μM ruthenium red was added to eliminate a leak of Ca2+ via this pathway. Then 20 mM NaCl, NMDG Cl, or 30 mM LiCl were added as indicated. For all single-cell imaging experiments, traces of averaged responses, recorded from 5 to 20 cells in each experiment, were analyzed and plotted using KaleidaGraph. The rate of ion transport was calculated from each graph (summarizing an individual experiment) by a linear fit of the change in the fluorescence (ΔF) over a period of 30 s following initiation of apparent efflux/influx (ΔF/dt). Rates from n experiments (as mentioned in legends to the figures) were averaged and displayed in bar graph format. Analysis with the t test was performed to determine significance when relevant.
Immunoelectron Microscopy. Brain sections were processed and imaged as described in SI Materials and Methods. Analysis of the distribution of NCLX in control and NCLX-overexpressing cells was performed blindly using the gold-labeled preparations for the EM. Each mitochondrion containing at least one gold particle was digitally-captured at ×25,000 magnification. Only one mitochondrion was imaged per cell section, with 20 cells overall (i.e., 20 mitochondria): 10 overexpressing and 10 controls. Only mitochondria that exhibited a clear outer membrane and cristae were selected for the analysis. Each cell was counted as an event, and we then compared the gold particle number for each event. Similar numbers of extramitochondrial gold particles (n = 1–2) were observed in approximately 40% of the sections obtained either from control or NCLX-overexpressing cells.
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21. Kar P, Chakraborti T, Samanta K, Chakraborti S (2008) mu-Calpain mediated cleavage of the Na+/Ca2+ exchanger in isolated mitochondria under A23187 induced Ca2+ stimulation. Arch Biochem Biophys . 22. Nicholls DG, Chalmers S (2004) The integration of mitochondrial calcium transport and storage. J Bioenerg Biomembr 36:277–281. 23. Hilgemann DW, Yaradanakul A, Wang Y, Fuster D (2006) Molecular control of cardiac sodium homeostasis in health and disease. J Cardiovasc Electrophysiol 17 (Suppl 1): S47–S56. 24. Ginger RS, et al. (2008) SLC24A5 encodes a trans-Golgi network protein with potassium-dependent sodium-calcium exchange activity that regulates human epidermal melanogenesis. J Biol Chem 283:5486–5495. 25. Schultz BD, Frizzell RA, Bridges RJ (1999) Rescue of dysfunctional deltaF508-CFTR chloride channel activity by IBMX. J Membr Biol 170:51–66. 26. Xie X, Wu G, Lu ZH, Rohowsky-Kochan C, Ledeen RW (2004) Presence of sodiumcalcium exchanger/GM1 complex in the nuclear envelope of non-neural cells: nature of exchanger-GM1 interaction. Neurochem Res 29:2135–2146. 27. Addya S, et al. (1997) Targeting of NH2-terminal-processed microsomal protein to mitochondria: a novel pathway for the biogenesis of hepatic mitochondrial P450MT2. J Cell Biol 139:589–599. 28. Robin MA, et al. (2000) Vesicular transport of newly synthesized cytochromes P4501A to the outside of rat hepatocyte plasma membranes. J Pharmacol Exp Ther 294: 1063–1069. 29. De Marchi U, et al. (2009) Intermediate conductance Ca2+-activated potassium channel (KCa3.1) in the inner mitochondrial membrane of human colon cancer cells. Cell Calcium 45:509–516. 30. Rudolf R, Mongillo M, Magalhães PJ, Pozzan T (2004) In vivo monitoring of Ca(2+) uptake into mitochondria of mouse skeletal muscle during contraction. J Cell Biol 166: 527–536. 31. Balemba OB, Bartoo AC, Nelson MT, Mawe GM (2008) Role of mitochondria in spontaneous rhythmic activity and intracellular calcium waves in the guinea pig gallbladder smooth muscle. Am J Physiol Gastrointest Liver Physiol 294:G467–G476. 32. Poburko D, Liao CH, van Breemen C, Demaurex N (2009) Mitochondrial regulation of sarcoplasmic reticulum Ca2+ content in vascular smooth muscle cells. Circ Res 104:104–112. 33. Tinel H, et al. (1999) Active mitochondria surrounding the pancreatic acinar granule region prevent spreading of inositol trisphosphate-evoked local cytosolic Ca(2+) signals. EMBO J 18:4999–5008. 34. Gandhi S, et al. (2009) PINK1-associated Parkinson's disease is caused by neuronal vulnerability to calcium-induced cell death. Mol Cell 33:627–638. 35. Filippin L, Magalhães PJ, Di Benedetto G, Colella M, Pozzan T (2003) Stable interactions between mitochondria and endoplasmic reticulum allow rapid accumulation of calcium in a subpopulation of mitochondria. J Biol Chem 278: 39224–39234. 36. Frieden M, et al. (2004) Ca(2+) homeostasis during mitochondrial fragmentation and perinuclear clustering induced by hFis1. J Biol Chem 279:22704–22714. 37. Lee B, et al. (2003) Inhibition of mitochondrial Na+-Ca2+ exchanger increases mitochondrial metabolism and potentiates glucose-stimulated insulin secretion in rat pancreatic islets. Diabetes 52:965–973.
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ACKNOWLEDGMENTS. The authors thank Dr. Ronit Ben-Romano, Salah Abu-Hamad, Rina Jaeger, Elizabeth Weinfeld, and Nichole Kahn for their technical help; Dr. Jonathan Lytton for the mouse NCLX isoform and NCKX4 plasmids; Dr. Atsushi Miyawaki for the RP-mt–encoding plasmid; and Dr. Claude Brodski for his critical reading of the manuscript. This work was partially supported by Israel Science Foundation (ISF) Grant 985/05 and German Israeli Foundation Grant 917-119.1/2006 (to I.S. and C.N.); ISF Grant 450/03 (to M.H.), and FIRB 2003 and PRIN 2006 grants from the Ministry of Research and Education of Italy (S.L.S.). R.P. was supported by the Adams Fellowship Program of the Israel Academy of Sciences and Humanities.
Supporting Information Palty et al. 10.1073/pnas.0908099107
Supporting Information Corrected 05/07/2010 SI Materials and Methods Cell Culture and Transfection Procedures. HEK-293-T, HEK-293,
SHSY-5Y, and CHO cells were cultured in DMEM as previously described (1). Transfection of HEK-293 or SHSY-5Y cells was performed using Ca2PO4 as described previously (1). CHO cells were transfected using Lipofectamine 2000 (Invitrogen) or Transfect-it CHO reagent (Mirus) according to the manufacturer's protocol. Fluorescent ion measurements or cell harvesting were conducted 30 to 72 h after transfection. Plasmid and siRNA Preparation. Preparation of the human NCLXencoding plasmid was described previously (1). NCLXS468T- and NCLX-6His-encoding plasmids were generated by replacing a NotI fragment from the α2 S273T or WT α2-6His plasmid (2), respectively, with the corresponding fragment of the NCLX-encoding plasmid. The murine NCLX variant and the NCKX4-encoding plasmids were provided by Jonathan Lytton (Calgary, AB, Canada) (3). The RP-mt plasmid was provided by Atsushi Miyawaki (Wako, Japan) (4). Double-stranded siRNAs used to silence NCLX expression was obtained from Ambion (Applied Biosystems). The sequence of 21 nucleotides corresponding to the sense strands used for the NCLX siRNA was AACGGCCACUCAACUGUCUtt and that for the control siRNA was AACGCGCAUCCAACUGUCUtt. The human NCLX shRNA plasmid was obtained from Sigma (Mission TRC shRNA Target Set TRCN-5045). Fluorescent Measurements of Intracellular Ions and Mitochondrial Membrane Potential. Changes in inner mitochondrial membrane
potential (ΔΨm) were monitored by detecting microfluorimetric changes in fluorescence (ΔF) of the membrane potential–sensitive probes TMRE (5) (for HEK293 cells). HEK293 cells loaded with 0.05 μM TMRE were washed in a static bath of Ringer solution containing the probe and then exposed to 10 μM of CCCP to obtain maximal loss of ΔΨm.. TMRE-loaded cells were excited with a 530-nm (±30) band-pass filter and emission monitored at greater than 590 nm. TMRE ΔF was calculated as fluorescence values at baseline and after exposure to CCCP. Flow Cytometry Analysis of ΔΨm. To evaluate ΔΨm in SHSY-5Y cells, 5 × 105 cells were incubated with 500 nM of 5,5′,6,6′-tetrachloro1,1′,3,3′-tetraethyl benzimidazolyl carbocyanine iodide (JC1; Molecular Probes) in the growth medium for 10 min at 37°C in the dark and immediately analyzed by flow cytometry using the FACSCalibur instrument (BD Bioscience). Data were processed with FlowJo software and presented as the ratio between mean fluorescence intensities measured with the FL1 (green) and FL2 (red) photomultipliers. Generation of Anti-NCLX Antibodies. The generation of the antibody used in Figs. 1 and 2 in the main text was previously described (1). Additional antibodies used in the experiment described in Fig. S1 was raised against peptide CSRSHTEVKLEDPDGL (CSR antibody), located close to the C terminus of NCLX, as previously described (1). Cell Fractionation, Immunoprecipitation, and Immunoblot Analysis.
Subcellular fractionation of ER/sarcoplasmic reticulum or mitochondrial fractions from HEK293 (6) cells or from rat heart using a small-scale protocol (7, 8) were performed as previously described. Briefly, rat ventricular tissue (approximately 20 g) was homogenized 3 times in Polytron, PT3000 (9,000 rpm × 30 s) with ice-cold TMS buffer (0.25 M sucrose/20 mM Tris-Cl, pH 7.4, 1 mM MgCl2, 1 mM PMSF, and proteinase inhibitor mixture). The lysates were further homogenized with a glass Douncer and then centrifuged at Palty et al. www.pnas.org/cgi/content/short/0908099107
800 × g for 15 min to remove the contractile proteins and nuclei. The supernatant was centrifuged twice at 8,000 × g for 20 min to pellet the mitochondrial fraction. The mitochondrial fraction was separately washed whereas the collected supernatants were centrifuged at 100,000 × g for 60 min to pellet the microsomal fraction. The pellet was suspended in the TMS buffer and the sarcoplasmic reticulum and sarcolemma membranes were separated by sucrose gradient as previously described (9). The subcellular fractions were resuspended in 0.25 M sucrose/20 mM Tris-Cl, pH 7.4, and stored at −20°C. Purification of the plasma membrane–enriched fraction was performed using a cell surface protein isolation kit (Pierce) according to the manufacturer's instructions. Protein concentration was determined by the bicinchoninate assay (Pierce), and 20 μg of protein was separated by SDS/PAGE and immunoblotted. In the case of the plasma membrane fraction, protein samples and BSA standards were precipitated in 80% acetone, suspended in ddH2O, and then assayed by the bicinchoninate method. Extracted proteins (20 μg) were separated by 10% SDS/PAGE and transferred onto a polyvinylidene difluoride membrane (Amersham). Membranes were probed using the following antibodies: anti-NCLX [1:500 (2)], anti-ANT (1:100, Santa Cruz Biotechnology), anti-Sec62 (1:5,000; provided by T. Sommer, Berlin, Germany), anti-Na/K ATPase [1:500 (10)], and anti–N-cadherin (1:1,000; BD Biosciences; donated by Frank Kirchner, Berlin, Germany), all diluted into 5% milk in tris-buffered saline solution with Tween 20. Isolation of mitochondria from transfected HEK293-T cells or from native mouse tissues was performed as previously described (11), with minor modifications. Briefly, cells were homogenized in isolation buffer containing (in mM): mannitol 225, sucrose 75, EDTA 0.5, HEPES 10, with the pH being adjusted to 7.4 with KOH. The crude homogenate samples were centrifuged at 1,500 × g for 5 min in 4°C, the supernatant was collected, and the samples were washed with fresh homogenization buffer and recentrifuged. Pellet was saved (crude membranes) and the supernatant collected and recentrifuged for 4 min at 12,000 × g at 4°C. Supernatant containing ER fragments and cytosolic proteins was saved (cytosolic fraction). The pellet, containing the mitochondria, was then washed 3 times with homogenization buffer. All samples were analyzed for protein concentration using the Bradford method (Bio-Rad). Equal amounts of protein were then resolved by SDS/PAGE and transferred onto nitrocellulose membranes for Western blot analysis. Immunoprecipitation procedure was performed using the ExactaCruz kit (Santa Cruz Biotechnology) according to manufacturer's protocol. Briefly, total cell lysate from HEK-293-T cells expressing NCLX bearing a c-Myc tag, NCLX bearing a 6His-tag, or both were incubated with 20 μg of an antibody targeted against the 6His epitope (Santa Cruz Biotechnology) overnight at 4°C. Subsequently, 50 μL of immunoprecipitation matrix was added to the lysate and beads were sedimented and further processed according to manufacturer's instructions. Immunoblot analysis was carried out as described previously (1). Antibodies used in this study included NCLX-antiserum (1:1,000–2,000 dilution), anti–β-actin (1:40,000; Sigma), monoclonal anti-VDAC antibodies against the N-terminal region of 31HL human porin (12) (1:10,000; Calbiochem), anti-ANT (1:100; Santa Cruz Biotechnology), anti-c-Myc (1:500; Santa Cruz Biotechnology), and anti-FLAG (1:1,000; VWR). Immunoelectron Microscopy. Brains sections were processed for preembedding immunoelectron microscopy according to a protocol 1 of 7
described previously (13). The NCLX antibody was used at a dilution of 1:200. The postembedding immunoelectron microscopy protocol was a modification of the method of Tokuyasu (14). Briefly, control or NCLX-overexpressing SHSY-5Y cells were fixed with 2% paraf-
ormaldehyde, 0.25% glutaraldehyde in 0.1 M phosphate buffer. Cryosections were cut on a Leica UltraCut UCT Ultramicrotome equipped with a low-temperature sectioning system. Analysis was performed on a JEOL JEM-1230 electron microscope equipped with a Tietz TemCam F214 CCD camera (TVIPS, Gauting, Germany).
1. Palty R, et al. (2004) Lithium-calcium exchange is mediated by a distinct potassiumindependent sodium-calcium exchanger. J Biol Chem 279:25234–25240. 2. Palty R, et al. (2006) Single alpha-domain constructs of the Na+/Ca2+ exchanger, NCLX, oligomerize to form a functional exchanger. Biochemistry 45:11856–11866. 3. Cai X, Lytton J (2004) Molecular cloning of a sixth member of the K+-dependent Na+/Ca2+ exchanger gene family, NCKX6. J Biol Chem 279:5867–5876. 4. Nagai T, Sawano A, Park ES, Miyawaki A (2001) Circularly permuted green fluorescent proteins engineered to sense Ca2+. Proc Natl Acad Sci USA 98:3197–3202. 5. Farkas DL, Wei MD, Febbroriello P, Carson JH, Loew LM (1989) Simultaneous imaging of cell and mitochondrial membrane potentials. Biophys J 56:1053–1069. 6. Bozidis P, Williamson CD, Colberg-Poley AM (2007) Isolation of endoplasmic reticulum, mitochondria, and mitochondria-associated membrane fractions from transfected cells and from human cytomegalovirus-infected primary fibroblasts. Curr Protoc Cell Biol 37: 3.27.1–3.27.23. 7. Khananshvili D, Shaulov G, Weil-Maslansky E, Baazov D (1995) Positively charged cyclic hexapeptides, novel blockers for the cardiac sarcolemma Na(+)-Ca2+ exchanger. J Biol Chem 270:16182–16188. 8. Pallotti F, Lenaz G (2007) Mitochondria, eds Pon LA, Schon EA , (Elsevier, New York), Vol 80, pp 3–44. 9. Baazov D, Wang X, Khananshvili D (1999) Time-resolved monitoring of electrogenic Na+–Ca2+ exchange in the isolated cardiac sarcolemma vesicles by using a rapidresponse fluorescent probe. Biochemistry 38:1435–1445.
10. Patchornik G, Goldshleger R, Karlish SJ (2000) The complex ATP-Fe(2+) serves as a specific affinity cleavage reagent in ATP-Mg(2+) sites of Na,K-ATPase: altered ligation of Fe(2+) (Mg(2+)) ions accompanies the E(1)—>E(2) conformational change. Proc Natl Acad Sci USA 97:11954–11959. 11. Vergun O, Reynolds IJ (2005) Distinct characteristics of Ca(2+)-induced depolarization of isolated brain and liver mitochondria. Biochim Biophys Acta 1709:127–137. 12. Babel D, et al. (1991) Studies on human porin. VI. Production and characterization of eight monoclonal mouse antibodies against the human VDAC “Porin 31HL” and their application for histotopological studies in human skeletal muscle. Biol Chem Hoppe Seyler 372:1027–1034. 13. Elgazar V, et al. (2005) Zinc-regulating proteins, ZnT-1, and metallothionein I/II are present in different cell populations in the mouse testis. J Histochem Cytochem 53:905–912. 14. Tokuyasu KT (1986) Application of cryoultramicrotomy to immunocytochemistry. J Microsc 143:139–149. 15. Ren X, Nicoll DA, Galang G, Philipson KD (2008) Intermolecular cross-linking of Na+-Ca2 + exchanger proteins: evidence for dimer formation. Biochemistry 47:6081–6087. 16. Filippin L, Magalhães PJ, Di Benedetto G, Colella M, Pozzan T (2003) Stable interactions between mitochondria and endoplasmic reticulum allow rapid accumulation of calcium in a subpopulation of mitochondria. J Biol Chem 278: 39224–39234. 17. Frieden M, et al. (2004) Ca(2+) homeostasis during mitochondrial fragmentation and perinuclear clustering induced by hFis1. J Biol Chem 279:22704–22714.
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Fig. S1. The dimeric form of NCLX and its cellular and subcellular distribution. (a) Immunoblot analysis of NCLX expression levels using the CSR-antibody (see SI Methods) in total cellular and mitochondrial fractions prepared from HEK-293 cells. Note that NCLX distribution detected with this antibody is similar to that seen with the antibody used in Fig. 1 in the main text. (b) Based on the well documented tendency of NCX proteins to form oligomers (15), we suggest that the 100-kDa form is a dimer of NCLX. To test this hypothesis, immunoblot analysis of rat brain mitochondrial fractions was performed before (control) and 1 h after incubation in 2× concentrated sample buffer at 50°C (heat denaturate). The apparent NCLX distribution shifts from a dominant 100-kDa form to a 50-kDa polypeptide following this denaturation procedure. This supports our hypothesis that the 100-kDa form is an SDS-stable dimer of NCLX. (c) Lysates of cells transfected to express NCLX-cMyc, NCLX-6His, or both, as indicated, were immunoprecipitated with anti-6His antibodies and immunoblots were performed with antibodies against c-Myc (see Materials and Methods in the main text). Note that the c-Myc–tagged NCLX was coprecipitated with the 6His-tagged NCLX, further substantiating our hypothesis that NCLX is found as a dimer. (d) Representative mitochondria used for the quantitative analysis of the Immunogold labeling of overexpressed (NCLX) or endogenous (control) NCLX in SHSY-5Y cells (Arrows point to Immunogold particles). Note that NCLX labeling is localized to the inner cristae of the mitochondria.
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Fig. S2. Subcellular distribution of NCLX and the NCLX-S468T mutant versus the plasma membrane–targeted NCKX4. (a) Immunoblot analysis using anti-FLAG antibodies (see Materials and Methods in the main text) of cellular fractions from HEK-293-T cells transfected with a NCKX4-FLAG–encoding plasmid or control (vector). NCKX4-FLAG staining was found in plasma membrane fraction whereas the mitochondrial protein VDAC was found mostly in the mitochondrial fraction. (b) HEK-293-T cells transfected to express either murine NCLX or NCKX4 or transfected with control plasmids were loaded with the cytoplasmic Ca2+-sensitive dye Fura-2 and superfused with the indicated Na+-containing and Na+-free (replaced by NMDG+) Ringer solution containing 0.5 mM Ca2+. Changes in cytosolic Ca2+, mediated by the reverse and forward Na+/Ca2+ exchange modes, were monitored in cells expressing NCKX4 but not in cells expressing the murine form of NCLX- or vector-transfected cells. This finding is consistent with previous studies showing lack of plasma membrane activity or localization of murine NCLX (3), and strongly indicates that changes in mitochondrial Ca2+ transport are mediated by mitochondrial NCLX and are not related to an indirect plasma membrane activity of this protein. (c–f) Immunoblot analysis of cellular fractions from HEK-293 cells transfected with control (c), human NCLX- (d), NCLX-S468T-encoding (e), or the murine NCLX isoform (f) plasmids. Note that the expression of endogenous and ectopically expressed NCLX is highly enriched in the mitochondrial fraction.
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Fig. S3. Ca2+ and pH values of intact and permeabilized cells. (a) Average of initial fluorescent signal (F430) in the indicated cells expressing RP-mt. No significant differences in steady state Ca2+ levels are observed 48 h following the transfection with the overexpression or silencing constructs of NCLX. (b) Representative image of HEK-293 cells expressing RP-mt (green) and stained with Mitotracker (red, magnification ×40). Projection of the images (Right) shows colocalization of RP-mt with Mitotracker and indicate that, in agreement with previous studies (1), RP-mt is strictly localized in the mitochondria. (c) HEK293 cells loaded with CFDA were subjected to permeabilization procedure as described in Fig. 5 in the main text (see Materials and Methods in the main text) and analyzed by flow cytometry. Bar graph depicting means of CFDA fluorescence intensities (n = 5, **P < 0.01) in control and permeabilized cells is shown (Left). (Right) Histogram overlay obtained in a typical experiment. CFDA fluorescence measured before and after digitonin treatment is represented by red and green curves, respectively. Gray curve shows a background fluorescence emitted by cells not labeled by CFDA (y axis depicts cell numbers). (d) HEK-293 cells expressing RP-mt alone (control) or together with NCLX were permeabilized and loaded with Ca2+ as described in Fig. 4A in the main text. RP-mt fluorescence (F430) was measured following Ca2+ loading. Note that, following Ca2+ loading, the Ca2+ level was similar in NCLX-expressing and control cells, thus allowing for accurate comparison between NCLX-expressing and control cells. (e) RP-mt F485 measured in SHSY-5Y cells overexpressing NCLX or not (control) using the same experimental procedure as in Fig. 3A in the main text. (f) Cells were treated with 2 μM of the protonophore FCCP as indicated to diminish the mitochondrial pH gradient, and RP-mt F485 was measured. (g) Average values of fluorescence at 485 nm before and after application of either ATP or FCCP (taken from E and F). Note that application of both ATP and FCCP triggered a similar fluorescent change in the F485 signal of the RP-mt. Because of the concern that these fluorescent changes may be related to pH change, and consistent with previous studies (16, 17), we relied on only the Ca2+ sensitive, but pH-insensitive, 430-nm wavelength in our Ca2+ measurements.
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Fig. S4. The effect of NCLX overexpression or silencing on steady-state Ca2+ levels, membrane potential, and cytoplasmic Ca2+ response. (a) SHSY-5Y cells transfected with NCLX-encoding or control vectors were loaded with JC1 (see SI Methods) and green and red fluorescent intensities were measured by flow cytometry in the steady state (resting, Left) and after application of 5 μM FCCP (Right). (b) Quantitative analysis of the ratio between red and green florescent intensities measured in control or NCLX-expressing cells (n = 9). No change in the JC1 red and green fluorescent ratio was apparent between control and NCLXexpressing cells, suggesting similar mitochondrial membrane potential levels in both cell types. (c) Mitochondrial membrane potential was determined with TMRE. HEK-293 cells that were transfected with the indicated constructs and loaded with TMRE were superfused with Ringer solution and subsequently with the same solution containing 10 μM CCCP while monitoring TMRE fluorescence. Data are presented as ΔF fluorescence of TMRE (difference of fluorescence values at baseline resting and fluorescence values after exposure to CCCP) versus control. Note that the mitochondria of HEK293 transfected with the NCLX S468T or NCLX siRNA constructs were slightly hyperpolarized compared with control. (d) Cytosolic Ca2+ responses following the application of ATP (40 μM) in Ca2+-free Ringer solution using Fura-2 loaded HEK-293 cells transfected with either NCLX siRNA or control siRNA, as described in Fig. 3 in the main text. No difference in the ATP-dependent cytosolic Ca2+ response was found following NCLX expression or silencing, indicating that, using this experimental paradigm, mitochondrial NCLX did not change the cytosolic Ca2+ response. (e) The same experiment as in d in SHSY-5Y cells expressing either NCLX or the shRNA construct aimed to silence NCLX. (f) ATP-dependent cytosolic Ca2+ response in HEK-293 cells expressing NCLX or controls that were superfused with Ca2+-containing Ringer solution, Ca2+ was then removed from the superfusing solution and was then readded to monitor store-operated Ca2+ influx. (g) Average cytosolic Ca2+ levels at distinct time points (arrow, f) (n = 7, *P < 0.05). Note that, in the presence of Ca2+-containing Ringer solution, expression of NCLX was associated with elevated cytosolic Ca2+ levels, consistent with the role of the mitochondrial exchanger in elevating cytosolic Ca2+ and activation of SOC.
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Fig. S5. Activity of human NCLX and the NCLX-S468T mutant, and the effect of endogenous NCLX knockdown on mitochondrial Na+- or Li+-dependent Ca2+ efflux. (a) HEK-293 cells transfected with either NCLX-S468T–encoding or control plasmids and expressing RP-mt were superfused with ATP (40 μM) containing Ringer solution while monitoring mitochondrial Ca2+ fluorescence (as in Fig. 3A in the main text). Note that expression of NCLX-S468T exerted a dominant negative effect on mitochondrial Ca2+ efflux and inhibited endogenous activity, similar to the effect shown in Fig. 3 in the main text. (b) The average of mitochondrial Ca2+ efflux rates (n = 11, *P < 0.05). (c) The same experimental paradigm as in a repeated with cells loaded with Fura-2. Similar cytosolic Ca2+ changes were triggered by ATP in both control and NCLX-S468T-expressing cells. (d) Endogenous NCLX expression was assessed by immunoblot analysis of CHO cells transfected with siNCLX or siControl constructs; β-actin served as loading marker (40 μg). (e) Mitochondrial Ca2+ responses triggered by ATP (40 μM, as in Fig. 3A in the main text) were measured in cells transfected with siNCLX or siControl together with the RP-mt-encoding plasmid. (f) The average rates of mitochondrial Ca2+ efflux are shown (n = 7, *P < 0.05). (g) RP-mt fluorescence was monitored in digitonin-permeabilized cells transfected with siNCLX or siControl. Mitochondrial Ca2+ uptake was induced by superfusion with Na+-free solution containing Ca2+ (60 μM). Ca2+ efflux was then monitored following superfusion in Ca2+-free solution in the presence of Na+. (h) The same experimental paradigm as described in G but replacing Na+ with Li+, as indicated. Note that endogenous NCLX mediates both Na+ or Li+-dependent Ca2+ efflux. (i) Average initial rates of mitochondrial Ca2+ efflux (n = 8 for all groups, *P < 0.05).
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