The P2X7 receptor is a key modulator of aerobic glycolysis

Ability to adapt to conditions of limited nutrient supply requires a reorganization of the metabolic pathways to balance energy generation and production of biosynthetic intermediates. Several fast-growing cells overexpress the P2X7 receptor (P2X7R) for extracellular ATP. A feature of this receptor is to allow growth in the absence of serum. We show here that transfection of P2X7R allows proliferation of P2X7R-transfected HEK293 (HEK293-P2X7) cells not only in the absence of serum but also in low (4 mM) glucose, and increases lactate output compared with mock-transfected HEK293 (HEK293-mock) cells. In HEK293-P2X7, lactate output is further stimulated upon addition of exogenous ATP or the mitochondrial uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP). In the human neuroblastoma cell line ACN, lactate output is also dependent on P2X7R function. P2X7R-expressing cells upregulate (a) the glucose transporter Glut1, (b) the glycolytic enzymes glyceraldehyde 3-phosphate dehydrogenase (G3PDH), (c) phosphofructokinase (PFK), (d) pyruvate kinase M2 (PKM2) and (e) pyruvate dehydrogenase kinase 1 (PDHK1); furthermore, P2X7R expression (a) inhibits pyruvate dehydrogenase (PDH) activity, (b) increases phosphorylated Akt/PKB and hypoxia-inducible factor 1α (HIF-1α) expression and (c) enhances intracellular glycogen stores. In HEK293-P2X7 cells, glucose deprivation increases lactate production, expression of glycolytic enzymes and ph-Akt/PKB level. These data show that the P2X7R has an intrinsic ability to reprogram cell metabolism to meet the needs imposed by adverse environmental conditions.     

The Na+-driven Cl-/HCO3- exchanger. Cloning, tissue distribution, and functional characterization

The Na(+)-driven Cl(-)/HCO(3)(-) exchanger is an important regulator of intracellular pH in various cells, but its molecular basis has not been determined. We show here the primary structure, tissue distribution, and functional characterization of Na(+)-driven chloride/bicarbonate exchanger (designated NCBE) cloned from the insulin-secreting cell line MIN6 cDNA library. The NCBE protein consists of 1088 amino acids having 74, 72, and 55% amino acid identity to the human skeletal muscle, rat smooth muscle, and human kidney sodium bicarbonate cotransporter, respectively. The protein has 10 putative membrane-spanning regions. NCBE mRNA is expressed at high levels in the brain and the mouse insulinoma cell line MIN6 and at low levels in the pituitary, testis, kidney, and ileum. Functional analyses of the NCBE protein expressed in Xenopus laevis oocytes and HEK293 cells demonstrate that it transports extracellular Na(+) and HCO(3)(-) into cells in exchange for intracellular Cl(-) and H(+), thus raising the intracellular pH. Thus, we conclude that NCBE is a Na(+)-driven Cl(-)/HCO(3)(-) exchanger that regulates intracellular pH in native cells.     

OAT2 catalyses efflux of glutamate and uptake of orotic acid

OAT (organic anion transporter) 2 [human gene symbol SLC22A7 (SLC is solute carrier)] is a member of the SLC22 family of transport proteins. In the rat, the principal site of expression of OAT2 is the sinusoidal membrane domain of hepatocytes. The particular physiological function of OAT2 in liver has been unresolved so far. In the present paper, we have used the strategy of LC (liquid chromatography)-MS difference shading to search for specific and cross-species substrates of OAT2. Heterologous expression of human and rat OAT2 in HEK (human embryonic kidney)-293 cells stimulated accumulation of the zwitterion trigonelline; subsequently, orotic acid was identified as an excellent and specific substrate of OAT2 from the rat (clearance=106 μl·min?1·mg of protein?1) and human (46 μl·min?1·mg of protein?1). The force driving uptake of orotic acid was identified as glutamate antiport. Efficient transport of glutamate by OAT2 was directly demonstrated by uptake of [3H]glutamate. However, because of high intracellular glutamate, OAT2 operates as glutamate efflux transporter. Thus expression of OAT2 markedly increased the release of glutamate (measured by LC-MS) from cells, even without extracellular exchange substrate. Orotic acid strongly trans-stimulated efflux of glutamate. We thus propose that OAT2 physiologically functions as glutamate efflux transporter. OAT2 mRNA was detected, after laser capture microdissection of rat liver slices, equally in periportal and pericentral regions; previous reports of hepatic release of glutamate into blood can now be explained by OAT2 activity. A specific OAT2 inhibitor could, by lowering plasma glutamate and thus promoting brain-to-blood efflux of glutamate, alleviate glutamate exotoxicity in acute brain conditions.     

Spontaneous autocrine release of protons activates ASIC-mediated currents in HEK293 cells

When examining HEK293 cells by whole-cell patch-clamp electrophysiology we found spontaneous currents, present in almost all cells. These currents were carried by Na(+) ions, were inhibited by amiloride and by cells exposure to acidic (pH 6.3) extracellular solutions. These properties (ion carrier, amiloride-sensitivity, and inactivation by constant lowering of extracellular pH) were similar to the properties of proton-activated currents measured from the same cells. Spontaneous currents required intracellular ATP, were completely inhibited by intracellular Ca(2+) buffering with BAPTA and were suppressed by intracellular administration of vesicular H(+)ATPase inhibitor bafilomycin. ATP-induced Ca(2+) influx through P2X receptors in HEK293 cells stably transfected with P2X(2), P2X(2/3) or P2X(4) purinoreceptor subunits transiently potentiated amplitude and frequency of spontaneous currents; this effect was antagonized by bafilomycin. We concluded that spontaneous currents represent activation of acid-sensitive ion channels (ASICs) by autocrine vesicular release of protons from HEK cells.     

A stable, inducible, dose-responsive ODC overexpression system in human cell lines

ODC is a labile protein subject to rapid turnover, and a conditional expression system providing long-term overexpression may be helpful in further understanding the biochemical properties of this enzyme and elucidating aspects of the polyamine biosynthetic pathway that have otherwise been difficult to study. HEK293 and LNCaP cell lines were engineered to stably and inducibly overexpress ODC using a Tet-on inducible construct. Clones from both cell lines were characterized by evaluating ODC mRNA expression, ODC activity, intracellular and extracellular polyamine levels, SSAT activity and growth kinetics. The ODC-inducible cell lines were time- and dose-responsive providing a mechanism to increase ODC and putrescine accumulation to a desired level in a flexible and controllable manner. The findings demonstrate that LNCaP ODC overexpressing cells maintained over a 100-fold increase in ODC activity and over a 10-fold increase in intracellular putrescine after 6 h. ODC induction at the highest levels was accompanied by a slight decline in intracellular spermidine and spermine levels and this observation was supported by the finding that SSAT activity was induced over 40-fold under these conditions. Growth rate remained unaffected following at least 12 h of ODC overexpression. Similar results were observed in the HEK293 ODC overexpressing cells.     

Na+ Sensitivity of ROMK1 K+ Channel: Role of the Na+/H+ Antiporter

To examine the extracellular Na⁺ sensitivity of a renal inwardly rectifying K⁺ channel, we performed electrophysiological experiments on Xenopus oocytes or a human kidney cell line, HEK293, in which we had expressed the cloned renal K⁺ channel, ROMK1 (Kir1.1). When extracellular Na⁺ was removed, the whole-cell ROMK1 currents were markedly suppressed in both the oocytes and HEK293 cells. Single-channel ROMK1 activities recorded in the cell-attached patch on the oocyte were not affected by removal of Na⁺ from the pipette solution. However, macro-patch ROMK1 currents recorded on the oocyte were significantly suppressed by Na⁺ removal from the bath solution. A blocker of Na⁺/H⁺ antiporters, amiloride, largely inhibited the Na⁺ removal-induced suppression of whole-cell ROMK1 currents in the oocytes. The pH-insensitive K80M mutant of ROMK1 was much less sensitive to Na⁺ removal. Na⁺ removal was found to induce a significant decrease in intracellular pH in the oocytes using H⁺-selective microelectrodes. Coexpression of ROMK1 with NHE3, which is a Na⁺/H⁺ antiporter isoform of the kidney apical membrane, conferred increased sensitivity of ROMK1 channels to extracellular Na⁺ in both the oocytes and HEK293 cells. Thus, it is concluded that the ROMK1 channel is regulated indirectly by extracellular Na⁺, and that the interaction between NHE transporter and ROMK1 channel appears to be involved in the mechanism of Na⁺ sensitivity of ROMK1 channel via regulating intracellular pH. Received: 13 April 1999/Revised: 15 July 1999     

Analysis of the suitability of calreticulin inducible HEK cells for adhesion studies: microscopical and biochemical comparisons

Calreticulin is a Ca²⁺-buffering ER chaperone that also modulates cell adhesiveness. In order to study the effect of calreticulin on the expression of adhesion-related genes, we created a calreticulin inducible Human Embryonic Kidney (HEK) 293 cell line. We found that fibronectin mRNA and both intra- and extra-cellular fibronectin protein levels increased following calreticulin induction. However, despite this increase in fibronectin, HEK293 cells did not assemble an extracellular fibrillar fibronectin matrix regardless of the level of calreticulin expression. Furthermore, HEK293 cells exhibited a poorly organized actin cytoskeleton, did not have clustered fibronectin receptors at the cell surface, and did not form focal contacts. This likely accounts for the lack of fibronectin matrix deposition by these cells regardless of calreticulin expression level. Vinculin abundance did not appreciably increase upon calreticulin induction and the level of active c-Src, a regulatory kinase of focal contacts, was found to be abundant and unregulated by calreticulin induction in these cells. The inability to form stable focal contacts and to commence fibronectin fibrillogenesis due to high c-Src activity may be responsible for the poor adhesive phenotype of HEK 293 cells. Thus, we show here that HEK293 cells are not suitable for microscopical studies of cell-substratum adhesions, but are best suited for biochemical studies. S. Papp and M. P. Fadel have contributed equally to this work.     

Latent membrane protein 1 is dispensable for Epstein-Barr virus replication in human embryonic kidney 293 cells

Epstein Barr Virus (EBV) replicates in oral epithelial cells and gains entry to B-lymphocytes. In B-lymphocytes, EBV expresses a restricted subset of genes, the Latency III program, which converts B-lymphocytes to proliferating lymphoblasts. Latent Membrane Protein 1 (LMP1) and the other Latency III associated proteins are also expressed during virus replication. LMP1 is essential for virus replication and egress from Akata Burkitt Lymphoma cells, but a role in epithelial cell replication has not been established. Therefore, we have investigated whether LMP1 enhances EBV replication and egress from HEK293 cells, a model epithelial cell line used for EBV recombinant molecular genetics. We compared wild type (wt) and LMP1-deleted (LMP1Δ) EBV bacterial artificial chromosome (BAC) based virus replication and egress from HEK293. Following EBV immediate early Zta protein induction of EBV replication in HEK293 cells, similar levels of EBV proteins were expressed in wt- and LMP1Δ-infected HEK293 cells. LMP1 deletion did not impair EBV replication associated DNA replication, DNA encapsidation, or mature virus release. Indeed, virus from LMP1Δ-infected HEK293 cells was as infectious as EBV from wt EBV infected HEK cells. Trans-complementation with LMP1 reduced Rta expression and subsequent virus production. These data indicate that LMP1 is not required for EBV replication and egress from HEK293 cells.     

A sodium zinc exchange mechanism is mediating extrusion of zinc in mammalian cells

Zinc influx, driven by a steep inward electrochemical gradient, plays a fundamental role in zinc signaling and in pathophysiologies linked to intracellular accumulation of toxic zinc. Yet, the cellular transport mechanisms that actively generate or maintain the transmembrane gradients are not well understood. We monitored Na+-dependent Zn2+ transport in HEK293 cells and cortical neurons, using fluorescent imaging. Treatment of the HEK293 cells with CaPO4 precipitates induced Na+-dependent Zn2+ extrusion, against a 500-fold transmembrane zinc gradient, or zinc influx upon reversal of Na+ gradient, thus indicating that Na+/Zn2+ exchange is catalyzing active Zn2+ transport. Depletion of intracellular ATP did not inhibit the Na+-dependent Zn2+ extrusion, consistent with a mechanism involving a secondary active transporter. Inhibitors of the Na+/Ca2+ exchanger failed to inhibit Na+-dependent Zn2+ efflux. In addition, zinc transport was unchanged in HEK293 cells heterologously expressing functional cardiac or neuronal Na+/Ca2+ exchangers, thus indicating that the Na+/Zn2+ exchange activity is not mediated by the Na+/Ca2+ exchanger. Sodium-dependent zinc exchange, facilitating the removal of intracellular zinc, was also monitored in neurons. To our knowledge, the Na+/Zn2+ exchanger described here is the first example of a mammalian transport mechanism capable of Na+-dependent active extrusion of zinc. Such mechanism is likely to play an important role, not only in generating the transmembrane zinc gradients, but also in protecting cells from the potentially toxic effects of permeation of this ion.     

Mechanisms of ATP release by human trabecular meshwork cells, the enabling step in purinergic regulation of aqueous humor outflow

Our guiding hypothesis is that ecto-enzymatic conversion of extracellular ATP to adenosine activates A(1) adenosine receptors, reducing resistance to aqueous humor outflow and intraocular pressure. The initial step in this purinergic regulation is ATP release from outflow-pathway cells by mechanisms unknown. We measured similar ATP release from human explant-derived primary trabecular meshwork (TM) cells (HTM) and a human TM cell line (TM5). Responses to 21 inhibitors indicated that pannexin-1 (PX1) and connexin (Cx) hemichannels and P2X(7) receptors (P2RX(7) ) were comparably important in modulating ATP release induced by hypotonic swelling, whereas vesicular release was insignificant. Consistent with prior studies of PX1 activity in certain other cells, ATP release was lowered by the reducing agent dithiothreitol. Overexpressing PX1 in HEK293T cells promoted, while partial knockdown (KD) in both HEK293T and TM5 cells inhibited hypotonicity-activated ATP release. Additionally, KD reduced the pharmacologically defined contribution of PX1 and enhanced those of Cx and P2RX(7) . ATP release was also triggered by raising intracellular Ca(2+) activity with ionomycin after a prolonged lag time and was unaffected by the PX1 blocker probenecid, but nearly abolished by P2RX(7) antagonists. We conclude that swelling-stimulated ATP release from human TM cells is physiologically mediated by PX1 and Cx hemichannels and P2X(7) receptors, but not by vesicular release. PX1 appears not to be stimulated by intracellular Ca(2+) in TM cells, but can be modulated by oxidation-reduction state. The P2RX(7) -dependent component of swelling-activated release may be mediated by PX1 hemichannels or reflect apoptotic magnification of ATP release, either through itself and/or hemichannels.     

Heparan sulfate proteoglycan synthesis in CHO DG44 and HEK293 cells

Chinese hamster ovary (CHO) and human embryonic kidney 293 (HEK293) cells are the most popular host cells for transient gene expression (TGE) of therapeutic proteins. These host cells require high transfection efficiency in order to enhance TGE. Heparan sulfate proteoglycan (HSPG) at the cell surface is known to regulate endocytosis for gene delivery. The HSPG expression in CHO DG44 and HEK293E cells was investigated in an effort to enhance the TGE. Immunostaining of HSPGs followed by confocal microscopy and flow cytometry analyses revealed that CHO DG44 cells possessed a higher amount of cell-surface and intracellular HSPGs than HEK293E cells. The mRNA levels of the representative enzymes involved in the HSPG biosynthesis in CHO DG44, which were determined by quantitative real time PCR, were quite different from those in HEK293E cells. Taken together, the results obtained here would be useful in improving TGE in CHO DG44 and HEK293E cells through genetic engineering of HSPG synthesis.     

Organic anion transporter 1 (OAT1/SLC22A6) enhances bioluminescence based on d-luciferin–luciferase reaction in living cells by facilitating the intracellular accumulation of d-luciferin

Bioluminescence (BL) imaging based on d-luciferin (d-luc)–luciferase reaction allows noninvasive and real-time monitoring of luciferase-expressing cells. Because BL intensity depends on photons generated through the d-luc–luciferase reaction, an approach to increase intracellular levels of d-luc could improve the detection sensitivity. In the present study, we showed that organic anion transporter 1 (OAT1) is useful, as a d-luc transporter, in boosting the BL intensity in luciferase-expressing cells. Functional screening of several transporters showed that the expression of OAT1 in HEK293?cells stably expressing Pyrearinus termitilluminans luciferase (HEK293/eLuc) markedly enhanced BL intensity in the presence of d-luc. When OAT1 was transiently expressed in HEK293?cells, intracellular accumulation of d-luc was higher than that in control cells, and the specific d-luc uptake mediated by OAT1 was saturable with a Michaelis constant (K_m) of 0.23?μM. The interaction between OAT1 and d-luc was verified using 6-carboxyfluorescein, a typical substrate of OAT1, which showed that d-luc inhibited the uptake of 6-carboxyfluorescein mediated by OAT1. BL intensity was concentration-dependent at steady states in HEK293/eLuc cells stably expressing OAT1, and followed Michaelis–Menten kinetics with an apparent K_m of 0.36?μM. In addition, the enhanced BL was significantly inhibited by OAT1-specific inhibitors. Thus, OAT1-mediated transport of d-luc could be a rate-limiting step in the d-luc–luciferase reaction. Furthermore, we found that expressing OAT1 in HEK293/eLuc cells implanted subcutaneously in mice also significantly increased the BL after intraperitoneal injection of d-luc. Our findings suggest that because OAT1 is capable of transporting d-luc, it can also be used to improve visualization and monitoring of luciferase-expressing cells.     

Identification of genotoxic stress in human cells by fluorescent monitoring of p53 expression.

The tumor suppressor protein p53 is induced upon DNA damage essentially by post-translational regulatory mechanisms, which lead to a substantial increase of p53 levels. To exploit this essential property of p53, we developed a novel reporter system for monitoring accumulation and subcellular translocation of p53 protein, which is able to function as a simple test for detecting mutagenic and genotoxic stress in human cells. For this purpose, we constructed a plasmid with a specific translational TP53::EGFP gene fusion and selected stable transfected clones in the human cell line HEK293, in which p53 is functionally stabilized due to the expression of the transgenic adenoviral E1A oncoproteins. HEK293-TP53::EGFP clones may be used as a living cell system for monitoring not only of the induction of p53 protein in the cell, but also of its subcellular localization. Using this human reporter cell system, we examined levels of p53 by fluorescence microscopy and by FACS analysis following treatment with several classes of genotoxic and carcinogenic compounds. All tested DNA damaging agents caused a significant increase of intracellular p53-EGFP levels in a concentration-dependent manner. On the other hand, non-genotoxic carcinogens and stress conditions that cannot damage DNA were not able to induce p53-EGFP accumulation. The induction effect caused by genotoxic stress was found to be dependent on the endogenous p53 status, because it was not observed in p53-deficient cell lines. This corroborates the notion that p53 may be used as an universal sensor for genotoxic stress and demonstrates the usefulness of HEK293-p53-EGFP cells as a reporter system for identification of mutagens and genotoxic carcinogens in human cells by means of visualizing and monitoring intracellular p53 levels and localization.     

Purification and characterization of the creatine transporter expressed at high levels in HEK293 cells

The bovine creatine transporter (CreaT) has been purified from membranes of HEK293 cells stably expressing high levels of the transporter. Membranes were solubilized with decyl maltoside and the CreaT was purified (90% pure) by affinity chromatography on wheat germ agglutinin (WGA)-Sepharose and gel-filtration. The CreaT was shown to be an approximately 70 kDa glycoprotein by SDS-polyacrylamide gel electrophoresis and Western blotting. Identification of the CreaT was confirmed by sequencing tryptic peptides by mass spectrometry. Laser light scattering showed the majority of the CreaT to be present as a 224 kDa species. Additional purification was obtained when the Creat was eluted from the WGA column and purified by gel-filtration in Fos-choline 12 instead of decyl maltoside, followed by a second WGA affinity step to exchange the detergent for sodium cholate. This resulted in a 30-fold purification (95% purity) of the approximately 70kDa CreaT, with a yield of 15%. From this, it is estimated that the CreaT comprises approximately 3% of total HEK293-CreaT membrane protein. Gel-filtration showed the transporter to migrate with an apparent molecular mass of 210 kDa. Circular dichroism showed a predominantly alpha-helical structure, consistent with the 12 transmembrane domains predicted for the transporter. This work has enabled the purification of the CreaT in amounts ( approximately 100 microg) that make it feasible to consider structural studies of a member of the Na(+)- and Cl(-)-dependent neurotransmitter transporter family.     

xCt cystine transporter expression in HEK293 cells: pharmacology and localization

xCT, the core subunit of the system x(c)(-) high affinity cystine transporter, belongs to a superfamily of glycoprotein-associated amino acid transporters. Although xCT was shown to promote cystine transport in Xenopus oocytes, little work has been done with mammalian cells (Sato, H., Tamba, M., Ishii, T., and Bannai, S. J. Biol. Chem. 274, 11455-11458, 1999). Therefore, we have constructed mammalian expression vectors for murine xCT and its accessory subunit 4F2hc and transfected them into HEK293 cells. We report that this transporter binds cystine with high affinity (81 microM) and displays a pharmacological profile expected for system x(c)(-). Surprisingly, xCT transport activity in HEK293 cells is not dependent on the co-expression of the exogenous 4F2hc. Expression of GFP-tagged xCT indicated a highly clustered plasma membrane and intracellular distribution suggesting the presence of subcellular domains associated with combating oxidative stress. Our results indicate that HEK293 cells transfected with the xCT subunit would be a useful vehicle for future structure-function and pharmacology experiments involving system x(c)(-).     

RNA沉默抑制子p19调控细胞周期相关蛋白表达

番茄丛矮病毒的P19蛋白不仅是一个重要的病毒致病因子,而且还可作为RNA干扰(RNAi)的抑制子．这种作用是通过限制细胞内的小RNA,比如小干扰RNA(siRNAs)和微RNA(miRNAs)来实现．但是目前对P19蛋白在哺乳动物细胞上的作用还未见报道．构建了一株p19稳定表达的293细胞系,即293-p19．流式细胞仪分析发现在293细胞中过量表达P19蛋白可显著引发细胞周期的G2/M阻滞．细胞增殖实验显示,293-p19细胞的DNA复制及细胞生长均受到显著的抑制． 此外,研究还发现p19可使人胚肾293细胞内的细胞周期调控子的表达谱发生改变． 其中包括上调cyclin A1,CDK2,CDK4,CDK6,p18,cyclin D2,p19INK4d和E2F1,及下调p15,cyclin A,cyclin B1和cyclin E1的表达．上述研究结果提示,p19有可能靶向多个G2/M调控蛋白从而引发细胞的G2/M阻滞．     

Extracellular ATP causes ROCK I-dependent bleb formation in P2X7-transfected HEK293 cells

The P2X7 ATP receptor mediates the cytotoxic effect of extracellular ATP. P2X7-dependent cell death is heralded by dramatic plasma membrane bleb formation. Membrane blebbing is a complex phenomenon involving as yet poorly characterized intracellular pathways. We have investigated the effect of extracellular ATP on HEK293 cells transfected with the cytotoxic/pore-forming P2X7 receptor. Addition of ATP to P2X7-transfected, but not to wt P2X7-less, HEK293 cells caused massive membrane blebbing within 1-2 min. UTP, a nucleotide incapable of activating P2X7, had no early effects on cell shape and bleb formation. Bleb formation triggered by ATP was reversible and required extracellular Ca2+ and an intact cytoskeleton. Furthermore, it was completely prevented by preincubation with the P2X blocker oxidized ATP. It was recently observed that the ROCK protein is a key determinant of bleb formation. Preincubation of HEK293-P2X7 cells with the ROCK blocker Y-27632 completely prevented P2X7-dependent blebbing. Although ATP triggered cleavage of the ROCK I isoform in P2X7-transfected HEK293 cells, the wide range caspase inhibitor z-VAD-fluoromethylketone had no effect. These observations suggest that P2X7-dependent plasma membrane blebbing depends on the activation of the serine/threonine kinase ROCK I.     

Ca(2+) signaling by TRPC3 involves Na(+) entry and local coupling to the Na(+)/Ca(2+) exchanger

TRPC3 has been suggested as a key component of phospholipase C-dependent Ca(2+) signaling. Here we investigated the role of TRPC3-mediated Na(+) entry as a determinant of plasmalemmal Na(+)/Ca(2+) exchange. Ca(2+) signals generated by TRPC3 overexpression in HEK293 cells were found to be dependent on extracellular Na(+), in that carbachol-stimulated Ca(2+) entry into TRPC3 expressing cells was significantly suppressed when extracellular Na(+) was reduced to 5 mm. Moreover, KB-R9743 (5 microm) an inhibitor of the Na(+)/Ca(2+) exchanger (NCX) strongly suppressed TRPC3-mediated Ca(2+) entry but not TRPC3-mediated Na(+) currents. NCX1 immunoreactivity was detectable in HEK293 as well as in TRPC3-overexpressing HEK293 cells, and reduction of extracellular Na(+) after Na(+) loading with monensin resulted in significant rises in intracellular free Ca(2+) (Ca(2+)(i)) of HEK293 cells. Similar rises in Ca(2+)(i) were recorded in TRPC3-overexpressing cells upon the reduction of extracellular Na(+) subsequent to stimulation with carbachol. These increases in Ca(2+)(i) were associated with outward membrane currents at positive potentials and inhibited by KB-R7943 (5 microm), chelation of extracellular Ca(2+), or dominant negative suppression of TRPC3 channel function. This suggests that Ca(2+) entry into TRPC3-expressing cells involves reversed mode Na(+)/Ca(2+) exchange. Cell fractionation experiments demonstrated co-localization of TRPC3 and NCX1 in low density membrane fractions, and co-immunoprecipitation experiments provided evidence for association of TRPC3 and NCX1. Glutathione S-transferase pull-down experiments revealed that NCX1 interacts with the cytosolic C terminus of TRPC3. We suggest functional and physical interaction of nonselective TRPC cation channels with NCX proteins as a novel principle of TRPC-mediated Ca(2+) signaling.