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Transient receptor potential melastatin 7 (TRPM7) channels are novel Ca2+-permeable non-selective cation channels ubiquitously expressed. Activation of TRPM7 channels has been shown to be involved in cellular Mg2+ homeostasis, diseases caused by abnormal magnesium absorption, and in Ca2+-mediated neuronal injury under ischemic conditions. Here we show strong evidence suggesting that TRPM7 channels also play an important role in cellular Zn2+ homeostasis and in Zn2+-mediated neuronal injury. Using a combination of fluorescent Zn2+ imaging, small interfering RNA, pharmacological analysis, and cell injury assays, we show that activation of TRPM7 channels augmented Zn2+-induced injury of cultured mouse cortical neurons. The Zn2+-mediated neurotoxicity was inhibited by nonspecific TRPM7 blockers Gd3+ or 2-aminoethoxydiphenyl borate, and by knockdown of TRPM7 channels with small interfering RNA. In addition, Zn2+-mediated neuronal injury under oxygen-glucose deprivation conditions was also diminished by silencing TRPM7. Furthermore, we show that overexpression of TRPM7 channels in HEK293 cells increased intracellular Zn2+ accumulation and Zn2+-induced cell injury, while silencing TRPM7 by small interfering RNA attenuated the Zn2+-mediated cell toxicity. Thus, TRPM7 channels may represent a novel target for neurological disorders where Zn2+ toxicity plays an important role.
Calcium toxicity is one of the key factors responsible for neuronal cell death associated with neurological disorders (1). Following brain ischemia, for example, intracellular calcium concentration ([Ca2+]i)2 increases dramatically, either through extracellular calcium entry or intracellular calcium release. Excessive elevation of [Ca2+]i activates destructive signaling cascades leading to neuronal cell death.
Despite a number of studies that have clearly demonstrated the role of Ca2+ toxicity in ischemic neuronal death, clinical trials targeting the Ca2+ entry pathways, e.g. by using glutamate antagonists, have had inconclusive results (2, 3). Although the importance of [Ca2+]i accumulation in neuronal cell death cannot be denied, certain results with [Ca2+]i measurement have been questionable. For example, previous studies reported that some indicators commonly used for calcium imaging, e.g. Calcium Green-1 and fura-2, are responsive to zinc with an extremely high affinity, and that specific zinc chelators reduced the intensity of calcium indicators (4,–7). These findings suggest that some of the biological effects previously assumed to be mediated by Ca2+ may be mediated, at least partially, by zinc ions. Like calcium, recent studies have demonstrated that zinc ions play an important role in neuronal injuries associated with various neurological conditions (8, 9). The exact pathways mediating intracellular zinc accumulations and toxicity are, however, not clear.
Zinc is one of the most crucial trace metals in cells. For example, zinc is required for the function of a broad range of enzymes involved in transcription, protein synthesis, and signal transductions (10). Although there are low levels of free zinc in cells, most zinc ions are bound to intracellular proteins (11). The mechanisms that affect the free zinc concentration are, therefore, pivotal for maintaining normal brain function. Although the extracellular fluid may contain up to several micromolar of zinc, intracellular zinc concentration ([Zn2+]i) is generally maintained at 10−9–10−10 m (10, 12, 13). This steep gradient across the cell membrane is maintained primarily by zinc extrusion systems such as zinc transporters (ZnTs). At least 10 members of ZnTs, with different tissue distribution, have been identified in the ZnTs family. They promote the efflux of intracellular zinc into extracellular space or uptake of zinc into vesicles (14, 15). In contrast to ZnTs, Zrt- and Irt-like proteins, or ZIPs, are known to transport zinc into the cells (14, 15). In addition, some calcium channels, e.g. voltage-dependent calcium channels (VDCCs), N-methyl-d-aspartate (NMDA) receptors, and amino-3-hydroxy-5-methyl-4-isoxazol propionate (AMPA)/kinate receptors have been reported to be zinc permeable (16, 17). The activities of these channels thus affect intracellular zinc homeostasis and toxicity. Unfortunately, clinical trials using the antagonists of these channels failed to provide satisfactory neuroprotection (2, 3).
Transient receptor potential melastatin 7 (TRPM7) is a member of the large TRP channel superfamily expressed in almost every tissue and cell type (18,–20). Increasing evidence suggests that activation of TRPM7 channels contributes to various physiological and pathophysiological processes (21,–23). Notably, we demonstrated that activation of TRPM7 channels by oxygen-free radicals plays a critical role in hypoxia-induced, glutamate-independent, neuronal injury (24). In addition to well established Ca2+ permeability, TRPM7 is, at present, the only known zinc permeable channel among the TRP family of ion channels (18, 25). It is reported that zinc permeability for TRPM7 channels is 4-fold higher than Ca2+ (25). Despite these facts, it has not been established whether TRPM7 channels play a role in intracellular zinc dynamics at physiological/pathological relevant concentrations, and more importantly, in zinc-mediated neurotoxicity. Using a combination of fluorescent zinc imaging, metal response element-based reporter gene assay, cell injury analysis, and small interfering RNA techniques, we show strong evidence suggesting that TRPM7 channels represent a novel pathway for intracellular zinc accumulation and zinc-mediated neurotoxicity.
Mouse cortical neurons were cultured as described previously (26). The protocol for the use of mice for neuronal cultures was reviewed and approved by the Institutional Animal Care and Use Committee of Legacy Clinical Research and Technology Center. Briefly, time-pregnant (embryonic day 16) Swiss mice (Charles River Laboratories) were anesthetized with halothane followed by cervical dislocation. Brains of fetuses were removed rapidly and placed in Ca2+/Mg2+-free cold phosphate-buffered saline. Cerebral cortices were dissected under a dissection microscope and incubated with 0.05% trypsin-EDTA for 10 min at 37 °C, followed by trituration with fire-polished glass pipettes. Cells were counted and plated in poly-l-ornithine-coated culture dishes or 24-well plates at a density of 1 × 106 cells/dish or 2 × 105 cells/well, respectively. Neurons were cultured with Neurobasal medium supplemented with B-27 (Invitrogen), and maintained at 37 °C in a humidified 5% CO2 atmosphere incubator. Cultures were fed twice a week. Neurons were used for the experiments between days 11 and 15 in vitro.
Human embryonic kidney (HEK293) cells, with inducible expression of human TRPM7 channels (HEK:TRPM7 cells), were cultured in minimal essential medium supplemented with 10% fetal bovine serum and antibiotics (19). For the induction of TRPM7, the cells were treated with 1 μg/ml of tetracycline, as described in our previous studies (22).
Immunoblotting was performed as described previously (27). Cells cultured in 35-mm dishes or 6-well plates were lysed in lysis buffer (50 mm Tris-HCl, pH 7.5, 100 mm NaCl, 1% Triton X-100, and protease inhibitors (Roche Diagnostics)). After centrifugation at 12,000 × g at 4 °C for 30 min the lysates were collected. The aliquots were then mixed with Laemmli sample buffer and incubated at 37 °C for 1 h. The samples were resolved by 7.5% SDS-PAGE, followed by electrotransfer to polyvinylidene difluoride membranes. For visualization, blots were probed with antibodies against TRPM7 (1:250; Abgent) or β-actin (1:2000; Abcam), and detected using horseradish peroxidase-conjugated secondary antibodies (1:1000; Cell Signaling) and an ECL kit (GE Healthcare). The intensity of the protein band was densitometrically quantified.
The plasmid containing short hairpin RNA (shRNA) for silencing human TRPM7 was described previously (22).
To construct the plasmid for silencing mouse TRPM7, two oligonucleotides were annealed and inserted into pSilencer 1.0-U6 (Ambion) according to manufacturer's instructions. The target sequence for TRPM7 corresponded to coding region 5152–5172 (GenBank accession number NM021450 (28)). A fragment cut with BamHI was excised and inserted into the BamHI site of pCAGGS-eGFP (kindly provided by Dr. J. Miyazaki, Division of Stem Cell Regulation Research, Osaka University Medical School, Osaka, Japan) to express both enhanced green fluorescence protein (eGFP) and shRNA (TRPM7-shRNA/eGFP) (29). For the negative control, a fragment cut with BamHI from pSilencer 1.0-U6 was inserted into pCAGGS-eGFP (control/eGFP).
For transfection, FuGENE HD (Roche Diagnostics) and NeuroFect (Genlantis) were used for HEK:TRPM7 cells, and for cortical neurons (between days 8 and 11 in vitro) in accordance with the manufacturer's instructions. The transfection efficiencies, as determined by eGFP-positive cells, were 2–5% for cultured mouse cortical neurons.
LDH measurement was performed as described in our previous studies (30, 31). Cells grown on 24-well plates were first washed with phosphate-buffered saline. 50 μl of the culture medium was taken from each well and placed into a 96-well plate for background LDH measurement. Cells were then treated with zinc containing medium for 1 or 2 h, followed by a wash and incubation in normal culture medium for 23 or 22 h, respectively. 50 μl of the medium was transferred from each well to 96-well plates for measurement of injury-mediated LDH release. For measurement of the maximal releasable LDH, cells were incubated with Triton X-100 (final concentration 0.5%) for 30 min at the end of each experiment. 50 μl of assay reagent from the cytotoxicity detection kit (Roche Diagnostics) was added to each sample according to the manufacturer's instructions. 30 min later, the absorbance at 492 and 620 nm was examined by a spectrometer (SpectraMax Plus, Molecular Devices). The values of the absorbance at 492 nm were subtracted by those at 620 nm to yield the value of LDH release.
Cells grown on 24-well plates were transfected with p(MREa)6-Luc (kindly provided by Dr. C. Seguin, Centre de Recherche en Cancerologie de l'Universite Laval) together with pRL-TK (Promega) to measure transfection efficiency (32). Expression plasmids were transfected in the following amounts per well: 0.3 μg of p(MREa)6-Luc and 0.1 μg of pLR-TK for HEK:TRPM7 cells; 0.8 μg of shRNA-containing pCAGGS-eGFP, 0.9 μg of p(MREa)6-Luc, and 0.3 μg of pLR-TK for cortical neurons. Luciferase assays were performed using the Dual-Luciferase Reporter System (Promega) with a microplate luminometer (VeritasTM, Turner Biosystems), in which relative firefly luciferase activity was calculated by normalizing the transfection efficiency according Renilla luciferase activity. Relative firefly luciferase activity detected in the cell lysates was presented (33).
The intracellular zinc level of HEK:TRPM7 cells or mouse cortical neurons was imaged using a zinc-sensitive fluorescent dye, FluoZin-3 (Invitrogen). Cells were incubated with 5 μm FluoZin-3-AM in standard extracellular fluid (ECF) for 30 min at 37 °C, followed by de-esterification of the dye for another 30 min at room temperature (22–25 °C). The coverslips containing dye-loaded cells were held in a recording chamber placed on the stage of an inverted microscope (Eclipse TE2000-U; Nikon), and superfused with standard ECF at room temperature for at least 10 min prior to experiments. FluoZin-3 was excited at a wavelength of 490 nm, and emitted light was filtered with a 500–550-nm band pass filter. Zinc fluorescence was detected with a ×40 objective lens (Super Fluor ×40, numerical aperture = 0.90; Nikon) and a CCD camera (CoolSNAP ES2; Photometrics), using Imaging Workbench software (INDEC BioSystems). Fluorescence intensities (ΔF) were normalized to the resting values (F, average of five-data points recorded immediately before the change of solutions).
For electrophysiology recording and zinc imaging, cells were superfused with a standard ECF containing (in mm) 140 NaCl, 5.4 KCl, 2 CaCl2, 1 MgCl2, 33 glucose, 20 HEPES (pH 7.4, adjusted with NaOH, and 320–335 mosmol with sucrose). For cortical neurons, MK-801 (10 μm; Sigma), 6-cyano-7-nitroquinoxaline-2,3-dione (20 μm; Sigma), and nimodipine (5 μm; Sigma) were included in the extracellular solutions to block potential zinc entry through NMDA and AMPA/kinate receptors, and VDCCs.
OGD was performed as described in our previous studies (30). The cultured cells were treated with glucose-free ECF containing (in mm) 140 NaCl, 5.4 KCl, 2 CaCl2, 1 MgCl2, 33 N-methyl-d-glucamine (or N-methyl-d-glucammonium at physiological pH), and 20 HEPES (pH 7.4, 320–335 mosmol), and then transferred to an anaerobic chamber containing 5% CO2 and 95% N2 atmosphere for 1 h. Cells were then washed with phosphate-buffered saline and placed in a normal cell culture incubator. All OGD experiments were carried out at 37 °C.
Cortical neurons were cultured on etched coverslips (Bellco Biotechnology) for monitoring the same cells before and after treatments. Three days after transfection, cells were treated with the indicated conditions for 1 h and then incubated in normal culture medium for 23 h. PI was added to the culture medium to a final concentration of 5 μg/ml. Images of the eGFP-positive cells were taken with the same fluorescent microscope used for zinc imaging. PI-positive cells were regarded as injured cells. PI was excited at a wavelength of 540 nm, and emitted light was filtered with a 550–660-nm band pass filter. 6–20 cells were evaluated in each culture, and 3–5 independent cultures were included for each group.
Whole cell patch clamp recordings were performed as described previously (22, 34). 3–4 days after transfection, cells were set on the stage of the microscope and continuously superfused with ECF at room temperature. Patch electrodes were fabricated from borosilicate capillary tubing of 1.5-mm diameter (WPI) using a Narishige PP-83 vertical puller (Narishige). The electrode resistance ranged from 3 to 4 megohms when filled with the intracellular solution. Membrane currents were recorded using an Axopatch 200B amplifier. Data were filtered at 2 kHz and digitized at 5 kHz using a Digidata 1322A data acquisition system (Molecular Devices). Pipettes were filled with a solution containing (in mm) 140 CsF, 11 EGTA, 5 tetraethylammonium chloride, and 10 HEPES (pH 7.3, adjusted with CsOH). A multibarrel perfusion system (SF-77B, Warner Instrument) was employed for rapid exchange of solutions.
Data were expressed as mean ± S.E. Groups were compared using one-way analysis of variance followed by Dunnett's test or unpaired Student's t test as appropriate. p < 0.05 was regarded as statistically significant (*, p < 0.05; **, p < 0.01).
Functional TRPM7 channels are expressed in neuronal cells where they play important roles in both physiological and pathological conditions (24, 35). Also, zinc ions have been reported to permeate through TRPM7 channels in heterologous expression systems (25). However, whether TRPM7 channels play a role in neuronal zinc dynamics and zinc-mediated neurotoxicity has never been explored. Therefore, we first examined whether promoting the activation of these channels affects zinc-induced injury of cultured mouse cortical neurons. In all experiments, the blockers for NMDA receptors, AMPA/kinate receptors, and VDCCs were included in the extracellular solutions to eliminate the potential zinc entry through these pathways (see “Experimental Procedures”). When 30 μm zinc was added to the extracellular solution to mimic brain ischemia (12, 36, 37), mouse cortical neurons showed a slight increase of cell injury as demonstrated by morphological changes (Fig. 1A) and increased LDH release measured at 24 h after zinc incubation (Fig. 1B). This zinc-induced cell injury was, however, dramatically exacerbated by Ca2+/Mg2+ removal (Fig. 1, A and B), a condition known to activate TRPM7 channels (22, 24, 28). These results suggest that activation of TRPM7 channels promotes zinc toxicity in native neurons. Consistent with an involvement of TRPM7 channels, addition of Gd3+ and 2-aminoethoxydiphenyl borate, nonspecific inhibitors of TRPM7 channels (22, 24, 25, 28, 34), both attenuated zinc-induced neuronal injury (Fig. 1C).
We next examined whether altering the function of TRPM7 channels affects intracellular zinc accumulation in cultured mouse cortical neurons using a zinc indicator FluoZin-3 (38). As shown in Fig. 2, A and B, application of 30 μm zinc in normal ECF slightly increased FluoZin-3 fluorescence intensity in cortical neurons (Fig. 2, A and B, blue circle). Activation of TRPM7 channels by removing Ca2+/Mg2+ from the ECF induced a dramatic increase of FluoZin-3 fluorescence intensity (Fig. 2, A and B, red circle). Addition of Gd3+ (10 μm) dramatically attenuated the increase of FluoZin-3 fluorescence induced by Ca2+/Mg2+ removal (Fig. 2, A and B, green triangle). In contrast, addition of Gd3+ did not affect the baseline FluoZin-3 fluorescence intensity (n = 9, supplemental Fig. S1A). Addition of (2-pyridylmethyl)ethylenediamine (TPEN), a high affinity zinc chelator (31), completely eliminated the increase of FluoZin-3 fluorescence intensity (supplemental Fig. S2A). Together, these data suggest that activation of TRPM7 channels can cause increases of [Zn2+]i in mouse cortical neurons.
To provide further evidence supporting the contribution of TRPM7 channels to zinc toxicity, we investigated if changing the expression levels of TRPM7 channels influences the zinc-mediated cell injury. We used HEK293 cells with inducible expression of TRPM7 channels (19, 22). In the absence of induced expression of TRPM7 channels (TRPM7(−) cells), incubation of HEK293 cells with 30 μm zinc for 1 h did not induce an increase of LDH release, although incubation with higher concentrations of zinc (100–1000 μm) did induce a significant increase of LDH release (Fig. 3A). However, when overexpression of the TRPM7 channel was induced by adding tetracycline (TRPM7(+) cells), incubation of HEK293 cells with zinc induced a greater increase of LDH release, suggesting that increased expression of TRPM7 channels exacerbates zinc-induced cell damage.
To confirm that the enhancement of zinc-mediated cell injury was indeed due to TRPM7 overexpression, RNA interference was employed to silence TRPM7 gene expression. Transfection of TRPM7(+) cells with TRPM7-shRNA reduced the TRPM7 protein level by ~50% at 48 h post-induction, as compared with cells transfected with a mock vector (Fig. 3B). This reduction in the protein level of TRPM7 was accompanied by a significant attenuation of zinc-induced LDH release (Fig. 3C), further supporting a role of TRPM7 channels in zinc-induced injury of HEK293 cells. In this experiment, a high concentration of zinc (1000 μm) was used to induce more cell death, which allows for easier detection of the reduction of cell injury by TRPM7-shRNA treatment.
To determine whether changes of TRPM7 expression affect [Zn2+]i, zinc imaging was performed in HEK293 cells. As shown in Fig. 4, A and B, when 30 μm zinc was added to the extracellular solution, FluoZin-3 intensity gradually increased in both TRPM7(+) and TRPM7(−) cells. However, TRPM7(+) cells showed a much steeper increment in FluoZin-3 intensity than that of TRPM7(−) cells (Fig. 4B). Consistent with an involvement of TRPM7 activation, the increase of FluoZin-3 intensity in TRPM7(+) cells was largely attenuated by Gd3+ in a dose-dependent manner. Addition of Gd3+ did not affect the baseline intensity of FluoZin-3 fluorescence in the absence of added zinc (n = 17, supplemental Fig. S1B). Similar to neuronal cells, the increment of FluoZin-3 fluorescence intensity in TRPM7(+) cells was completely eliminated by the addition of TPEN (supplemental Fig. S2B). These results suggest that the level of [Zn2+]i is largely determined by the level of TRPM7 channel expression.
To provide additional biochemical evidence that TRPM7 channels play an important role in determining the level of [Zn2+]i, we performed a reporter gene assay, in which a metal regulatory element (MRE) was involved upstream of firefly luciferase. MREs are known to be activated by binding metal-regulatory transcription factors in the presence of heavy metals such as zinc (32, 39). Thus, by measuring the MRE-dependent reporter activity, intracellular zinc levels can be indirectly evaluated (32, 39). As shown in Fig. 4C, TRPM7(+) cells were dramatically more responsive to zinc application than TRPM7(−) cells, suggesting a higher intracellular zinc level in TRPM7(+) cells. In addition to zinc application, TRPM7(+) cells also showed a higher baseline reporter activity (Fig. 4C), consistent with a higher basal level of [Zn2+]i in these cells. A zinc-induced increase of reporter activity was also blocked by the addition of TPEN (supplemental Fig. S3).
To further test our hypothesis that TRPM7 channels contribute to zinc accumulation in neurons, TRPM7-shRNA/eGFP was used to determine whether knocking down the expression of TRPM7 channels reduces the [Zn2+]i. As shown in Fig. 5A, cells transfected with TRPM7-shRNA/eGFP for 3–4 days significantly reduced the density of TRPM7-like current activated by Ca2+/Mg2+ removal (28), as compared with cells transfected with control/eGFP. TRPM7-like currents in both control/eGFP and TRPM7-shRNA/eGFP-transfected cells were attenuated by 10 μm Gd3+, suggesting that they were carried by TRPM7 channels. However, the difference between the current before Gd3+ and the current after Gd3+ is smaller in TRPM7-shRNA/eGFP-transfected cells, indicating suppression of the TRPM7 current by TRPM7-shRNA (Fig. 5A, panel b). In control/eGFP-transfected neurons, addition of 10 μm Gd3+ reduced the current density from 5.7 ± 1.1 to 2.0 ± 0.5 pA/pF (n = 5). In TRPM7-shRNA/eGFP-transfected cells, addition of 10 μm Gd3+ reduced the current density from 2.9 ± 0.6 to 1.4 ± 0.3 pA/pF (n = 6). The difference between the Gd3+-sensitive current in mock-transfected cells and TRPM7-shRNA-transfected cells (3.7 versus 1.5 pA/pF) was statistically significant (p < 0.05). We also examined the effect of shRNA on TRPM7 expression in neurons with immunocytochemistry. However, despite three different antibodies used, we were unable to obtain high quality TRPM7 immunostaining of cultured mouse cortical neurons. The overall staining of TRPM7 is weak in these neurons (not shown), making the quantitative comparison between control and TRPM7-shRNA-transfected cells unreliable.
Similar to the TRPM7-like current, silencing TRPM7 with TRPM7-shRNA significantly reduced the [Zn2+]i in neurons as demonstrated by reduced MRE-dependent reporter activity (Fig. 5B). These results strongly suggest that TRPM7 channels play an important role in zinc accumulation in mouse cortical neurons.
To determine whether TRPM7 channels are involved in zinc-mediated neurotoxicity, the degree of zinc-induced injury of cultured mouse cortical neurons grown on etched coverslips was compared between TRPM7-shRNA/eGFP-transfected and control/eGFP-transfected cells. eGFP-positive neurons were recorded before and 24 h after a 1-h incubation with 30 μm zinc. As shown in Fig. 6, A and B, incubation of control/eGFP neurons with 30 μm zinc induced ~50% of cell death. However, the same treatment induced less than 30% injury of neurons transfected with TRPM7-shRNA/eGFP (Fig. 6, A and B). Similar to control/eGFP-transfected cells, 1 h incubation with 30 μm zinc induced ~50% of cell death in eGFP-negative cells. Thus, TRPM7 channels play an important role in zinc-induced injury of mouse cortical neurons.
Finally, we determined whether TRPM7-mediated zinc toxicity is also involved in the OGD condition, an in vitro model of ischemia. Consistent with our previous studies (24), 1-h OGD, in the presence of the blockers of glutamate receptors and VDCCs, did not induce much damage to neurons. However, when 30 μm zinc was added, cell injury was dramatically increased (Fig. 7A). This damage was inhibited by Gd3+, suggesting the involvement of TRPM7 channels. Further supporting this claim, TRPM7-shRNA/eGFP-tranfected cells were resistant to injury by zinc-OGD compared with control/eGFP-transfected cells (Fig. 7, B and C). Thus, TRPM7 is involved in zinc toxicity under ischemic conditions.
The significance of zinc homeostasis in normal brain function and in brain pathology has been increasingly recognized (8, 9, 12, 40, 41). The role of zinc in neuronal cell death associated with various neurological disorders, for example, has been studied. In ischemic conditions, it has been reported that extracellular chelation of zinc reduces cell injury (9, 12, 42). In seizure conditions, zinc acts either as an anticonvulsant or proconvulsant depending on the type of seizure (12, 43, 44). In addition, the potential involvement of zinc in the pathology of Alzheimer disease has been documented. For example, it has been shown that the amyloid plaque is highly enriched with chelatable zinc and the expression level of ZnTs is altered in patients with Alzheimer disease (12, 40, 45, 46). Although both extra- and intracellular zinc are tightly regulated at low levels under physiological conditions, they change dramatically under pathological conditions (12, 40). Levels of extracellular zinc at tens of micromolar, for example, have been reported following brain ischemia (12, 36, 37). The increased level of extracellular zinc is accompanied by a dramatic increase in intracellular zinc and resultant neurotoxicity, although the exact zinc entry pathways have not been clearly delineated.
Recent studies have demonstrated that some ion channels and transporters, e.g. VDCCs and glutamate receptor-gated channels, are zinc permeable (16, 17). Activation of these channels is therefore expected to play a role in zinc homeostasis and toxicity. Unfortunately, clinical trials showed little effectiveness of their blockers in protecting the ischemic brain (3). Searching for additional pathways involved in intracellular zinc accumulation and toxicity may help for establishing new and effective neuroprotective strategies. In this study, we provided strong evidence suggesting that TRPM7 is an important player for intracellular zinc accumulation and zinc-induced neurotoxicity. Based on their non-desensitizing nature and potentiation by acidosis (see below), TRPM7 channels likely play an important role in zinc accumulation associated with ischemic neuronal injury.
Studies by Monteilth-Zoller et al. (25) first indicated zinc permeation through recombinant TRPM7 channels, using electrophysiological measurements. The studies were performed using millimolar concentrations of zinc, which are several orders of magnitude higher than the physiological/pathophysiological concentrations of zinc (25). In the present studies, we explored the role of native TRPM7 channels in intracellular zinc accumulation at physiological/pathological relevant concentrations (12), and more importantly, in zinc-induced neuronal injury.
Using fluorescent zinc imaging, we demonstrated direct increases in stimulated [Zn2+]i in cortical neurons, and that this increase of [Zn2+]i was suppressed by Gd3+, a commonly used nonspecific blocker of TRPM7 channels. We then demonstrated that activation of TRPM7 channels augmented zinc-induced cell injury. In contrast, knockdown of the expression of TRPM7 channels with TRPM7-shRNA, or blocking the function of TRPM7 channels with Gd3+, both reduced zinc-mediated cell injury. Interestingly, cell injury caused by Ca2+/Mg2+ removal in the absence of added zinc was not affected by TPEN (data not shown), indicating that residual zinc in the extracellular solution, which is in the 20–50 nm range (26), is not responsible for cell injury. Therefore, the underlying mechanism for this baseline cell injury is not clear at present. We suspect that increased Na+ entry through TRPM7 channels could be partially responsible for increased neuronal toxicity upon removing Ca2+/Mg2+ in the absence of added zinc. Similar to cortical neurons, we show that increased TRPM7 expression is associated with elevated [Zn2+]i in HEK293 cells and zinc-induced cell injury. This zinc-mediated cell toxicity was also inhibited by TRPM7-shRNA.
Activation of TRPM7 channels has been shown to contribute to Ca2+ toxicity in neurons (24, 28). Using Ca2+/Mg2+ removal to activate TRPM7 channels eliminates Ca2+ entry and thus Ca2+-dependent toxicity through these channels. Under this condition, zinc entry through TRPM7 channels can still induce dramatic cell injury. Therefore, targeting Ca2+ alone may not be sufficient to protect neurons from injury.
Following brain ischemia, increased production of reactive oxygen species, reduced concentration of extracellular Ca2+ ([Ca2+]o), and the change of cellular ATP/ADP content, all facilitate the opening of TRPM7 channels resulting in toxic Ca2+ loading (24, 47, 48). Compared with other Ca2+-permeable cation channels involved in ischemic Ca2+ loading, e.g. NMDA channels, VDCCs, and recently identified acid-sensing ion channels, TRPM7 channels do not show clear desensitization over time (see Fig. 5A, for example), meaning that the channels remain open whenever the activators (e.g. reactive oxygen species, reduced [Ca2+]o, and acidosis, see below) are present. This characteristic likely makes the TRPM7 channels unique and one of the important players in ischemic neuronal injury.
Although Ca2+ entry through TRPM7 channels plays an important role in neuronal injury, our findings suggest that zinc entry through these channels could be equally important in TRPM7-mediated neuronal injury. It is worth mentioning that [Ca2+]o decreases dramatically during ischemia to as low as 0.1 mm (49). This reduction of [Ca2+]o should dramatically reduce the driving force for Ca2+ thus limiting Ca2+ entry, e.g. through TRPM7 channels. In contrast to the reduction of [Ca2+]o, extracellular zinc concentration increases following brain ischemia (50,–52). This should facilitate the entry of zinc through zinc-permeable channels, thus promoting the zinc-mediated neuronal injury.
During ischemia, a significant drop in brain pH also takes place. A reduced pH is known to inhibit the activity of NMDA receptors and VDCCs (53,–55), two major zinc permeable channels (16). Thus, zinc entry through these channels would be limited. In contrast, acidosis has been shown to potentiate, rather than inhibit, the activation of TRPM7 channels (48). Based on all these factors, TRPM7 channels may represent an important zinc entry pathway in the ischemic brain. Therefore, targeting these channels could be a novel neuroprotective strategy for stroke patients, particularly following the failure of clinical trials using glutamate antagonists (3).
We thank Dr. J. Miyazaki for providing pCAGGS-eGFP, Dr. C. Seguin for providing p(MREa)6-Luc, and Dr. A. Scharenberg (University of Washington) for providing HEK293 cells with inducible expression of TRPM7.
2The abbreviations used are: