Address correspondence to Andrea Fleig, Center for Biomedical Research, The Queen's Medical Center University, Tower 8, 1301 Punchbowl Street, Honolulu, HI 96813. Fax: (808) 537-7899; E-mail: afleig{at}hawaii.edu
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ABSTRACT |
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Key Words: zinc magnesium nucleotide trp channel cation channel
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INTRODUCTION |
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Although considerable progress has been made toward understanding intracellular Ca2+ homeostasis, transport processes, and homeostatic mechanisms, the regulation of intracellular concentrations of other divalent metal ions remains poorly understood (Hediger, 1997; Nelson, 1999
; Romani and Scarpa, 2000
). So far, only few proteins with established roles in trace metal ion homeostasis are known: (a) the natural resistanceassociated macrophage proteins (NRAMPs),* a family of proton-coupled divalent cation transporters of varying selectivity (Gunshin et al., 1997
; Knopfel et al., 2000
; Picard et al., 2000
) and two more zinc-specific families of proteins, (b) the CDFs (cation diffusion facilitator), a family that includes the ZnT proteins, which are thought to be involved in zinc export and vesicular zinc transport (Cousins and McMahon, 2000
; Harris, 2002
), and the ZIPs (ZRT/IRT-related protein), a family that functions in zinc influx into cytoplasm (Gaither and Eide, 2000
, 2001
). However, there may be additional metal ion transport mechanisms available to cells.
Most divalent ions block ion fluxes through Ca2+-permeable ion channels, with the exception of Sr2+ and Ba2+, which are usually more permeable than Ca2+. Nevertheless, various ion channels have been implicated in transporting divalent ions other than Ca2+. For example, early reports on insect muscle membranes indicated permeation of Mn2+, Cd2+, Zn2+, and beryllium (Be2+) but not Co2+, Ni2+, or Mg2+ through calcium channels (Fukuda and Kawa, 1977). Later, endplate channels were shown to conduct trace metal ions including Co2+, Ni2+, Mg2+, and Cd2+ (Adams et al., 1980
). Furthermore, voltage-dependent and receptor-mediated influx pathways may allow some Mn2+ influx, which can be detected as fluorescence quench of fura-2 (Merritt et al., 1989
; Jacob, 1990
; Shibuya and Douglas, 1992
; Fasolato et al., 1993a
,b
). The store-operated Ca2+ current ICRAC has a very small Mn2+ permeability that can only be resolved electrophysiologically when using isotonic Mn2+ solutions (Hoth and Penner, 1993
). Zn2+ permeation has been suggested for AMPA/kainate glutamate receptors (Weiss and Sensi, 2000
) and voltage-dependent Ca2+ channels have been reported to carry small Zn2+ currents (Kerchner et al., 2000
). Of the TRP-related ion channels, the vanilloid receptor VR1 has been suggested to have some limited Mg2+ permeability (Caterina et al., 1997
) and the epithelial calcium channel ECaC1 (CaT2) has been reported to conduct Cd2+ (Peng et al., 2000
). However, in most of these cases, the transport rates of divalent cations are a fraction of those achieved by the main permeant ion species and rarely large enough to be resolved by electrophysiological recordings.
TRPM7 (Nadler et al., 2001; Runnels et al., 2001
), a widely expressed member of the TRPM family of ion channels (Harteneck et al., 2000
; Montell et al., 2002
) is a cation channel that is regulated by intracellular levels of Mg·ATP and is strongly activated when Mg·ATP falls below 1 mM, thereby producing a current that we have designated MagNuM for magnesium-nucleotideregulated metal ion current (Nadler et al., 2001
; Hermosura et al., 2002
). Our initial analyses demonstrated that TRPM7 is similarly permeable to both of the dominant divalent cations Ca2+ and Mg2+. We here report that TRPM7 is generally permeable to metal ions, exhibiting particularly high permeation of Zn2+, but also permeating other essential divalent cations, including Mg2+, Mn2+, and Co2+ as well as the nonphysiological or toxic metal ions Ba2+, Sr2+, Ni2+, and Cd2+. These results suggest that TRPM7 acts as a ubiquitous metal ion influx pathway, the existence of which may have important implications for many physiological and pathological contexts.
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MATERIALS AND METHODS |
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Solutions
Cells grown on glass coverslips were transferred to the recording chamber and kept in a standard modified Ringer's solution of the following composition (in mM): NaCl 145, KCl 2.8, CaCl2 1, MgCl2 2, glucose 10, HEPES·NaOH 10, pH 7.2, with osmolarity typically ranging from 298308 mOsm. In some experiments, CaCl2 and MgCl2 concentrations were adjusted as indicated in the text and figure legends. Intracellular pipette-filling solutions contained (in mM): Cs-glutamate 145, NaCl 8, MgCl2 1, Cs-BAPTA 10, HEPES·CsOH, pH 7.2 adjusted with CsOH. In some experiments, 10 mM Cs-BAPTA was replaced with 200 µM fura-2 and in others 3 mM Mg·ATP was added. For equimolar ion-substitution experiments, cells were bathed in a standard Ringer's solution containing 10 mM CaCl2 and no added MgCl2. This solution was temporarily replaced by an identical solution that contained 10 mM of the metal-chloride salt of the relevant test ion. Isotonic solutions were prepared from Cl- salts of the relevant metal ions in distilled water at concentrations of 120 mM (osmolarities, 295310 mOsm; pH, 5.15.5). The effects of pH were assessed in TRPM7-overexpressing cells by applying isotonic choline-Cl solutions (150 mM) without divalent ions and buffered to various pH values using 10 mM TRIS and HCl. These solutions completely inhibited inward currents through TRPM7 down to pH values of 4 (n = 5, unpublished data), indicating that the tested isotonic metal solutions (pH = 5.15.5) are not contaminated with proton currents. For the case of Zn2+, we also prepared a choline-Clbased solution that lacked any monovalent cations. To obtain an adequately buffered, nonprecipitating Zn2+ solution, a 150 mM choline-Cl solution was pH adjusted to 7.6 using TRIS base and HCl, followed by addition of 10 mM ZnCl2, yielding a final pH of 5.5. We also performed Zn2+-substitution experiments where the cells were kept in a solution containing (in mM): choline-Cl 160, TRIS 10, and CaCl2 1, pH 7.3. Cells were then superfused with an identical solution, except that 1 mM CaCl2 was replaced by 1 mM ZnCl2 (Fig. 5 A, open circles). Solution changes were performed by pressure ejection from a wide-tipped pipette.
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Fluorescence Measurements and Divalent-induced Quench of Fura-2
Cytosolic calcium concentration of patch-clamped cells was monitored at a rate of 5 Hz with a photomultiplier-based system using a monochromatic light source (TILL Photonics) tuned to excite fura-2 fluorescence at 360 and 390 nm for 20 ms each. Emission was detected at 450550 nm with a photomultiplier whose analogue signals were sampled and processed by the X-Chart software package (HEKA). Fluorescence ratios were translated into free intracellular calcium concentration based on calibration parameters derived from patch-clamp experiments with calibrated calcium concentrations. A standard Ringer's solution was used except that Ca2+ was increased to 2 mM. In patch-clamp experiments, 200 µM Fura-2 was added to the standard intracellular solution without BAPTA. In some cases, 3 mM Mg·ATP was added to suppress TRPM7. For Mn2+-, Co2+-, or Ni2+-induced fluorescence quenching, intact cells were loaded with 5 µM fura-2-AM for 4560 min at 37°C. Fluorescence recordings were initially made in an external solution that was nominally Mg2+ free but contained 3 mM extracellular Ca2+. Quenching of the 360 nm signal (the Ca2+-independent wavelength of fura-2) was assessed during application of a Ringer's solution containing either 1 mM of MnCl2, CoCl2, or NiCl2 plus both CaCl2 and MgCl2 (1 mM each) via a wide-tipped, pressure-controlled application pipette.
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RESULTS |
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In the experiments described above, where we substituted 10 mM Ca2+ with other divalent ions, one would expect the reversal potential (Erev) to shift at least to some degree toward depolarized potentials. Indeed, this could be observed for all ion species investigated. Compared with control Erev values (-7.2 ± 2.7 mV; n = 39), Ba2+ and Ni2+ had the strongest effect in shifting Erev by 19 ± 7, (n = 5) and 15 mV ± 3 (n = 6), respectively. At 10 mM concentration, the other ion species investigated had less dramatic effects, ranging between 2 mV ± 0.7 mV for Cd2+ (n = 5) and 8.5 mV ± 4.7 mV for Co2+ (n = 5). Example I-V curves are shown in Fig. 2 C. Here a cell was superfused with a 10 mM Ni2+-containing extracellular Mg2+-free solution. The currents just before application (at 200 s whole-cell time) and during application (at 230 s) are shown. It can be seen that there is a remarkable increase in inward currents with some suppression of the outward current. This is accompanied by a clear depolarizing shift of Erev, as detailed by the magnified inset. However, as will be discussed later, a quantitative assessment of reversal potentials under these conditions is rather difficult and error prone, because the current is highly nonlinear and has a very shallow slope around Erev, so that relatively small changes in TRPM7 current and/or Ca2+-dependent conductances may shift it.
Permeation Profile of Transition Metals in Isotonic Solutions
The above experiments reveal the surprising finding that TRPM7 is not only permeable to Ca2+ ions, but in fact even more permeable to a range of divalent transition metal ions and their permeation yields considerable macroscopic inward currents at negative membrane potentials. It should be noted, however, that the above experiments cannot unequivocally rule out possible ionion interactions that would allow the cotransport of, e.g., Na+ ions along with divalent ions. To avoid such complications, we next studied permeation of divalent ions in isotonic extracellular solutions of each metal ion. Isotonic solutions of 120 mM of each divalent ion species yielded appropriate osmolarities within 10% of the standard bath and pipette solutions and pH values in the range of 5.1 to 5.5, which do not compromise accurate assessment of inward currents carried by TRPM7 (see MATERIALS AND METHODS). However, Zn2+ and Cd2+ had to be excluded from this analysis, as isotonic solutions of Zn2+ yielded pH values below 3 and 120 mM Cd2+ solutions were hypo-osmotic.
In this series of experiments, the cells were initially bathed in the standard external solution containing 1 mM Ca2+ and 2 mM Mg2+. At 200 s, when TRPM7 current had reached its full amplitude, isotonic solutions of each metal ion were applied for 60 s via a puffer pipette. The development of inward and outward currents before, during, and after application of selected isotonic divalent cation solutions are shown in Fig. 3 A for the case of Ca2+ and Mg2+. The experiments illustrate that isotonic Ca2+ and Mg2+ solutions induce an increase in inward currents through TRPM7, while at the same time there is strong suppression of outward currents. We next performed essentially identical experiments for a range of divalent metal ions and illustrate the average responses in Fig. 3, BD. Example I-V curves measured before (200 s whole-cell time) and during isotonic Ni2+ application (at 230 s) are shown in Fig. 3 E. Note the dramatic increase in inward currents, the substantial reduction of the outward currents, and the significant depolarizing shift of Erev, which is detailed in the data inset of the graph. When analyzing these data for the relative increase of inward currents, it can be seen that, with the exception of Mg2+, the rank order of permeation under isotonic conditions (Fig. 4, A and B) is virtually identical with the permeation sequence observed in the 10 mM ion substitution experiments (Fig. 2 D).
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Physiological Ca2+ Levels Do Not Impede Trace Metal Permeation Through TRPM7
TRPM7 mediates low levels of homeostatic plasma membrane Ca2+ fluxes and several Ca2+ influx pathways are known to also allow permeation of Mn2+ ions (Shuttleworth and Thompson, 1999). Therefore, we hypothesized that TRPM7, if it carried Mn2+ as well, may underlie experimental observations of unstimulated Mn2+ entry that lead to fluorescence quenching of fura-2 in resting cells (Mertz et al., 1990
; Shuttleworth and Thompson, 1996
). If this were the case, it would also hand us a tool to test whether the presence of physiological Ca2+ and Mg2+ concentrations, the major divalent ion species in extracellular fluids, affect the permeation of other divalent ions through TRPM7 channels.
To test these hypotheses, HEK-293 cells overexpressing TRPM7 as well as control cells were loaded with fura-2-AM and bathed in the standard external solution containing 3 mM Ca2+ and 0 mM Mg2+. Fluorescence was monitored at 360 nm excitation (the isosbestic or Ca2+-independent wavelength of fura-2) and after 20 s, an external solution that maintained the total divalent concentration but contained 1 mM Mn2+, 1 mM Ca2+, and 1 mM Mg2+ was applied for 180 s. As shown in Fig. 4 D, the application of 1 mM Mn2+ caused a pronounced quench of the 360 nm signal in HEK-293 cells induced to overexpress TRPM7 (n = 3), whereas Mn2+-induced quench of the 360-nm signal in uninduced cells, which express only low levels of endogenous TRPM7, was much less dramatic (n = 3). Linear regression over the initial 60 s of Mn2+ exposure yielded a fourfold higher rate of Mn2+-induced quench of fura-2 fluorescence in induced cells (28%/min) as compared with uninduced cells (6.6%/min). Similar experiments were performed for Ni2+, where an external solution containing 1 mM Ni2+, 1 mM Ca2+, and 1 mM Mg2+ was applied for 180 s. Fig. 4 E confirms that Ni2+ too can permeate TRPM7 in the presence of Ca2+ and Mg2+. Here, the fura-2-induced fluorescence quench increases about twofold from 10%/min in uninduced cells (n = 3) to 20%/min in TRPM7 overexpressing cells (n = 4). Finally, using Co2+ in the presence of Ca2+ and Mg2+ also increases the rate of fura-2 fluorescence quenching (by about threefold) in TRPM7 overexpressing cells (17%, n = 3; Fig. 4 F) versus uninduced controls (5.4%, n = 3). These results clearly suggest that basal, unstimulated TRPM7 activity in intact cells constitutes an important pathway for Mn2+, Co2+, and Ni2+ entry that is significantly augmented by TRPM7 overexpression and can occur in the presence of physiological levels of Ca2+ and Mg2+.
Permeation of Zn2+and Trivalent Ion Species
Based on its physiological and pathophysiological importance, we wanted to ascertain that Zn2+ permeation of TRPM7 occurs under experimental conditions in which it was the only permeable ion species available to carry inward currents through TRPM7 channels. As the pH of isotonic Zn2+ solutions precluded such experiments, we used the same experimental approach as in Fig. 2, but tested Zn2+ permeation by applying 10 mM Zn2+ in a choline-Clbased extracellular solution (see MATERIALS AND METHODS). As illustrated in Fig. 5 A, this resulted in a significant enhancement of inward currents in HEK-293 cells overexpressing TRPM7, indicating that Zn2+ is indeed a highly permeable ion species of TRPM7. The current-voltage relationships of TRPM7 before and during Zn2+ application are illustrated in Fig. 5 B, demonstrating the increase in inward current at negative potentials and the decrease in outward currents at positive potentials, consistent with significant Zn2+ permeation. To further confirm Zn2+ permeation in the absence of other permeant ion species, we performed experiments where TRPM7-overexpressing cells were kept in a choline-Clbased solution supplemented with 1 mM Ca2+ instead of our standard external solution (see MATERIALS AND METHODS; Fig. 5 A, open circles, n = 5). At the time indicated, patch-clamped cells were then superfused with 1 mM Zn2+ and 0 mM Ca2+ in an otherwise identical choline-Cl solution. The results show that even at 1 mM concentration, Zn2+ alone is more effective in supporting inward current than equimolar Ca2+.
We also attempted to measure Zn2+ permeation under more physiological conditions, i.e., in the presence of physiological Ca2+ and Mg2+ concentrations. To this end, we allowed TRPM7 currents to develop in standard extracellular solutions containing 1 mM Ca2+ and 2 mM Mg2+ and applied the same solution, but additionally contained 1 mM Zn2+. Under these conditions, we again observed a clear increase in inward current and a concomitant small decrease in outward current (Fig. 5 C), suggesting additive permeation of Zn2+ ions in the presence of Ca2+ and Mg2+.
Finally, to address the issue of whether Zn2+ permeation is also characteristic of native, endogenously expressed TRPM7, we recorded MagNuM in wild-type (WT) HEK-293 cells under identical conditions as the ion-substitution experiments in Fig. 2, with 10 mM Zn2+ as the replacement ion. Since Zn2+ inhibits resting background currents unrelated to TRPM7, we corrected for this effect by first measuring Zn2+ effects in cells where MagNuM was completely suppressed by pipette solutions containing 4 mM Mg2+ (n = 5). These data were averaged and subtracted from the average dataset obtained from cells where MagNuM was activated by omission of pipette Mg·ATP (n = 12). As can be seen in Fig. 5 D, we observed a significant increase in inward current when applying Zn2+, indicating that Zn2+ permeation is a genuine physiological property of native TRPM7 channels.
We also tested for permeation of trivalent metal ions La3+ and Gd3+ (Fig. 5 E). We first assessed concentrations that normally completely suppress voltage- or store-operated Ca2+ channels and found that 10 µM of either Gd3+ or La3+ were rather ineffective in suppressing inward or outward currents carried by TRPM7 (<5% inhibition, n = 34, unpublished data). However, at 10 mM, both ions almost completely inhibited inward currents (n = 5 each), although the effects on outward currents were markedly different. Provided that the inhibitory action of Gd3+, and to a smaller degree that of La3+, is mediated at the cytosolic side, there may in fact be a very small permeation of this ion that cannot be unambiguously resolved at the macroscopic current level.
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DISCUSSION |
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TRPM7 Constitutes a Significant Ca2+ Entry Pathway
Previous electrophysiological characterizations of TRPM7 have established that the channel is capable of sustaining small inward currents carried by Ca2+ ions (Nadler et al., 2001; Runnels et al., 2001
). We have extended this analysis in the present work by combined fluorescence and patch-clamp measurements (see Fig. 1), which demonstrate that TRPM7 can drive significant changes in [Ca2+]i after depletion of intracellular Mg·ATP in cells that overexpress the protein. In native cells, where expression levels of endogenous TRPM7 is smaller, the contribution of MagNuM may be more subtle and may not lead to measurable changes of free [Ca2+]i. However, given that TRPM7 is constitutively active to a small degree, even in the presence of physiological Mg·ATP, it can well serve a function of a homeostatic mechanism for basal, background influx of Ca2+ that could be used to maintain the filling state of intracellular stores. The notion that MagNuM constitutes a background pathway for divalent cation fluxes is supported by the fluorescence quenching experiments illustrated in Fig. 4, where a small basal entry of Mn2+, Co2+, and Ni2+ is observed in intact uninduced cells, which is presumably due to native MagNuM and is augmented significantly in cells overexpressing TRPM7. From this, it seems reasonable to assume that the same pathway can support a steady, low-level transport of Ca2+ ions, which under extreme conditions, such as prolonged ischemia, can lead to a persistent strong activation of TRPM7 that ultimately may contribute to Ca2+ overload and cell death.
Equimolar Substitution of 10 mM Extracellular Ca2+ by Transition Metals
In general, all tested divalent metal ions were at least as efficient and some even considerably more so than Ca2+ in permeating TRPM7 at negative membrane potentials. Zn2+ and Ni2+ caused the largest increases of the inward current, which were greater than threefold compared with Ca2+. We also observed significant Ba2+ inward currents, while Co2+, Mg2+, and Mn2+ exhibit more modest (less than double) increases of inward currents and Sr2+ and Cd2+ still supported slightly larger inward currents than Ca2+ itself. Taken together, the changes in inward current observed under 10 mM ion substitution experiments are most consistent with the interpretation that TRPM7 is as or more permeable to each of the divalent cations than Ca2+.
At the same time, most of these ions produced a significant block of outward currents, the strongest one caused by Mg2+, which we have shown previously is a potent intracellular suppressor of MagNuM currents (Nadler et al., 2001; Hermosura et al., 2002
). In contrast, Ba2+, Sr2+, and Cd2+, while still supporting inward currents, actually increase outward currents. The outward currents at positive potentials represent currents of monovalent ions at potentials where divalent ions do not experience sufficient driving force to enter the cell and therefore no longer impede monovalent outward fluxes (Hille, 1992
). Their behavior is a complex function of several factors including (a) the Nernst equilibrium potential of divalent ions, which determines the degree of permeation block imposed by divalent ions at positive membrane potentials, and (b) inhibitory effects of the relevant divalent ion at the intracellular side of the channel after it has gained access to the cytosol. The latter effects are particularly relevant for Mg2+, which plays an important role as a cofactor in the gating of TRPM7 and therefore can inactivate MagNuM if allowed to accumulate intracellularly. A similar inhibition is also seen with Ca2+, which is the second major physiological divalent cation to permeate TRPM7. However, under our experimental conditions, the inhibitory action of [Ca2+]i may be more limited due to the inclusion of 10 mM BAPTA and may be only noticeable under the weakly buffered conditions during calcium signaling measurements (e.g., Fig. 1). Based on the inhibitory actions of a number of divalent ions, we propose putative divalent binding site(s) at the cytosolic side of TRPM7 that modify channel gating and/or cause channel block. Different divalent ions may possess different affinities to this site, resulting in the observed inhibition of outward currents. Thus, the efficacy of any divalent ion to block outward currents would be the net result of its degree of permeation, its ability to accumulate in the cytosol in the presence of BAPTA, and its inhibitory potency on the channel.
Permeation of Transition Metals in Isotonic Solutions
In general agreement with the 10 mM substitution experiments, application of isotonic divalent solutions resulted in increased inward currents and inhibition of outward currents. The permeation sequence of divalent ions, based on peak inward currents, is similar to the sequence we observed with 10 mM ion-substitution experiments. Thus, in the absence of data for isotonic Zn2+, Co2+ and Mn2+ are the best-permeating physiologically relevant metal ions, followed by Ca2+ and Mg2+. Among the toxic or nonphysiologic metals, Ni2+ is the most permeable, followed by Ba2+ and Sr2+. This permeation sequence, in combination with the facilitated transport of trace metals over Ca2+ and Mg2+, is unprecedented for any ion channel presently known. TRPM7 must therefore be considered an important influx pathway for both essential and toxic metal ions. With respect to outward currents, isotonic solutions invariably caused a complete or almost complete inhibition, with the exception of Ba2+ and Sr2+, which were less effective in doing so. As in the equimolar substitution experiments, here too the efficacy of permeation and its rank order would be a net result of the three factors discussed above (permeation, intracellular ion accumulation and negative feedback on the channel). In most cases, the currents recovered close to their original sizes within 150 s, suggesting that none of the divalent ion species exert any lasting effect on channel integrity.
Together, both datasets (10 mM equimolar replacements and isotonic substitutions) are complementary in that they address interpretation issues that arise under either experimental condition. For example, the acidic nature of isotonic divalent solutions prevented us to conduct experiments with isotonic Zn2+. This was not a concern with the 10 mM replacement experiments. Most importantly, however, the two datasets show that in the case of TRPM7, the rank order of the permeation sequence is essentially independent of the divalent ion concentration and the presence of monovalent cations (Na+). The latter aspect is in marked contrast to observations made with other channels that are able to conduct divalent ions other than Ca2+.
Trace Metalinduced Current Augmentation Shifts Erev
Ion permeation ratios are usually calculated based on reversal potentials using Nernst and Goldmann-Hodgkin-Katz (GHK) equations (Hille, 1992), which make the important implicit assumption that ions can permeate independently. Applying this formalism to TRPM7 currents, which normally reverse around 0 mV, would lead to the conclusion that the channel is nonselective (Runnels et al., 2001
). However, since TRPM7 conducts divalent ions into the cell and monovalent ions out of the cell (Nadler et al., 2001
), the independence rule is questionable and, therefore, correct permeability ratios based on GHK-based calculations do not adequately describe this channel's permeability. Nevertheless, if other ions preferentially permeate TRPM7 over Ca2+, one would expect a depolarizing shift in the reversal potential when applying the 10 mM equimolar or isotonic replacement solutions. Indeed, analyses of reversal potentials revealed that all divalent ions caused a shift in Erev toward more positive potentials. The rank order of the Erev values under isotonic conditions fairly well reflected the rank order observed for the inward currents, with Ni2+ inducing the strongest change (35 mV) and Mg2+ the weakest (10 mV). This indicates that unlike observations made in other channels (Adams et al., 1980
) the apparent permeability of TRPM7 for divalent ions is independent of their concentration.
Reversal potential measurements of TRPM7 are clearly very difficult, because the current is highly nonlinear and has a very shallow slope around Erev. Therefore, even small changes in the activity of other ion currents may have an effect on Erev. In particular, we have noticed that the current magnitude as well as Erev of TRPM7 is influenced by intracellular Ca2+ concentrations. In experiments where intracellular Ca2+ remained largely unbuffered (e.g., Fig. 1 B), there is a slight shift of the apparent Erev to more hyperpolarized potentials (-33 mV) compared with standard conditions in which [Ca2+]i is buffered to zero using 10 mM Cs-BAPTA (here, Erev values are around -7.2 mV, Fig. 2 C). We have not assessed the reasons for this shift, but we suspect that elevated [Ca2+]i may cause small increases in Ca2+-activated K+ or Cl- channels, which may account for the observed changes in Erev.
TRPM7-mediated Trace Metal Entry Occurs at Physiological Levels of Ca2+ and Mg2+
Our present work extends our previous observation that TRPM7 sustains Ca2+ and Mg2+ currents under isotonic conditions (Nadler et al., 2001) by demonstrating other divalent ions to do so. An important question is whether these divalent ions permeate TRPM7 in the presence of physiological levels of extracellular Ca2+ and Mg2+? Our results presented in Fig. 4 demonstrate that physiological concentrations of Ca2+ and Mg2+ do not impede the permeation of Mn2+, Ni2+, or Co2+, providing strong evidence that neither Ca2+ nor Mg2+ interfere with the permeation of trace metal ions and that divalent ions permeate independently. Thus, the probability of a trace metal ion to pass through the channel would depend on its relative permeability and relative concentration levels. For example, the data in Fig. 2 D show that Zn2+ has a fourfold higher chance over Ca2+ to permeate TRPM7. Thus, one can presume that under physiological divalent concentrations (i.e., 1 mM Ca2+, 1 mM Mg2+, and 10 µM Zn2+ plasma levels; Perveen et al., 2002
),
1 in every 50 ions passing through the channel will be a Zn2+ ion.
Permeation of Zn2+and Trivalent Ion Species
Zn2+ plays an important physiological role in many cellular functions. Our results under experimental conditions where Zn2+ is the only permeable ion species available to support inward currents through TRPM7 channels demonstrate that Zn2+ permeates TRPM7 at least twice as well as Ca2+, indicating unequivocally that Zn2+ is indeed a highly permeable ion species of TRPM7. Moreover, this finding could be confirmed in native TRPM7-like MagNuM currents. In the brain, Zn2+ levels can reach 100500 µM during ischemia, making TRPM7 an attractive candidate for mediating at least part of the severe neurotoxic effects observed with this ion (Choi and Koh, 1998; Koh, 2001
), particularly in view of the fact that Mg·ATP levels during ischemia are reduced and would favor activation of MagNuM.
Although TRPM7 clearly allows passage of divalent ions, the situation is less clear for trivalent ion influx. Generally, trivalent ions such as Gd3+ or La3+ are known to efficiently inhibit voltage- or store-operated Ca2+ channels at µM concentrations. However, when testing these two ion species, we found that 10 µM of either Gd3+ or La3+ were rather ineffective in suppressing inward or outward currents carried by TRPM7. At 10 mM concentrations, both ions completely inhibited inward currents with no sign of significant permeation. Interestingly, Gd3+ caused a complete suppression of outward currents. It remains to be determined whether this is due to some limited Gd3+ entry that causes block of outward currents from the cytosolic side. By contrast, the same concentration of La3+, while potently and persistently inhibiting inward currents, was far less effective in suppressing outward currents, which might be interpreted such that its action is limited to the extracellular side of the plasma membrane.
Conclusions
The present study establishes that the TRPM7 ion channel possesses the so far unique ability to efficiently conduct many nutritionally essential trace metal ions, along with several toxic metal ions as well. This ability in conjunction with its widespread expression and constitutive activity suggests that TRPM7 underlies a ubiquitous mechanism for entry of metal ions into cells. A remarkable finding is that trace divalent metal ions are significantly more permeable through TRPM7 than the dominant physiological divalent metal ions Ca2+ and Mg2+. Such a property would help ensure that trace metals are able to effectively compete with these ions and enter into cells despite their concentration disadvantage relative to the millimolar amounts of Ca2+ and Mg2+ typically present in extracellular fluids. Furthermore, the discovery of the general nature of metal ion permeation via TRPM7 has important implications for our understanding of pathophysiological situations in which toxicity of TRPM7-permeant ions is thought to play a role. Due to its transport capacity of this and other metal ions, TRPM7 may represent an intriguing target pathway for therapeutic intervention in these situations.
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FOOTNOTES |
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* Abbreviation used in this paper: NRAMP, natural resistanceassociated macrophage protein.
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ACKNOWLEDGMENTS |
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David Gadsby served as editor.
Submitted: 29 October 2002
Revised: 10 December 2002
Accepted: 11 December 2002
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REFERENCES |
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