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TRPM7 (transient-receptor-potential melastatin 7) is an ion channel with α-kinase function. TRPM7 is divalent-selective and regulated by a range of receptor-stimulated second messenger pathways, intracellular Mg-nucleotides, divalent and polyvalent cations and pH. TRPM7 is ubiquitously found in mammalian cells, including kidney, the responsible organ for osmolyte regulation, posing the question whether the channel is osmosensitive. Recent reports investigated the sensitivity of native TRPM7-like currents to cell swelling with contradictory results. Here, we assess the sensitivity of TRPM7 to both hypo- and hyperosmotic conditions and explored the involvement of the channel's kinase domain. We find that hypotonicity facilitates TRPM7 at elevated intracellular magnesium and Mg·ATP (3–4 mm), but has no effect in the absence of these solutes. Hypertonic conditions, in contrast, inhibit TRPM7 with an IC50 of 430 mosmol l−1. This inhibitory effect is maintained in the complete absence of intra- and extracellular divalent ions, although shifted to higher osmolarities (IC50= 510 mosmol l−1). TRPM7 senses osmotic gradients rather than ionic strength and this is independent of cAMP or not affected by cytochalasin D treatment. Furthermore, the kinase-domain deletion mutant of TRPM7 shows a similar behaviour to osmolarity as the wild-type protein, both in the presence and absence of divalent ions. This indicates that at least part of the osmosensitivity resides in the channel domain. Physiologically, TRPM7 channels do not seem to play an active role in regulatory volume changes, but rather those volume changes modulate TRPM7 activity through changes in the cytosolic concentrations of free Mg, Mg-nucleotides and a further unidentified factor. We conclude that TRPM7 senses osmotically induced changes primarily through molecular crowding of solutes that affect channel activity.
TRPM7, a member of the melastatin-related transient-receptor-potential ion channel TRPM subfamily, is a ubiquitously expressed bifunctional plasma-membrane protein with both ion channel and α-kinase domains (Penner & Fleig, 2007). TRPM7 has a unique selectivity profile (Monteilh-Zoller et al. 2003) allowing cellular divalent ion influx, including the permeation of the physiologically two most abundant ions Ca2+ and Mg2+. Channel activity is regulated by a variety of second messengers (Runnels et al. 2002; Takezawa et al. 2004; Langeslag et al. 2007), intracellular Mg-nucleotides (Nadler et al. 2001; Hermosura et al. 2002; Demeuse et al. 2006), divalent and polyvalent ions and pH (Nadler et al. 2001; Kerschbaum et al. 2003; Jiang et al. 2005; Kozak et al. 2005; Demeuse et al. 2006). TRPM7 plays a central role in cellular Mg2+ homeostasis (Schmitz et al. 2003, 2005) and has been implicated in anoxic calcium overload and cell death (Aarts et al. 2003). Recent reports implicate TRPM7 function in synaptic transmission of sympathetic neurons (Krapivinsky et al. 2006), cell adhesion (Clark et al. 2006; Su et al. 2006) and cell growth (Hanano et al. 2004). Known downstream targets of TRPM7's kinase domain include annexin I, a protein involved in many of the cellular morphological changes induced by glucocorticoids (Dorovkov & Ryazanov, 2004), and myosin IIA heavy chain, linking TRPM7 to cell shape maintenance and formation (Clark et al. 2006). Maintenance of cellular morphology is essential for cell function, but is influenced by many physiological processes such as cell division, exocytosis and ionic exchange. Osmotic gradients also cause changes in cell morphology, which ultimately lead to changes in cell volume due to cellular water loss or uptake. Cell swelling is counteracted by an efflux of osmolytes followed by water and resulting in restoration of cell volume (regulatory volume decrease, RVD). Conversely, cell shrinkage activates osmolyte influx followed by water influx and compensatory swelling (regulatory volume increase, RVI). Several signalling pathways have been proposed to underlie RVD and RVI, including the involvement of ionic strength, membrane stretch-activated ion channels and transporters, changes in intracellular Ca2+ and Mg2+ concentration, phosphorylation of transporters and changes in macromolecular crowding (Lang et al. 1998; Pasantes-Morales et al. 2000; Okada et al. 2001, 2004; Sardini et al. 2003; Wehner et al. 2003).
Some members of the TRP superfamily are regulated or modulated by osmotic pressure (Harteneck & Reiter, 2007). TRPV4 and TRPM3 have been reported to increase Ca2+ conductance in response to hypotonic stress (Strotmann et al. 2000; Grimm et al. 2003), while TRPC1 responds to direct mechanical stress with increased Ca2+ conductance (Maroto et al. 2005). Endogenous TRPM7-like currents have been measured in many cell types and were originally called magnesium-nucleotide-regulated metal currents (MagNuM) due to their sensitivity to not only intracellular magnesium ions but also strong dependence on Mg-nucleotides (Nadler et al. 2001; Hermosura et al. 2002; Demeuse et al. 2006). Recent publications investigated the sensitivity of MagNuM to hypertonic environments reaching divergent conclusions. Jiang et al. (2003) probed the response of MagNuM to hypertonic extracellular challenges in rat brain microglia without any obvious effect on current activity. In contrast, Numata and colleagues observed stretch-induced activation of a 23 pS cationic conductance in human epithelial cells that was not observed after treatment with small interfering RNA (siRNA) targeted against TRPM7 (Numata et al. 2007b). They observed a similar 26 pS conductance in HEK293 cells overexpressing TRPM7 (Numata et al. 2007a).
We here report dose-dependent inhibition of native MagNuM and heterologously expressed TRPM7 channels. Our results demonstrate that channel regulation by osmolarity largely resides in the channel domain, is partially linked to intracellular Mg2+ and Mg-nucleotide concentration and is not altered by exposure of cells to cytochalasin D, a toxin of the actin cytoskeleton. We show that TRPM7 is not involved in RVD or RVI, but rather mediates osmolarity-induced changes in intracellular Ca2+ concentration, and possibly mediating cell detachment or adherence following volume changes.
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TRPM7 is a homeostatic divalent cation influx pathway, whose basal activity is regulated in synergy by intracellular Mg-nucleotides and Mg2+ (Nadler et al. 2001; Monteilh-Zoller et al. 2003; Demeuse et al. 2006). Consequently, dilution or concentration of these membrane-impermeant intracellular solutes will affect channel activity, implicating a role for TRPM7 in cell volume physiology. We set forth to test this hypothesis and find that increasing cell volume by an extracellular hypertonic challenge facilitates TRPM7 currents but only in the presence of either physiological intracellular Mg·ATP or high Mg2+ concentration. In the absence of Mg·ATP the facilitatory effect is lost.
On the other hand, hypertonicity inhibits TRPM7 in a dose-dependent manner. This is only partially dependent on the presence of divalent ions and is linked to the protein's channel domain, since the TRPM7 kinase-deletion mutant is similarly sensitive to hyperosmotic conditions as wild-type channels, even in the complete absence of divalent ions. Inhibition by hypertonic conditions seems independent of the solute used to increase the osmolarity (NaCl, glucose or sucrose) and neither perturbing the f-actin cytoskeleton by cytochalasin-D nor interfering with the PKA signalling pathway using cAMP alters TRPM7's inhibitory response to hyperosmolarity.
TRPM7 does not seem to be directly involved in either RVD or RVI, because overexpression of the protein does not alter the onset or recovery from osmosis-induced cell volume changes when compared with wild-type cells expressing native TRPM7 channels. Rather, it seems that cell-volume-induced changes in TRPM7 activity lead to changes of intracellular calcium concentrations, since suppressing TRPM7 during hypertonic solution applications concomitantly reduces cytosolic calcium concentrations.
A recent study reported shear stress-induced recruitment of TRPM7 ion channels to the plasma membrane via an exocytotic event (Oancea et al. 2006). While this does not implicate TRPM7 as a mechano-sensitive ion channel, it has to be considered whether the current facilitation observed in our study is due to shear stress rather than osmolarity. Several observations argue against this mechanism in our cells. Superfusion of control (isotonic) extracellular solution at a constant application pipette pressure of 12 cmH2O did not alter the size of fully developed TRPM7 currents nor did it change the cell size as assessed by capacitance (23 ± 2 pF, n= 5), which one would expect to see upon integration of additional membrane by vesicle fusion events (Fernandez et al. 1984). Furthermore, although our wide-mouthed application pipettes had similar diameters (5–10 μm) and hydrostatic pressure was identical for all application conditions used in this study, differential effects on current behaviour could be seen when either using hypertonic or hypotonic solutions or when removing intracellular Mg·ATP. This would not be expected if current changes were caused by vesicular insertion.
Two reports have tested the response of native TRPM7-like MagNuM to hypotonic gradients. Jiang and colleagues found that reduction of extracellular osmolarity to 75% of normal values did not alter MagNuM in rat brain microglia cells (Jiang et al. 2003), an observation that our results confirm in wild-type HEK293 cells. Numata and colleagues conducted a detailed analysis of a cationic conductance in HeLa cells that could be activated by either suction-induced membrane stretch or exposure of cells to hypotonic solutions (Numata et al. 2007b). Single channel activity of the cationic conductance was absent in HeLa cells transfected with siRNA targeted against TRPM7 and in HEK293 cells transiently overexpressing TRPM7, they described a similar conductance (Numata et al. 2007a). The authors concluded that the stretch-induced channels represent TRPM7. However, several biophysical properties of these channels are incompatible with this conclusion. A number of reports confirm a single channel conductance of 40 pS for MagNuM and TRPM7 (Kerschbaum & Cahalan, 1999; Nadler et al. 2001; Kerschbaum et al. 2003), which is significantly different from the 23 pS reported for the stretch-induced single channels in HeLa cells (Numata et al. 2007b) and the 26 pS conductance seen in HEK-TRPM7 cells (Numata et al. 2007a). Furthermore, the suction-induced conductance has a low open probability at negative potentials (−50 mV and below), whereas many studies confirm high open probabilities independent of voltage (Kerschbaum & Cahalan, 1999; Fomina et al. 2000; Nadler et al. 2001; Hermosura et al. 2002; Kozak et al. 2002, 2005; Prakriya & Lewis, 2002; Kerschbaum et al. 2003; Monteilh-Zoller et al. 2003; Schmitz et al. 2003). The current is blocked by Gd3+ in a voltage-independent manner, while in our studies we did not see any effects of Gd3+ on MagNuM at +80 mV (Hermosura et al. 2002). Finally, the standard experiments in both HeLa and HEK293 cells were conducted under intracellular and extracellular divalent-free conditions. Using these conditions, the current–voltage relationship of TRPM7 currents would be almost linear (Schmitz et al. 2003 and Fig. 5), irrespective of whether cells were kept in isotonic or hypotonic extracellular conditions. However, this is clearly not the case for the conductance reported in HeLa cells, whose appearance is strongly outwardly rectifying under control conditions and assumes a linear shape only upon induction of cell swelling. Taken together, it seems unlikely that the proposed stretch-activated conductance is mediated by TRPM7, although MagNuM seems to play a role in hypotonicity-induced volume regulation due to the significant delay of volume decrease in intact cells treated with TRPM7-siRNA (Numata et al. 2007b). It should be noted, however, that TRPM7 siRNA experiments may also down-regulate other ion channels, e.g. TRPM2 (Aarts et al. 2003).
We show here that under physiological divalent conditions hypotonic gradients facilitated TRPM7 only when using high osmotic intracellular conditions, or in the presence of intracellular Mg·ATP when decreasing extracellular osmolarity. Hypotonic solutions had no effect on TRPM7 activity in the absence of Mg·ATP even when the intracellular solution was supplemented with 0.9 mm Mg2+. These deviating results argue against a stretch-activated mechanism, as this would be expected to be independent of intracellular solutes and direction of osmotic gradients. On the other hand, effects of hyperosmotic conditions have not yet been studied in TRPM7. Our results show a dose-dependent inhibition of the current induced by hypertonicity. The inhibitory effectiveness is enhanced in the presence of Ca2+ and Mg2+ ions, left-shifting the IC50 from 510 mosmol l−1 to 430 mosmol l−1. Thus our data indicate that TRPM7 senses a wide range of osmotic gradients.
The osmosensitivity of TRPM7 could be due to several mechanisms. Several TRP channels, including TRPA1, TRPV1 and TRPC1, have been reported to be mechanosensitive (Mutai & Heller, 2003; Barritt & Rychkov, 2005); however, membrane stress-induced activation seems an unlikely major mechanism for TRPM7 since the mere absence of intracellular Mg·ATP at physiological free Mg2+ (0.9 mm) abolishes hypotonicity-induced current facilitation. Furthermore, application of extracellular pressure via the application pipette (12 cmH2O) had no effect on current size despite increasing cell volume to a similar extent as hypotonic solutions. Mechano-sensitive cation channels have been studied using the balloon-patch technique (Hamill & McBride, 1997), but TRPM7 currents were not significantly affected by inflating the cell through application of intracellular pressure. Interference with the f-actin cytoskeleton has been reported to increase activity of stretch-activated BK and Cl channels (Sakai et al. 1999; Piao et al. 2003). However, in our hands overnight treatment of TRPM7-overexpressing cells with cytochalasin-D did not enhance current sizes nor was hypertonicity-induced inhibition alleviated.
The activity of many transporters that are involved in cell volume regulation depend on phosphorylation events using cAMP and cAMP-independent protein kinases during RVI, whereas dephosphorylation events cause RVD (Wehner et al. 2003). TRPM7 is regulated through Gs/Gi receptor stimulation involving PKA-dependent cAMP production (Takezawa et al. 2004). However, perfusion of TRPM7-overexpressing cells with maximal cAMP concentrations (100 μm) had no effect on the inhibitory action of hypertonic stress, indicating that these two regulatory pathways may act on different parts of the protein. This is further strengthened by the observation that cAMP-induced current enhancement requires a functional kinase domain (Takezawa et al. 2004), whereas the effect of hypertonicity does not.
Volume-activated Cl− channels (VRACs) respond to changes in ionic strength caused by altering osmolarity (Nilius et al. 2000). However, our data show that relative osmotic pressure or osmolyte concentration, rather than ionic strength, seems to cause TRPM7 inhibition, as the inhibitory effect of hypertonic solutions is similar whether NaCl, glucose or sucrose are used to increase osmolarity. Another argument against TRPM7's osmosensitivity being due to sensing ionic strength can be made from our results that TRPM7 is facilitated or inhibited by osmotic changes irrespective of the direction of the osmotic gradient. One interesting observation here is that creating relative hypo-osmotic conditions by increasing intracellular osmolarity facilitates TRPM7 currents even in the absence of intracellular Mg·ATP, unlike the result when intracellular tonicity remains constant and the cell is superfused with a 200 mosmol l−1 solution.
The most straightforward interpretation of how TRPM7 increases or decreases channel activity in response to changes in osmolarity is through volume-induced concentration changes of solutes that interfere with channel activity, such as Mg-nucleotides, Mg2+ or polyvalent cations (Nadler et al. 2001; Hermosura et al. 2002; Kozak et al. 2005; Demeuse et al. 2006). Most of our data support this conclusion. Nevertheless, TRPM7's sensitivity to hyperosmotic conditions is retained to some extent even in the complete absence of divalent ions or the kinase domain. This could be due to concentration changes of a hitherto unidentified cellular factor or process that resists washout imposed by cell perfusion. Indeed, Kozak et al. (2002) have previously noted that MagNuM develops slower than anticipated for the time it would take to washout relatively small solutes such as divalents or Mg-nucleotides (Pusch & Neher, 1988).
Numata and colleagues observed delayed volume regulation upon hypotonic challenge in intact cells treated with siRNA against TRPM7 (Numata et al. 2007b), which was dependent on the presence of extracellular Ca2+. In our hands, overexpression of TRPM7, which causes enhanced TRPM7 activity in intact cells (Monteilh-Zoller et al. 2003), did not alter the cells' ability to perform RVD or RVI compared with control. On the other hand, we could observe a marked decrease in cytosolic calcium levels when exposing TRPM7-expressing HEK293 cells to hypertonic conditions, consistent with an inhibition of Ca2+ influx through TRPM7 when the channels close during cell shrinkage. When exposing cells to hypotonic solutions, we observed only small calcium increases, although TRPM7 activity was probably enhanced. While TRPM7 currents could not be resolved due to the concomitant development of Cl− currents, the TRPM7-mediated influx of Ca2+ resulted in a relatively small increase in global free Ca2+, presumably due to the presence of high ATP levels, which may help remove cytosolic Ca2+ through enhanced pump activity. Since many studies have reported the importance of extracellular Ca2+ in TRPM7's physiology, it could be that relative Ca2+ increases through hypotonic TRPM7 facilitation are concentrated locally around the channel pore and are too small to be detected globally.
In our first report on TRPM7 (Nadler et al. 2001), we had noted that overexpression of TRPM7 causes HEK293 cells to swell and detach from the substrate. This was recently confirmed and reported to be due to TRPM7 regulating cell adhesion through controlling the calcium-dependent protease calpain (Su et al. 2006), where Ca2+ influx through TRPM7 promotes activation of m-calpain leading to decreases in peripheral adhesion complexes. In contrast to this, another study observed an increase in cell adhesion and cell spreading in response to ‘mild’ overexpression of TRPM7 in N1E-115 neuroblastoma (Clark et al. 2006). These differing results may be due to the two different cell types investigated or related to the relative expression levels of TRPM7. Nevertheless, in the light of our results, it is tempting to speculate that regulation of TRPM7 channel activity by osmolarity will lead to enhanced or reduced Ca2+ influx, ultimately affecting the integrity of the cytoskeleton. Thus, TRPM7 would promote regulated substrate detachment under hypotonic stress and reduced or ablated m-calpain activity under hypertonic stress. Hence, we can hypothesize that in physiological settings, osmotic stress will cause cellular volume changes, which secondarily regulate TRPM7 activity by either increasing or decreasing the cytosolic concentrations of free Mg2+, Mg-nucleotides and a further unidentified factor. The resulting changes in the influx rates of divalent cations such as Mg2+ and Ca2+ may then regulate processes that control cytoskeletal functions. Deregulation of TRPM7 activity in either direction will cause the deleterious effects observed following TRPM7 overexpression or knockdown.