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CELLULAR METABOLISM
1Department of Cell Physiology, National Institute for Physiological Sciences and 2Department of Physiological Sciences, School of Life Science, Graduate University for Advanced Studies (SOKENDAI), Okazaki; and 3Japan Society for the Promotion of Science, Tokyo, Japan
Submitted 6 July 2006 ; accepted in final form 28 August 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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regulatory volume decrease
Since osmotic swelling has been reported to activate a number of members of the transient receptor potential (TRP) channel family such as TRP vanilloid (TRPV)4 (24, 46, 47, 51), TRPV2 (31), TRP canonical (TRPC)1 (4), and TRP melastatin (TRPM)3 (10), there is a possibility that some of these channels play a volume-regulatory role. Actually, TRPV4 was recently shown to play a role in RVD in tracheal epithelial cells (2) and keratinocyte HaCaT cells (3). It is known, however, that TRPV4 is an osmosensitive, not mechanosensitive, cation channel (46). Also, it must be noted that expression of TRPV4 may rather work against cell volume regulation in some conditions, because almost complete downregulation of volume-regulatory Cl– channels was observed in TRPV4-transfected cells (34). Thus the purpose of the present study was to identify the molecule corresponding to the SSAC channel that is endogenously expressed and plays an essential role in cell volume regulation in human epithelial HeLa cells. Here we demonstrate that TRPM7 represents the endogenous SSAC channel and is involved in RVD in HeLa cells.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Patch-clamp experiments.
Whole cell and single-channel recordings were performed at room temperature (22–26°C). The patch electrodes prepared from borosilicate glass capillaries had a resistance of 
2 M
for whole cell recordings and 7 M for single-channel recordings. Currents were recorded with an Axopatch 200B amplifier (Axon Instruments, Union City, CA). Current signals were filtered at 5 kHz with a four-pole Bessel filter and digitized at 20 kHz. pCLAMP software (version 9.0.2; Axon Instruments) was used for command pulse control, data acquisition, and analysis. To minimize K+ and anion currents, all recordings were carried out with low-Cl–, Cs+-rich intracellular solution and Cl–-free, Cs+-rich extracellular solution.
Single-channel recordings were performed in the cell-attached and excised inside-out configurations. The amplitude of single-channel currents was measured as the peak-to-peak distance in Gaussian fits of the amplitude histogram. The open probability (Po) of single channels was calculated by dividing the total time spent in the open state by the total time of continuous recording (30–200 s) in the patches containing one active channel. For cell-attached recordings, cells were exposed to bathing solution containing (in mM) 100 KCl, 2 CaCl2, 1 MgCl2, 5 Na-HEPES, 10 HEPES, 5 glucose, and 100 mannitol (pH 7.4). The pipette solution was composed of (in mM) 100 Cs-aspartate, 10 HEPES, 0.5 CsCl, and 100 mannitol (pH 7.4). For inside-out recordings, the intracellular side was perfused with a Cl–-free bath solution (300 mosmol/kgH2O) consisting of (in mM) 100 Cs-aspartate, 1 EGTA, 10 HEPES, and 100 mannitol (pH 7.4 adjusted with CsOH), and an extracellular (pipette) solution that contained (in mM) 100 Cs-aspartate, 1 EGTA, 10 HEPES, 0.5 CsCl, and 100 or 180 mannitol (pH 7.4) was used. To observe effects of intracellular or extracellular free Mg2+, 1 mM EGTA was replaced with 1 mM EDTA, and an appropriate amount of MgSO4 was added to give various concentrations (0.0001–10 mM) of free Mg2+. The free Mg2+ concentration was calculated with CaBuf software (provided by Dr. G. Droogmans, Katholieke Universiteit Leuven, Leuven, Belgium). To test the effects of mechanical stretch, patch membranes were subjected to a pulse of negative pressure applied to the back of the patch pipette. The pressure level was monitored with a manometer.
In whole cell recordings, series resistance (<5 M) was compensated (to 70–80%) to minimize voltage errors. Step pulses were applied from a prepotential of +80 mV to test potentials (50-ms duration) of +80 to –80 mV in 20-mV decrements after steady currents were attained. Ramp pulses were applied every 4 s from +100 mV to –100 mV or from –100 mV to +100 mV at a speed of 4 mV/ms. Osmotic cell swelling was induced by applying hypertonic low-Cl– intracellular solution. The isotonic (300 mosmol/kgH2O) and hypertonic (370 mosmol/kgH2O) low-Cl– intracellular (pipette) solutions contained (in mM) 100 Cs-aspartate, 1 EGTA, 10 HEPES, 0.5 CsCl, and 100 or 170 mannitol, respectively (pH 7.4). In some experiments, 1 mM MgSO4 was added to the intracellular solution. The isotonic Cl–-free bath solution (320 mosmol/kgH2O) contained (in mM) 100 Cs-aspartate, 1 EGTA, 10 HEPES, and 120 mannitol (pH 7.4). When required, 0.1 mM MgSO4 was added to the bath solution. Experiments were performed after perfusion of bath solution was stopped, except when the effects of perfusion-induced mechanical stress were observed.
RNA isolation and RT-PCR. Total cellular RNA was extracted from HeLa cells by using Sepasol RNA-I reagent (Nacalai Tesque, Kyoto, Japan) according to the protocol supplied by the manufacturer. RNA samples were reverse-transcribed at 42°C for 50 min with Maloney murine leukemia virus reverse transcriptase (Invitrogen) and oligo(dT) primers (Invitrogen), according to the manufacturer's protocols. Gene-specific primers used for PCR were designed with Primer3 software (www.genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi) and NCBI BLAST (www.ncbi.nlm.nih.gov/blast/) to identify complementary sequences in the human genome. Two sets of primers were used for TRPM7 (GenBank accession no. NM_017672). The sequences of the forward and reverse primers were 5'-AAGTGGCTTTTGCAACTTGG-3' and 5'-ACACTCACTGCCCAGAAAGC-3' (product size 503 bp), respectively, and 5'-CCACCCCCAATCAATATTAAACAC-3' and 5'-GGCAGTCAAAATTTTCCTCAGT-3' (867 bp), respectively. PCR was performed with 1.25 U of Ex Taq (Takara, Shiga, Japan) in 10x Ex Taq buffer (Takara) containing 0.2 mM dNTPs. Amplification was carried out in a thermal cycler (Gene Amp PCR System 9600, Perkin Elmer Life Sciences, Boston, MA) under the following conditions: initial heating at 94°C for 4 min, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and then final extension at 72°C for 1 min. Negative control experiments were performed with RNA that had not been reverse-transcribed. As a positive control, we amplified the glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) sequence with a specific set of primers (5'-ACCACAGTCCATGCCATCAC-3' and 5'-TCCACCACCACCCTGTTGCTGTA-3'). The products of RT-PCR were electrophoresed on a 2% agarose gel and, after purification with the Wizard SV Gel and PCR Clean-Up System (Promega), were cloned into pGEM-T Easy vector (Promega). Plasmids were purified with the Wizard Plus Minipreps DNA Purification System (Promega) and used as templates for sequencing with the ABI PRISM 310 genetic analyzer (Applied Biosystems, Foster City, CA).
Small interfering RNA transfection. HeLa cells were transfected with 5 µg of small interfering RNA (siRNA) purchased from Qiagen (Chatsworth, CA) and RNAiFect Transfection Reagent (Qiagen), following the manufacturer's instructions. In brief, siRNA and RNAiFect Transfection Reagent were each diluted to 100 µl with EC-R buffer, mixed together, and incubated at room temperature for 15 min. Cells on a six-well plate were fed with 1.5 ml of fresh culture medium and overlaid with the transfection mixture. After 48–72 h, transfected cells were used for experiments. To determine transfection efficiency, we used mock siRNA and siRNA against TRPM7, which were both Alexa 488 conjugated. The mock siRNA was a commercially available nonsilencing sequence (Qiagen). The order-made TRPM7 siRNA corresponded to nucleotides 1318–1338 in the coding region and has been reported to suppress the level of TRPM7 mRNA (1). No other known genes, including those of other TRP channels, exhibit sequence homology to this target sequence. For patch-clamp experiments and single-cell size measurements, cells exhibiting Alexa 488 fluorescence were selected.
Immunoblotting. Crude membranes were prepared from control and siRNA-transfected HeLa cells as well as from TRPM7-transfected HEK293T (HEK/TRPM7) cells by Dounce homogenization followed by differential centrifugation. In brief, cells were washed twice with phosphate-buffered saline (PBS), collected by a cell scraper, and homogenized with 20 strokes of a Dounce homogenizer in 10 vols of homogenization buffer, which contained (mM) 250 sucrose, 5 EDTA, 5 EGTA, and 10 HEPES (pH 7.4). The resulting homogenate was centrifuged at 9,100 g for 10 min. The supernatant was subsequently centrifuged at 100,000 g for 1 h to pellet the microsomal fraction, and the pellet was solubilized with sample buffer containing 2% sodium dodecyl sulfate (SDS), 10% glycerol, 0.063 mM Tris·HCl (pH 6.8), 0.002% bromophenol blue, and 5% 2-mercaptoethanol. All these steps were carried out at 4°C. An aliquot of membrane protein (150 or 30 µg for HeLa or HEK293T cells, respectively) was subjected to one-dimensional SDS-PAGE using a 7% polyacrylamide gel (Daiichikagaku, Tokyo, Japan). The proteins on the gel were electrophoretically transferred to a polyvinylidene difluoride sheet, which was subsequently incubated for 1 h with a 1:1,000 dilution of an affinity-purified polyclonal rabbit antibody (a gift from Dr. Y. Mori and Dr. Y. Hara, Kyoto University) raised against a peptide corresponding to amino acids 1816–1835 of human TRPM7 (12). A purified mouse monoclonal antibody recognizing human actin (Chemicon, Temecula, CA) was used for positive control experiments. Antibody detection was performed with an Amersham biotin-streptavidin system with biotinylated anti-rabbit or anti-mouse IgG and the nitro blue tetrazolium-5-bromo-4-chloro-3-indolyl phosphate system (Amersham Life Science, Little Chalfont, UK).
Mean cell volume measurements. Mean cell volume was measured at room temperature by electronic sizing with a Coulter-type cell size analyzer (CDA-500; Sysmex, Kobe, Japan), as previously described (13). The mean volume of the cell population was calculated from the cell volume distribution measured after the machine was calibrated with latex beads of known volume. Isotonic (310 mosmol/kgH2O) and hypotonic (200 mosmol/kgH2O) solutions contained (in mM) 95 NaCl, 4.5 KCl, 1 MgCl2, 1 CaCl2, 110 or 0 mannitol, respectively, and 5 HEPES (pH 7.3). Ca2+-free and Mg2+-free solutions were made by removing CaCl2 or MgCl2 and adding 1 mM EGTA or EDTA, respectively.
Single-cell size measurements. For siRNA-transfected cells, application of the electronic sizing technique was found to be unsuitable because of limited transfection efficiency. Single-cell size measurements were therefore performed at room temperature, as previously reported (22). Spherical cells exhibiting Alexa 488 fluorescence were selected and held under gigaseal in the cell-attached mode by a patch pipette. Cell images were recorded through a charge-coupled device camera (C2400, Hamamatsu Photonics, Hamamatsu, Japan) and analyzed with ImageJ software (version 1.25 s; freeware at http://rsb.info.nih.gov/ij/ provided by Dr. W. Rasband, National Institutes of Health, Bethesda, MD). The relative cell size was calculated as the cube of the ratio of diameters ([d/d0]3 where d and d0 represent the cell diameter at a given time and the initial cell diameter, respectively.). The isotonic and hypotonic solutions used were the same as those used in mean cell volume measurements.
Statistical analysis. Data are presented as means ± SE of n observations. Statistical differences of the data were evaluated by paired or unpaired Student's t-test and were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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3 cmH2O. As shown in Fig. 1B, the single-channel current exhibited ohmic dependence on voltage (Fig. 1B, top), with a slope conductance of 22.7 ± 0.01 pS (n = 22–34) (Fig. 1B, middle) in the voltage range of –40 to +80 mV and 37 pS at –80 to –60 mV. The steady-state Po increased with depolarization at a constant negative pressure of 8 cmH2O (Fig. 1B, bottom).
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0.6 mM for Mg2+-inhibited cation (MIC) channels, which are believed to be endogenous TRPM7, in Jurkat T cells (41). The concentration-inhibition curve for blocking by extracellular Mg2+ at a negative potential of –40 mV was shifted toward lower concentrations compared with the curve at a positive potential of +80 mV (Fig. 2B), suggesting open channel block by extracellular Mg2+. The IC50 for extracellular Mg2+ was 3.2 µM at –40 mV and 0.11 mM at +80 mV, showing that block was sharply dependent on voltage. These IC50 values are comparable to those previously observed for MIC channels in Jurkat T cells (1 µM at –40 mV and 30 µM at +40 mV; Ref. 20).
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Abolition of stretch-activated single-cation channel currents by siRNA-induced knockdown of TRPM7. Since sensitivity of stretch-activated cation channels in HeLa cells to Mg2+ and Gd3+ is essentially the same as that of TRPM7 to Mg2+ (32, 44) and to Gd3+ (1, 16), we next conducted RT-PCR studies to check for the expression of TRPM7. Robust amplification of PCR products of expected size (503 bp) from reverse-transcribed RNA was seen with two separate sets of TRPM7-specific primers, as shown in Fig. 3A, top left, for one set of primers. Also, no PCR product was amplified when reverse transcriptase was omitted from the reaction (data not shown). The nucleotide sequences of the PCR products obtained with TRPM7-specific primers were completely identical to the corresponding sequences in human TRPM7 (42). Treatment of HeLa cells with TRPM7 siRNA for 48–72 h eliminated expression of TRPM7 mRNA almost completely, whereas mock siRNA had no effect. Treatment not only with mock siRNA but also with TRPM7 siRNA did not affect expression of mRNA for the housekeeping gene GAPDH (Fig. 3A, top right), indicating that the siRNA-induced silencing was specific.
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220 kDa, and this band (but not other nonspecific bands) was almost completely abolished when HeLa cells were treated with siRNA for TRPM7. In HEK/TRPM7 cells, the band reactive to anti-TRPM7 antibody was also detected at 220 kDa (Fig. 3A, bottom), and this band disappeared when antibodies were preabsorbed with antigen peptides (blocking peptides). In these experiments, actin bands of a molecular mass of 42 kDa were detected at equal levels (data not shown). The siRNA-induced inhibition of TRPM7 expression led to the abolition of single-channel activity of the mechanosensitive cation channel in HeLa cells, as shown in Fig. 3B. In contrast, in HeLa cells treated with mock siRNA, we consistently observed typical single-channel events of the stretch-activated cation channel. We thus conclude that TRPM7 is the channel corresponding to the stretch-activated cation channel in HeLa cells.
Sensitivity of swelling-activated whole cell cation currents to Mg2+, Gd3+, and siRNA-induced knockdown of TRPM7. As shown in Fig. 4A, top, intracellular dialysis with Mg2+- and ATP-free, Cs+-rich solution induced spontaneous activation of whole cell currents in HeLa cells. The current-voltage (I-V) relationship for steady-state currents showed strong outward rectification under ramp-clamp (Fig. 4A, trace 1). When osmotic cell swelling was induced by intracellular hypertonicity, spontaneously activated whole cell currents became larger (Fig. 4A, top). Since swelling-induced augmentation was more marked at negative potentials, the I-V relationship became less outwardly rectified and almost linear (Fig. 4A, trace 2).
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Sensitivity of RVD to Mg2+, Gd3+, and siRNA-induced knockdown of TRPM7.
An increase in the intracellular free Ca2+ concentration is known to be involved in RVD in many cell types (26), including human epithelial cells (14, 38). Because a SSAC channel has been reported to serve as the volume-regulatory Ca2+ influx pathway in epithelial cells (5, 37), we tested the possible involvement of the TRPM7-like channel in RVD by measuring the mean cell volume in HeLa cells. As shown in Fig. 5A, top, control HeLa cells responded quickly to a hypotonic challenge (65% osmolality) with osmotic swelling and then with an RVD that resulted in an 70% recovery toward the original volume within 30 min after exposure to the hypotonic solution (containing 1 mM Ca2+ and 1 mM Mg2+). The rate of RVD was significantly diminished when swelling-activated cation channel currents were blocked by Gd3+ (30 µM) or when Ca2+ influx through the channel was prevented by removal of extracellular Ca2+ ions. In contrast, the rate of RVD was significantly facilitated when Ca2+ influx mediated by swelling-activated cation channels was augmented by elimination of extracellular Mg2+. These data are summarized in Fig. 5A, bottom. RR (100 µM) also prominently inhibited the RVD in HeLa cells (Fig. 5A, bottom).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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In prokaryotes and invertebrates, a number of molecules such as mechanosensitive channel large (MscL), Mid1, NOMPC, and members of the degenerin (DEG) family have been reported to be stretch-activated cation channels (11). No homologs of MscL or Mid1 have ever been identified in vertebrates. It has been suggested that the mammalian homolog of DEG, ENaC, serves as a stretch-activated, amiloride-sensitive cation channel in vertebrates (21). However, HeLa cells lack expression of ENaC (48), and in the present study the stretch-activated cation channel in HeLa cells was shown to be amiloride insensitive. The heterodimer of TRP polycystin (TRPP)1 and TRPP2 was proposed to be a mechanosensitive cation channel expressed in the primary cilia of mouse kidney epithelium (33). In HeLa cells, however, our preliminary studies showed that treatment with siRNA targeted against TRPP2 abolished expression of TRPP2 mRNA but failed to affect swelling-activated whole cell cation currents (T. Numata and Y. Okada, unpublished observations). Also, TRP ankyrin (TRPA)1 has recently been suggested as a candidate for the mechanosensitive transduction channel in mouse and zebra fish hair cells (6). However, the mechanosensitivity of TRPA1 has never been directly tested by electrophysiological experiments. Similarly, direct evidence is still lacking for the mechanosensitivity of TRPC6, TRPM4, and NOMPC. TRPC6 and TRPM4 were suggested to be mechanosensitive channels involved in the myogenic tone of arteries (7, 50), and NOMPC was reported to be a mechanosensory channel in Drosophila (9) and zebra fish larvae (45). Evidence that TRPC1 forms a component of a frog mechanosensitive cation channel has been obtained recently by electrophysiological and molecular biological experiments (25). However, molecular expression of TRPC1 was not observed by RT-PCR in human epithelial HeLa cells (T. Numata, unpublished observations).
TRPM7 is a member of the recently emerging TRPM subfamily of TRP proteins and has been characterized as a bifunctional protein consisting of a channel and protein kinase (29, 42). TRPM7 is expressed in a wide variety of tissues including brain and hematopoietic tissues (27), as well as in kidney and heart (30). TRPM7 has been implicated in cell proliferation and survival (12, 44), cell Mg homeostasis (44), regulation of anoxic neuronal cell death (1), and the uptake of trace metal ions (28). Oancea et al. (35) showed that shear stress upregulates TRPM7 currents by augmenting exocytotic insertion of TRPM7 into the plasma membrane in vascular smooth muscle A7R5 cells and HEK/TRPM7 cells. However, it has not been shown that the TRPM7 channel is a cation channel that can be directly activated by membrane stretch. The present study indicates that TRPM7 is the mechanosensitive cation channel endogenously expressed in human epithelial HeLa cells. First, the endogenous activity in HeLa cells of the stretch-activated cation channel that had exquisite mechanosensitivity exhibited sensitivity to Mg2+ and Gd3+ typical of TRPM7 (1, 16, 32, 44). Second, the endogenous stretch-activated single-cation channel current was abolished by treatment of HeLa cells with siRNA targeted against TRPM7.
In epithelial cells, the stretch-activated cation channel was shown to be activated by osmotic cell swelling, thereby serving as a route of volume-regulatory Ca2+ influx (5, 37). However, the molecular identity of the SSAC channel has not been known. The present study demonstrated that swelling-activated whole cell cation currents exhibit sensitivity to Mg2+, Gd3+, and treatment with TRPM7 siRNA in HeLa cells. Also, all these properties of swelling-activated whole cell current were found, in the present study, to match well with those of stretch-activated single-channel current in HeLa cells. Thus it appears that TRPM7 represents the SSAC channel in HeLa cells.
In the present study, it was found that RVD in HeLa cells was largely inhibited by extracellular Ca2+ removal and extracellular application of Gd3+. Also, it was found that RVD was facilitated by extracellular Mg2+ removal. These effects could be explained well by the suppression or augmentation, resulting from these manipulations, of Ca2+ influx mediated by TRPM7 or MIC channels, which are known to be permeable to Ca2+ (1, 12, 19, 28, 32, 42). It was found, in fact, that siRNA-induced knockdown of the TRPM7 channel largely inhibited RVD. Thus it appears that TRPM7-mediated Ca2+ influx plays an essential role in RVD, which is known to require both Ca2+ release and Ca2+ influx. Also, there is a possibility that TRPM7-mediated Mg2+ influx might somehow affect the RVD process.
In conclusion, TRPM7 represents the SSAC channel endogenously expressed in human epithelial HeLa cells, and it is essentially involved in RVD by serving as a swelling-induced, volume-regulatory Ca2+ influx pathway.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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), 100 µM extracellular Mg2+ (Mg
), 30 µM extracellular Gd3+, and 100 µM extracellular RR on swelling-activated cation currents. The relative macroscopic currents (I) were calculated by dividing the whole cell steady-state currents recorded at +80 and –80 mV in the presence of blockers by the values obtained in the absence of blockers; n values are in parentheses. C: suppression of swelling-activated whole cell cation currents after treatment with TRPM7 siRNA but not after treatment with mock siRNA. *Significantly different from mock siRNA values at the same voltages.