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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Identification and characterization of a novel family of membrane magnesium transporters, MMgT1 and MMgT2

Department of Medicine, University of British Columbia, Vancouver, British Columbia, Canada

Submitted 6 June 2007 ; accepted in final form 28 November 2007

	ABSTRACT

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Magnesium is an essential metal, but few selective transporters have been identified at the molecular level. Microarray analysis was used to identify two similar transcripts that are upregulated with low extracellular Mg²⁺. The corresponding cDNAs encode proteins of 131 and 123 amino acids with two predicted transmembrane domains. The two separate gene products comprise the family that we have termed "membrane Mg²⁺ transporters" (MMgTs), because the proteins reside in the membrane and mediate Mg²⁺ transport. When expressed in Xenopus laevis oocytes, MMgT1 and MMgT2 mediate Mg²⁺ transport as determined with two-electrode voltage-clamp analysis and fluorescence measurements. Transport is saturable Mg²⁺ uptake with Michaelis constants of 1.47 ± 0.17 and 0.58 ± 0.07 mM, respectively. Real-time RT-PCR demonstrated that MMgT mRNAs are present in a wide variety of cells. Subcellular localization with immunohistochemistry determined that the MMgT1-hemagglutinin (HA) and MMgT2-V5 fusion proteins reside in the Golgi complex and post-Golgi vesicles, including the early endosomes in COS-7 cells transfected with the respective tagged constructs. Interestingly, MMgT1-HA and MMgT2-V5 were found in separate populations of post-Golgi vesicles. MMgT1 and MMgT2 mRNA increased by about threefold, respectively, in kidney epithelial cells cultured in low-magnesium media relative to normal media and in the kidney cortex of mice maintained on low-magnesium diets compared with those animals consuming normal diets. With the increase in transcripts, there was an apparent increase in MMgT1 and MMgT2 protein in the Golgi and post-Golgi vesicles. These experiments suggest that MMgT proteins may provide regulated pathways for Mg²⁺ transport in the Golgi and post-Golgi organelles of epithelium-derived cells.

microarray analysis; two-electrode voltage clamp; fluorescence; Xenopus oocytes

We have shown that cellular Mg²⁺ control is predominantly through differential gene expression leading to synthesis of Mg²⁺-responsive proteins (20). Indeed, we used this approach to identify genes encoding novel Mg²⁺ transport proteins (8). In the present study, we used microarray analysis to screen for magnesium-regulated transcripts in epithelial cells. This revealed two transcripts whose relative levels were dramatically altered by extracellular magnesium. Thus, these transcripts potentially represented a species of RNA whose synthesis was regulated by changes in magnesium. The corresponding cDNAs showed that they represented a novel gene family comprising two members that we have termed membrane Mg²⁺ transporter 1 (MMgT1) and 2 (MMgT2) because they are membrane proteins and mediate Mg²⁺ uptake when expressed in Xenopus laevis oocytes. Subcellular localization with immunohistochemistry demonstrated that MMgT1 and MMgT2 fusion proteins are principally found in the Golgi complex and post-Golgi vesicles. Consistent with the observation that low magnesium increases MMgT1 and MMgT2 transcripts, there was an apparent increase in the respective proteins in the Golgi and post-Golgi vesicles. Also evident was the observation that MMgT1 and MMgT2 partially sorted to different post-Golgi organelles. We conclude that the differential expressions of MMgT1 and MMgT2 are involved in the control of Mg²⁺ metabolism within the Golgi complex and post-Golgi vesicles.

	MATERIALS AND METHODS

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Animal preparation and cell culture. Male mice were maintained for 3 days on a low-magnesium diet (ICN diet no. 902205, Nutritional Biochemicals, Cleveland, OH) or on this diet supplemented with 0.05% MgSO₄, which was comparable with commercial mouse chow. The plasma magnesium concentrations of the two groups were not different (low magnesium: 0.87 ± 0.06 mM vs. normal magnesium: 0.88 ± 0.07 mM), but the mean urinary magnesium concentration was significantly lower (3.9 ± 2.9 mM) in animals maintained on the magnesium-restricted diet compared with control mice (15.8 ± 1.9 mM). This is in agreement with our early study (26). Urinary volumes and sodium and calcium excretions were not different between the two groups. The protocol was approved by the Committee on Animal Care of the University of British Columbia.

Mouse distal convoluted tubule (MDCT) cells were derived and immortalized by Pizzonia and colleagues (19). The MDCT cell line has been extensively used by us to study the hormonal and nonhormonal control of renal magnesium transport (4). Either MDCT cells or monkey kidney COS-7 cells were grown in basal DMEM-Ham's F-12 (1:1) media (GIBCO) supplemented with 10% FCS (Flow Laboratories, McLean, VA), 1 mM glucose, 5 mM L-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin in a humidified environment of 5% CO₂- 95% air at 37°C. Where indicated, subconfluent MDCT cells were cultured in Mg²⁺-free media (Stem Cell Technologies, Vancouver, BC, Canada) for 16 h before being harvested. Other constituents of the Mg²⁺-free culture media were similar to the complete media.

Oligonucleotide microarray analysis. Microarray analysis was performed according to the protocol recommended by Affymetrix (http://www.affymetric.com) using MG U74 Bv2 and MG U74 Cv2 arrays (Affymetrix, Santa Clara, CA) as described previously by us (8). DNA fragments representing transcripts that were upregulated with low magnesium were selected and prioritized according to properties characteristic of membrane transport proteins.

Quantitative analysis of MMgT transcripts by real-time RT-PCR. PCR products were quantified continuously with AB7000 (Applied Biosystems) using SYBR green fluorescence according to the manufacturer's instructions. The primer sets for mouse MMgT1 were 5'-GCTTGTAGGTGTCGGGCTT-3' (forward) and 5'-TGAAGGCCGGAACAGCACTC-3' (reverse), and those for mouse MMgT2 were 5'-AGAGACAGGGATGCCACTTCG-3' (forward) and 5'-ACAGTCGGTAGCCAGAACGGTG-3' (reverse). Relative amounts of MMgT RNA were normalized to mouse β-actin transcripts.

Genomic sequence analysis. MMgT cDNA sequences were determined by standard methods. Database searching and alignments were performed using BLAST. Nonredundant and expressed sequence tag databases were sourced. Protein homology searches were performed by comparing the amino acid query sequence against the SWISSPROT database. Full-length MMgT cDNA sequences have been deposited in the GenBank database (Accession Nos. human: Eu069461, mouse: Eu069461).

Protein motifs were identified using BLASTP and the SWISSPROT database. Membrane topology was predicted by SOSUI and Kyte-Doolittle hydrophobicity analysis.

Plasmid construction and generation of expression constructs. Mouse MMgT1 cDNA was purchased from RIKEN (Rik960048L06), and MMgT2 was from IMAGE (IRAV 4459181). MMgT1 contained the entire coding region of the mMMgT1 cDNA flanked by 47 bp of untranslated 5'-nucleotide sequence and 205 bp of untranslated 3'-sequence, and MMgT2 possessed 122 5'-untranslated and 96 3'-untranslated sequences. Isolated clones were sequenced to confirm sequence integrity.

For immunolocalization experiments, MMgT1 cDNA was subcloned into the pcDNA3.1 vector containing a COOH-terminal hemagglutinin (HA) tag, and MMgT2 was subcloned into the pcDNA3.1 vector with a COOH-terminal V5 tag. PCRs were performed using 5'-CAAGGTACCTGAATCAGTGCGCCGTCG-3' (5MG1KPN1) and 5'-AATCATGATACGGCGCAGTGAGTC-3' (3MG1) primers for MMgT1 and 5'-TTCGGTACCATGGTGGCGTGGCTG-3' (5MG2KPN1) and 5'-AATCATGATAAACTTCAACGGTAA-3' (3MG2) primers for MMgT2.

Expression of mouse MMgT constructs in Xenopus oocytes and characterization of Mg²⁺ transport. For Xenopus oocyte expression, cRNA was synthesized from the mMMgT1 cDNA construct, linearized, and then transcribed with T7 polymerase in the presence of the ^m7GpppG cap using the mMESSAGE MACHINE T7 kit (Ambion) and mMMgT2 with SP6 polymerase and the mMESSAGE MACHINE SP6 kit (Ambion) transcription system. The preparation of oocytes, injection with cRNA, and two-electrode voltage clamp were as previously described (8) and performed at 21°C. Oocytes were studied at 3–5 days following injection. Permeability ratios were calculated using the Nernstian relation and apparent K_m and V_max values with nonlinear regression analysis (10).

Epifluorescence microscopy was used to measure Mg²⁺ flux into single oocytes using the Mg²⁺-responsive mag-fura-2 fluorescence dye (10). Oocytes were injected with 50 µM mag-fura-2 acid (Molecular Probes) to a final concentration of 5 µM 20 min prior to experimentation. The chamber (0.5 ml) was mounted on an inverted Nikon Diaphot-TMD microscope with a fluor x10 objective, and the current-voltage association was determined. Subsequently, oocytes were clamped at –70 mV for fluorescence measurements for the indicated times. Fluorescence was continuously recorded using a dual-excitation wavelength spectrofluorometer (Delta-scan, Photon Technologies) with excitation for mag-fura-2 at 340 and 385 nm (chopper speed set at 100 Hz) and emission at 505 nm. Results are presented as 340-to-385-nm ratios, which reflect the intracellular Mg²⁺ concentrations. All experiments were performed at 23°C.

Immunofluorescence confocal microscopy. COS-7 cells were transiently transfected with MMgT1-HA and/or MMgT2-V5 fusion constructs using Lipofectamine 2000 (Invitrogen). Coverslips of cultured COS-7 cells were fixed at room temperature for 10 min in 2% paraformaldehyde. Cells were washed three times with PBS containing 0.3% Triton X-100 (PBST) before each antibody incubation. The following primary antibodies were used: a cis-Golgi matrix protein (GM130) and a GTP-binding protein (Rab5) that were raised in the mouse (BD Transduction Labs). Alexa 488- and Alexa 568-conjugated secondary antibodies were obtained from Molecular Probes. All antibody reactions were performed in blocking solution composed of 2% normal goat serum in PBST for 1.5 h at room temperature. Alexa 350-conjugated phalloidin (Molecular Probes) was used to stain for actin in the indicated experiments to aid in delimiting peripheral membrane ruffles. After cells had been stained, coverslips were then mounted on slides with Fluoromount-G glycerol-based mounting media (Southern Biotechnology).

Oocytes were mounted in OCT cryostat medium and flash frozen in isopentane cooled in liquid nitrogen. Ten-micrometer-thick sections were cut through frozen oocytes and mounted directly onto superfrost plus slides (Fisher). Sections were fixed in –20°C methanol and processed for immunohistochemistry using HA or V5 antibodies and anti-rabbit Alexa 488- or Alexa 568-conjugated secondary antibodies.

All images were taken using a x63 water-immersion lens affixed to a Zeiss LSM 510 Meta microscope and AxioVision (epifluorescent) or LSM 510 Meta (confocal) software. Cells were selected from 10 to 12 fields of view and used for the assessment of colocalization of antibody staining.

	RESULTS

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Identification of MMgT1 and MMgT2 as magnesium-responsive genes. With the knowledge that differential gene expression is involved with selective control of epithelial cell magnesium conservation, our strategy was to use microarray analysis to identify cDNAs that were upregulated with low magnesium (8). Two similar cDNA fragments were identified by increases in transcripts that conformed to full DNA sequences that might encode related hypothetical proteins. Based on electrophysiological and fluorescence properties and the subcellular location of the encoded proteins, we designated proteins as MMgT1 or MMgT2, respectively. In confirmation of the microarray data, MMgT1 mRNA was increased 2.5-fold in the kidney cortex of hypomagnesemic mice (n = 10 separate animals) and 3.5-fold in immortalized MDCT epithelial cells (n = 11 independent preparations) cultured in low magnesium compared with normal animals and cells, respectively. The respective increases in MMgT2 transcripts were a 1.5-fold increase in the kidney cortex and 3.0-fold increase in MDCT cells.

Characterization of MMgT genes. Full-length mouse MMgT cDNAs were identified by BLAST searches of the GenBank database. Full-length MMgT1 possessed 80% amino acid similarity to MMgT2 (Fig. 1A). MMgT1 and MMgT2 comprise 131 and 123 amino acids, respectively (Fig. 1A). Both MMgTs were highly conserved among the human, mouse, rat, and pig. To date, there is no reported MMgT2 in the human genome. Mouse MMgT1 is 97% similar to human MMgT1, and mouse MMgT2 is 81% similar to human MMgT1. Hydrophobicity plots using the SOUSI program predicted only two putative transmembrane helixes (Fig. 1, B and C). Human MMgT1 is located on chromosome Xq26.3. The respective chromosomal locations in the mouse were XA5 for MMgT1 and 11B2 for MMgT2, and those in the rat were Xq36 for MMgT1 and 10q23 for MMgT2.

View larger version (37K): [in this window] [in a new window]   Fig. 1. Molecular characterization of membrane Mg2+ transporters (MMgTs). A: amino acid sequences of mouse MMgT1 and MMgT2. Common amino acids are in bold, and predicted transmembrane segments are underlined. B: predicted membrane topology and domain structure of mouse MMgT1. The hydrophobicity plot was performed according to Kyte-Doolittle using the SOUSI program. This analysis predicted two transmembrane regions. C: predicted secondary structure of MMgT2. D: quantitative analysis of MMgT1 and MMgT2 transcripts in mouse tissues by real-time RT-PCR. Expression levels of MMgT1 and MMgT2 mRNAs were normalized with those of the β-actin transcript, which was measured on the same cDNAs. Indicated values are means ± SE and were obtained from 10 separate tissue preparations.

MMgT mediates Mg²⁺ transport in expressing Xenopus oocytes. To determine if the MMgTs cDNA encoded functional Mg²⁺ transporters, we prepared the respective cRNA, injected it into Xenopus oocytes, and performed two-microelectrode voltage-clamp analysis and microfluorescence experiments. The electrophysiological data gave evidence for a rheogenic process with large inward currents in either MMgT1 (Fig. 2A) or MMgT2 (Fig. 2B) cRNA-injected oocytes, whereas there were no appreciable currents in control H₂O- or total poly(A)⁺ RNA-injected cells from the same batch of oocytes. The reversal potentials shifted right with increasing external Mg²⁺ concentrations, similar to that predicted by the Nernstian relationship for Mg²⁺ transport (indicated by arrows; Fig. 2, A and B). Mouse MMgT-mediated Mg²⁺-induced uptake was saturable: the K_m for MMgT1 was 1.47 ± 0.17 mM (n = 32; Fig. 2C), and the K_m for MMgT2 was 0.58 ± 0.07 mM (n = 29; Fig. 2D). K_m values did not vary with voltage in each case. In support of the conclusions using voltage-clamp analysis, we performed flux experiments with fluorescence spectrometry using the Mg²⁺-sensitive dye mag-fura-2 (10). Currents were initially measured at resting potentials with oocytes in Mg²⁺-free solutions and then in solutions containing 2.0 mM MgCl₂to confirm that oocytes were expressing the respective MMgTs. Oocytes were then voltage clamped at –70 mV (Fig. 2E). Fluorescence was determined throughout the experiment. The 340-to-385-nm fluorescence ratio reflects the change in free Mg²⁺ concentration within the oocyte. The 340-to-385-nm ratio, i.e., intracellular Mg²⁺ content, increased in MMgT1- and MMgT2-expressing oocytes but not in control H₂O-injected oocytes (Fig. 2E). Moreover, the increase in Mg²⁺ concentration was associated with the simultaneously measured currents, indicating that Mg²⁺-evoked currents were mediated by Mg²⁺ flux for both MMgT1 (Fig. 2F) and MMgT2 (Fig. 2G).

View larger version (29K): [in this window] [in a new window]   Fig. 2. Mg2+-mediated transport into Xenopus oocytes expressing MMgTs. A: current-voltage (I-V) relationships obtained from linear voltage steps from –150 to +25 mV in the presence of Mg2+-free solutions or those containing the indicated concentrations of MgCl2. Oocytes were clamped at a holding potential of –15 mV and stepped from –150 to +25 mV in 25-mV increments for - s sweeps at each of the concentrations indicated. Shown are average I-V curves obtained from control H2O-injected (n = 3) or MMgT1-expressing (n 3) oocytes. Note the positive shifts in reversal potential with increments in magnesium concentrations. Reversal potentials (arrows) were –32 mV at 0.35 mM, –24 mV at 0.7 mM, and –18 mV at 2.0 mM. In the experiments shown, currents were corrected for changes in membrane resistance caused by the respective divalent cation using values from H2O-injected oocytes. Values are means ± SE of observations measured at the end of each voltage sweep for the respective Mg2+ concentration. B: I–V relationship of MMgT2-mediated currents in expressing oocytes. The methods used were as described for A. Reversal potentials (arrows) were 33 mV at 0.35 mM, 21 mV at 0.7 mM, and 14 mV at 2.0 mM. C: summary of concentration-dependent Mg2+-evoked currents in MMgT1-expressing oocytes using a holding potential of –125 mV. The Km, as determined with nonlinear regression analysis, was 1.47 ± 0.17 mM. D: summary of concentration-dependent Mg2+-evoked currents in MMgT2-expressing oocytes using a holding potential of –125 mV. The Km was 0.58 ± 0.07 mM. E: Mg2+ flux into MMgT1- and MMgT2-expressing oocytes. Currents were measured in control and MMgT1-expressing oocytes, at resting potentials, in solutions consisting of 2.0 mM MgCl2. Oocytes were subsequently voltage clamped at a holding potential of –70 mV where indicated. Mg2+ fluxes were determined with fluorescence using the Mg2+-sensitive dye mag-fura-2. Results are presented as 340-to-385-nm excitation ratios determined with 505-nm emission. Results are means of tracings performed with 5 different oocyte preparations for each experiment. F: association of Mg2+ flux measured with fluorescence and Mg2+-induced currents determined with voltage clamp performed on the same oocyte expressing MMgT1. Note the close correlation of the results with the two independent methods. G: association of Mg2+ flux and Mg2+-induced currents performed on the same oocyte expressing MMgT2. H: summary of substrate specificity of MMgT1 and MMgT2 following application of Ca2+ (2.0 mM) and other test cations (0.2 mM) in the absence of external Mg2+. Oocytes were clamped at a holding potential of –15 mV and stepped from –150 to +25 mV in 25-mV increments for 2 s for each of the cations. Values are means ± SE of permeability ratios measured at the end of each voltage sweep for the respective divalent cations. There were no detectable currents in water-injected oocytes with each of the indicated divalent cations. I: MMgT2 mediates Mn2+ transport in expressing oocytes, whereas Mn2+ is not a substrate for MMgT1-mediated flux. Oocytes were initially voltage clamped at –70 mV in the presence of Mg2+, and Mg2+ was then replaced with Mn2+, a cation that quenches mag-fura-2 fluorescence at both 340 and 385 nm. Note the intensity determined at 340 nm diminished in MMgT2-expressing cells but not in MMgT1-expresssing oocytes. J: surface expression of MMgT1-HA and MMgT2-V5 fusion proteins in Xenopus laevis oocytes determined with immunofluorescence using HA or V5 antibodies and anti-rabbit Alexa 488- or Alexa 568-conjugated secondary antibodies. Magnification: x200.

A variety of other divalent cations was used to determine the selectivity of the expressed MMgT-mediated transport. MMgT1 mediated Sr²⁺, Fe²⁺, Co²⁺, and Cu²⁺ transport, whereas MMgT2 mediated Sr²⁺, Co²⁺, Cu²⁺, Ba²⁺, Mn²⁺, and Ni²⁺ transport in addition to Mg²⁺ (Fig. 2D). None of the divalent cations tested elicited detectable currents in water-injected oocytes. Notably, Mn²⁺ elicited currents in MMgT2-expressing oocytes but not in MMgT1-expressing cells. The differential selectivity for Mn²⁺ was also evident using fluorescence measurements (Fig. 2E). Mn²⁺ did not quench mag-fura-2 fluorescence in MMgT1-expressing cells but markedly quenched fluorescence in MMgT2-expressing oocytes, as would be expected if MMgT2 but not MMgT1 mediated Mn²⁺ transport. These experiments clearly indicate that MMgT1 and MMgT2 mediate Mg²⁺ transport in MMgT1- and MMgT2-expressing oocytes. Additional electrophysiological experiments demonstrated that 0.2 mM Mn²⁺ inhibited Mg²⁺-mediated currents in MMgT1-expressing oocytes, whereas Ni²⁺ and Gd³⁺ did not affect Mg²⁺ transport (data not given). Accordingly, MMgT-mediated transport is substrate selective and inhibitible, confirming properties of membrane transporters. Of the cation substrates tested here, only Mg²⁺ is found at the physiological concentrations used in these experiments, so we conclude that MMgT1 and MMgT2 are principally Mg²⁺ transporters.

Finally, we used immunofluorescence to show that MMgT1 and MMgT2 fusion proteins are localized in surface membranes of MMgT1- and MMgT2-expressing oocytes (Fig. 2J). The surface staining was consistent with the results of the functional experiments. No staining was present in native water-injected oocytes.

Subcellular localization of MMgT. To investigate the subcellular localization of MMgT1 and MMgT2 proteins, we performed immunofluorescence using anti-HA and anti-V5 antibodies in kidney COS-7 cells transfected with MMgT1-HA and MMgT2-V5 constructs, respectively. The MMgT1-HA fusion protein extensively colocalized with GM130 (a cis-Golgi matrix protein), suggesting that MMgT1 is principally resident in the Golgi complex (Fig. 3A). However, there was also apparent staining of post-Golgi vesicles with partial overlap with Rab5, a marker of early recycling endosomes (Fig. 3B). There was no colocalization with endoplasmic reticulum (ER) or late endosome/lysosome markers. The MMgT2 fusion protein demonstrated a similar distribution, predominately in the Golgi, with appreciable amounts in post-Golgi vesicles, including partial staining of the punctate structures representing the early recycling endosomes (Fig. 4).

View larger version (61K): [in this window] [in a new window]   Fig. 3. Subcellular localization of MMgT1. Immunofluorescence staining of MMgT1-hemagglutinin (HA)-transfected COS-7 cells. A: cells were cultured in media containing normal magnesium concentrations, fixed, and incubated with HA antibody (left) and the Golgi marker GM130 (middle). The overlay is shown on the right. The enlarged areas at 2-fold magnification (insets) show post-Golgi vesicles that were not stained with the GM130 marker. B: normal cells stained with HA (left) and the endosome marker Rab5 (middle). The merged image is shown on the right. The images showing colocalization with GM130 and Rab5 indicate that MMgT1 protein is localized to the Golgi complex and post-Golgi vesicles including the early endosomes. C: COS-7 cells were cultured in low magnesium, nominally magnesium free, for 12 h, fixed, and incubated with HA antibody (left) and the Golgi marker GM130 (middle). The overlay is shown on the right. The enlarged areas (insets) highlight the presence of MMgT1 in post-Golgi vesicles. There was an apparent increase in MMgT1 protein in the Golgi and post-Golgi vesicles in magnesium-deprived cells compared with those cultured in normal magnesium.

View larger version (76K): [in this window] [in a new window]   Fig. 4. Subcellular localization of MMgT2. Immunofluorescence staining of MMgT2-V5 transfected COS-7 cells. A and B: cells were cultured in media containing normal magnesium concentrations, fixed, and incubated with V5 antibody (left) and GM130 (A, middle) or Rab5 (B, middle). The respective overlays are shown on the right. MMgT2 protein was distributed to the Golgi complex and post-Golgi vesicles including in the partial labeling of early endosomes. A similar pattern of MMgT2 distribution was observed as with the MMgT1 protein, as shown in Fig. 3. C: COS-7 cells were cultured in low magnesium, fixed, and incubated with HA antibody (left) and GM130 (middle). The overlay is shown on the right. As with MMgT1, there was an apparent increase in MMgT2 protein in the Golgi and post-Golgi vesicles in magnesium-deprived cells.

Finally, we determined if there was colocalization of MMgT1 and MMgT2 proteins within epithelium-derived cells. COS-7 cells were transfected with both fusion constructs, and the subcellular localization was determined with immunofluorescence. As expected, there was colocalization of MMgT1 and MMgT2 fusion proteins within the Golgi complex (Fig. 5A). However, it was of interest that there was only partial colocalization within the post-Golgi vesicles, which suggests that they might traffic to separate compartments. A similar pattern was observed with cells cultured in low-magnesium media (Fig. 5B). The major change with magnesium deficiency appears to be amount of protein in Golgi and post-Golgi vesicles rather than a change in trafficking intracellular compartments.

View larger version (41K): [in this window] [in a new window]   Fig. 5. Subcellular colocalization of MMgT1 and MMgT2 fusion proteins. COS-7 cells were transfected with both MMgT1-HA and MMgT1-V5 epitope-tagged constructs, and immunofluorescence staining was performed with HA (left) and V5 (middle) antibodies. Merged images are presented on the right. A: cells cultured in normal media with magnesium. B: cells cultured in low magnesium. Both proteins were evident in the Golgi complex, but, of note, they appeared to localize to different post-Golgi vesicles in normal and Mg2+-depleted cells.

	DISCUSSION

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Intracellular Mg²⁺ concentration is in the order of 0.5 mM, but it is not homogeneously distributed across the cell. Using spatial imaging with mag-fura-2, we have shown that there are relatively higher levels in the perinuclear region comprising the ER, Golgi, and other organelles. Mg²⁺ plays an important role in the ER and Golgi as it is essential in protein assembly, lipid biosynthesis, and vesicle trafficking. These biochemical processes are catalyzed by specific enzymes that require specific luminal Mg²⁺ levels for optimal function. It is now known that the Golgi and post-Golgi vesicles possess numerous ionic transporters to maintain ideal Ca²⁺, Mn²⁺, Zn²⁺, H⁺, K⁺, and ATP levels (1, 2, 6, 11, 13–15, 17, 27, 32). Accordingly, it is not surprising that there are dedicated transporters to maintain optimal Mg²⁺ concentrations in these organelles. The novel MMgT1 and MMgT2 proteins described here provide the first Mg²⁺ transporters to be identified in the Golgi complex and post-Golgi vesicles.

Numerous mammalian divalent metal transporters have been molecularly identified and extensively studied, but few have been shown to mediate Mg²⁺transport. The first mammalian Mg²⁺ transporter to be identified was MRS2, a mitochondrial protein encoded by nuclear DNA (33). In the transient receptor potential melastatin (TRPM) family of cation channels, the ubiquitous TRPM7 has been shown to mediate plasma membrane Mg²⁺ transport and to be essential for cellular viability (16, 18). Another member of this family, TRPM6, forms a major Mg²⁺ transporter in the plasma membrane of intestinal and kidney epithelial cells (3, 25, 29). Using differential gene expression, we (8) have recently identified a novel family of Mg²⁺ transporters, designated MagT, that was regulated at the transcriptional and protein levels by cellular Mg²⁺ balance (personnel observations). More recently, by applying the same microarray platform, we (7) have shown that the solute carrier SLC41, a family of proteins comprising three gene products (SLC41A1, SLC41A2, and SLC41A3), mediates Mg²⁺ transport when expressed in Xenopus oocytes. Additionally, we have shown that the ancient conserved domain protein (ACDP) family, composed of ACDP1, ACDP2, ACDP3, and ACDP4, is differentially expressed in response to Mg²⁺ content and the expressed proteins mediate Mg²⁺ transport (9). Mutations in ACDP1 are thought to underlie urofacial syndrome, although the alteration has not been identified (30). Finally, we (10) have shown that the nonimprinted in Prader-Willi/Angelman (NIPA) family of genes encode plasma membrane Mg²⁺ transport proteins (10). Mutations in NIPA1 lead to a loss of function defect that provides the basis for hereditary spastic paraplegia (22, 23). The role of each of these transporter families in the control of cell Mg²⁺ is currently under active investigation. Nevertheless, there is now abundant evidence for a number of unique mammalian Mg²⁺ transporters. In the present study, we report the identification of another family of Mg²⁺ transporters that appear to be predominately located in the Golgi complex but might play an important role in post-Golgi vesicles.

	GRANTS

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This work was supported by Canadian Institutes of Health Research Grant MOP-53288 (to G. A. Quamme).

	FOOTNOTES

 

Address for reprint requests and other correspondence: G. A. Quamme, Dept. of Medicine, Vancouver Hospital, Koerner Pavilion, 2211 Wesbrook Mall, Vancouver, BC, Canada V6T 1Z3 (e-mail: quamme{at}interchange.ubc.ca)

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.

	REFERENCES

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1. al-Awqati Q. Chloride channels of intracellular organelles. Curr Opin Cell Biol 7: 504–508, 1995.[CrossRef][Web of Science][Medline]

2. Borrelly G, Boyer JC, Touraine B, Szponarski W, Rambier M, Gibrat R. The yeast mutant vps5Delta affected in the recycling of Golgi membrane proteins displays an enhanced vacuolar Mg²⁺/H⁺ exchange activity. Proc Natl Acad Sci USA 98: 9660–9665, 2001.[Abstract/Free Full Text]

3. Chubanov V, Schlingmann KP, Waring J, Heinzinger J, Kaske S, Waldegger S, Schnitzler MM, Gudermann T. Hypomagnesemia with secondary hypocalcemia due to a missense mutation in the putative pore-forming region of TRPM6. J Biol Chem 282: 7656–7667, 2007.[Abstract/Free Full Text]

4. Dai Lj Ritchie G, Kerstan D, Kang HS, Cole DEC, Quamme GA. Magnesium transport in the distal nephron plays an important role in determining normal and abnormal renal magnesium balance. Physiol Rev 81: 51–84, 2001.[Abstract/Free Full Text]

5. Flatman PW. Magnesium transport across cell membranes. J Membr Biol 80: 1–14, 1984.[CrossRef][Web of Science][Medline]

6. Futai M, Oka T, Sun-Wada G, Moriyama Y, Kanazawa H, Wada Y. Luminal acidification of diverse organelles by V-ATPase in animal cells. J Exp Biol 203: 107–116, 2000.[Abstract]

7. Goytain A, Quamme GA. Functional characterization of human SLC41A1, a Mg²⁺ transporter with similarity to prokaryotic MgtE Mg²⁺ transporters. Physiol Genomics 21: 337–342, 2005.[Abstract/Free Full Text]

8. Goytain A, Quamme GA. Identification and characterization of a novel mammalian Mg²⁺ transporter with channel-like properties. BMC Genomics 6: 48, 2005.[CrossRef][Medline]

9. Goytain A, Quamme GA. Functional characterization of ACDP2 (ancient conserved domain Mg²⁺ transporter that is differentially regulated by magnesium. Physiol Genomics 22: 382–389, 2005.[Abstract/Free Full Text]

10. Goytain A, Hines RM, El-Husseini A, Quamme GA. NIPA1 (SPG6), the basis for autosomal dominant form of hereditary spastic paraplegia, encodes a functional Mg²⁺ transporter. J Biol Chem 282: 8060–8068, 2007.[Abstract/Free Full Text]

11. Johnson JD, Chang JP. Function- and agonist-specific Ca²⁺ signalling: the requirement for and mechanism of spatial and temporal complexity in Ca²⁺ signals. Biochem Cell Biol 78: 217–240, 2000.[CrossRef][Web of Science][Medline]

12. Jung DW, Panzeter E, Baysal K, Brierley GP. On the relationship between matrix free Mg²⁺ concentration and total Mg²⁺ in heart mitochondria. Biochim Biophys Acta 1320: 310–320, 1997.[Medline]

13. Mahieu F, Owsianik G, Verbert L, Janssens A, De Smedt H, Nilius B, Voets T. TRPM8-independent menthol-induced Ca²⁺ release from endoplasmic reticulum and Golgi. J Biol Chem 282: 3325–3336, 2007.[Abstract/Free Full Text]

14. McCarron JG, Chalmers S, Bradley KN, MacMillan D, Muir TC. Ca²⁺ microdomains in smooth muscle. Cell Calcium 40: 461–93, 2006.[CrossRef][Medline]

15. Missiaen L, Dode L, Vanoevelen J, Raeymaekers L, Wuytack F. Calcium in the Golgi apparatus. Cell Calcium 41: 405–416, 2007.[CrossRef][Web of Science][Medline]

16. Nadler MJ, Hermosura MC, Inabe K, Perraud AL, Zhu Q, Stokes AJ, Kurosaki T, Kinet JP, Penner R, Scharenberg AM, Fleig A. LTRPC7 is a Mg.ATP-regulated divalent cation channel required for cell viability. Nature 411: 590–595, 2001.[CrossRef][Medline]

17. Nagasawa M, Kanzaki M, Iino Y, Morishita Y, Kojima I. Identification of a novel chloride channel expressed in the endoplasmic reticulum, Golgi apparatus, and nucleus. J Biol Chem 276: 20413–20418, 2001.[Abstract/Free Full Text]

18. Penner R, Fleig A. The Mg²⁺ and Mg²⁺-nucleotide-regulated channel-kinase TRPM7. Handb Exp Pharmacol: 313–328, 2007.

19. Pizzonia JH, Gesek FA, Kennedy SM, Coutermarsh BA, Bacskai BJ, Friedman PA.Immunomagnetic separation, primary culture and characterization of cortical thick ascending limb plus distal convoluted tubule cells from mouse kidney. In Vitro Cell Dev Biol 27A: 409–416, 1991.[CrossRef]

20. Quamme GA. Renal magnesium handling: new insights in understanding old problems. Kidney Int 52: 1180–1195, 1997.[Web of Science][Medline]

21. Quamme GA, Dai LJ, Rabkin SW. Dynamics of intracellular free Mg²⁺ changes in a vascular smooth muscle cell line. Am J Physiol Heart Circ Physiol 265: H281–HH288, 1993.[Abstract/Free Full Text]

22. Rainer S, Chai JH, Tokarz D, Nicholls RD, Fink JK. NIPA1 gene mutations cause autosomal dominant hereditary spastic paraplegia (SPG6). Am J Hum Genet 3: 967–971, 2003.

23. Reed JA, Wilkinson PA, Patel H, Simpson MA, Chatonnet A, Robay D, Patton MA, Crosby AH, Warner TT. A novel NIPA1 mutation associated with a pure form of autosomal dominant hereditary spastic paraplegia. Neurogenetics 6: 79–84, 2005.[CrossRef][Web of Science][Medline]

24. Romani AM, Scarpa A. Regulation of cellular magnesium. Front Biosci 5: D720–D734, 2000.[Web of Science][Medline]

25. Schlingmann KP, Weber S, Peters M, Niemann Nejsum L, Vitzthum H, Klingel K, Kratz M, Haddad E, Ristoff E, Dinour D, Syrrou M, Nielsen S, Sassen M, Waldegger S, Seyberth HW, Konrad M. Hypomagnesemia with secondary hypocalcemia is caused by mutations in TRPM6, a new member of the TRPM gene family. Nat Genet 31: 166–170, 2003.[Web of Science]

26. Shafik IM, Quamme GA. Early adaptation of renal magnesium reabsorption in response to magnesium restriction. Am J Physiol Renal Fluid Electrolyte Physiol 257: F974–F977, 1989.[Abstract/Free Full Text]

27. Thompson RJ, Akana HC, Finnigan C, Howell KE, Caldwell JH. Anion channels transport ATP into the Golgi lumen. Am J Physiol Cell Physiol 290: C499–C514, 2006.[Abstract/Free Full Text]

28. Touyz RM. Magnesium in clinical medicine. Front Biosci 9: 1278–1293, 2004.[Web of Science][Medline]

29. Walder RY, Landau D, Meyer P, Shalev H, Tsolia M, Borochowitz Z, Boettger MB, Beck GE, Englehardt RK, Carmi R, Sheffield VC. Mutation of TRPM6 causes familial hypomagnesemia with secondary hypocalcemia. Nat Genet 31: 171–174, 2002.[CrossRef][Web of Science][Medline]

30. Wang CY, Shi JD, Huang YQ, Cruz PE, Ochoa B, Hawkins-Lee B, Davoodi-Semiromi A, She JX. Construction of a physical and transcript map for a 1-Mb genomic region containing the urofacial (Ochoa) syndrome gene on 10q23-q24 and localization of the disease gene within two overlapping BAC clones (<360 kb). Genomics 60: 12–19, 1999.[CrossRef][Web of Science][Medline]

31. Weglicki W, Quamme G, Tucker K, Haigney M, Resnick L. Potassium, magnesium, and electrolyte imbalance and complications in disease management. Clin Exp Hypertens 27: 95–112, 2005.[CrossRef][Web of Science][Medline]

32. Zhou M, Tanaka O, Suzuki M, Sekiguchi M, Takata K, Kawahara K, Abe H. Localization of pore-forming subunit of the ATP-sensitive K⁺-channel, Kir6.2, in rat brain neurons and glial cells. Brain Res Mol Brain Res 101: 23–32, 2002.[Medline]

33. Zsurka G, Gregan J, Schweyen RJ. The human mitochondrial Mrs2 protein functionally substitutes for its yeast homologue, a candidate magnesium transporter. Genomics 72: 158–168, 2001.[CrossRef][Web of Science][Medline]

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