Identification and characterization of a novel mammalian Mg2+ transporter with channel-like properties
© Goytain and Quamme; licensee BioMed Central Ltd. 2005
Received: 23 November 2004
Accepted: 01 April 2005
Published: 01 April 2005
Intracellular magnesium is abundant, highly regulated and plays an important role in biochemical functions. Despite the extensive evidence for unique mammalian Mg2+ transporters, few proteins have been biochemically identified to date that fulfill this role. We have shown that epithelial magnesium conservation is controlled, in part, by differential gene expression leading to regulation of Mg2+ transport. We used this knowledge to identify a novel gene that is regulated by magnesium.
Oligonucleotide microarray analysis was used to identify a novel human gene that encodes a protein involved with Mg2+-evoked transport. We have designated this magnesium transporter (MagT1) protein. MagT1 is a novel protein with no amino acid sequence identity to other known transporters. The corresponding cDNA comprises an open reading frame of 1005 base pairs encoding a protein of 335 amino acids. It possesses five putative transmembrane (TM) regions with a cleavage site, a N- glycosylation site, and a number of phosphorylation sites. Based on Northern analysis of mouse tissues, a 2.4 kilobase transcript is present in many tissues. When expressed in Xenopus laevis oocytes, MagT1 mediates saturable Mg2+ uptake with a Michaelis constant of 0.23 mM. Transport of Mg2+ by MagT1 is rheogenic, voltage-dependent, does not display any time-dependent inactivation. Transport is very specific to Mg2+ as other divalent cations did not evoke currents. Large external concentrations of some cations inhibited Mg2+ transport (Ni2+, Zn2+, Mn2+) in MagT1-expressing oocytes. Ca2+and Fe2+ were without effect. Real-time reverse transcription polymerase chain reaction and Western blot analysis using a specific antibody demonstrated that MagT1 mRNA and protein is increased by about 2.1-fold and 32%, respectively, in kidney epithelial cells cultured in low magnesium media relative to normal media and in kidney cortex of mice maintained on low magnesium diets compared to those animals consuming normal diets. Accordingly, it is apparent that an increase in mRNA levels is translated into higher protein expression.
These studies suggest that MagT1 may provide a selective and regulated pathway for Mg2+ transport in epithelial cells.
Magnesium is the second most abundant cation within the cell and plays an important role in many intracellular biochemical functions . Despite the abundance and importance of magnesium, little is known about how eukaryotic cells regulate their magnesium content.
Intracellular free Mg2+ concentration is in the order of 0.5 mM which is 1–2% of the total cellular magnesium . Accordingly, intracellular Mg2+ is maintained below the concentration predicted from the transmembrane electrochemical potential. Intracellular Mg2+ concentration is finely regulated likely by precise controls of Mg2+ entry, Mg2+ efflux, and intracellular storage compartments . The transporters comprising these pathways have only begun to be identified.
Few magnesium transporters have been identified at the molecular level. Schweyen and colleagues have demonstrated that the mitochondrial RNA splicing2 (Mrs2) gene encodes a protein that is present in yeast and mammalian inner mitochondrial membranes [4, 5]. Mrs2 mediates high capacity Mg2+ influx in isolated yeast mitochondria driven by the inner membrane potential but also transports a range of divalent cations such as Ni2+, Co2+, and Cu2+ . Overexpression of Mrs2 increases influx while deletion of the gene abolishes uptake suggesting that it is the major mitochondrial system. This data suggests that Mrs2 protein may mediate Mg2+ transport in mammalian mitochondria. Nadler et al first identified TRPM7, a widely expressed member of the transient receptor potential melastatin (TRPM) ion channel family, that produces a Mg2+ current in a wide variety of cells . TRPM7 is regulated by intracellular Mg·ATP levels and is similarly permeable to both major divalent cations, Ca2+ and Mg2+, but also many of the trace elements, such as Zn2+, Mn2+, and Co2+ . Using a positional cloning approach, Schlingmann et al  and Walder et al  found that hypomagnesemia with secondary hypocalcemia (HSH) was caused by mutations in TRPM6, a new member of the TRPM family. HSH is an inherited disease affecting both intestinal and renal Mg2+ absorption . The functional characteristics of the TRPM6 transporter have not been fully elucidated [11, 12]. Other magnesium transporters have been functionally described but they have not been characterized at the molecular level [13–18]. It is disparaging that, despite the significance of cellular Mg2+, only three specific magnesium transporters have been described in mammalian cells to date.
Mammalian magnesium homeostasis is a balance of epithelial intestinal magnesium absorption and renal magnesium excretion. The kidney plays a major role in control of vertebrate magnesium balance, in part, by active magnesium transport within the distal tubule of the nephron . Using the Madin-Darby canine kidney (MDCK) cell line obtained from canine distal tubules and immortalized mouse distal convoluted tubule cells (MDCT), we have shown that Mg2+ entry is through specific and regulated magnesium pathways that are controlled by a variety of hormonal influences . However these hormones do not provide selective control as they also affect calcium and in some cases sodium and potassium transport . Selective and sensitive control of cellular Mg2+ transport is regulated by intrinsic mechanisms so that culture in media containing low magnesium results in upregulation of Mg2+ uptake in these cells. This adaptive increase in Mg2+ entry was shown to be dependent on de novo transcription since prior treatment of the epithelial cells with actinomycin D prevented the adaptation to low extracellular magnesium . The data suggest that epithelial cells can somehow sense the environmental magnesium and through transcription- and translation-dependent processes alter Mg2+ transport and maintain magnesium balance. These conclusions using isolated epithelial cells are consonant with our views of magnesium conservation in the intact kidney .
In an attempt to identify genes underlying cellular changes resulting from adaptation to low extracellular magnesium, we used oligonucleotide microarray analysis to screen for magnesium-regulated transcripts in epithelial cells. This approach revealed one transcript whose relative level was dramatically altered by extracellular magnesium. Thus, this transcript potentially represented a species of mRNA whose synthesis was regulated by changes in cell magnesium. In this study, we describe the identification and characterization of this novel transcript referred to as MagT1. Our data indicate MagT1 may mediate Mg2+ transport in a wide variety of cells and may play a role in control of cellular magnesium homeostasis.
Identification of MagT1
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 candidates that were up-regulated with low magnesium. Using Affymetrix GeneChipR technology, we showed that 116 DNA fragments were significantly increased (p < 0.0002) from the 24,000 mouse ESTs represented on the chips. The RNA of one of these was significantly increased, greater than 2-fold, n = 3, determined by real-time RT-PCR. The full length human cDNA was identified from clone DKFZp564K142Q3 obtained from RZPD Resource Center, Berlin, in pAMP1 vector and bidirectionally sequenced at NAPS, University of British Columbia. Based on the cDNA sequence, electrophysiological properties and cation selectivity of the encoded protein, we designated it as MagT1 for Magnesium Transport protein, subtype 1. MgT was not used to avoid confusion with the bacterial MgtA/B and MgtE magnesium transporters [21, 22].
Primary structure of MagT1
MagT1 is a novel gene located at Xq13.1–13.2
A BLAST search yielded a number of poorly characterized proteins with similar amino acid sequences to MagT1 (Fig. 1). Using the BESTFIT sequence alignment program, MagT1 shows 100% identity to a human unnamed protein (GenBank™ CAB66571.1, BAC11592.1), 88% to a mouse implantation associated protein (GenBank™ NP_080228.1, BAB28739.1, BAB31313.1, AAH03881.1), 87% to a rat implantation associated protein (GenBank™ IAG2_RAT, NP_446398.1, AAB63294.2), 66% (first 131 amino acids) to a human implantation associated protein (GenBank™ XP_497668) and to an unknown protein MGC:56218 from the zebra fish (AAH46002.1). MagT1 shares some similarity (65–67%) to the human (GenBank™ AAH10370.1, AAB18376.1, AAB18374.1, G02297, N33_HUMAN, NP_006756.1, AAB18375.1), mouse (GenBank™ BAC25795.1), and rat (GenBank™ XP_214356.1) putative prostate cancer tumor suppressor protein. There is also some similarity (23–54%) to a number of un-characterized proteins in Anopheles (GenBank™ EAA13927.1), Drosophila melanogaster (GenBank™ AAL68198.1, AAF52636.2, NP_609204.2), Ochlerotatus trisertiatus (GenBank™ AF275675.1), and Caenorhabditis elegans (GenBank™ NP_498691.1, AAA28222.1, S44911, Y013_CAEEL). None of these proteins, with similar amino acid sequences to MagT1, are sufficiently characterized to suggest a common functional purpose. MagT1 has a more distant relationship (P = 3 × 10-12) to the OST3 gene of Saccharomyces cervisiae that encodes a regulatory subunit of the endoplasmic reticulum oligosaccharyltransferase complex . A gapped alignment of these sequences showed only 21% identical residues between the hMagT1 and OST3 sequences extending throughout most of both proteins.
Tissue distribution of MagT1 expression
Northern analysis of cultured mouse distal convoluted tubule cells and tissues harvested from mice revealed a single strong transcript of about 2.4 kb (Fig. 2). The kidney, colon, heart and liver possessed relatively high levels of MagT1 mRNA and smaller amounts were found in intestine, spleen, brain, and lung (Fig. 2). Accordingly, MagT1 mRNA appears to be widely expressed among tissues but the transcript is variably expressed among these tissues.
The MagT1 antibody recognized two protein bands, 35 and 38 kDa, in tissues expressing the MagT1 transcript (Fig. 3). Two bands were apparent in kidney and liver tissue whereas one was evident in heart, colon, and brain. The molecular size of MagT1 calculated from cDNA is 38 kDa. A significant difference in the calculated molecular size and that the smaller band found by immunoblot analysis raises the possibility that MagT1 may be cleaved to yield the 35 kDa carboxyl-terminal protein detected by MagT1 antibody. There was very little MagT1 protein in the small intestine (Fig. 3). Other than liver tissue, there appears to be a good correlation between the respective amounts of transcripts and the protein content. The discrepancy between the levels of MagT1 mRNA and protein expression in liver (abundant mRNA detected but little protein detected) suggests that a posttranslational mechanism may play a role in tissue-specific expression of the MagT1 protein. In summary, the 38 kDa MagT1 protein is expressed to a variable extent in all of the tissues sampled (Fig. 3) but the 35 kDa band appears to be present in only some of the tissues. Although this is a limited survey of tissues, the results suggest that MagT1 is expressed in many tissues with an apparent correlation of mRNA and protein but expression may be post-translationally modified in a tissue-specific fashion such as the liver.
The specificity of the affinity-purified polyclonal anti-MagT1 antibody was assessed by Western blots of the total protein extract from the MDCT cells probed with a preimmune serum. No protein of the predicted size (~35 kDa) was detected when the preimmune serum was used (Fig. 3). Taken together, the results indicate that the affinity-purified anti-MagT1 antibody specifically reacts with the endogenous MagT1 protein.
Human MagT1 elicits Mg2+-evoked currents in Xenopus oocytes
Large concentrations (2 mM) of Ca2+, or its analogs, Sr2+ and Ba2+ or the other divalent cations tested, Fe2+, Cu2+, Co2+, Zn2+, Mn2+, and Ni2+, did not produce appreciable currents in the absence of Mg2+ in hMagt1-expressing oocytes (Fig. 9). In the experiments shown, the permeability ratios (Erev for tested cation relative to Erev for Mg2+) were corrected for changes in membrane resistance caused by the respective divalent cation using values from H2O-injected oocytes (Fig. 9).
MagT1 expression is responsive to magnesium
The rationale for these studies is based on the observation that renal magnesium conservation is principally regulated by differential expression of genes encoding magnesium transport proteins. Accordingly, we determined the response of MagT1 to changes in magnesium at the messenger and protein levels. These studies were performed with distal tubule epithelial cells, MDCT, cultured in media containing normal (1.0 mM) or low (nominally magnesium-free) magnesium concentrations for 16 h and on kidney cortex tissue harvested from mice maintained on either normal or magnesium-restricted diets for 5 days. The mRNA and protein expression was relatively stronger in cells cultured in low magnesium media and in tissue of mice maintained on low magnesium diets (urine and plasma magnesium concentration, 1.1 ± 0.3 and 0.13 ± 0.01 mM, respectively) compared to normal cells and tissue of animals on normal diets (urine and plasma magnesium, 13.2 ± 1.2 and 0.75 ± 0.09 mM, respectively). MDCT and tissue mMagT1 mRNA, as measured by real-time RT-PCR was increased by 2.1-fold and 2.3-fold, respectively (Figure 12). In association with the increases in mRNA, MagT1 protein was increased by 31 ± 12% in the cultured epithelial cells and 33 ± 6 % in kidney cortex with low magnesium relative to the respective controls (Figure 13). Accordingly, it is apparent that an increase in mRNA levels is translated into higher protein expression and by inference leads to greater magnesium transport (the latter conclusion is based on the urinary magnesium excretion of animals maintained on low magnesium relative to normal diets).
Despite the extensive evidence for unique mammalian Mg2+ transporters, few proteins have been biochemically identified to date that fulfill this role. Moreover, functional characterization has not been fully investigated for those that have been reported [11, 12]. With the knowledge that the kidney, particularly the distal tubule, regulates magnesium conservation through transcriptional mechanisms, we used oligonucleotide microarray analysis to identify MagT1, a novel Mg2+ transporter [2, 19]. The MagT1 transcript is a 2.4-kb mRNA that encodes a protein comprising a relatively long N-terminal segment, a putative region of four TM domains, and a short C-terminal sequence. The cytoplasmic segments possess a number of characteristic phosphorylation motifs. MagT1 shows no structural similarity to any known transporter. Functional expression of MagT1 in oocytes results in large Mg2+-evoked currents with little permeability to other divalent cations. However, some divalent cations, Ni2+, Zn2+, and Mn2+inhibit Mg2+-evoked currents at relatively large external concentrations. These cations are not found in the extracellular or intracellular fluid at the concentrations used here, 0.2 mM. The other major extracellular divalent cation, Ca2+, was neither transported nor were Mg2+-evoked currents inhibited by extracellular Ca2+. MagT1 is widely distributed among tissues particularly those of epithelial structure. Finally, MagT1 expression is regulated in these tissues by external magnesium as predicted by our starting premise. Accordingly, MagT1 fulfills the role of a dedicated mammalian magnesium transporter. The function of MagT1 in cellular Mg2+ balance remains to be determined.
The electrophysiological characteristics of MagT1 expressed in Xenopus oocytes are reminiscent of our observations of Mg2+ transport in intact renal epithelial cells measured by microfluorescence . There is not a suitable isotope of Mg2+ for use in physiological experiments so that we have used fura-mag-2 fluorescence to investigate Mg2+ transport . We have shown that Mg2+ uptake in a variety of epithelial cells is driven by the electrochemical gradient of Mg2+. Membrane hyperpolarization stimulates Mg2+ transport whereas depolarization abrogates uptake (19). There was no evidence in renal distal tubule cells for coupling of apical Mg2+ entry to other ions such as Na+, Cl-, or H+ . Magnesium transport in immortalized mouse distal convoluted tubule (MDCT) cells is dependent on the transmembrane concentration gradient and uptake is saturable, as determined by fluorescence. The apparent affinity constant is in the order of 0.5 mM that is similar to that observed for MagT1 expressed in Xenopus oocytes (Fig. 5). This affinity is appropriate for a physiological role of the transporter in cellular Mg2+ conservation . Mg2+-evoked currents in oocytes expressing MagT1 is highly specific for Mg2+, an observation that is again consonant with our views of Mg2+ transport in MDCT cells and in vivo kidney . The microfluorescence experiments suggest that there may be some variability in cationic inhibition of Mg2+ uptake depending on the cell-type used so that other transporters may be present with differing selectivity that are tissue specific . Relatively large concentrations of nitrendipine, a 1,4-dihydropyridine channel blocker, inhibited Mg2+-evoked currents in MagT1-expressing oocytes not unlike the inhibition of Mg2+ entry into distal epithelial cells . Intriguingly, nifedipine did not influence Mg2+-induced currents in MagT1-expressing oocytes that is similar to our previous reports using MDCT cells . Although both antagonists are dihydropyridines, they have differing efficacy based on their structural differences . Again, reminiscent of our observations using MDCK and MDCT epithelial cells, the channel agonist, BAY 8644, increased Mg2+-evoked currents . The 1,4-dihydropyridines analogues are not highly selective channel blockers/activators but these findings support the notion that Mg2+ entry into MagT1-expressed oocytes or distal epithelial cells is via channel-like proteins. Two other characteristics are noteworthy. First, Mg2+-evoked currents in MagT1-expressed oocytes are greater at physiological pH values relative to acidic pH. This is also true for Mg2+ uptake in distal tubule epithelial cells and magnesium conservation by the intact kidney in vivo . Magnesium reabsorption is greater and urinary excretion is less in metabolic alkalosis than acidosis. Indeed, magnesium wasting may be sufficient in chronic metabolic acidosis to lead to hypomagnesemia . Second, the presence of multiple putative protein kinase A and C phosphorylation sites in MagT1 may suggest phosphorylation-dependent regulation. We have shown that Mg2+ entry into epithelial cells is stimulated by peptide hormones, such as parathyroid hormone, glucagon and calcitonin, that act through protein kinases A and C . Further studies are needed to elucidate the mechanisms underlying these phenomena. On balance, many of the functional characteristics of MagT1 expressed in oocytes are harmonious with our earlier physiological observations using kidney distal convoluted tubule cells.
MagT1 is a membrane protein that may comprise ER, early and late endosomes or apical and basolateral plasma membrane fractions. The role of each of these structures in cellular magnesium homeostasis is poorly understood. Using single cell spatial imaging, we have previously shown that intracellular ionized Mg2+ concentration is heterogenously distributed across the cell . The ER or sarcoplasmic reticulum normally contains high concentrations of Mg2+, ranging from 0.4–2.0 mM, relative to the cytosolic concentration, 0.5 mM, and nucleus, 0.32 mM. It is clear that Mg2+ is transported into and out of a variety of intracellular compartments and there is likely dedicated magnesium transporters for each event. Further studies are required to establish the subcellular localization and intracellular trafficking of Mg2+ and the role of MagT1 protein.
Our evidence is that the expression of MagT1 mRNA and protein is responsive to cellular magnesium. The ability of epithelial cells to selectively respond to the availability of essential nutrients, such as Zn2+ and Fe2+, is not unique but the cellular mechanisms are unknown [29, 30]. Presumably epithelial cells may sensitively sense intracellular nutrient concentration and through transcriptional and post-translational mechanisms adjust transport rates appropriately [19, 29, 30]. Our studies indicate that this response within the cell is the basis for sensitive and selective control of magnesium balance in the kidney .
Epithelial cells comprising the intestine and kidney are primarily involved with dietary magnesium absorption, urinary magnesium excretion, and total body magnesium homeostasis . Accordingly, MagT1 may, in part, be responsible for intestinal and renal tubular Mg2+ conservation. In support of this is the observation that the MagT1 transcript is present in these tissues (Fig. 2). However, magnesium is necessary in all cells and the wide-spread distribution of the MagT1 transcript may suggest a housekeeping role for this transporter. It is also germane to note that MagT1 mRNA is regulated in all cells investigated. Further studies are needed to define the function of MagT1 in intestine and kidney and the role in overall cellular magnesium balance.
We have identified a novel magnesium transporter, probably a channel, that is regulated by extracellular magnesium. To our knowledge this is the first report of a highly selective Mg2+ transporter. Its role in cellular magnesium homeostasis and transepithelial magnesium absorption is unknown but our evidence from our differential gene expression studies indicate that it plays an important in cellular magnesium homeostasis.
Cell culture and oligonucleotide microarray analysis
Mouse distal convoluted tubule (MDCT) cells were isolated from kidneys and immortalized by Pizzonia et al (31). The MDCT cell line has been extensively used by us to study renal magnesium transport . Cells were grown in Basal Dulbecco's minimal essential medium (DMEM)/Ham's F-12, 1:1, media (GIBCO) supplemented with 10% fetal calf serum (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 Inc., Vancouver, BC) for 4 h. Other constituents of the Mg2+-free culture media were similar to the complete media.
Microarray analysis was performed according to the protocol recommended by Affymetrix http://www.affymetric.com. Poly(A)+ RNA was extracted with Poly(A)Pure (Ambion) from cells cultured in high and low magnesium media. Twenty Fg RNA was used for cDNA synthesis followed by in vitro transcription. The cRNA was biotin-labeled, fragmented, and the probes hybridized to Affymetrix MG U74 Bv2 and MG U74 Cv2 arrays (Affymetrix, Santa Clara) representing approximately 24,000 mouse transcripts. Detailed protocols for data analysis, documentation of sensitivity, reproducibility and other aspects of the quantitative microarray analysis are those given by Affymetrix. Gene categorization was based on the NetAffx Database.
Northern blot analysis
Cells were harvested by scraping and total RNA isolated using TRIzol (Life Technologies, Inc.). In some experiments poly(A)+ RNA was isolated using the Poly(A)Pure mRNA isolation system (Ambion) following the manufacturer's instructions. Samples of total RNA (20 μg) or poly(A)+ RNA (8 μg) were denatured in 2.2 M formaldehyde, 50% (v/v) formamide buffer and electrophoresed on 0.8% agarose 3 M formaldehyde, 0.02 M MOPS, 8 mM Na acetate, 1 mM EDTA, pH 7.0 gels. The size-fractionated RNA was transferred to GeneScreen nylon membranes (NEN) by downward alkali transfer and UV crosslinked (Stratagene Stratalinker 1800). Membranes were probed with 32P-labelled probes made from gene specific inserts represented in the microarray analytical results. The probe templates were prepared from PCR products representing inserts using specific primers on cDNA prepared from MDCT cells. The inserts were ligated into pGEM-t vector (Promega) following QiaexII gel (Qiagen) purification. Blots were prehybridized in 50% formamide, 5 X SSPE, 100 μg/ml denatured sonicated salmon sperm DNA, 5 X Denhardt's solution, 0.1% SDS for 1 h at 42EC in a rotating hybridization oven (Tyler HI-16000). Probe was heated to 95EC for 5 min, then added to the prehybridization solution. Membranes were hybridized for 16 h at 42EC then washed at high stringency sequentially: 2X [1X SSPE, 0.2% SDS, 28EC] 2X [1X SSPE, 0.4% SDS, 37EC] 1X [0.1X SSPE, 0.2% SDS, 55EC]. Membranes were exposed on Kodak X-AR-2 film. In most cases, after images were obtained, membranes were incubated at 95°C for 1 h in 0.1% SDS to remove the bound probe and hybridized with a 32P-labelled β-actin probe in order to normalize loading.
Quantitative analysis of MagT1 transcripts by real-time RT PCR
Total RNA of cells was extracted by TRIzol (Invitrogen). Genomic DNA contamination was removed by DNA-free™ kit (Ambion) prior to making first strand cDNA. Standard curves were constructed by serial dilution of a linear pGEM-T vector (Promega) containing the MagT gene. The primer set of mouse MagT1 was: forward, 5'-CCAAAGGGGCTGATACATA-3' and reverse, 5'-ATAGAAGAACGATGTGTG-3' and the human MagT1: forward, 5'-GCAAACTCCTGGCGATACTCC-3' and the human reverse 5'-ACTGGGCTTGACTGCTTCC-3'. PCR products were quantified continuously with AB7000™ (Applied Biosystems) using SYBR Green™ fluorescence according to the manufacturer's instructions. The relative amounts of MagT1 RNA were normalized to the respective human and mouse β-actin transcripts.
Genomic sequence analysis
The MagT1 cDNA sequence was determined by standard methods. Data base searching and alignments were performed using BLAST. The nonredundant and EST data bases were sourced. Protein homology searches were performed by comparing the amino acid query sequence against SWISSPROT data base. The full-length MagT1 cDNA sequence has been deposited in the GenBank™ data base (accession human DQ000004, mouse DQ000005).
Western blot analysis
A rabbit polyclonal antibody, anti-MagT1, was raised against the N-terminal domain of the final cleaved human MagT1 protein using a synthetic peptide, INFPAKGKPKRGDTYELQV (amino acid residues 140–158), coupled to keyhole limpet hemocyanin. Affinity-purified rabbit anti-human MagT1 antibody was diluted in TBS (Tris-buffered saline, 20 mM Tris, 200 mM NaCl, pH 7.6) containing 0.5 % BSA at a final concentration about 0.7 μl/ml. For subcellular fractionation, cells were suspended in lysis buffer (0.25 M sucrose, 10 mM triethanolamine-acetic acid pH 7.6, 1 mM EDTA) containing protease inhibitors (1 mM PMSF, 2 μg/ml leupeptin, 2 μg/ml aprotinin). Protein concentrations were determined using Bio-Rad protein assay reagent. SDS-PAGE was performed according to Laemmli. For immunobblotting, the proteins were electrophoretically transferred to polyvinylidene difluoride membranes (HybondR, Amersham Biosciences) by semidry electroblotting for 45 min. Western analysis was performed by incubating the blots with antiMagT1 antibody or anti-MagT1 antibody preabsorbed with 50 × antigen peptide (control for antibody specificity) overnight at 4EC followed by three washes with TBS/0.1% Tween-20, 10 min each. The blots were then incubated with 1/10,000 horseradish peroxidase-conjugated donkey anti-rabbit secondary (Sigma Aldrich) antibody for 1 h. After washing three times with TBS/Tween-20, 10 min each, the blots were visualized with ECL (Amersham Biosciences) according to the manufacturer's instructions.
Expression of MagT1 in Xenopus oocytes and current measurements
The cDNA comprising the open reading frame (ORF) of MagT1 was amplified from the pAMP1 vector using the cloning primers (sense: 5'-GATTGGTACCGTGAACATGGCCTC-3'; antisense: 5'-CTTGTCGACCCTCTTTAACTCATC-3') and was subcloned into the KpnI and ApaI restriction sites of the pEYFP-N1 expression vector. The constructs were linearized and then transcribed with SP6 polymerase in the presence of m7GpppG cap using the mMESSAGE MACHINE™ SP6 KIT (Ambion) transcription system. Oocytes were injected with MagT1 complementary RNA (cRNA) or for control observations, H2O or kidney total poly(A)+ RNA; no Mg2+-induced currents were detected in the latter.
Xenopus oocytes were prepared and injected with cRNA and electrophysiological recordings were preformed according to previously described techniques . Briefly, defolliculated stage V-VI oocytes were typically injected with 25 ng cRNA in 50 nl H2O. Oocytes were incubated at 18°C for 3–6 days in multiwell tissue culture plates containing Barth's solution (88 mM NaCl, 1.0 mM KCl, 2.4 mM NaHCO3, 1.0 mM MgSO4, 1.0 mM CaCl2, 0.3 mM Ca(NO3)2, 10 mM Hepes-NaOH, pH 7.6, 2.5 mM Na-pyruvate, 0.1 % BSA, 10,000 U/l penicillin, 100 mg/l streptomycin). To record expressed membrane currents, the oocytes were placed in a recording chamber (0.3 ml) and perfused with modified Barth's (96 mM NaCl, 10 mM Hepes-NaOH) containing various concentrations of MgCl2, as indicated, in substitution for osmotically equivalent amounts of NaCl. All experiments were performed at room temperature (21°C).
Steady-state membrane currents were recorded with the two-microelectrode voltage-clamp technique using a Geneclamp 500 amplifier (Axon Instruments, Inc.). Electrophysiology consisted of a voltage clamp step profile consisting of a holding potential of -15 mV, followed by 8 episode series of +25 mV steps of 2 s duration, from -150 mV to +25 mV within an episode duration of 6.14 sec. Each episode recorded 1536 data points collected at 4 ms intervals. The data was filtered at the appropriate frequency before digitization. In order to assess the permeability of different divalent cations, we used the shift in the reversal potentials of the respective cation from the reversal potentials of Mg2+ currents, ΔErev, and calculated by the permeability ratio by:
Px/PMg = exp/(ΔErev X F/RT)
where R, T, and F have their standard meanings. Voltage clamp episodes in the presence of extracellular test cations were corrected against episodes in the absence of external test cations.
All experimental conditions were performed on oocytes harvested from a minimum of 3 different animals.
This work was supported by a research grants from the Canadian Institutes of Health Research, MOP-53288, and the Kidney Foundation of Canada. We acknowledge Genomic Sciences Center, Riken Yokohama Institute, Japan for EST clones A530029P05, A330056M18, and A530032I23 and RZPD Deutsches Ressourcenzentrum für Genomforschung GmbH, Berlin, Germany for clone DKFZp564K142Q3.
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