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Identification and characterization of a novel mammalian Mg2+ transporter with channel-like properties Goytain, Angela; Quamme, Gary A Apr 1, 2005

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ralssBioMed CentBMC GenomicsOpen AcceResearch articleIdentification and characterization of a novel mammalian Mg2+ transporter with channel-like propertiesAngela Goytain and Gary A Quamme*Address: Department of Medicine University of British Columbia Vancouver, B.C. CanadaEmail: Angela Goytain - angeking@interchange.ubc.ca; Gary A Quamme* - quamme@interchange.ubc.ca* Corresponding author    AbstractBackground: Intracellular magnesium is abundant, highly regulated and plays an important role inbiochemical 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 thatepithelial magnesium conservation is controlled, in part, by differential gene expression leading toregulation of Mg2+ transport. We used this knowledge to identify a novel gene that is regulated bymagnesium.Results: Oligonucleotide microarray analysis was used to identify a novel human gene thatencodes a protein involved with Mg2+-evoked transport. We have designated this magnesiumtransporter (MagT1) protein. MagT1 is a novel protein with no amino acid sequence identity toother known transporters. The corresponding cDNA comprises an open reading frame of 1005base 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 onNorthern analysis of mouse tissues, a 2.4 kilobase transcript is present in many tissues. Whenexpressed in Xenopus laevis oocytes, MagT1 mediates saturable Mg2+ uptake with a Michaelisconstant of 0.23 mM. Transport of Mg2+ by MagT1 is rheogenic, voltage-dependent, does notdisplay any time-dependent inactivation. Transport is very specific to Mg2+ as other divalent cationsdid 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-timereverse transcription polymerase chain reaction and Western blot analysis using a specific antibodydemonstrated 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 kidneycortex of mice maintained on low magnesium diets compared to those animals consuming normaldiets. Accordingly, it is apparent that an increase in mRNA levels is translated into higher proteinexpression.Conclusion: These studies suggest that MagT1 may provide a selective and regulated pathway forMg2+ transport in epithelial cells.Published: 01 April 2005BMC Genomics 2005, 6:48 doi:10.1186/1471-2164-6-48Received: 23 November 2004Accepted: 01 April 2005This article is available from: http://www.biomedcentral.com/1471-2164/6/48© 2005 Goytain and Quamme; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 18(page number not for citation purposes)BackgroundMagnesium is the second most abundant cation withinthe cell and plays an important role in many intracellularbiochemical functions [1]. Despite the abundance andBMC Genomics 2005, 6:48 http://www.biomedcentral.com/1471-2164/6/48importance of magnesium, little is known about howeukaryotic cells regulate their magnesium content.Intracellular free Mg2+ concentration is in the order of 0.5mM which is 1–2% of the total cellular magnesium [2].Accordingly, intracellular Mg2+ is maintained below theconcentration predicted from the transmembrane electro-chemical potential. Intracellular Mg2+ concentration isfinely regulated likely by precise controls of Mg2+ entry,Mg2+ efflux, and intracellular storage compartments [3].The transporters comprising these pathways have onlybegun to be identified.Few magnesium transporters have been identified at themolecular level. Schweyen and colleagues have demon-strated that the mitochondrial RNA splicing2 (Mrs2) geneencodes a protein that is present in yeast and mammalianinner mitochondrial membranes [4,5]. Mrs2 mediateshigh capacity Mg2+ influx in isolated yeast mitochondriadriven by the inner membrane potential but also trans-ports a range of divalent cations such as Ni2+, Co2+, andCu2+ [6]. Overexpression of Mrs2 increases influx whiledeletion of the gene abolishes uptake suggesting that it isthe major mitochondrial system. This data suggests thatMrs2 protein may mediate Mg2+ transport in mammalianmitochondria. Nadler et al first identified TRPM7, awidely expressed member of the transient receptor poten-tial melastatin (TRPM) ion channel family, that producesa Mg2+ current in a wide variety of cells [7]. TRPM7 is reg-ulated by intracellular Mg·ATP levels and is similarly per-meable to both major divalent cations, Ca2+ and Mg2+, butalso many of the trace elements, such as Zn2+, Mn2+, andCo2+ [8]. Using a positional cloning approach, Schling-mann et al [9] and Walder et al [10] found that hypomag-nesemia with secondary hypocalcemia (HSH) was causedby mutations in TRPM6, a new member of the TRPM fam-ily. HSH is an inherited disease affecting both intestinaland renal Mg2+ absorption [3]. The functional characteris-tics of the TRPM6 transporter have not been fully eluci-dated [11,12]. Other magnesium transporters have beenfunctionally described but they have not been character-ized at the molecular level [13-18]. It is disparaging that,despite the significance of cellular Mg2+, only three spe-cific magnesium transporters have been described inmammalian cells to date.Mammalian magnesium homeostasis is a balance of epi-thelial intestinal magnesium absorption and renal mag-nesium excretion. The kidney plays a major role in controlof vertebrate magnesium balance, in part, by active mag-nesium transport within the distal tubule of the nephron[2]. Using the Madin-Darby canine kidney (MDCK) cellline obtained from canine distal tubules and immortal-lated magnesium pathways that are controlled by a varietyof hormonal influences [19]. However these hormonesdo not provide selective control as they also affect calciumand in some cases sodium and potassium transport [19].Selective and sensitive control of cellular Mg2+ transport isregulated by intrinsic mechanisms so that culture inmedia containing low magnesium results in upregulationof Mg2+ uptake in these cells. This adaptive increase inMg2+ entry was shown to be dependent on de novo tran-scription since prior treatment of the epithelial cells withactinomycin D prevented the adaptation to low extracel-lular magnesium [20]. The data suggest that epithelialcells can somehow sense the environmental magnesiumand through transcription- and translation-dependentprocesses alter Mg2+ transport and maintain magnesiumbalance. These conclusions using isolated epithelial cellsare consonant with our views of magnesium conservationin the intact kidney [2].In an attempt to identify genes underlying cellularchanges resulting from adaptation to low extracellularmagnesium, we used oligonucleotide microarray analysisto screen for magnesium-regulated transcripts in epithe-lial cells. This approach revealed one transcript whose rel-ative level was dramatically altered by extracellularmagnesium. Thus, this transcript potentially represented aspecies of mRNA whose synthesis was regulated bychanges in cell magnesium. In this study, we describe theidentification and characterization of this novel transcriptreferred to as MagT1. Our data indicate MagT1 may medi-ate Mg2+ transport in a wide variety of cells and may playa role in control of cellular magnesium homeostasis.ResultsIdentification of MagT1With the knowledge that differential gene expression isinvolved with selective control of epithelial cell magne-sium conservation, our strategy was to use microarrayanalysis to identify candidates that were up-regulated withlow magnesium. Using Affymetrix GeneChipR technol-ogy, we showed that 116 DNA fragments were signifi-cantly increased (p < 0.0002) from the 24,000 mouseESTs represented on the chips. The RNA of one of thesewas significantly increased, greater than 2-fold, n = 3,determined by real-time RT-PCR. The full length humancDNA was identified from clone DKFZp564K142Q3obtained from RZPD Resource Center, Berlin, in pAMP1vector and bidirectionally sequenced at NAPS, Universityof British Columbia. Based on the cDNA sequence, elec-trophysiological properties and cation selectivity of theencoded protein, we designated it as MagT1 for Magne-sium Transport protein, subtype 1. MgT was not used toavoid confusion with the bacterial MgtA/B and MgtE mag-Page 2 of 18(page number not for citation purposes)ized mouse distal convoluted tubule cells (MDCT), wehave shown that Mg2+ entry is through specific and regu-nesium transporters [21,22].BMC Genomics 2005, 6:48 http://www.biomedcentral.com/1471-2164/6/48Primary structure of MagT1MagT1 cDNA comprises 2241-base pairs (bp) with anopen reading frame of 1005 bp that predicts a protein of335 amino acids with a relative molecular mass of 38,036Da (Fig. 1). Hydropathy profile analysis suggested thatMagT1 is an integral membrane protein containing fivehydrophobic transmembrane-spanning (TM) α helicalN-linked glycosylation site at residue 215 located in thefirst extracellular loop. The N-terminal region of MagT1contains four putative cAMP-dependent protein kinasephosphorylation sites at residues S73, S108, T153 andS162 and four possible protein kinase C phosphorylationsites at residues S38, T48, S103, T111. The short C-termi-nal cytoplasmic region does not possess any cAMP-Primary amino acid sequence of human hMagT1.Figure 1Primary amino acid sequence of human hMagT1.  Human MagT1 was aligned with human candidate tumor suppressor sequence, N33, and the human implantation associated protein, designated IAP. The six predicted transmembrane domains are overlined and numbered. The amino acid numbers corresponding to the MagT1 protein are shown on the left side.  Page 3 of 18(page number not for citation purposes)regions, the first of which is likely cleaved to form the finalproduct with four TM domains (Fig. 1). MagT1 contains adependent or protein kinase C phosphorylation sites. Thepresence of putative phosphorylation sites for proteinBMC Genomics 2005, 6:48 http://www.biomedcentral.com/1471-2164/6/48kinase A and protein kinase C in the cytoplasmic domainsuggests that transport might be regulated byphosphorylation.MagT1 is a novel gene located at Xq13.1–13.2The human origin, chromosomal location, and intron-exon organization of the MagT1 gene were deduced fromthe expressed sequence tag (EST) database and the humangenome data. There may be an alternative splicing ofMagT1 but only one transcript could be seen on theNorthern blot (Fig. 2). Mouse mMagT1 gene is comprisedof 10 exons spanning 41,680 bp located on the X chromo-some (unplaced). The human hMagT1 gene is composedof 11 exons spanning 69,137 bp and is also on the X chro-mosome (Xq13.1–13.2).A BLAST search yielded a number of poorly characterizedproteins with similar amino acid sequences to MagT1(Fig. 1). Using the BESTFIT sequence alignment program,MagT1 shows 100% identity to a human unnamed pro-tein (GenBank™ CAB66571.1, BAC11592.1), 88% to amouse 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 131amino acids) to a human implantation associated protein(GenBank™ XP_497668) and to an unknown proteinMGC:56218 from the zebra fish (AAH46002.1). MagT1shares some similarity (65–67%) to the human (Gen-Bank™ 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 suppressorprotein. There is also some similarity (23–54%) to anumber of un-characterized proteins in Anopheles (Gen-Bank™ EAA13927.1), Drosophila melanogaster (Gen-Bank™ AAL68198.1, AAF52636.2, NP_609204.2),Ochlerotatus trisertiatus (GenBank™ AF275675.1), andCaenorhabditis elegans (GenBank™ NP_498691.1,AAA28222.1, S44911, Y013_CAEEL). None of these pro-teins, with similar amino acid sequences to MagT1, aresufficiently characterized to suggest a common functionalpurpose. MagT1 has a more distant relationship (P = 3 ×10-12) to the OST3 gene of Saccharomyces cervisiae thatencodes a regulatory subunit of the endoplasmic reticu-lum oligosaccharyltransferase complex [23]. A gappedalignment of these sequences showed only 21% identicalresidues between the hMagT1 and OST3 sequencesextending throughout most of both proteins.Tissue distribution of MagT1 expressionNorthern analysis of cultured mouse distal convolutedtubule cells and tissues harvested from mice revealed aof MagT1 mRNA and smaller amounts were found inintestine, spleen, brain, and lung (Fig. 2). Accordingly,MagT1 mRNA appears to be widely expressed among tis-sues but the transcript is variably expressed among thesetissues.The MagT1 antibody recognized two protein bands, 35and 38 kDa, in tissues expressing the MagT1 transcript(Fig. 3). Two bands were apparent in kidney and liver tis-sue whereas one was evident in heart, colon, and brain.The molecular size of MagT1 calculated from cDNA is 38kDa. A significant difference in the calculated molecularsize and that the smaller band found by immunoblotanalysis raises the possibility that MagT1 may be cleavedto yield the 35 kDa carboxyl-terminal protein detected byMagT1 antibody. There was very little MagT1 protein inthe small intestine (Fig. 3). Other than liver tissue, thereappears to be a good correlation between the respectiveamounts of transcripts and the protein content. The dis-crepancy between the levels of MagT1 mRNA and proteinexpression in liver (abundant mRNA detected but littleprotein detected) suggests that a posttranslational mecha-nism may play a role in tissue-specific expression of theMagT1 protein. In summary, the 38 kDa MagT1 protein isexpressed to a variable extent in all of the tissues sampled(Fig. 3) but the 35 kDa band appears to be present in onlysome of the tissues. Although this is a limited survey of tis-sues, the results suggest that MagT1 is expressed in manytissues with an apparent correlation of mRNA and proteinbut expression may be post-translationally modified in atissue-specific fashion such as the liver.The specificity of the affinity-purified polyclonal anti-MagT1 antibody was assessed by Western blots of the totalprotein extract from the MDCT cells probed with a preim-mune 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 theendogenous MagT1 protein.Human MagT1 elicits Mg2+-evoked currents in Xenopus oocytesThe functional properties of MagT1-evoked Mg2+ currentswere characterized using two-microelectrode voltageclamp analysis in Xenopus oocytes injected with hMagT1cRNA. The electrophysiological data gave evidence for arheogenic process with inward currents in hMagT1 cRNA-injected oocytes whereas there were no appreciable cur-rents in control H2O- or total poly(A)+RNA-injected cellsfrom the same batch of oocytes (Fig. 4). Human MagT1-mediated Mg2+-evoked uptake was linear for at least 20min and did not display any time-dependent decay duringPage 4 of 18(page number not for citation purposes)single strong transcript of about 2.4 kb (Fig. 2). The kid-ney, colon, heart and liver possessed relatively high levelsrepetitive stimulation with voltage steps (data notshown). The reversal potential was significantly shifted toBMC Genomics 2005, 6:48 http://www.biomedcentral.com/1471-2164/6/48Tissue distribution of mMagT1 mRNA. F g re 2Tissue distribution of mMagT1 mRNA. A, Northern blot analysis of mMagT1 mRNA in MDCT cells or mouse tissues. Tissues were harvested and poly(A)+ RNA prepared by standard techniques. Each lane was loaded with 8 µg of poly(A)+ RNA. The same blot was stripped and hybridized with 32P-labeled β-actin as a control for loading. B, real-time reverse transcription PCR analysis of mMagT1 RNA in tissues harvested from mice maintained on normal magnesium diet.  mMagT1 and murine β-actin RNA was measured with Real-Time RT PCR (AB7000TM, Applied Biosystems) using SYBR GreenTM fluorescence. Standard curves for MagT1 and  β-actin were generated by serial dilution of each plasmid DNA. The expression level of the mMagT1 transcript was normalized to that of the mouse  β-actin transcript measured in the same 1.0 µg RNA sample. Results are nor-malized to the small intestine and expressed as fold-difference. Mean mRNA levels of kidney, colon, heart, brain, lung, and liver Page 5 of 18(page number not for citation purposes)tissues were significantly greater, p>0.01, than small intestine ans spleen.   BMC Genomics 2005, 6:48 http://www.biomedcentral.com/1471-2164/6/48the right as would be expected of a magnesium transporter(Fig. 5). In consonant with the notion that MagT1 proteinmediates the observed Mg2+ currents is the association ofthe magnitude of the Mg2+-evoked current with the quan-tity of MagT1 protein in oocytes injected with MagT1cRNA (Fig. 6). In this study oocytes were selected accord-ing to the size of the Mg2+-evoked current and Westernblotting performed on the same oocyte. Both 38 and 35kDa molecular size bands were correlated with the meas-ured currents. Steady-state Mg2+-evoked currents were sat-urable (Fig. 7). The Michaelis constant (Km) was 0.23 mM,n = 29, when measured at -125 mV holding potential (Fig.7, insert). The Michaelis constant was independent of theVm used to determine the saturation kinetics. The Michae-lis constants (Km) were +25 mV, 0.22 mM; -50 mV, 0.19mM; -75, 0.20 mM; -100 mV, 0.19 mM; -125 mV, 0.23; -150 mV, 0.23 mM (data not shown).The Mg2+-evoked currents were not altered with deletionof external sodium by substitution with choline (89 ± 9%, n = 3, of control currents) or replacement of chloridewith nitrate (100 ± 1 %, n = 3, of control) suggesting thattransport does not depend on extracellular Na+ or Cl-(data not shown). Niflumic acid (0.5 mM), a Cl- channelantagonist, did not affect Mg2+ currents (data not shown).Next, we determined the effect of transmembrane H+ gra-dients on Mg2+-evoked currents in MagT1-injected oocytes(Fig. 8). Currents are maximal at physiological pH, 7.4,and diminished with acidic and alkaline pH values (Fig.8). Moreover, amiloride (0.1 mM), a Na+/K+ exchangeinhibitor, did not influence expressed Mg2+ currents inoocytes (data not shown). This data suggests that Mg2+-evoked currents are not coupled to H+ movement but aresensitive to external pH. On balance, these data indicatethat Mg2+-evoked currents in MagT1-injected oocytes areTissue distribution of mMagT1 protein.F g re 3Tissue distribution of mMagT1 protein. A. Western blots of membrane proteins from tissue extracts. Extracts were pre-pared from tissues as described under “Experimental Proce-dures”. MagT1 bands were probed with anti-MagT1antibody. Molecular sizes are expressed in kDa of pre-stained stand-ards are shown on the left of each of the representative blots. B, summary of 38 kDa MagT1 protein in 15 µg total protein from various mice tissues. Data were obtained from 3 different mice and are indicated as the mean ± SEM. C, spe-cificity of anti-MagT1 antibody. The fractions isolated from normal and magnesium-depleted MDCT cells were blotted with MagT1 antibody and MagT1 antibody preadsorbed with excess antigen peptide. The signal was reduced to back-ground levels when preadsorbed antibody was used indicat-ing that the antibody was specific to MagT1. Page 6 of 18(page number not for citation purposes)not coupled to Na+, Cl-, or H+ but are influenced by exter-nal pH values.Figure 3BMC Genomics 2005, 6:48 http://www.biomedcentral.com/1471-2164/6/48Large 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 appreciablecurrents in the absence of Mg2+ in hMagt1-expressingoocytes (Fig. 9). In the experiments shown, the permeabil-ity ratios (Erev for tested cation relative to Erev for Mg2+)were corrected for changes in membrane resistance causedby the respective divalent cation using values from H2O-injected oocytes (Fig. 9).Some divalent cations inhibited Mg2+-evoked currents atrelatively large concentrations of the respective inhibitor,0.2 mM, in the presence of 2.0 mM MgCl2 (Fig. 10). Thecations Ni2+and Zn2+ markedly shifted the ∆Erev to the leftwhereas Mn2+was less effective and Gd3+, Cd2+, Co2+ andCu2+ were modest inhibitors (Fig. 10). The multivalentcation, Gd3+, is a nonselective channel blocker thatinhibits most Ca2+-permeable channels and known TRPchannels [24]. The presence of 0.2 mM (Fig. 10) or 10.0mM Ca2+, 98 ± 8 % (data not shown), was without effect2+ 2+ ance, these data indicate that hMagT1 cRNA-inducedtransport in oocytes is highly selective for Mg2+. Otherdivalent cations may be blockers but our evidence is thatthey are at most very weak agonists.We have shown that relatively high concentrations of 1,4-dihydropyridine analogues, organic blockers of L-typeCa2+ channels, inhibit Mg2+ entry into distal tubule epithe-Mg2+-evoked currents in Xenopus oocytes expressing hMagT1 RNA transcripts.Fi ur  4Mg2+-evoked currents in Xenopus oocytes expressing hMagT1 RNA transcripts. Current was continuously moni-tored in a single oocyte expressing hMagT1 clamped at -100 mV and superfused for the period indicated, first with modi-fied Barth’s solution containing 0 mM magnesium then with 2.0 mM magnesium and finally returning to magnesium-free solution. Mg2+-evoked currents in Xenopus oocytes expressing hMagT1.Fi ur  5Mg2+-evoked currents in Xenopus oocytes expressing hMagT1. Current-voltage relationships obtained from linear voltage steps from -150 mV to +25 mV in the presence of Mg2+-free solutions or those containing the indicated con-centrations of MgCl2. Oocytes were clamped at a holding potential of -15 mV and stepped from -150 mV to +25 mV in 25 mV increments for 2 s at each of the concentrations indi-cated. Shown are average I-V curves obtained from control H2O-injected (n = 13) or MagT1-expressing (n =/>7) oocytes. Note, the positive shift in reversal potential, indi-cated by arrows, with increments in magnesium concentra-tion. Values are mean ± SEM of observations measured at the end of each voltage sweep for the respective Mg2+ concen-tration. Page 7 of 18(page number not for citation purposes)on the amplitude of Mg -evoked currents. Fe had noinfluence on MagT1-mediated currents (Fig. 10). On bal-lial cells [19,20]. In the present experiments, nifedipine(10 µM) did not inhibit Mg2+-evoked currents (0.61 ±BMC Genomics 2005, 6:48 http://www.biomedcentral.com/1471-2164/6/480.08 µA at -125 mV, n = 5) but its analogue nitrendipine(10 µM) was an effective inhibitor (0.15 ± 0.02 µA, n = 7)in MagT1 expressed oocytes (Fig. 11). Control Mg2+-induced currents were 0.59 ± 0.09 µA, n = 6, in this seriesof experiments (Fig. 11). These findings were similar toour experience with MDCK and MDCT epithelial cells[19,20]. Again in consonant with our previous studies, thechannel agonist, BAY K8644 (10 µM) stimulated Mg2+-evoked currents in expressing oocytes (0.80 ± 0.18 µA, n =5) supporting the above electrophysiological data thatMagT1 is a channel-like protein (Fig. 11).MagT1 expression is responsive to magnesiumThe rationale for these studies is based on the observationthat renal magnesium conservation is principallyregulated by differential expression of genes encodingmagnesium transport proteins. Accordingly, we deter-mined the response of MagT1 to changes in magnesium atthe messenger and protein levels. These studies were per-formed with distal tubule epithelial cells, MDCT, culturedin media containing normal (1.0 mM) or low (nominallymagnesium-free) magnesium concentrations for 16 h andon kidney cortex tissue harvested from mice maintainedin cells cultured in low magnesium media and in tissue ofmice maintained on low magnesium diets (urine andplasma magnesium concentration, 1.1 ± 0.3 and 0.13 ±0.01 mM, respectively) compared to normal cells and tis-sue of animals on normal diets (urine and plasma magne-sium, 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 increasesin mRNA, MagT1 protein was increased by 31 ± 12% inthe cultured epithelial cells and 33 ± 6 % in kidney cortexwith low magnesium relative to the respective controls(Figure 13). Accordingly, it is apparent that an increase inmRNA levels is translated into higher protein expressionand by inference leads to greater magnesium transport(the latter conclusion is based on the urinary magnesiumexcretion of animals maintained on low magnesium rela-tive to normal diets).DiscussionDespite the extensive evidence for unique mammalianMg2+ transporters, few proteins have been biochemicallyidentified to date that fulfill this role. Moreover,functional characterization has not been fully investigatedfor those that have been reported [11,12]. With theknowledge that the kidney, particularly the distal tubule,regulates magnesium conservation through transcrip-tional mechanisms, we used oligonucleotide microarrayanalysis to identify MagT1, a novel Mg2+ transporter[2,19]. The MagT1 transcript is a 2.4-kb mRNA thatencodes a protein comprising a relatively long N-terminalsegment, a putative region of four TM domains, and ashort C-terminal sequence. The cytoplasmic segmentspossess a number of characteristic phosphorylationmotifs. MagT1 shows no structural similarity to anyknown transporter. Functional expression of MagT1 inoocytes results in large Mg2+-evoked currents with littlepermeability to other divalent cations. However, somedivalent cations, Ni2+, Zn2+, and Mn2+inhibit Mg2+-evokedcurrents at relatively large external concentrations. Thesecations are not found in the extracellular or intracellularfluid at the concentrations used here, 0.2 mM. The othermajor extracellular divalent cation, Ca2+, was neithertransported nor were Mg2+-evoked currents inhibited byextracellular Ca2+. MagT1 is widely distributed among tis-sues particularly those of epithelial structure. Finally,MagT1 expression is regulated in these tissues by externalmagnesium as predicted by our starting premise. Accord-ingly, MagT1 fulfills the role of a dedicated mammalianmagnesium transporter. The function of MagT1 in cellularMg2+ balance remains to be determined.The electrophysiological characteristics of MagT1Association of Mg2+ currents with the expression of 38 kDa MagT1 protein in Xenopus oocytes inj cted w th MagT1 cRNA.Figure 6Association of Mg2+ currents with the expression of 38 kDa MagT1 protein in Xenopus oocytes injected with MagT1 cRNA. Oocytes were selected from one frog according to the expressed Mg2+ currents as shown. Results illustrated is representative of four oocyte preparations from different animals. The relative amplitude of Mg2+ currents was associ-ated with the amount of MagT1 protein determined by Western blot analysis.  Page 8 of 18(page number not for citation purposes)on either normal or magnesium-restricted diets for 5 days.The mRNA and protein expression was relatively strongerexpressed in Xenopus oocytes are reminiscent of our obser-vations of Mg2+ transport in intact renal epithelial cellsBMC Genomics 2005, 6:48 http://www.biomedcentral.com/1471-2164/6/48measured by microfluorescence [19]. There is not a suita-ble isotope of Mg2+ for use in physiological experimentsso that we have used fura-mag-2 fluorescence to investi-gate Mg2+ transport [25]. We have shown that Mg2+ uptakein a variety of epithelial cells is driven by the electrochem-ical gradient of Mg2+. Membrane hyperpolarization stim-ulates Mg2+ transport whereas depolarization abrogatesuptake (19). There was no evidence in renal distal tubulecells for coupling of apical Mg2+ entry to other ions suchas Na+, Cl-, or H+ [19]. Magnesium transport in immortal-and uptake is saturable, as determined by fluorescence.The apparent affinity constant is in the order of 0.5 mMthat is similar to that observed for MagT1 expressed inXenopus oocytes (Fig. 5). This affinity is appropriate for aphysiological role of the transporter in cellular Mg2+ con-servation [19]. Mg2+-evoked currents in oocytes express-ing MagT1 is highly specific for Mg2+, an observation thatis again consonant with our views of Mg2+ transport inMDCT cells and in vivo kidney [19]. The microfluores-cence experiments suggest that there may be some varia-2+ Summary of concentration-dependent Mg2+-evoked currents in MagT1-expressing oocytes using a holding potential of -125 mV.Figure 7Summary of concentration-dependent Mg2+-evoked currents in MagT1-expressing oocytes using a holding potential of -125 mV. Mean ± SEM values are those given in Fig. 1A. Inset illustrates an Eadie-Hofstee plot of concentration-dependent Mg2+-evoked currents demonstrating a Michaelis constant of 0.23 mM.  Page 9 of 18(page number not for citation purposes)ized mouse distal convoluted tubule (MDCT) cells isdependent on the transmembrane concentration gradientbility in cationic inhibition of Mg uptake depending onthe cell-type used so that other transporters may beBMC Genomics 2005, 6:48 http://www.biomedcentral.com/1471-2164/6/48Characterization of Mg2+-evoked currents in Xenopus oocytes expressing hMagT1.Figure 8Characterization of Mg2+-evoked currents in Xenopus oocytes expressing hMagT1. A, effect of pH on Mg2+-evoked currents. Currents were measured in standard solutions containing 2.0 mM MgCl2 at the pH values indicated. B, summary of mean cur-rents with external pH at a holding potential of -125 mV. Mg2+ did not evoke currents in H2O-injected oocytes at any of the Page 10 of 18(page number not for citation purposes)pH values tested.BMC Genomics 2005, 6:48 http://www.biomedcentral.com/1471-2164/6/48Substrate specificity of MagT1 following application of test cations, 2.0 mM, in the absence of external Mg2+.Figure 9Substrate specificity of MagT1 following application of test cations, 2.0 mM, in the absence of external Mg2+. For clarity, only Mg2+,Cu2+, Mn2+,  and Sr2+ are represented in panel A. Oocytes were clamped at a holding potential of -15 mV and stepped from -150 mV to +25 mV in 25 mV increments for 2 s for each of the cations. Values are mean ± SEM of currents measured at the end of each voltage sweep for the respective divalent cation. B, summary of permeabilities of the tested divalent cations. Page 11 of 18(page number not for citation purposes)Figure illustrates average permeability ratios (Erev for tested cation relative to  Erev for Mg2+) given in Fig. 9A. BMC Genomics 2005, 6:48 http://www.biomedcentral.com/1471-2164/6/48Inhibition of MagT1-mediated currents.Figure 10Inhibition of MagT1-mediated currents. A,inhibition of Mg2+-evoked currents with 0.2 mM test cation in the presence of exter-nal 2.0 mM Mg2+.  For clarity, only Cu2+, Mn3+, and Zn2+ relative to Mg2+ are represented. Values are mean ± SEM of currents measured at the end of each voltage sweep for the respective cation. B, summary of inhibition by multivalent cations of Mg2+ currents based on the change in Erev represented in Fig. 10A. The inhibitor was added with MgCl2 and voltage-clamp was per-Page 12 of 18(page number not for citation purposes)formed about 5 min later.  BMC Genomics 2005, 6:48 http://www.biomedcentral.com/1471-2164/6/48Effect of voltage-dependent channel antagonists on MagT1-mediated currents.Figure 11Effect of voltage-dependent channel antagonists on MagT1-mediated currents. A, the antagonists nifedipine (10 µM) and nitren-dipine (10 µM), or the agonist, Bay K8644 (10 µM), were added prior to determining Mg2+-evoked currents. B, summary of mean currents (I µA) with the respective inhibitors at a holding potential (Vm) of -125 mV (n=7). The analogues were added 5 Page 13 of 18(page number not for citation purposes)min prior to voltage-clamping.  BMC Genomics 2005, 6:48 http://www.biomedcentral.com/1471-2164/6/48present with differing selectivity that are tissue specific[26]. Relatively large concentrations of nitrendipine, a1,4-dihydropyridine channel blocker, inhibited Mg2+-evoked currents in MagT1-expressing oocytes not unlikethe inhibition of Mg2+ entry into distal epithelial cells[19]. Intriguingly, nifedipine did not influence Mg2+-induced currents in MagT1-expressing oocytes that is sim-ilar to our previous reports using MDCT cells [19].Although both antagonists are dihydropyridines, theyhave differing efficacy based on their structural differences[27]. Again, reminiscent of our observations using MDCKand MDCT epithelial cells, the channel agonist, BAY8644, increased Mg2+-evoked currents [19]. The 1,4-dihy-dropyridines analogues are not highly selective channelblockers/activators but these findings support the notionthat Mg2+ entry into MagT1-expressed oocytes or distalepithelial cells is via channel-like proteins. Two othercharacteristics are noteworthy. First, Mg2+-evoked currentsin MagT1-expressed oocytes are greater at physiologicalpH values relative to acidic pH. This is also true for Mg2+uptake in distal tubule epithelial cells and magnesiumconservation by the intact kidney in vivo [19]. Magnesiumreabsorption is greater and urinary excretion is less in met-abolic alkalosis than acidosis. Indeed, magnesium wast-ing may be sufficient in chronic metabolic acidosis to leadto hypomagnesemia [2]. Second, the presence of multipleputative protein kinase A and C phosphorylation sites inMagT1 may suggest phosphorylation-dependent regula-tion. We have shown that Mg2+ entry into epithelial cellsis stimulated by peptide hormones, such as parathyroidhormone, glucagon and calcitonin, that act through pro-tein kinases A and C [19]. Further studies are needed toelucidate the mechanisms underlying these phenomena.On balance, many of the functional characteristics ofMagT1 expressed in oocytes are harmonious with our ear-lier physiological observations using kidney distal convo-luted tubule cells.MagT1 is a membrane protein that may comprise ER, earlyand late endosomes or apical and basolateral plasmamembrane fractions. The role of each of these structures incellular magnesium homeostasis is poorly understood.Using single cell spatial imaging, we have previouslyshown that intracellular ionized Mg2+ concentration isheterogenously distributed across the cell [28]. The ER orsarcoplasmic reticulum normally contains high concen-trations of Mg2+, ranging from 0.4–2.0 mM, relative to thecytosolic concentration, 0.5 mM, and nucleus, 0.32 mM.It is clear that Mg2+ is transported into and out of a varietyof intracellular compartments and there is likelydedicated magnesium transporters for each event. Furtherstudies are required to establish the subcellularlocalization and intracellular trafficking of Mg2+ and theOur evidence is that the expression of MagT1 mRNA andprotein is responsive to cellular magnesium. The ability ofepithelial cells to selectively respond to the availability ofessential nutrients, such as Zn2+ and Fe2+, is not uniquebut the cellular mechanisms are unknown [29,30]. Pre-sumably epithelial cells may sensitively sense intracellularnutrient concentration and through transcriptional andpost-translational mechanisms adjust transport ratesappropriately [19,29,30]. Our studies indicate that thisresponse within the cell is the basis for sensitive and selec-tive control of magnesium balance in the kidney [19].Epithelial cells comprising the intestine and kidney areprimarily involved with dietary magnesium absorption,urinary magnesium excretion, and total body magnesiumhomeostasis [2]. Accordingly, MagT1 may, in part, beresponsible for intestinal and renal tubular Mg2+ conser-vation. In support of this is the observation that theMagT1 transcript is present in these tissues (Fig. 2). How-ever, magnesium is necessary in all cells and the wide-spread distribution of the MagT1 transcript may suggest ahousekeeping role for this transporter. It is also germaneto note that MagT1 mRNA is regulated in all cells investi-MagT1 mRNA expression is responsive to magnesium.Figure 12MagT1 mRNA expression is responsive to magnesium. Where indicated MDCT cells were cultured in normal (1.0 mM) or low (<0.01 mM) magnesium media for 16 h. Kidney cortical tissue was harvested from mice on normal (0.05% by weight) or low magnesium (<0.01%) diets for 5 days. MagT1 and murine β-actin RNA was measured with Real-Time RT PCR (AB7000TM, Applied Biosystems) using SYBR GreenTM fluorescence. Data is from 10-12 PCRs performed on five separate cultures or animals in each group maintained on low and normal magnesium.Page 14 of 18(page number not for citation purposes)role of MagT1 protein. gated. Further studies are needed to define the function ofBMC Genomics 2005, 6:48 http://www.biomedcentral.com/1471-2164/6/48MagT1 in intestine and kidney and the role in overall cel-lular magnesium balance.ConclusionWe have identified a novel magnesium transporter, prob-ably a channel, that is regulated by extracellular magne-sium. To our knowledge this is the first report of a highlyselective Mg2+ transporter. Its role in cellular magnesiumhomeostasis and transepithelial magnesium absorption isunknown but our evidence from our differential geneexpression studies indicate that it plays an important incellular magnesium homeostasis.MethodsCell culture and oligonucleotide microarray analysisMouse distal convoluted tubule (MDCT) cells were iso-lated from kidneys and immortalized by Pizzonia et al(31). The MDCT cell line has been extensively used by usto study renal magnesium transport [21]. Cells weregrown in Basal Dulbecco's minimal essential medium(DMEM)/Ham's F-12, 1:1, media (GIBCO) supplementedwith 10% fetal calf serum (Flow Laboratories, McLean,VA), 1 mM glucose, 5 mM L-glutamine, 50 U/ml penicil-lin, and 50 µg/ml streptomycin in a humidified environ-ment of 5% CO2- 95% air at 37°C. Where indicated,subconfluent MDCT cells were cultured in Mg2+-freemedia (Stem Cell Technologies Inc., Vancouver, BC) for 4h. Other constituents of the Mg2+-free culture media weresimilar to the complete media.Microarray analysis was performed according to the pro-tocol recommended by Affymetrix http://www.affymetric.com. Poly(A)+ RNA was extracted with Poly(A)Pure(Ambion) from cells cultured in high and low magnesiummedia. Twenty Fg RNA was used for cDNA synthesis fol-lowed by in vitro transcription. The cRNA was biotin-labeled, fragmented, and the probes hybridized toAffymetrix MG U74 Bv2 and MG U74 Cv2 arrays (Affyme-trix, Santa Clara) representing approximately 24,000mouse transcripts. Detailed protocols for data analysis,documentation of sensitivity, reproducibility and otheraspects of the quantitative microarray analysis are thosegiven by Affymetrix. Gene categorization was based on theNetAffx Database.Northern blot analysisCells were harvested by scraping and total RNA isolatedusing TRIzol (Life Technologies, Inc.). In some experi-ments poly(A)+ RNA was isolated using the Poly(A)PuremRNA isolation system (Ambion) following the manu-facturer's instructions. Samples of total RNA (20 µg) orpoly(A)+ RNA (8 µg) were denatured in 2.2 Mformaldehyde, 50% (v/v) formamide buffer and electro-size-fractionated RNA was transferred to GeneScreennylon membranes (NEN) by downward alkali transferand UV crosslinked (Stratagene Stratalinker 1800). Mem-branes were probed with 32P-labelled probes made fromgene specific inserts represented in the microarray analyt-ical results. The probe templates were prepared from PCRproducts representing inserts using specific primers oncDNA prepared from MDCT cells. The inserts were ligatedinto pGEM-t vector (Promega) following QiaexII gel (Qia-gen) purification. Blots were prehybridized in 50% forma-mide, 5 X SSPE, 100 µg/ml denatured sonicated salmonsperm DNA, 5 X Denhardt's solution, 0.1% SDS for 1 h at42EC in a rotating hybridization oven (Tyler HI-16000).Probe was heated to 95EC for 5 min, then added to theprehybridization solution. Membranes were hybridizedfor 16 h at 42EC then washed at high stringency sequen-tially: 2X [1X SSPE, 0.2% SDS, 28EC] 2X [1X SSPE, 0.4%SDS, 37EC] 1X [0.1X SSPE, 0.2% SDS, 55EC]. Membraneswere exposed on Kodak X-AR-2 film. In most cases, afterimages were obtained, membranes were incubated at95°C for 1 h in 0.1% SDS to remove the bound probe andhybridized with a 32P-labelled β-actin probe in order tonormalize loading.Quantitative analysis of MagT1 transcripts by real-time RT PCRTotal 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 alinear pGEM-T vector (Promega) containing the MagTgene. The primer set of mouse MagT1 was: forward, 5'-CCAAAGGGGCTGATACATA-3' and reverse, 5'-ATAGAA-GAACGATGTGTG-3' and the human MagT1: forward, 5'-GCAAACTCCTGGCGATACTCC-3' and the human reverse5'-ACTGGGCTTGACTGCTTCC-3'. PCR products werequantified continuously with AB7000™ (Applied Biosys-tems) using SYBR Green™ fluorescence according to themanufacturer's instructions. The relative amounts ofMagT1 RNA were normalized to the respective humanand mouse β-actin transcripts.Genomic sequence analysisThe MagT1 cDNA sequence was determined by standardmethods. Data base searching and alignments were per-formed using BLAST. The nonredundant and EST databases were sourced. Protein homology searches were per-formed by comparing the amino acid query sequenceagainst SWISSPROT data base. The full-length MagT1cDNA sequence has been deposited in the GenBank™ database (accession human DQ000004, mouse DQ000005).Western blot analysisPage 15 of 18(page number not for citation purposes)phoresed on 0.8% agarose 3 M formaldehyde, 0.02 MMOPS, 8 mM Na acetate, 1 mM EDTA, pH 7.0 gels. TheA rabbit polyclonal antibody, anti-MagT1, was raisedagainst the N-terminal domain of the final cleaved humanBMC Genomics 2005, 6:48 http://www.biomedcentral.com/1471-2164/6/48MagT1 protein expression is responsive to magnesium.Figure 13MagT1 protein expression is responsive to magnesium. Western blots of membrane proteins from cells and tissues as described under “Experimental Procedures”. MagT1 bands were probed with anti-MagT1antibody. Data are from four West-Page 16 of 18(page number not for citation purposes)ern blots performed on five separate cultures or animals in each group maintained on low and normal magnesium.  BMC Genomics 2005, 6:48 http://www.biomedcentral.com/1471-2164/6/48MagT1 protein using a synthetic peptide, INFPAKGKP-KRGDTYELQV (amino acid residues 140–158), coupledto keyhole limpet hemocyanin. Affinity-purified rabbitanti-human MagT1 antibody was diluted in TBS (Tris-buffered saline, 20 mM Tris, 200 mM NaCl, pH 7.6) con-taining 0.5 % BSA at a final concentration about 0.7 µl/ml. For subcellular fractionation, cells were suspended inlysis buffer (0.25 M sucrose, 10 mM triethanolamine-ace-tic acid pH 7.6, 1 mM EDTA) containing protease inhibi-tors (1 mM PMSF, 2 µg/ml leupeptin, 2 µg/ml aprotinin).Protein concentrations were determined using Bio-Radprotein assay reagent. SDS-PAGE was performed accord-ing to Laemmli. For immunobblotting, the proteins wereelectrophoretically transferred to polyvinylidene difluo-ride membranes (HybondR, Amersham Biosciences) bysemidry electroblotting for 45 min. Western analysis wasperformed by incubating the blots with antiMagT1 anti-body or anti-MagT1 antibody preabsorbed with 50 × anti-gen peptide (control for antibody specificity) overnight at4EC 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. Afterwashing three times with TBS/Tween-20, 10 min each, theblots were visualized with ECL (Amersham Biosciences)according to the manufacturer's instructions.Expression of MagT1 in Xenopus oocytes and current measurementsThe cDNA comprising the open reading frame (ORF) ofMagT1 was amplified from the pAMP1 vector using thecloning primers (sense: 5'-GATTGGTACCGTGAACAT-GGCCTC-3'; antisense: 5'-CTTGTCGACCCTCTTTAACT-CATC-3') and was subcloned into the KpnI and ApaIrestriction sites of the pEYFP-N1 expression vector. Theconstructs were linearized and then transcribed with SP6polymerase in the presence of m7GpppG cap using themMESSAGE MACHINE™ SP6 KIT (Ambion) transcriptionsystem. Oocytes were injected with MagT1 complemen-tary RNA (cRNA) or for control observations, H2O or kid-ney total poly(A)+ RNA; no Mg2+-induced currents weredetected in the latter.Xenopus oocytes were prepared and injected with cRNAand electrophysiological recordings were preformedaccording to previously described techniques [32]. Briefly,defolliculated stage V-VI oocytes were typically injectedwith 25 ng cRNA in 50 nl H2O. Oocytes were incubated at18°C for 3–6 days in multiwell tissue culture plates con-taining Barth's solution (88 mM NaCl, 1.0 mM KCl, 2.4mM NaHCO3, 1.0 mM MgSO4, 1.0 mM CaCl2, 0.3 mMCa(NO3)2, 10 mM Hepes-NaOH, pH 7.6, 2.5 mM Na-pyruvate, 0.1 % BSA, 10,000 U/l penicillin, 100 mg/land perfused with modified Barth's (96 mM NaCl, 10 mMHepes-NaOH) containing various concentrations ofMgCl2, as indicated, in substitution for osmotically equiv-alent amounts of NaCl. All experiments were performedat room temperature (21°C).Steady-state membrane currents were recorded with thetwo-microelectrode voltage-clamp technique using aGeneclamp 500 amplifier (Axon Instruments, Inc.). Elec-trophysiology consisted of a voltage clamp step profileconsisting of a holding potential of -15 mV, followed by 8episode series of +25 mV steps of 2 s duration, from -150mV to +25 mV within an episode duration of 6.14 sec.Each episode recorded 1536 data points collected at 4 msintervals. The data was filtered at the appropriate fre-quency before digitization. In order to assess the permea-bility of different divalent cations, we used the shift in thereversal potentials of the respective cation from thereversal potentials of Mg2+ currents, ∆Erev, and calculatedby the permeability ratio by:Px/PMg = exp/(∆Erev X F/RT)where R, T, and F have their standard meanings. Voltageclamp episodes in the presence of extracellular test cationswere corrected against episodes in the absence of externaltest cations.All experimental conditions were performed on oocytesharvested from a minimum of 3 different animals.Authors' contributionsAuthors contributed equally in all parts of this study. Allauthors read and approved the final manuscript.AcknowledgementsThis 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.References1. Flatman PW: Magnesium transport across cell membranes. JMembr Biol 1984, 80:1-14.2. Quamme GA: Renal magnesium handling: New insights inunderstanding old problems. Kidney Int 1997, 52:1180-1195.3. Cole DEC, Quamme GA: Inherited disorders of renal magne-sium handling. J Am Soc Nephrol 2000, 11:1937-1947.4. Bui DM, Gregan J, Jarosch E, Ragnini A, Schweyen RJ: The bacterialmagnesium transporter CorA can functionally substitute forits putative homologue Mrs2p in the yeast inner mitochon-drial membrane. J Biol Chem 1999, 274:20438-20443.5. Zsurka G, Gregan J, Schweyen RJ: The human mitochondrialMrs2 protein functionally substitutes for its yeast homo-logue, a candidate magnesium transporter. Genomics 2001,72:158-168.Page 17 of 18(page number not for citation purposes)streptomycin). To record expressed membrane currents,the oocytes were placed in a recording chamber (0.3 ml)6. Kolisek M, Zsurka G, Samaj J, Weghuber J, Schweyen RJ, Schweigel M:Mrs2p is an essential component of the major electro-Publish with BioMed Central   and  every scientist can read your work free of charge"BioMed Central will be the most significant development for disseminating the results of biomedical research in our lifetime."Sir Paul Nurse, Cancer Research UKYour research papers will be:available free of charge to the entire biomedical communitypeer reviewed and published immediately upon acceptancecited in PubMed and archived on PubMed Central BMC Genomics 2005, 6:48 http://www.biomedcentral.com/1471-2164/6/48phoretic Mg2+ influx system in mitochondria. EMBO J 2003,22:1235-1244.7. Nadler MJS, Hermosura MC, Inabe K, Perraud A-L, Zhu Q, Stokes AJ,Kurosaki T, Kinet J-P, Penner R, Scharenberg AM, Fleig A:Hypomagnesemia with secondary hypocalcemia is caused bymutations in TRPM6, a new member of the TRPM genefamily. Nature 2001, 411:590-595.8. Monteilh-Zoller MK, Hermosura MC, Nadler MJS, Scharenberg AM,Penner R, Fleig A: TRPM7 provides an ion channel mechanismfor cellular entry of trace metal ions. J Gen Physiol 2003,121:49-60.9. Schlingmann KP, Weber S, Peters M, Nejsums LN, Vitzthum H, Klin-gel K, Kratz M, Haddad E, Ristoff E, Dinour D, Syrrou M, Nielsen S,Sassen M, Waldegger S, Seyberth HW, Konrad M: Hypomag-nesemia with secondary hypocalcemia is caused by muta-tions in TRPM6, a new member of the TRPM gene family. NatGenet 2002, 31:166-171.10. Walder YW, Landau D, Meyer P, Shalev H, Tsolia M, Borochowitz Z,Boettger MB, Beck GE, Englehardt RK, Carmi R, Sheffield VC: Muta-tion of TRPM6 causes familial hypomagnesemia with sec-ondary hypocalcemia. Nat Genet 2002, 31:171-174.11. Voets T, Nilius B, Hoefs S, van der Kemp AW, Droogmans G, BindelsRJ, Hoenderop JG: TRPM6 forms the Mg2+ influx channelinvolved in intestinal and renal Mg2+ absorption. J Biol Chem2004, 279:19-25.12. Chubanov V, Waldegger S, Mederos y, Schnitzler M, Vitzthum H, Sas-sen MC, Seyberth HW, Konrad M, Gudermann T: Disruption ofTRPM6/TRPM7 complex formation by a mutation in theTRPM6 gene causes hypomagnesemia with secondaryhypocalcemia. Proc Nat Acad Sci U S A 2004, 101:2894-2899.13. Cefaratti C, Romani A, Scarpa A: Differential localization andoperation of distinct Mg(2+) transporters in apical and baso-lateral sides of rat liver plasma membrane. J Biol Chem 2000,275:3772-3780.14. Günther T: Mechanisms and regulation of Mg2+ efflux andMg2+ influx. Miner Electrolyte Metab 1993, 19:259-265.15. Schweigel M, Vormann J, Martens H: Mechanisms of Mg2+ trans-port in cultured ruminal epithelial cells. Am J Physiol 2000,278:G400-G408.16. Rasgado-Flores H, Gonzales-Serratos H: Plasmalemmal trans-port of magnesium in excitable cells. Front Biosci 2000,5:D866-D879.17. Touyz RM, Mercure C, Reudelhuber TL: Angiotensin II type Ireceptor modulates intracellular free Mg2+ in renallyderived cells via Na+-dependent Ca2+-independentmechanisms. J Biol Chem 2001, 276:13657-13663.18. Tashiro M, Konishi M, Iwamoto T, Shigekawa M, Kurihara S: Trans-port of magnesium by two isoforms of the Na+-Ca2+exchanger expressed in CCL39 fibroblasts. Pflügers Archiv Eur JPhysiol 2000, 440:819-827.19. Dai L-j, Ritchie G, Kerstan D, Kang HS, Cole DEC, Quamme GA:Magnesium transport in the renal distal convoluted tubule.Physiol Rev 2001, 81:51-84.20. Dai L-j, Quamme GA: Intracellular Mg2+ and magnesium deple-tion in isolated renal thick ascending limb cells. J Clin Invest1991, 88:1255-1264.21. Smith RL, Thompson LJ, Maguire ME: Cloning and characteriza-tion of MgtE, a putative new class of Mg2+ transporter fromBacillus firmus OF4. J Bacteriol 1995, 177:1233-1238.22. Moncrief MB, Maguire ME: Magnesium transport inprokaryotes. J Biol Inorg Chem 1999, 4:523-527.23. Knauer R, Lehle L: The oligosaccharyltransferase complexfrom Saccharomyces cerevisiae. Isolation of the OST6 gene,its synthetic interaction with OST3, and analysis of thenative complex. J Biol Chem 1999, 274:17249-17256.24. Lee N, Chen J, Sun L, Wu S, Gray KR, Rich A, Huang M, Lin J-H, FederJN, Janovitz EB, Levesque PC, Blanar MA: Expression and charac-terization of human transient receptor potential melastatin3 (hTRPM3). J Biol Chem 2003, 278:20890-20897.25. Quamme GA, Dai L-j: Presence of a novel influx pathway forMg2+ in MDCK cells. Am J Physiol 1990, 258:C521-C525.26. Quamme GA, Rabkin SW: Cytosolic free magnesium in cardiacmyocytes: Identification of a Mg2+ influx pathway. Biochim Bio-phys Res Comm 1990, 167:1406-1412.27. Hockerman GH, Peterson BZ, Johnson BD, Catterall WA: Molecu-lar determinants of drug binding and action on L-type cal-cium channels. Ann Rev Pharmacol Toxicol 1997, 37:361-396.28. Quamme GA, Dai L-j, Rabkin SW: Dynamics of intracellular freeMg2+ changes in vascular smooth muscle cells. Am J Physiol1993, 265:H281-H288.29. Eide DJ: The SLC39 family of metal ion transporters. PflügersArchiv Eur J Physiol 2004, 447:796-800.30. Roy CN, Andrews NC: Recent advances in disorders of ironmetabolism: mutations, mechanisms and modifiers. Hum MolGen 2001, 10:2181-2186.31. Pizzonia JH, Gesek FA, Kennedy SM, Coutermarsh BA, Bacskai BJ,Friedman PA: Immunomagnetic separation, primary cultureand characterization of cortical thick ascending limb plusdistal convoluted tubule cells from mouse kidney. In Vitro CellDev Biol 1991, 27A:409-416.32. Quamme GA: Chlorpromazine activates a chloride current inXenopus oocytes. Biochem Biophys Acta 1997, 1324:18-26.yours — you keep the copyrightSubmit your manuscript here:http://www.biomedcentral.com/info/publishing_adv.aspBioMedcentralPage 18 of 18(page number not for citation purposes)

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