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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
1Department of Medicine and 2Department of Psychiatry, University of British Columbia, Vancouver, British Columbia, Canada
Submitted 15 February 2008 ; accepted in final form 28 July 2008
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ABSTRACT |
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 TOP ABSTRACT EXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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nonimprinted in Prader-Willi/Angelman syndrome
Renal magnesium handling is regulated, in part, by a large number of hormones and, in part, by differential gene expression leading to changes in expression of proteins mediating magnesium transport (8, 21). We have used the latter observation to design a strategy to identify novel gene products that are differentially expressed in response to magnesium and intimately involved in epithelial Mg2+ transport. Microarray analysis was used to determine gene transcripts that are upregulated in DCT cells with low extracellular magnesium concentration (11). To date, we have identified four novel membrane Mg2+ transporters with this approach (11–15).
In the present study, we describe another family of Mg2+ transporters that was identified by its response to magnesium. The initial candidate gene selected conformed to NIPA2 (nonimprinted in Prader-Willi/Angelman syndrome) subtype 2 [variants: NM-030922, NM-001008860, NM-001008892, and NM-001008894] (2). In addition to NIPA2, the NIPA family comprise the paralogs NIPA1, NIPA3, and NIPA4 that have an overall similarity of about 40%. NIPA2 is located among about 30 imprinted genes linked to chromosome 15q11-q13 (SPG6 locus) involved in the Prader-Willi syndrome (4). Chai and colleagues (4) identified a number of genes in this region, one of which was the novel gene NIPA2. As NIPA2 encoded a putative polypeptide with nine transmembrane domains, they suggested that it might function as a receptor or transporter. Accordingly, it was of interest that NIPA2 was differentially upregulated by low magnesium in our microarray assays. To determine its function in the present study, we expressed NIPA2 cRNA in Xenopus oocytes and performed voltage-clamp and fluorescence studies to characterize encoded protein-mediated ion transport. The data indicate that NIPA2-mediated transport was highly selective to the Mg2+ as no other cation elicited transport in expressing oocytes. NIPA2 is only the second transporter reported that is selective for Mg2+. Furthermore, we have localized the NIPA2 protein to the early endosomes and have shown that cellular distribution is regulated by magnesium. This notion is consonant with our observations of other members of the NIPA family of proteins, but unlike NIPA2-mediated transport, NIPA1, NIPA3, and NIPA4 are not selective to Mg2+ (10).
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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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. Genomic sequence analysis. The NIPA2 cDNA sequence was determined by standard methods (24). Data base searching and alignments were performed using BLAST. Protein homology searches were performed by comparing the amino acid query sequence against SWISSPROT data base. The full-length NIPA2 cDNA sequence has been deposited in the GenBank data base (accession human no. NM-030922, mouse no. NM-023647). The respective accession numbers were NIPA3, listed as NIPA-like (NPAL1) NM-207330, (AK014427) and NIPA4 (NIPAL2) NM-024759, and NM-145469.
Animal preparation and cell culture. Male mice were maintained for 5 days on a low-magnesium diet (ICN diet no. 902205, Nutritional Biochemicals) or on this diet supplemented with 0.05% MgSO4, which is comparable to normal mouse chow. The plasma concentrations were 0.13 ± 0.01 and 0.75 ± 0.09 mM, respectively, confirming that the mice consuming a magnesium-restricted diet were relatively magnesium deficient (27).
Mouse distal convoluted tubule (MDCT) cells were derived and immortalized by Pizzonia and colleagues (20). The MDCT cell line has been extensively used by us to study the hormonal and nonhormonal control of renal epithelial cell magnesium transport (8). 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), 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) for 16 h before harvest. Other constituents of the Mg2+-free culture media were similar to the complete media.
Quantitative analysis of NIPA 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) before making first-strand cDNA. Standard curves were constructed by serial dilution of a linear pGEM-T vector (Promega) containing the NIPA2 gene. The primer set of mouse NIPA2 was the following: forward, 5'-ATGTGCCCGTTTGATACCAT-3' and reverse, 5'-TACAGGAGACAAGGTGACGCAA-3'. PCR products were quantified continuously with AB7000 (Applied Biosystems) using SYBR Green fluorescence according to the manufacturer's instructions. The relative amounts of NIPA2 mRNA were normalized to the mouse-actin transcripts. Similarly, the NIPA3 and NIPA4 transcripts were quantified with real-time RT PCR. The primers for NIPA3 were forward 5'-TGTATGTGGGCTTGGTATT-3' and reverse 5'-TTGCACTTATGAGAACGCT-3' and NIPA4 forward 5'-GCTTCGTCACCATCATCCT-3' and reverse 5'-TAGTCACCAAGGCTGTATCTC-3'.
Plasmid construction and generation of expression constructs. A mouse NIPA2 cDNA clone was purchased from RIKEN no. 3830408P04 and subcloned into the XbaI and SacII restriction sites of the pcms-GFP expression vector. Isolated clones were subcloned and sequenced to confirm sequence integrity. Standard protocols were used for Escherichia coli cloning procedures and DNA sequencing (24). NIPA3 (no. 3830408610) and NIPA4 (no. 953006K23) were purchased from RIKEN and processed in the same way as NIPA2.
In vitro transcription and expression of NIPA in xenopus oocytes. To synthesize complementary RNA (cRNA), the cDNA constructs were linearized and then transcribed with T7 polymerase in the presence of m7GpppG cap using the mMESSAGE MACHINE T7 (Ambion) transcription system. Xenopus oocytes were prepared and injected with cRNA, and electrophysiological recordings were preformed according to techniques previously described (11). Briefly, stage V–VI oocytes were treated with collagenase (2 mg/ml) for 2 h and manually defolliculated. Twenty-four hours after defolliculation, the oocytes were typically injected with 25 ng cRNA in 50 nl H2O. Oocytes were incubated at 18°C for 3–5 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, and 100 mg/l streptomycin changed daily].
Electrophysiological studies. To record expressed membrane currents, 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 interfaced to an IBM-compatible PC via a Digidata 1200 A/D converter and controlled by pCLAMP software (Axon Instruments). Electrophysiology consisted of a voltage-clamp step profile consisting of a holding potential of –15 mV, followed by eight episode series of +25-mV steps of 2 s duration, from –150 mV to +25 mV within an episode duration of 6.14 s. Each episode recorded 1,536 data points collected at 4-ms intervals. The data were filtered at the appropriate frequency before digitization. To assess the permeability of different divalent cations, we used the shift in the reversal potentials of the respective cation in relation to the reversal potentials observed for Mg2+ currents. Voltage-clamp episodes in the presence of extracellular test cations were corrected against episodes in the absence of external test cations.
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 (
Erev) and calculated by the permeability (P) ratio by:
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Mean data (±SE) from the electrophysiology experiments are presented as Mg2+-evoked currents from four or more oocytes from the same batch of oocytes used on the same day. Each experiment was repeated at least twice on oocytes from different frogs. No Mg2+-evoked currents were detected in oocytes injected with water alone, demonstrating that the currents in NIPA2-producing oocytes were specific to the expressed transporter.
Measurement of Mg2+ influx. Epifluorescence microscopy was used to monitor changes in intracellular Mg2+ content within single oocytes using the Mg2+-responsive mag-fura-2 fluorescence dye (10). Oocytes were injected with 50 µM mag-fura-2 acid (Molecular Probes) 20 min before experimentation. The chamber (0.5 ml) was mounted on an inverted Nikon Diaphot-TMD microscope with a Fluor x10 objective and voltage clamped as described above. Subsequently, they 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 the 340/385 ratio, which reflects the Mg2+ concentration. All experiments were performed at 23°C. Each experiment was repeated at least three times using oocytes from different frogs.
Immunolocalization of NIPA2 protein in expressing oocytes. 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 the hemagglutinin (HA) primary antibody and anti-rabbit Alexa 568 secondary antibody. Oocytes images were taken using a x20 dry objective affixed to a Zeiss LSM 510 Meta microscope and LSM Image software.
Subcellular localization of NIPA2 in MDCK and COS7 epithelial cells. A COOH-terminal HA tag was added to NIPA2 cDNA by PCR with an oligo(5N2Bg1: 5'-AGATCTGAACGAAATGAGCCTG-3', 3N2HA: 5'-CTAAGCCTAATCTGGAACATCGTATGGGTATCGTCGGAAAAAGATGGC-3') containing the full HA. NIPA2-HA was cloned into the pcDNA 3.1/V5-HIS TOPO TA expression vector (Invitrogen) resulting in the plasmid pcDNA-NIPA2-HA. The MDCK and COS7cells were grown to subconfluence on coverslips for 6 h and transfected with the plasmid using Lipofectamine 2000 for 5 h in Opti-MEM media (Invitrogen). After 14–16 h recovery in F-12 DMEM, the cells were fixed with 4% paraformaldehyde and immunostained with rat monoclonal anti-HA antibody (Roche) followed by Alexa Flour 568 goat anti-rat secondary antibody (Molecular Probes). Cells were washed three times with phosphate-buffered saline containing 0.3% Triton X-100 (PBST) before each antibody incubation. Primary antibodies of Rab5 (GTP-binding proteins) that were raised in the mouse (BD Transduction Labs) were used to localize the early endosomes. Phalloidin (Molecular Probes) was used to stain for actin in the indicated experiments to aid in delimiting membrane ruffles. Alexa 350 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 h room temperature. After staining was completed, coverslips were then mounted on slides with Fluoromount-G glycerol-based mounting media (Southern Biotechnology).
All images were taken using a x63 water lens affixed to a Zeiss LSM 510 Meta microscope and AxioVision (epifluorescent) software (512 x 512 pixel resolution; excitation 1, argon 488 nm at 10%; emission 1, Bandpass Filter 505–530; excitation 2, HeNe 543 nm at 40%; emission 2, Longpass Filter 560). Cells were selected from 10 to 12 fields of view and used for analysis of colocalization of antibody staining using Northern Eclipse software. Confocal imaging was used to assess the distribution of NIPA2-HA staining following alterations in culture media magnesium. Cells were selected from 5 to 10 fields of view and distribution was assessed using a line scan across the cell in the middle z-section using ImageJ software.
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RESULTS |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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An in silico search identified two related genes: NIPA3 and NIPA4. NIPA2, NIPA3, and NIPA4 have greater amino acid similarity, about 66%, than they have with NIPA1 (Fig. 1A). NIPA1 is the least similar member of the family even though it is located in tandem with NIPA2 on human chromosome 15q and mouse 7B. The human NIPA1 and mouse NIPA1 exhibited between 36% and 43% identity to the other three respective human and mouse NIPA forms. Genes encoding close homologues (83%-91% amino acid sequence identity) are present in the chicken, orangutan, and chimpanzee genomes, indicating that divergence of the NIPA1 from NIPA2, NIPA3, and NIPA4 forms was an early event in vertebrate evolution (Fig. 1B). NIPA3 is located on human chromosome 4p12 and mouse 5 chromosome. NIPA4 is on human chromosome 5q33 and on mouse 11. Because NIPA3 and NIPA4 are similar to NIPA2, we thought it prudent to test these proteins for transport function.
NIPAs mediate Mg2+ transport in expressing Xenopus oocytes. To determine whether NIPA2 encodes a functional Mg2+ transporter, we prepared cRNA, injected it into Xenopus oocytes, and characterized Mg2+ transport using two-microelectrode voltage-clamp analysis and fluorescence measurements. The electrophysiological data gave evidence for a rheogenic process with inward currents in NIPA2 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 (Fig. 2A). The reversal potential was significantly shifted to the right with increases in magnesium concentration as would be expected of a Mg2+ transporter (Fig. 2A). In support of this conclusion, we performed flux studies with fluorescence spectrometry using the Mg2+-sensitive dye mag-fura-2 (10). Currents were initially measured at resting potentials with the oocytes in Mg2+-free solutions and then in solutions containing 2.0 mM MgCl2. Oocytes were subsequently voltage clamped at –70 mV (Fig. 2B). Fluorescence was determined throughout the experiment. The 340/385 fluorescence ratio, a reflection of the free Mg2+ concentration within the oocyte, increased in NIPA2-expressing oocytes but not in the control water-injected oocytes (Fig. 2B). The increase in cytosolic Mg2+ concentration with time (Mg2+ flux rate) was linearly associated with the simultaneously measured currents, indicating that the Mg2+-elicited currents was due to the movement of Mg2+ cations. In these experiments a number of oocytes expressing variable amounts of NIPA2 protein were clamped at –70 mV, and the mag-fura-2 340/345 ratio and electrical currents were simultaneously measured (Fig. 2C). The increase in cytosolic Mg2+ concentration observed after readdition of extracellular Mg2+, taken as an index of plasma membrane Mg2+ transport, was linearly associated with the simultaneously measured currents, consistent with the concept that the Mg2+-elicited currents were due to the movement of Mg2+. Consistent with the prediction from early studies that apical Mg2+ uptake is not energy dependent, NIPA2-mediated transport did not appear to be coupled to Na+, Cl–, or H+. Substitution of Na+ or Cl– with choline or cyclamate, respectively, did not alter NIPA2-mediated currents (data not shown). Acid pH diminished Mg2+ uptake in a manner opposite to what would be expected if Mg2+ was coupled to H+ (data not shown). Also consistent with ionic transport was the observation that mouse NIPA2-mediated Mg2+ uptake was saturable with a demonstrated mean Michaelis constant (Km), which was 0.31 ± 0.07 mM, n = 29 (Fig. 1D). As the extracellular Mg2+ concentration is in the order of 0.5 mM, the affinity of NIPA2-mediated transport is consistent with a physiological role in cellular Mg2+ metabolism.
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We then determined whether these cations would inhibit Mg2+-evoked currents. The apparent potency sequence of inhibition was Cd2+, 2% (IMg2+ in the presence of 0.2 mM putative inhibitor as a percentage of control IMg2+ in the absence of inhibitor for each oocyte); Ni2+, 3%; Mn2+, 4%; and Gd3+, 7% (Fig. 1F). The other cations tested did not significantly effect Mg2+-evoked currents. Accordingly, although NIPA2-mediated transport was very selective for Mg2+, it may be inhibited by relatively large concentrations of some metals.
Immunofluorescence using an HA antibody shows predominantly surface localization of NIPA2-HA protein in NIPA2-expressing oocytes (Fig. 2G), whereas there was no staining in control, water-injected oocytes (Fig. 1H).
Using the same approaches, we showed that NIPA3 and NIPA4 also mediated Mg2+ transport (Fig. 3). Both transporters were rheogenic, voltage dependent, and saturable with Km values of 0.90 ± 0.08 and 0.36 ± 0.02 mM, respectively (Fig. 3, A and B). NIPA3 transported Mg2+, 100%; Sr2+, 62% (relative to Mg2+); Ba2+, 51%; Fe2+, 32%; Mn2+, 32%; Cu2+, 20%; Co2+, 20%, and NIPA4 transported Mg2+, 100%; Ba2+, 49%; Sr2+, 39%; Mn2+, 32%; Co2+, 20% (Fig. 3E). Accordingly, NIPA3 and NIPA4 are able to mediate the transport a number of divalent cations in addition to Mg2+. It should be noted that these apparent permeabilities were determined in the absence of Mg2+ and at relatively large concentrations. None of the NIPA family members mediated Ca2+ transport nor was Mg2+ uptake inhibited by Ca2+. In summary, NIPA2 was selective for Mg2+, whereas NIPA3 and NIPA4 mediated the transport of a number of other cations.
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To investigate the subcellular localization of NIPA2 protein, we constructed a tagged NIPA2-HA, transfected in MDCK and COS7 cells, and performed immunofluorescence using the specific antibody to HA (Fig. 4). In normal transfected MDCK cells, the NIPA2-HA-fusion protein was predominately localized with Rab5, indicting that it was an early endosome protein (Fig. 4A). Furthermore, the punctate peripheral staining pattern of NIPA2-HA is consistent with the presence of NIPA2 in early endosomes. In addition to the endosomal localization, NIPA2-HA staining was widely dispersed across the cell surface at the membrane ruffles as evidenced by phaloidin staining of actin (Fig. 4E). This pattern of distribution is consistent with the notion that NIPA2 translocates via the early endosomes to the plasma membrane. The suggestion that endogenous NIPA2 is present at the surface membrane is in keeping with the functional studies performed with heterologous-expressing oocytes.
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DISCUSSION |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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Based on our present studies, we conclude that the undefined orphan NIPA2 protein is a transporter that mediates selective Mg2+ flux. Expression of NIPA2 in Xenopus oocytes produces Mg2+-evoked currents with channel-like properties measured with voltage-clamp conditions. The reversible potential shifts to the right with a magnitude of 28 mV as predicted by the Nernst relationship with decade increases in magnesium concentration (11). Mg2+ currents are concentration dependent, saturable, reversible, and inhibitable as would be expected of a cationic transporter. In support of the electrophysiological findings, NIPA2 mediates only Mg2+ flux as determined by fluorescence with the Mg2+-sensitive mag-fura-2 dye. These two independent approaches resulted in the same conclusion. The evidence that NIPA1 is a selective Mg2+ transporter is persuasive.
Within the cell, the NIPA2 was principally localized to the early endosomal compartment and the peripheral surface. This localization suggests that the NIPA2 protein plays a role at the surface membrane. In support of this notion is the redistribution of NIPA2 in response to magnesium. There was an apparent trafficking of NIPA2 to the periphery with low magnesium. Consonant with the mRNA analysis, there was an apparent increase in total protein in epithelial cells grown in low magnesium and an apparent clustering of NIPA2 protein in early endosomes giving a punctate appearance. These changes in protein expression likely account, in part, for the associated changes in Mg2+ transport.
By similar methods, we have recently showed that NIPA1, the NIPA2-related gene to NIPA2 within the 15q11-q13 chromosome region, is also a Mg2+ transporter (10). Both NIPA1 and NIPA2 locate to the early endosomes and plasma membrane. Although there are functional similarities between NIPA2 and NIPA1, there are some remarkable differences between currents mediated by these proteins. First, both NIPA1- and NIPA2-mediated transport is saturable but the Michaelis constants differed; NIPA1 was 0.66 ± 0.08 mM compared with NIPA2, 0.31 ± 0.07 mM. Second, the substrate specificity of the two transporters are different in that NIPA2 is very specific for Mg2+, whereas NIPA1 transported Sr2+, Fe2+, and Co2+albeit to a much less extent than Mg2+ (10). In summary, NIPA1 is more permissive in substrate specificity than NIPA2. It is unknown whether these divergent electrophysiological properties translate into differences in intracellular function. As NIPA1 and NIPA2 genes are in tandem on chromosome 15q, they are likely paralogs; unlike orthologs that commonly retain the same function in the course of evolution, paralogs evolve new functions, albeit related to the original one. Accordingly, it is of interest that NIPA2 possesses different properties to NIPA1 but they both mediate Mg2+ transport. The relationship of NIPA1 to NIPA2 remains to be determined. As remarked by Chai and colleagues (4), both NIPA1 and NIPA2 polypeptides each display 98% identity between the respective human and mouse orthologs, and these have a 55% identity to the respective Fugu NIPAs, but there is only 32–36% identity between NIPA1 and NIPA2 in each of the species. NIPA2 shows greater common identity in its ancestral invertebrate polypeptides than it does to NIPA1. The phylogenetic distance between NIPA1 and NIPA2 paralogs is as great as that between the orthologs. This observation led Chai et al. (4) to suggest that NIPA1 and NIPA2 have evolved related but different functions. However, both NIPA1 and NIPA2 transcripts and proteins are upregulated with low magnesium indicating similar regulation of the individual gene products, suggesting that they are somehow involved in Mg2+ metabolism. Thus it was of interest that both NIPA1 and NIPA2 localize to the early endosomes and the plasma membrane. Mutations in NIPA1 (SPG6 locus) have been shown to be the basis of autosomal dominant hereditary spastic paraplegia (HSP) [OMIM no. 608145 [OMIM] , no. 600363] (4, 5, 9). Two missense NIPA1 mutations, G100R and T45R, have been reported to be the basis of HSP (22, 23). We have shown that these mutations lead to a loss-of-function of NIPA1 suggesting that G100 and T45 sites are important, if not essential, in processing of the NIPA1 Mg2+ transport protein (10). Heterologous expression in COS7 cells demonstrate that these mutants were retained in the endoplasmic reticulum rather than trafficking to the plasma membrane. Moreover, Gly106 and T45 are conserved among ortholog NIPA1 channels supporting the notion that the mutation is strongly pathogenetic. There are no similar consensus sites in the other members of the NIPA family, either the paralog NIPA2 or the related NIPA3 and NIPA4 transporters, so that protein folding is likely different between these transporters. Because the disruption of NIPA1 led to neuropathy in the HSP patients where NIPA2 is presumably normal, NIPA2 cannot functionally replace NIPA1. This might be related to their additional expression in other compartments or so-far-unknown functional differences.
Of the Mg2+ transporters identified to date, only MagT1 and NIPA2 are selective for Mg2+. The other identified mammalian proteins (Mrs2, TRPM6/7, SLC41, ACDP, and MMgT) transport a variety of divalent metals including in some cases Ca2+ (12, 14, 15, 19, 26, 30, 31). Interestingly, the other members of the MagT and NIPA families are not cationic selective. The second member of the MagT family, N33, mediates Mg2+, Fe2+, Cu2+, and Mn2+. In addition to Mg2+, NIPA1 transports Sr2+, Co2+; NIPA3 transports Sr2+, Ba2+, Fe2+, Cu2+; and NIPA4 transports Sr2+, Ba2+ (Fig. 3E). Thus substrate selectivity of the identified Mg2+ transporters cannot be predicted by phylogenetic profiling (Fig. 1B) nor does evolutionary divergence predicate Mg2+ selectivity. Moreover, there are no obvious similarities in the secondary amino acid structures of MagT1 and NIPA2, suggesting that there are different means of making a selective Mg2+ transporter.
Prader-Willi syndrome [MIM 176270 [OMIM] ] is a genetic disorder, affecting 1 in 15,000 newborns, that presents as a complex developmental and multisystem disorder. It is characterized by infantile hypotonia, gonadal hypoplasia, moderate mental retardation, short statue, obsessive/compulsive behavior, feeding problems, and later hyperphagia leading to obesity (2, 3, 17). At least 12 imprinted genes have been identified in the 15q11-q13 region that are candidates for Prader-Willi syndrome. Angelman syndrome [MIM 105830 [OMIM] ] also maps to 15q-q13 but is now thought to be caused by a single maternally expressed gene (UBE3A) and is a clinically distinct neurological disorder from Prader-Willi syndrome (16). The role of NIPA2 in the etiology of Prader-Willi syndrome is obscure. Prader-Willi patients do not have a single gene mutation, suggesting that it is a contiguous gene syndrome resulting from the loss of function of several paternally expressed, maternal uniparental disomy, or imprinted genes (1, 6, 29). Although NIPA1 and NIPA2 reside within a cluster of expressed genes in chromosome 15q11–13 that are thought to cause the Prader-Willi phenotype, there is no evidence that they are directly involved with the many complexities of this disorder (17, 18). There are a number of mouse models of Prader-Willi that demonstrate similar phenotypes of severe failure-to-thrive and early postnatal lethality, but studies of these models have not directly implicated NIPA1 and NIPA2 in the disorder (28). Stefan et al. (28) generated a transgenic mouse model of Prader-Willi syndrome (designated TgPWS) that has a deletion of 13 known imprinted and 10 nonimprinted genes including NIPA2. Microarray analysis of the brains to ascertain the primary transcriptional changes failed to demonstrate a change in NIPA1 and NIPA2 expression leading these authors to conclude that these were unlikely to be involved in this disorder (28). Further studies are warranted to determine role of NIPA2 in Prader-Willi syndrome and the disturbances, if any, of cellular magnesium metabolism in this disorder.
In summary, we show here that NIPA2 is a very selective Mg2+ transporter. NIPA2 protein is localized to the early endosomes/plasma membrane and is differentially increased with low magnesium. NIPA2 is only one-of-two selective Mg2+ transporters that have been reported in the mammalian cell.
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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