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J Biol Chem
2007 Mar 16;28211:8060-8. doi: 10.1074/jbc.M610314200.
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NIPA1(SPG6), the basis for autosomal dominant form of hereditary spastic paraplegia, encodes a functional Mg2+ transporter.
Goytain A
,
Hines RM
,
El-Husseini A
,
Quamme GA
.
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Mutations in the NIPA1(SPG6) gene, named for "nonimprinted in Prader-Willi/Angelman" has been implicated in one form of autosomal dominant hereditary spastic paraplegia (HSP), a neurodegenerative disorder characterized by progressive lower limb spasticity and weakness. However, the function of NIPA1 is unknown. Here, we show that reduced magnesium concentration enhances expression of NIPA1 suggesting a role in cellular magnesium metabolism. Indeed NIPA1 mediates Mg2+ uptake that is electrogenic, voltage-dependent, and saturable with a Michaelis constant of 0.69+/-0.21 mM when expressed in Xenopus oocytes. Subcellular localization with immunofluorescence showed that endogenous NIPA1 protein associates with early endosomes and the cell surface in a variety of neuronal and epithelial cells. As expected of a magnesium-responsive gene, we find that altered magnesium concentration leads to a redistribution between the endosomal compartment and the plasma membrane; high magnesium results in diminished cell surface NIPA1 whereas low magnesium leads to accumulation in early endosomes and recruitment to the plasma membrane. The mouse NIPA1 mutants, T39R and G100R, corresponding to the respective human mutants showed a loss-of-function when expressed in oocytes and altered trafficking in transfected COS7 cells. We conclude that NIPA1 normally encodes a Mg2+ transporter and the loss-of function of NIPA1(SPG6) due to abnormal trafficking of the mutated protein provides the basis of the HSP phenotype.
FIGURE 1.
Molecular characterization of NIPA1. A, phylogenetic tree constructed from a multiple alignment of the following Homo sapiens (h), Mus musculus (m), Rattus norvegicus (r), Gallus gallus (g), Pan troglodytus (pt), Pongo pygmaeus (p), Bos tauris (b), and Canis familiaris (c) sequences using ClustalX version 1.88 (21) and the neighbor-joining method of Saitou and Nei (22) using Phylo Draw, version 0.8 (Graphics Application Laboratory, Pusan National University). B, predicted secondary structure of mouse NIPA1. The two mouse mutation sites, T39R and G100R, corresponding to the respective human T45R and G106R mutations are illustrated. C, current-voltage (I-V) relationships obtained from linear voltage steps from -150 mV to +25 mV in the presence of Mg2+-free solutions or those containing the indicated concentrations of MgCl2. Oocytes were clamped at a holding potential of -15 mV and stepped from -150 mV to +25 mV in 25-mV increments for 2 s at each of the concentrations indicated. Shown are average I-V curves obtained from control H2O-injected (n = 3) or NIPA1-expressing (n =>3) oocytes. Note the positive shift in reversal potential with increments in Mg2+ concentration. Values are mean ± S.E. of observations measured at the end of each voltage sweep for the respective Mg2+ concentration. D, summary of concentration-dependent Mg2+-evoked currents in NIPA1-expressing oocytes using a holding potential of -125 mV. Mean ± S.E. values are those given in Fig. 1C. The Michaelis constant determined with nonlinear regression anaylsis was 0.69 mm. The Michaelis constant was independent of the respective holding potential. E, Mg2+ flux into NIPA1-expressing oocytes. Mag-fura-2 fluorescence ratios were measured in control and NIPA1-expressing oocytes, at resting potentials, in solutions consisting of nominally magnesium-free solutions and then with 2.0 mm MgCl2 with interruption as indicated. Oocytes were subsequently voltage-clamped at a holding potential of -70 mV, where indicated. Mg2+ fluxes were determined with fluorescence using the Mg2+-sensitive dye, mag-fura-2. CaCl2, 2.0 mm, was added and removed where indicated. Results are presented as the 340/385 excitation ratio that reflect changes in divalent cation concentration. Results are mean of tracings performed with three different oocyte preparations. F, surface expression of NIPA1 protein in X. laevis oocytes determined with immunofluorescence. Left panel, control water-injected oocytes tested with affinity-purified NIPA1-specific antibody. The arrows indicate membrane surface (×200 magnification). Right panel, NIPA1-injected oocyte treated with NIPA1 antibody showing intense surface staining. The measured current for this oocyte was 0.12 μA with 2.0 mm external MgCl2 concentration clamped at -70 mV.
FIGURE 2.
Tissue distribution of endogenous mouse NIPA1 protein expression. Left, Western blotting of endogenous NIPA1 protein in various mouse tissues. An aliquot of protein (50 μg) from the indicated mouse tissues was applied to each lane and labbeled with a specific polyclonal antibody for NIPA1. The gel is representative of three separate tissue preparations. Right, NIPA1 protein increases in epithelial cells cultured in low magnesium media. Shown is a representative blot, one of seven performed on different cell preparations. The mean band density increased 163 ± 7% with low magnesium relative to those cultured with normal concentrations of magnesium.
FIGURE 3.
Subcellular localization of endogenous NIPA1. Immunofluorescence staining of COS7 cells is shown. A, cells were fixed and incubated with NIPA1 antibody and the early endosome markers, EEA1 (A, top panel), or the Golgi marker, GM130 (A, center panel), or the TGN-recycling endosome marker, Rab8 (A, bottom panel). B, Rab5 (B, top panel) or Rab5Q79L (B, bottom panel) were used to confirm endosomal localization. C, COS7 cells were stained with phaloidin to delineate the cell surface. Overlays are shown in the third column of each figure with enlarged close-ups in the far right column. The arrows indicate co-localization of NIPA1 protein with the respective marker. Similar subcellular distributions were found in MDCT, HEK293, and primary hippocampal brain cells.
FIGURE 4.
Subcellular redistribution of NIPA1 in response to magnesium. COS7 cells were cultured in high magnesium, 5.0 mm, normal magnesium, 0.8 mm, or low magnesium, nominally magnesium-free, for 12 h. A, immunofluorescence was performed with NIPA1 antibody and the surface marker, phalloidin. The merged images of labeled cells are also shown in the far right panel. B, summary of NIPA1 accumulation at the peripheral membrane in response to changes in magnesium.
FIGURE 5.
Loss-of-function mutations in NIPA1. A, summary of Mg2+-evoked currents in NIPA1T39R- and NIPA1G100R-expressing oocytes compared with wild-type NIPA1-expressing oocytes. Currents were measured with 2.0 mm MgCl2 at a holding voltage of 100 mV according to the methods given in the legend to Fig. 2, left. Results are mean ± S.E. for n = 7, 10, and 7 oocytes, respectively. B, Mg2+ flux, determined by mag-fura-2 fluorescence, into NIPA1T39R- and NIPA1G100R-expressing oocytes. Mg2+ concentration was measured with 2.0 mm MgCl2 according to the methods given in the legend to Fig. 2, right. Results are means of four oocytes. C, immunofluorescence images of NIPA1-HA-, NIPA1T39R-HA-, or NIPA1G100R-HA-expressing COS7 cells. D, summary of NIPA1T39R-HA and NIPA1T100R-HA retention in the endoplasmic reticulum (ER) relative to the wild-type NIPA1 protein. E, increase in endosomal size of NIPA1T39R-HA and NIPA1T100R-HA transfected relative to the wild-type NIPA1-HA-transfected COS7 cells. WT, wild type.
