Magnesium transporters

:"This page links directly from the magnesium in biological systems page."

All forms of life require magnesium, and yet the molecular mechanisms of Mg2+ uptake from the environment and the distribution (transport) of this vital element within the organism are only slowly being elucidated. In bacteria Mg2+ is probably mainly supplied by the CorA proteincite journal| last=Hmiel| first= S.P. | coauthors=Snavely, M.D., Miller, C.G., and Maguire, M.E.|year=1986| title= Magnesium transport in Salmonella typhimurium: characterisation of magnesium influx and cloning of a transport gene| journal=Journal of Bacteriology| volume= 168| pages=1444–1450] and, where the CorA protein is absent, by the MgtE proteincite journal| last=Townsend| first= D.E. | coauthors=Esenwine, A.J., Georgei, J.I., Bross, D., Maguire, M.E., and Smith, R.L.|year=1995| title= Cloning of the mgtE Mg2+ transporter from Providencia stuartii and the distribution of mgtE in gram-negative and gram-positive bacteria| journal=Journal of Bacteriology | volume=177| pages=5350–5354] cite journal| last=Smith| first= R.L. | coauthors=Thompson, L.J., and Maguire, M.E.|year=1995| title= Cloning and characterization of MgtE, a putative new class of Mg2+ transporter from Bacillus firmus OF4| journal=Journal of Bacteriology | volume=177| pages=1233–1238] . In yeast the initial uptake is via the Alr1p and Alr2p proteinscite journal| last=MacDiarmid| first= C.W. | coauthors=Gardner, R.C.|year=1998| title= Overexpression of the Saccharomyces cerevisiae magnesium transport system confers resistance to aluminum ion| journal=Journal of Biological Chemistry| volume= 273| pages=1727–1732| doi= 10.1074/jbc.273.3.1727| pmid=9430719] , but at this stage the only internal Mg2+ distributing protein identified is Mrs2pcite journal| last=Bui| first= D.M. | coauthors=Gregan, J., Jarosch, E., Ragnini, A., and Schweyen, R.J. | year=1999| title= The bacterial magnesium transporter CorA can functionally substitute for its putative homologue Mrs2p in the yeast inner mitochondrial membrane| journal=Journal of Biological Chemistry | volume=274| pages=20438–20443| doi= 10.1074/jbc.274.29.20438| pmid=10400670] . Within the protozoa only one Mg2+ transporter (XntAp) has been identifiedcite journal| last=Haynes| first= W.J. | coauthors=Kung, C., Saimi, Y., and Preston, R.R.|year=2002| title= An exchanger-like protein underlies the large Mg2+ current in Paramecium| journal=PNAS| volume= 99| pages=15717–15722| doi=10.1073/pnas.242603999] . In metazoa, Mrs2pcite journal| last=Zsurka| first= G. | coauthors=Gregan, J., and Schweyen, R.J.|year=2001| title= The human mitochondrial Mrs2 protein functionally substitutes for its yeast homologue, a candidate magnesium transporter| journal=Genomics | volume=72| pages=158–168| doi= 10.1006/geno.2000.6407] and MgtE homologuescite journal| last=Wabakken| first= T. | coauthors=Rian, E., Kveine, M., and Aasheim, H.-C.|year=2003| title= The human solute carrier SLC41A1 belongs to a novel eukaryotic subfamily with homology to prokaryotic MgtE Mg2+ transporters| journal=Biochemical and Biophysical Research Communications | volume=306| pages=718–724| doi=10.1016/S0006-291X(03)01030-1] have been identified, along with two novel Mg2+ transport systems TRPM6/TRPM7cite journal| last=Nadler| first= M.J.S. | coauthors=Hermosura, M.C., Inabe, K., Perraud, A.-L., Zhu, Q., Stokes, A.J., Kurosaki, T., Kinet, J.-P., Penner, R., Scharenberg, A.M., and Fleig, A.|year=2001| title= LTRPC7 is a Mg. ATP-regulated divalent cation channel required for cell viability| journal=Nature | volume=411| pages=590–595| doi= 10.1038/35079092] cite journal| last=Walder| first= R.Y. | coauthors=Landau, D., Meyer, P., Shalev, H., Tsolia, M., Borochowitz, Z., Boettger, M.B., Beck, G.E., Englehardt, R.K., Carmi, R., and Sheffield, V.C.|year=2002| title= Mutation of TRPM6 causes familial hypomagnesemia with secondary hypocalcemia| journal=Nature Genetics| volume= 31| pages=171–174| doi= 10.1038/ng901] and PCLN-1cite journal| last=Simon| first= D.B. | coauthors=Lu, Y., Choate, K.A., Velazquez, H., Al-Sabban, E., Praga, M., Casari, G., Bettinelli, A., Colussi, G., Rodriguez-Soriano, J., McCredie, D., Milford, D., Sanjad, S., and Lifton, R.P.|year=1999| title= Paracellin-1, a renal tight junction protein required for paracellular Mg2+ resorption| journal=Science| volume= 285| pages=103–106| doi=10.1126/science.285.5424.103| pmid=10390358] . Finally, in plants, a family of Mrs2p homologues has been identifiedcite journal| last=Schock| first= I. | coauthors=Gregan, J., Steinhauser, S., Schweyen, R., Brennicke, A., and Knoop, V.|year=2000| title= A member of a novel Arabidopsis thaliana gene family of candidate Mg2+ ion transporters complements a yeast mitochondrial group II intron-splicing mutant| journal=Plant Journal | volume=24| pages=489–501| doi=10.1046/j.1365-313x.2000.00895.x] cite journal| last=Li| first= L. | coauthors=Tutone, A.F., Drummond, R.S.M., Gardner, R.C., and Luan, S.|year=2001| title= A novel family of magnesium transport genes in Arabidopsis| journal=Plant Cell| volume= 13| pages=2761–2775| doi= 10.1105/tpc.13.12.2761] along with another novel protein, AtMHXcite journal| last=Shaul| first= O. | coauthors=Hilgemann, D.W., de-Almeida-Engler, J., Van, M.M., Inze, D., and Galili, G.|year=1999| title= Cloning and characterization of a novel Mg2+/H+ exchanger| journal=EMBO Journal | volume=18| pages=3973–3980| doi=10.1093/emboj/18.14.3973] .

The evolution of Mg2+ transport appears to have been rather complicated. Proteins apparently based on MgtE are present in bacteria and metazoa, but are missing from fungi and plants, whilst proteins apparently related to CorA are present in all of these groups. The two active-transport transporters present in bacteria, MgtA and MgtB, do not appear to have any homologues in the higher organisms. There are also Mg2+ transport systems that are found only in the higher organisms.

Clearly there are a large number of proteins yet to be identified that transport Mg2+. Even in the best studied eukaryote, yeast, Borrellycite journal| last=Borrelly| first= G. | coauthors=Boyer, J.-C., Touraine, B., Szponarski, W., Rambier, M., and Gibrat, R. | year= 2001| title= The yeast mutant vps5D affected in the recycling of Golgi membrane proteins displays an enhanced vacuolar Mg2+/H+ exchange activity| journal=PNAS| volume= 98| pages=9660–9665| doi=10.1073/pnas.161215198| pmid=11493679] have reported a Mg2+/H+ exchanger, without an associated protein, which is probably localised to the Golgi. At least one other major Mg2+ transporter in yeast still unaccounted for — that effecting Mg2+ transport into and out of the yeast vacuole. In higher, multicellular organisms it seems that many Mg2+ transporting proteins await discovery.

The CorA-domain-containing Mg2+ transporters (CorA, Alr-like and Mrs2-like) have a similar but not identical array of affinities for divalent cations. In fact, this observation can be extended to all of the Mg2+ transporters so far identified. This similarity suggests that the basic properties of Mg2+ strongly influence the possible mechanisms of recognition and transport. However, this observation also suggests that using other metal ions as tracers for Mg2+ uptake will not necessarily produce results comparable to the transporter’s ability to transport Mg2+. Ideally, Mg2+ should be measured directlycite journal| last=Tevelev| first= A. | coauthors=Cowan, J.A.|year=1995| title= Metal substitution as a probe of the biological chemistry of magnesium ion. In The Biological Chemistry of Magnesium, J.A. Cowan, ed (New York: VCH)] .

In a world where 28Mg2+ is practically unobtainable, much of the old data will need to be reinterpreted in terms of new tools for measuring Mg2+ transport, if different transporters are to be compared directly. The pioneering work of Kolisekcite journal| last=Kolisek| first= M. | coauthors=Zsurka, G., Samaj, J., Weghuber, J., Schweyen, R.J., and Schweigel, M.|year=2003| title= Mrs2p is an essential component of the major electrophoretic Mg2+ influx system in mitochondria| journal=EMBO Journal| volume= 22| pages=1235–1244| doi=10.1093/emboj/cdg122] and Froschauercite journal| last=Froschauer| first= E.M. | coauthors=Kolisek, M., Dieterich, F., Schweigel, M., and Schweyen, R.J.|year=2004| title= Fluorescence measurements of free [Mg2+] by use of mag-fura 2 in Salmonella enterica| journal=FEMS Microbiology Letters| volume= 237| pages=49–55] using mag-fura 2 has shown that free Mg2+ can be reliably measured "in vivo" in some systems. By returning to the analysis of CorA with this new tool, we have gained an important baseline for the analysis of new Mg2+ transport systems as they are discovered. However, it is important that the amount of transporter present in the membrane is accurately determined if comparisons of transport capability are to be made. This bacterial system might also be able to provide some utility for the analysis of eukaryotic Mg2+ transport proteins. However, the differences in biological systems between prokaryotes and eukaryotes will have to be considered as part of any experiment.

Comparing the functions of the characterised Mg2+ transport proteins is currently almost impossible. The proteins have been investigated in different biological systems using different methodologies and technologies. Finding a system where all the proteins can be compared directly would be a major advance. If the proteins could be shown to be functional in bacteria ("S. typhimurium") then a combination of the techniques of mag-fura 2, quantification of protein in the envelope membrane, and structure of the proteins (X-ray crystal or cryo-TEM) might allow the determination of the basic mechanisms involved in the recognition and transport of the Mg2+ ion. However, perhaps the best advance would be the development of methods allowing the measurement of the protein’s function in the patch-clamp system using artificial membranes.


Early research

In 1968 Luskcite journal| last=Lusk| first= J.E. | coauthors=Williams, R.J.P., and Kennedy, E.P.|year=1968| title= Magnesium and the growth of Escherichia coli| journal=Journal of Biological Chemistry| volume= 243| pages=2618–2624] described the limitation of bacterial ("Escherichia coli") growth on Mg2+-poor media, suggesting that bacteria required Mg2+ and were likely to actively take up this ion from the environment. The following year the same groupcite journal| last=Lusk| first= J.E. | coauthors=Kennedy, E.P.|year=1969| title= Magnesium transport in Escherichia coli| journal=Journal of Biological Chemistry | volume=244| pages=1653–1655] and another group, Silvercite journal| last=Silver| first= S.|year=1969| title= Active transport of magnesium in Escherichia coli| journal=PNAS | volume=62| pages=764–771| doi= 10.1073/pnas.62.3.764| pmid=4895213] , independently described the uptake and efflux of Mg2+ in metabolically active "E. coli" cells using 28Mg2+. By the end of 1971 two papers had been published describing the interference of Co2+, Ni2+ and Mn2+ on the transport of Mg2+ in "E. coli"cite journal| last=Nelson| first= D.L. | coauthors=Kennedy, E.P.|year=1971| title= Magnesium transport in Escherichia coli. Inhibition by cobaltous ion| journal=Journal of Biological Chemistry| volume=246| pages=3042–3049] and in Aerobacter aerogenes and Bacillus megateriumcite journal| last=Webb| first= M.|year=1970| title= Interrelationships between the utilization of magnesium and the uptake of other bivalent cations by bacteria| journal=Biochima et Biophysica Acta| volume= 222| pages=428–440] . In a last major development prior to the cloning of the genes encoding the transporters, it was discovered that there was a second Mg2+ uptake system that showed similar affinity and transport kinetics to the first system but with a different range of sensitivities to interfering cations. This system was also repressible by high extracellular concentrations of Mg2+ cite journal| last=Nelson| first= D.L. | coauthors=Kennedy, E.P.|year=1972| title= Transport of magnesium by a repressible and a nonrepressible system in Escherichia coli| journal=PNAS| volume= 69| pages=1091–1093| doi= 10.1073/pnas.69.5.1091| pmid=4556454] cite journal| last=Park| first= M.H. | coauthors=Wong, B.B., and Lusk, J.E.|year=1976| title= Mutants in three genes affecting transport of magnesium in Escherichia coli: genetics and physiology| journal=Journal of Bacteriology| volume= 126| pages=1096–1103] .


The CorA gene and its corresponding protein are the most exhaustively studied Mg2+ transport system in any organism. Most of the published literature on the CorA gene comes from the laboratory of M. E. Maguire. Recently the group of R. J. Schweyen has made a significant impact on the understanding Mg2+ transport by CorA. The gene was originally named after the cobalt-resistant phenotype in "E. coli" that was caused by the gene’s inactivation.

The gene was genetically identified in "E. coli" by Park "et al.", but wasn’t cloned until Hmiel "et al." isolated the "Salmonella enterica" serovar Typhimurium ("S. typhimurium") homologue. Later it would be shown by Smith and Maguirecite journal| last=Smith| first= R.L. | coauthors=Maguire, M.E.|year=1995a| title= Distribution of the CorA Mg2+ transport system in gram-negative bacteria| journal=Journal of Bacteriology | volume=177| pages=1638–1640] that the CorA gene was present in 17 gram-negative bacteria. With the large number of complete genome sequences now available for the prokaryotes, CorA has been shown to be virtually ubiquitous amongst the Eubacteria, as well as being widely distributed within the Archaeacite journal| last=Kehres| first= D.G. | coauthors=Lawyer, C.H., and Maguire, M.E.|year=1998| title= The CorA magnesium transporter gene family| journal=Microbial and Comparative Genomics| volume= 3| pages=151–169] . The CorA locus in "E. coli" contains a single open reading frame of 948 nucleotides, producing a protein 316 amino acids in size. This protein is well conserved amongst the Eubacteria and Archaea. Between "E. coli" and "S. typhimurium" the proteins are 98% identical, but in more distantly related species the similarity falls to between 15 and 20%. In the more distantly related genes the similarity is often restricted to the C-terminal part of the protein, and a short amino acid motif GMN within this region is very highly conserved. The CorA domain, also known as PF01544 in the pFAM conserved protein domain database (, is additionally present in a wide range of higher organisms and these transporters will be reviewed below.

The CorA gene is constitutively expressed in "S. typhimurium" under a wide range of external Mg2+ concentrationscite journal| last=Snavely| first= M.D. | coauthors=Gravina, S.A., Cheung, T.T., Miller, C.G., and Maguire, M.E.|year=1991a| title= Magnesium transport in "Salmonella typhimurium". Regulation of mgtA and mgtB expression| journal=Journal of Biological Chemistry | volume=266| pages=824–829] . However, recent evidence suggests that the activity of the protein may be regulated by the PhoPQ two-component regulatory systemcite journal| last=Chamnongpol| first= S. | coauthors=Groisman, E.A. | year=2002| title= Mg2+ homeostasis and avoidance of metal toxicity| journal=Molecular Microbiology| volume= 44| pages=561–571| doi=10.1046/j.1365-2958.2002.02917.x] . This sensor responds to low external Mg2+ during the infection process of "S. typhimurium" in humanscite journal| last=Groisman| first= E.A.|year=2001| title= The pleiotropic two-component regulatory system PhoP-PhoQ| journal=Journal of Bacteriology | volume=183| pages=1835–1842| doi= 10.1128/JB.183.6.1835-1842.2001| pmid=11222580] . In low external Mg2+ conditions the PhoPQ system was reported to suppress the function of CorA and it has been previously shown that the transcription of the alternative Mg2+ transporters MgtA and MgtB is activated in these conditions. Chamnongpol and Groisman suggest that this allows the bacteria to escape metal ion toxicity caused by the transport of other ions, particularly Fe(II), by CorA in the absence of Mg2+. For a conflicting report on the source of toxicity, see Papp and Maguirecite journal| last=Papp| first= K.M. | coauthors=Maguire, M.E.|year=2004| title= The CorA Mg2+ transporter does not transport Fe2+| journal=Journal of Bacteriology | volume=186| pages=7653–7658| doi=10.1128/JB.186.22.7653-7658.2004| pmid=15516579] .

The figure (not to scale) shows the originally published transmembrane (TM) domain topology of the "S. typhimurium" CorA protein, which was said to have three membrane-spanning regions in the C-terminal part of the protein (shown in blue), as determined by Smith "et al."cite journal| last=Smith| first= R.L. | coauthors=Banks, J.L., Snavely, M.D., and Maguire, M.E.|year=1993a| title= Sequence and topology of the CorA magnesium transport systems of Salmonella typhimurium and Escherichia coli: Identification of a new class of transport protein| journal=Journal of Biological Chemistry | volume=268| pages=14071–14080] . Evidence for CorA acting as a homotetramer was published by Warren "et al." in 2004cite journal| last=Warren| first= M.A. | coauthors=Kucharski, L.M., Veenstra, A., Shi, L., Grulich, P.F., and Maguire, M.E.|year=2004| title= The CorA Mg2+ transporter is a homotetramer| journal=Journal of Bacteriology | volume=186| pages=4605–4612| doi=10.1128/JB.186.14.4605-4612.2004| pmid=15231793] . In December 2005 the crystal structure of the CorA channel was posted to the RSCB protein structure database. The results show that the protein has two TM domains and exists as a homopentamer, in direct conflict with the eariler reports. [
] . The soluble intracellular parts of the protein are highly charged containing 31 positively charged and 53 negatively charged residues. Conversely, the TM domains contain only one charged amino acid , which has been shown to be unimportant in the activity of the transportercite journal| last=Smith| first= R.L. | coauthors=Szegedy, M.A., Kucharski, L.M., Walker, C., Wiet, R.M., Redpath, A., Kaczmarek, M.T., and Maguire, M.E.|year=1998a| title= The CorA Mg2+ transport protein of Salmonella typhimurium: Mutagenesis of conserved residues in the third membrane domain identifies a Mg2+ pore| journal=Journal of Biological Chemistry | volume=273| pages=28663–28669| doi=10.1074/jbc.273.44.28663| pmid=9786860] . From mutagenesis experiments it appears that the chemistry of Mg2+ transport relies on hydroxyl groups lining the inside of the transport pore; there is also an absolute requirement for the GMN motif (shown in red)cite journal| last=Szegedy| first= M.A. | coauthors=Maguire, M.E.|year=1999| title= The CorA Mg2+ transport protein of Salmonella typhimurium. Mutatgenesis of conserved residues in the second transmembrane domain| journal=Journal of Biological Chemistry | volume=274| pages=36973–36979| doi=10.1074/jbc.274.52.36973| pmid=10601252] .

Before the activity of CorA could be studied "in vivo", any other Mg2+ transport systems in the bacterial host had to be identified and inactivated/deleted (see below). A strain of "S. typhimurium" containing a functional CorA gene but lacking MgtA and MgtB was constructedcite journal| last=Hmiel| first= S.P. | coauthors=Snavely, M.D., Florer, J.B., Maguire, M.E., and Miller, C.G.|year=1989| title= Magnesium transport in Salmonella typhimurium: genetic characterization and cloning of three magnesium transport loci| journal=Journal of Bacteriology| volume= 171| pages=4742–4751] (see also below), and the uptake kinetics of the transporter were analysedcite journal| last=Snavely| first= M.D. | coauthors=Florer, J.B., Miller, C.G., and Maguire, M.E.|year=1989b| title= Magnesium transport in Salmonella typhimurium: 28/sup>Mg2+ transport by the CorA, MgtA, and MgtB systems| journal=Journal of Bacteriology | volume=171| pages=4761–4766] . This strain showed nearly normal growth rates on standard media (50 μM Mg2+), but the removal of all three genes created a bacterial strain requiring 100 mM external Mg2+ for normal growth.

Mg2+ is taken up into cells containing only the CorA transport system with similar kinetics and cation sensitivities as the Mg2+ uptake described in the earlier papers, and has additionally been quantified(see table). The uptake of Mg2+ was seen to plateau as in earlier studies, and although no actual mechanism for the decrease in transport has been determined, it has been assumed that the protein is inactivated. Co2+ and Ni2+ are toxic to "S. typhimurium" cells containing a functional CorA protein and this toxicity stems from the blocking of Mg2+ uptake (competitive inhibition) and the accumulation of these ions inside the cell. Co2+ and Ni2+ have been shown to be transported by CorA by using radioactive tracer analysiscite journal| last=Gibson| first= M.M. | coauthors=Bagga, D.A., Miller, C.G., and Maguire, M.E.|year=1991| title= Magnesium transport in Salmonella typhimurium: the influence of new mutations conferring Co2+ resistance on the CorA Mg2+ transport system| journal=Molecular Microbiology| volume= 5| pages=2753–2762| doi=10.1111/j.1365-2958.1991.tb01984.x] , although with lower affinities (Km) and velocities (Vmax) than for Mg2+ (see table). The Km values for Co2+ and Ni2+ are significantly above those expected to be encountered by the cells in their normal environment, so it is unlikely that the CorA transport system mediates the uptake of these ions under natural conditions. To date, the evidence for Mn2+ transport by CorA is limited to "E. coli".

The table shows a comparison of transport kinetics of MgtE and CorA. Key kinetic parameter values for MgtE and CorA are listed. As shown, the data has been generated at differing incubation temperatures. Km and Ki are not significantly altered by the differing incubation temperature. Conversely, Vmax shows a strong positive correlation with temperature, hence the value of Co2+ Vmax for MgtE is not directly comparable with the values for CorA.


Early research

The earliest research showing that yeast take up Mg2+ appears to be Schmidt "et al." (1949). However, these authors only showed altered yeast Mg2+ content in a table within the paper, and the report’s conclusions dealt entirely with the metabolism of phosphate. A series of experiments by Rothsteincite journal| last=Rothstein| first= A., Hayes| coauthors=A., Jennings, D., and Hooper, D.|year=1958| title= The active transport of Mg2+ and Mn2+ into the yeast cell| journal=Journal of General Physiology | volume=41| pages=585–594| doi=10.1085/jgp.41.3.585| pmid=13491823] cite journal| last=Fuhrmann| first= G.-F. | coauthors=Rothstein, A.|year=1968| title= The transport of Zn2+, Co2+ and Ni2+ into yeast cells| journal=Biochimica et Biophysica Acta | volume=163| pages=325–330| doi=10.1016/0005-2736(68)90117-X] shifted the focus more towards the uptake of the metal cations, showing that yeast take up cations with the following affinity series: Mg2+, Co2+, Zn2+ > Mn2+ > Ni2+ > Ca2+ > Sr2+. Additionally, it was suggested that the transport of the different cations is mediated by the same transport systemcite journal| last=Norris| first= P.R. | coauthors=Kelly, D.P.|year=1977| title= Accumulation of cadmium and cobalt by Saccharomyces cerevisiae| journal=Journal of General Microbiology | volume=99| pages=317–324] cite journal| last=Okorokov| first= L.A. | coauthors=Lichko, L.P., Kadomtseva, M., Kholodenko, V.P., Titovsky, V.T., and Kulaev, I.S.|year=1977| title= Energy-dependent transport of manganese into yeast cells and distribtuion of accumulated ions| journal=European Journal of Biochemistry| volume= 75| pages=373–377| doi= 10.1111/j.1432-1033.1977.tb11538.x] cite journal| last=Conklin| first= D.S. | coauthors=Kung, C., and Culbertson, M.R.|year=1993| title= The COT2 gene is required for glucose-dependent divalent cation transport in Saccharomyces cerevisiae| journal=Molecular and Cellular Biology | volume=13| pages=2041–2049] — a situation very much like that in bacteria.

In 1998, MacDiarmid and Gardner finally identified the proteins responsible for the observed cation transport phenotype in "Saccharomyces cerevisiae". The genes involved in this system and a second mitochondrial Mg2+ transport system, functionally identified significantly after the gene was cloned, are described in the sections below.

ALR1 and ALR2

Two genes, ALR1 and ALR2, were isolated in a screen for Al3+ tolerance (resistance) in yeast. Overexpression constructs containing yeast genomic DNA were introduced into wild type yeast and the transformants screened for growth on toxic levels of Al3+. ALR1 and ALR2 containing plasmids allowed the growth of yeast in these conditions.

The Alr1p and Alr2p proteins consist of 859 and 858 amino acids respectively, and are 70% identical. A region in the C-terminal half of these proteins is weakly similar to the full CorA protein. The computer-predicted TM topology of Alr1p is shown in the figure. The presence of a third TM domain was suggested by MacDiarmid and Gardner (1998) on the strength on sequence homology and more recently by Lee and Gardner (2006)cite journal |author=Lee J, Gardner R |title=Residues of the yeast ALR1 protein that are critical for magnesium uptake |journal=Curr Genet |volume=49 |issue=1 |pages=7–20 |year=2006 |pmid=16328501 |doi=10.1007/s00294-005-0037-y] , on the strength of mutagenesis studies, making the TM topology of these proteins more like that of CorA (see figure). Also of note is that Alr1p contains the conserved GMN motif at the outside end of TM 2 (TM 2') and that the mutation of the methionine (M) in this motif to a leucine (L) led to the loss of transport capability.

The figure shows the two possible TM topologies of Alr1p. Part A of the figure shows the computer-predicted membrane topology of the Alr1p protein in yeast. Part B shows the topology of Alr1p based on the experimental results of Lee and Gardner (2006). The GMN motif location is indicated in red and the TM domains in light blue. The orientation in the membrane and the positions of the N- and C-termini are indicated. The various sizes of the soluble domains are given in amino acids (AA). TM domains are numbered by their similarity to CorA. Where any TM domain is missing the remaining domains are numbered with primes. Not drawn to scale.A third ALR-like gene is present in "S. cerevisiae" and there are two homologous genes in both "Schizosaccharomyces pombe" and "Neurospora crassa". These proteins contain a GMN motif like that of CorA, with the exception of the second "N. crassa" gene. No ALR-like genes have been identified in species outside of the fungi.

Membrane fractionation and green fluorescent protein (GFP) fusion studies established that Alr1p is localised to the plasma membranecite journal| last=Graschopf| first= A. | coauthors=Stadler, J.A., Hoellerer, M.K., Eder, S., Sieghardt, M., Kohlwein, S.D., and Schweyen, R.J.|year=2001| title= The yeast plasma membrane protein Alr1 controls Mg2+ homeostasis and is subject to Mg2+-dependent control of its synthesis and degradation| journal=Journal of Biological Chemistry| volume= 276| pages=16216–16222| doi=10.1074/jbc.M101504200| pmid=11279208] . Interestingly, the localisation of the Alr1p was observed to be internalised and degraded in the vacuole in response to extracellular cations. Mg2+ at very low extracellular concentrations (100 μM; < 10% of the standard media Mg2+ content) and Co2+ and Mn2+ at relatively high concentrations (> 20× standard media) induced the change in Alr1p protein localisation and the effect was dependent on functional ubiquitination, endocytosis and vacuolar degradation. This mechanism allows a regulation of Mg2+ uptake by yeast. Whether this degradation is triggered by the binding of the ions to the transporter or by some less direct mechanism has not been determined.

A functional Alr1p (wild type) or Alr2p (overexpressed) is required for "S. cerevisiae" growth in standard conditions (4 mM Mg2+) and Alr1p can support normal growth at Mg2+ concentrations as low as 30 μM. 57Co2+ is taken up into yeast via the Alr1p protein with a Km of 77 – 105 μM (; C. MacDiarmid and R. C. Gardner, unpublished data), but the Ki for Mg2+ inhibition of this transport is currently unknown. The transport of other cations by the Alr1p protein was assayed by the inhibition of yeast growth. The overexpression of Alr1p led to increased sensitivity to Ca2+, Co2+, Cu2+, La3+, Mn2+, Ni2+ and Zn2+, an array of cations similar to those shown to be transported into yeast by a CorA-like transport system. The increased toxicity of the cations in the presence of the transporter is assumed to be due to the increased accumulation of the cation inside the cell.

The evidence that Alr1p is primarily a Mg2+ transporter is that the loss of Alr1p leads to a decreased total cell content of Mg2+ but not of the other cations, and that the regulation of the transporter occurs at environmental concentrations of Mg2+, but not the other cations tested. Additionally, two electrophysiological studies where Alr1p was produced in yeast or "Xenopus" oocytes showed a Mg2+-dependent current in the presence of the proteincite journal| last=Liu| first= G.J. | coauthors=Martin, D.K., Gardner, R.C., and Ryan, P.R.|year=2002| title= Large Mg2+-dependent currents are associated with the increased expression of ALR1 in Saccharomyces cerevisiae| journal=FEMS Microbiology Letters | volume=213| pages=231–237| doi=10.1111/j.1574-6968.2002.tb11311.x] ; Salih "et al.", in prep.).

The kinetics of Mg2+ uptake by Alr1p have been investigated by electrophysiology techniques on whole yeast cells. The results suggested that Alr1p is very likely to act as an ion-selective channel. In the same paper the authors reported that Mg2+ transport by Alr1p varied from 200 pA to 1500 pA, with a mean current of 264 pA. No quantification of the amount of protein producing the current was presented, and so the results lack comparability with the bacterial Mg2+ transport proteins.

The alternative techniques of 28Mg2+ radiotracer analysis and mag-fura 2 to measure Mg2+ uptake have not yet been used with Alr1p. 28Mg2+ is currently not available and the mag-fura 2 system is unlikely to provide simple uptake data in yeast. The yeast cell maintains a heterogeneous distribution of Mg2+cite journal| last=Zhang| first= A. | coauthors=Cheng, T.P., Wu, X.Y., Altura, B.T., and Altura, B.M.|year=1997| title= Extracellular Mg2+ regulates intracellular Mg2+ and its subcellular compartmentation in fission yeast, Schizosaccharomyces pombe| journal=Cellular and Molecular Life Sciences | volume=53| pages=69–72| doi=10.1007/PL00000581] suggesting that multiple systems inside the yeast are transporting Mg2+ into storage compartments. This internal transport will very likely mask the uptake process. The expression of ALR1 in "S. typhimurium" without Mg2+ uptake genes may be an alternative, but as stated earlier, the effects of a heterologous expression system would need to be taken into account.

MRS2 and Lpe10

Like the ALR genes the MRS2 gene was cloned and sequenced before it was identified as a Mg2+ transporter. The MRS2 gene was identified in the nuclear genome of yeast in a screen for suppressors of a mitochondrial gene RNA splicing mutationcite journal| last=Koll| first= H. | coauthors=Schmidt, C., Wiesenberger, G., and Schmelzer, C.|year=1987| title= Three nuclear genes suppress a yeast mitochondrial splice defect when present in high copy number| journal=Current Genetics | volume=12| pages=503–510| doi= 10.1007/BF00419559] , and was cloned and sequenced by Wiesenberger "et al." (1992)cite journal| last=Wiesenberger| first= G. | coauthors=Waldherr, M., and Schweyen, R.J.|year=1992| title= The nuclear gene MRS2 is essential for the excision of group II introns from yeast mitochondrial transcripts "in vivo"| journal=Journal of Biological Chemistry | volume=267| pages=6963–6969] . Mrs2p was not identified as a putative Mg2+ transporter until Bui "et al." (1999). Gregan "et al." (2001a) identified LPE10 by homology to MRS2 and showed that both LPE10 and MRS2 mutants altered the Mg2+ content of yeast mitochondria and affected RNA splicing activity in the organellecite journal| last=Gregan| first= J. | coauthors=Bui, D.M., Pillich, R., Fink, M., Zsurka, G., and Schweyen, R.J.|year=2001a| title= The mitochondrial inner membrane protein Lpe10p, a homologue of Mrs2p, is essential for magnesium homeostasis and group II intron splicing in yeast| journal=Molecular Genome and Genetics | volume=264| pages=773–781] cite journal| last=Gregan| first= J. | coauthors=Kolisek, M., and Schweyen, R.J.|year=2001b| title= Mitochondrial Mg2+ homeostasis is critical for group II intron splicing "in vivo"| journal=Genes & Development | volume=15| pages=2229–2237| doi=10.1101/gad.201301] . Mg2+ transport has been shown to be directly mediated by Mrs2p, but not for Lpe10p.

The Mrs2p and Lpe10p proteins are 470 and 413 amino acid residues in size, respectively, and a 250 – 300 amino acid region in the middle of the proteins shows a weak similarity to the full CorA protein. The TM topologies of the Mrs2p and Lpe10p proteins have been assessed using a protease protection assay and are shown in the figure. TM 1 and 2 correspond to TM 2 and 3 in the CorA protein. Note the conserved GMN motif at the outside end of the first TM domain. When the glycine (G) in this motif was mutated to a cysteine (C) in Mrs2p, Mg2+ transport was strongly reduced.

The figure shows the experimentally determined topology of Mrs2p and Lpe10p as adapted from Bui "et al." (1999) and Gregan "et al." (2001a). The GMN motif location is indicated in red and the TM domains in light blue. The orientation in the membrane and the positions of the N- and C-termini are indicated. The various sizes of the soluble domains are given in amino acids (AA). TM domains are numbered. Not drawn to scale.

Mrs2p has been localised to the mitochondrial inner membrane by subcellular fractionation and immunodetection and Lpe10p to the mitochondria. Mitochondria lacking Mrs2p do not show a fast Mg2+ uptake, but only a slow ‘leak’, and overaccumulation of Mrs2p leads to an increase in the initial rate of uptake. Additionally CorA, when fused to the mitochondrial leader sequence of Mrs2p, can partially complement the mitochondrial defect conferred by the loss of either Mrs2p or Lpe10p. Hence, Mrs2p and/or Lpe10p may be the major Mg2+ uptake system for mitochondria. An intriguing possibility is that the proteins form heterodimers, as neither protein (when overexpressed) can fully complement the loss of the other.

The characteristics of Mg2+ uptake into isolated mitochondria by Mrs2p were quantified using mag-fura 2. The uptake of Mg2+ by Mrs2p shared a number of attributes with CorA. First, Mg2+ uptake was directly dependent on the electric potential (ΔΨ) across the boundary membrane. Second, the uptake is saturated far below that which the ΔΨ theoretically permits. Thus, the transport of Mg2+ by Mrs2p is likely to be regulated in a similar manner to CorA, possibly by the inactivation of the protein. Third, Mg2+ efflux was observed via Mrs2p upon the artificial depolarisation of the mitochondrial membrane by valinomycin. Finally, the Mg2+ fluxes through Mrs2p are inhibited by cobalt (III) hexaammine.

The kinetics of Mg2+ uptake by Mrs2p were determined as in the Froschauer "et al." (2004) paper on CorA in bacteria. The initial change in free Mg2+ concentration was 150 μM s-1 for wild type and 750 μM s-1 for mitochondria from yeast overexpressing MRS2. No attempt was made to scale the observed transport to the amount of transporter present.

Protozoan ("Paramecium")

The transport of Mg2+ into Paramecium has been characterised largely by R. R. Preston and coworkers. Electrophysiological techniques on whole Paramecium were used to identify and characterise Mg2+ currents in a series of paperscite journal| last=Preston| first= R.R.|year=1990| title= A magnesium current in Paramecium| journal=Science | volume=250| pages=285–288| doi= 10.1126/science.2218533| pmid=2218533] cite journal| last=Preston| first= R.R. | coauthors=Kung, C.|year=1994a| title= Inhibition of Mg2+ current by single-gene mutation in Paramecium| journal=Journal of Membrane Biology | volume=137| pages=203–212] cite journal| last=Preston| first= R.R. | coauthors=Kung, C.|year=1994b| title= Isolation and characterization of paramecium mutants defective in their response to magnesium| journal=Genetics | volume=137| pages=759–769] cite journal| last=Preston| first= R.R.|year=1998| title= Transmembrane Mg2+ currents and intracellular free Mg2+ concentration in Paramecium tetraurelia| journal=Journal of Membrane Biology| volume= 164| pages=11–24| doi=10.1007/s002329900389] before the gene was cloned by Haynes "et al." (2002).

The open reading frame for the XNTA gene is 1707 bp in size, contains two introns and produces a predicted protein of 550 amino acids. The protein has been predicted to contain 11 TM domains and also contains the α1 and α2 motifs (see figure) of the SLC8 (Na+/Ca2+ exchangercite journal| last=Quednau| first= B.D. | coauthors=Nicoll, D.A., and Philipson, K.D.|year=2004| title= The sodium/calcium exchanger family—SLC8| journal=Pflügers Archiv European Journal of Physiology| volume= 447| pages=543–548| doi= 10.1007/s00424-003-1065-4] ) and SLC24 (K+ dependent Na+/Ca2+ exchangercite journal| last=Schnetkamp| first= P.P.M.|year=2004| title= The SLC24 Na+/Ca2+-K+ exchanger family: vision and beyond| journal=Pflügers Archiv European Journal of Physiology| volume= 447| pages=683–688| doi=10.1007/s00424-003-1069-0] ) human solute transport proteins. The XntAp is equally similar to the SLC8 and SLC24 protein families by amino acid sequence, but the predicted TM topology is more like that of SLC24. In any case, the similarity is at best weak and the relationship is very distant. It is interesting to note that the AtMHX protein from plants also shares a distant relationship with the SLC8 proteins.

The figure shows the predicted TM topology of XntAp. Adapted from Haynes "et al." (2002), this figure shows the computer predicted membrane topology of XntAp in "Paramecium". The orientation in the membrane was determined using HMMTOPcite journal| last=Tusnady| first= G.E. | coauthors=Simon, I.|year=1998| title= Principles governing amino acid composition of integral membrane proteins: application to topology prediction| journal=Journal of Molecular Biology | volume=283| pages=489–506| doi= 10.1006/jmbi.1998.2107] cite journal| last=Tusnady| first= G.E. | coauthors=Simon, I.|year=2001| title= The HMMTOP transmembrane topology prediction server| journal=Bioinformatics | volume=17| pages=849–850| doi= 10.1093/bioinformatics/17.9.849| pmid=11590105] . The TM domains are shown in light blue. The α1 and α2 domains are shown in green. The orientation in the membrane and the positions of the N- and C-termini are indicated. Not drawn to scale.

The Mg2+-dependent currents carried by XntAp are kinetically like that of a channel protein, and have an ion selectivity order of Mg2+ > Co2+, Mn2+ > Ca2+ — a series again very similar to that of CorA. Unlike the other transport proteins reported so far, XntAp is dependent on intracellular Ca2+. The transport was also dependent on ΔΨ, but again Mg2+ is not transported to equilibrium, being limited to approximately 0.4 mM free Mg2+ in the cytoplasm. The existence of an intracellular compartment with a much higher free concentration of Mg2+ (8 mM) was also supported by the results.


The investigation of Mg2+ in animals, including humans, has lagged behind that in bacteria and yeast. This is largely because of the complexity of the systems involved, but it is also due to the impression within the field that Mg2+ was maintained at high levels within all cells and was unchanged by external influences. Only in the last 25 years has a series of reports begun to challenge this view, with new methodologies finding that free Mg2+ content is maintained at levels where changes might influence cellular metabolismcite journal| last=Romani| first= A.M.P. | coauthors=Maguire, M.E.|year=2002| title= Hormonal regulation of Mg2+ transport and homeostasis in eukaryotic cells| journal=BioMetals | volume=15| pages=271–283| doi=10.1023/A:1016082900838] .


A bioinformatic search of the sequence databases identified one homologue of the MRS2 gene of yeast in a range of metazoans. The protein has a very similar sequence and predicted TM topology to the yeast protein, and the GMN motif is intact at the end of the first TM domain. The human protein, hsaMrs2p, has been localised to the mitochondrial membrane in mouse cells using a GFP fusion protein.

Very little is known with regard to the Mg2+ transport characteristics of the protein in mammals. However, Zsurka "et al." (2001) have shown that the human Mrs2p complements the mrs2 mutants in the yeast mitochondrial Mg2+ uptake system.

LC41 (MgtE)

The identification of this gene family in the metazoa began with a signal sequence trap method for isolating secreted and membrane proteins. Much of the identification has come from bioinformatic analyses. Three genes were eventually identified in humans, another three in mouse and three in "Caenorhabditis elegans", with a single gene in "Anopheles gambiae". The pFAM database lists the MgtE domain as pFAM01769 and additionally identifies an MgtE domain-containing protein in "Drosophila melanogaster". The proteins containing the MgtE domain can be divided into seven classes, as defined by pFAM using the type and organisation of the identifiable domains in each protein. Metazoan proteins are present in three of the seven groups. All of the metazoa proteins contain two MgtE domains. However, some of these have been predicted only by context recognition (Coin, Bateman and Durbin, unpublished. See the pFAM website for further details).

The human SLC41A1 protein contains two MgtE domains with 52% and 46% respective similarity to the PF01769 consensus sequence and is predicted to contain ten TM domains, five in each MgtE domain (see Figure). Incidentally, this structure suggests that the MgtE protein of bacteria may work as a dimer. Adapted from Wabakken "et al." (2003) and the pFAM database, the figure shows the computer predicted membrane topology of MgtE in "H. sapiens". The TM domains are shown in light blue. The orientation in the membrane and the positions of the N- and C-termini are indicated. The figure is not drawn to scale.

Wabakken "et al." (2003) found that the transcript of the SLC41A1 gene was expressed in all human tissues tested, but at varying levels. The tissues with the most abundant expression of the gene were the heart and testis. No explanation of the expression pattern has been suggested with regard to Mg2+-related physiology.

It has not been shown whether the SLC41 proteins transport Mg2+ or complement a Mg2+ transport mutation in any experimental system. However, it has been suggested that as MgtE proteins have no other known function, they are likely to be Mg2+ transporters in the metazoa as they are in the bacteria. This will need to be verified using one of the now standard experiment systems for examining Mg2+ transport.


The investigation of the TRPM genes and proteins in human cells is an area of intense recent study and, at times, debate. Montell "et al." (2002)cite journal| last=Montell| first= C. | coauthors=Birnbaumer, L., and Flockerzi, V.|year=2002| title= The TRP Channels, a remarkably functional family| journal=Cell | volume=108| pages=595–598| doi= 10.1016/S0092-8674(02)00670-0] have reviewed the research into the TRP genes, and a second review by Montell (2003)cite journal| last=Montell| first= C.|year=2003| title= Mg2+ Homeostasis: The Mg2+nificent TRPM Chanzymes| journal=Current Biology | volume=13| pages=R799–R801| doi=10.1016/j.cub.2003.09.048] has reviewed the research into the TRPM genes.

The TRPM family of ion channels has members throughout the metazoa. The TRPM6 and TRPM7 proteins are highly unusual, containing both an ion channel domain and a kinase domain (Figure 1.7). The role of the kinase domain brings about the most heated debate.

The activity of these two proteins has been very difficult to quantify. TRPM7 by itself appears to be a Ca2+ channelcite journal| last=Runnels| first= L.W. | coauthors=Yue, L., and Clapham, D.E.|year=2002| title= The TRPM7 channel is inactivated by PIP2 hydrolysis| journal=Nature Cell Biology| volume= 4| pages=329–336] but in the presence of TRPM6 the affinity series of transported cations places Mg2+ above Ca2+.cite journal| last=Monteilh-Zoller| first= M.K. | coauthors=Hermosura, M.C., Nadler, M.J.S., Scharenberg, A.M., Penner, R., and Fleig, A.|year=2003| title= TRPM7 provides an ion channel mechanism for cellular entry of trace metal ions| journal=Journal of General Physiology| volume= 121| pages=49–60| doi= 10.1085/jgp.20028740| pmid=12508053] The differences in reported conductance were caused by the expression patterns of these genes. TRPM7 is expressed in all cell types tested so far, while TRPM6 shows a more restricted pattern of expressioncite journal| last=Chubanov| first= V. | coauthors=Waldegger, S., Mederos y Schnitzler, M., Vitzthum, H., Sassen, M.C., Seyberth, H.W., Konrad, M., and Gudermann, T.|year=2004| title= Disruption of TRPM6/TRPM7 complex formation by a mutation in the TRPM6 gene causes hypomagnesemia with secondary hypocalcemia| journal=PNAS | volume=101| pages=2894–2899| doi= 10.1073/pnas.0305252101| pmid=14976260] . An unfortunate choice of experimental system by Voets "et al.", (2004)cite journal| last=Voets| first= T. | coauthors=Nilius, B., Hoefs, S., van der Kemp, A.W.C.M., Droogmans, G., Bindels, R.J.M., and Hoenderop, J.G.J.|year=2004| title= TRPM6 forms the Mg2+ influx channel involved in intestinal and renal Mg2+ absorption| journal=Journal of Biological Chemistry | volume=279| pages=19–25| doi=10.1074/jbc.M311201200| pmid=14576148] led to the conclusion that TRPM6 is a functional Mg2+ transporter. However, later work by Chubanov "et al." (2004) clearly showed that TRPM7 is required for TRPM6 activity, and that the results of Voets "et al." are explained by the expression of TRPM7 in the experimental cell line used by Voets "et al." in their experiments. Whether TRPM6 is functional by itself is yet to be determined.

The predicted TM topology of the TPRM6 and TRPM7 proteinshave been adapted from Nadler "et al." (2001), Runnels "et al." (2001)cite journal| last=Runnels| first= L.W. | coauthors=Yue, L., and Clapham, D.E.|year=2001| title= TRP-PLIK, a bifunctional protein with kinase and ion channel activities| journal=Science| volume= 291| pages=1043–1047| doi= 10.1126/science.1058519| pmid=11161216] and Montell "et al." (2002), this figure shows the computer predicted membrane topology of the TRPM6 and TRPM7 proteins in "Homo sapiens". At this time the topology shown should be considered a tentative hypothesis. The TM domains are shown in light blue, the pore loop in purple, the TRP motif in red and the kinase domain in green. The orientation in the membrane and the positions of the N- and C-termini are indicated. Not drawn to scale.

The conclusions of the Voets "et al." (2004) paper are probably incorrect in attributing the Mg2+ dependent currents to TRPM7 alone, and their kinetic data are likely to reflect the combined TRPM7/ TRPM6 channel. The report presents a robust collection of data consistent with a channel-like activity passing Mg2+, based on both electrophysiological techniques and also mag-fura 2 to determine changes in cytoplasmic free Mg2+.

Claudin-16 (Paracellin-1, PCLN-1)

This gene is the only known example of Mg2+ transport via the paracellular pathway; that is, it mediates the transport of the ion through the tight junctions between cells that form an epithelial cell layer. In particular, Claudin-16 allows the selective reuptake of Mg2+ in the human kidney.

The gene Claudin-16 was cloned by Simon "et al." (1999), again only after a series of reports describing the Mg2+ flux itself with no gene/proteincite journal| last=Shareghi| first= G.R. | coauthors=Agus, Z.S.|year=1982| title= Magnesium transport in the cortical thick ascending limb of Henle's loop of the rabbit| journal=Journal of Clinical Investigation | volume=69| pages=759–769| doi= 10.1172/JCI110514] cite journal| last=di Stefano| first= A. | coauthors=Roinel, N., de Rouffignac, C., and Wittner, M.|year=1993| title= Transepithelial Ca2+ and Mg2+ transport in the cortical thick ascending limb of Henle's loop of the mouse is a voltage-dependent process| journal=Renal Physiology and Biochemistry| volume= 16| pages=157–166] cite journal| last=de Rouffignac| first= C. | coauthors=Quamme, G.|year=1994| title= Renal magnesium handling and its hormonal control| journal=Physiology Reviews| volume= 74| pages=305–322] . The expression pattern of the gene was determined by RT-PCR, and was shown to be very tightly confined to a continuous region of the kidney tubule running from the medullary thick descending limb to the distal convoluted tubule. This localisation was consistent with the earlier reports for the location of Mg2+ re-uptake by the kidney. Following the cloning, mutations in the gene were identified in patients with familial hypomagnesaemia with hypercalciuria and nephrocalcinosiscite journal| last=Weber| first= S. | coauthors=Hoffmann, K., Jeck, N., Saar, K., Boeswald, M., Kuwertz-Broeking, E., Meij, I.I.C., Knoers, N.V.A.M., Cochat, P., Sulakova, T., Bonzel, K.E., Soergel, M., Manz, F., Schaerer, K., Seyberth, H.W., Reis, A., and Konrad, M.|year=2000| title= Familial hypomagnesaemia with hypercalciuria and nephrocalcinosis maps to chromosome 3q27 and is associated with mutations in the PCLN-1 gene| journal=European Journal of Human Genetics| volume= 8| pages=414–422| doi= 10.1038/sj.ejhg.5200475] cite journal| last=Weber| first= S. | coauthors=Schneider, L., Peters, M., Misselwitz, J., Roennefarth, G., Boeswald, M., Bonzel, K.E., Seeman, T., Sulakova, T., Kuwertz-Broeking, E., Gregoric, A., Palcoux, J.-B., Tasic, V., Manz, F., Schaerer, K., Seyberth, H.W., and Konrad, M.|year=2001| title= Novel paracellin-1 mutations in 25 families with familial hypomagnesemia with hypercalciuria and nephrocalcinosis| journal=Journal of the American Society of Nephrology | volume=12| pages=1872–1881] , strengthening the links between the gene and the uptake of Mg2+.


The current knowledge of the molecular mechanisms for Mg2+ transport is plants is very limited, with only three publications reporting a molecular basis for Mg2+ transport in plants. However, the importance of Mg2+ to plants has been well described, and physiological and ecophysiological studies into the effects of Mg2+ are numerous. This section will summarise the knowledge of a gene family identified in plants that is distantly related to CorA. Another gene unrelated to this gene family and to CorA has also been identified. This Mg2+/H+ exchanger (AtMHX), localised to the vacuolar membrane, is described last.

The AtMRS2 gene family

Schock "et al." (2000) identified and named the family AtMRS2 based on the similarity of the genes to the MRS2 gene of yeast. The authors also showed that the AtMRS2-1 gene could complement a Δmrs2 yeast mutant phenotype. Independently, Li "et al." (2001) published a report identifying the family and showing that two additional members could complement Mg2+ transport deficient mutants, one in "S. typhimurium" and the other in "S. cerevisiae".

The three genes that have been shown to transport Mg2+ are AtMRS2-1, AtMRS2-10 and AtMRS2-11, and these genes produce proteins 442, 443 and 459 amino acids in size, respectively. Each of the proteins is predicted to have two TM domains, and each shows significant similarity to Mrs2p of yeast and a weak similarity to CorA of bacteria. All three proteins contain the conserved GMN amino acid motif at the outside end of the first TM domain.

The AtMRS2-1 gene, when expressed in yeast from the MRS2 promoter and being fused C-terminally to the first 95 amino acids of the Mrs2p protein, was directed to the mitochondria, where it complemented a Δmrs2 mutant both phenotypically (mitochondrial RNA splicing was restored) and with respect to the Mg2+ content of the organelle. No data on the kinetics of the transport were presented. The AtMRS2-11 gene was analysed in yeast (in the alr1alr2 strain), where it was shown that expression of the gene significantly increased the rate of Mg2+ uptake into starved cells over the control, as measured using flame atomic absorption spectroscopy of total cellular Mg2+ content. However, Alr1p was shown to be significantly more effective at transporting Mg2+ at low extracellular concentrations, suggesting that the affinity of AtMRS2-11 for Mg2+ is lower than that of Alr1p. An electrophysiological (voltage clamp) analysis of the AtMRS2-11 protein in Xenopus oocytes also showed a Mg2+-dependent current at membrane potentials (ΔΨ) of –100 – –150 mV insidecite journal| last=Tutone| first= A.F.|year=2004| title= Cloning and chararcterisation of the Mg2+ transport gene from A. thaliana. In School of Biological Sciences (Auckland: University of Auckland)] . These values are physiologically significant, as several membranes in plants maintain ΔΨ in this range. However, the author had difficulty reproducing these results due to an apparent ‘death’ of oocytes containing the AtMRS2-11 protein, and therefore these results should be viewed with caution.

The AtMRS2-10 transporter has been analysed using radioactive tracer uptake analysis. 63Ni2+ was used as the substitute ion and Mg2+ was shown to inhibit the uptake of 63Ni2+ with a Ki of 20 μM. Uptake was also inhibited by Co(III)Hex and by other divalent cations. Only Co2+ and Cu2+ inhibited transport with Ki values less than 1 mM.

The AtMRS2-10 protein was fused to GFP, and shown to be localised to the plasma membrane. A similar experiment was attempted in the Schock "et al." (2000) paper, but the observed localisation was not significantly different from that seen with unfused GFP. The most likely reason for the lack of a definitive localisation of AtMRS2-1 in the Schock "et al." paper is that the authors removed the TM domains from the protein, thereby precluding its insertion into a membrane.

The exact physiological significance of the AtMRS2-1 and AtMRS2-10 proteins in plants has yet to be clarified. The AtMRS2-11 gene has been overexpressed (from the CaMV 35S promoter) in A. thaliana. The transgenic line has been shown to accumulate high levels of the AtMRS2-11 transcript. A strong Mg2+ deficiency phenotype (necrotic spots on the leaves, see Chapter 1.5 below) was recorded during the screening process (in both the T1 and T2 generations) for a homozygote line. However, this phenotype was lost in the T3 generation and could not be reproduced when the earlier generations were screened a second time. The author suggested that environmental affects were the most likely cause of the inconsistent phenotype.


The first magnesium transporter isolated in any multicellular organism, AtMHX shows no similarity to any previously isolated Mg2+ transport protein. The gene was initially identified in the A. thaliana genomic DNA sequence database, by its similarity to the SLC8 family of Na+/Ca2+ exchanger genes in humans.

The cDNA sequence of 1990 bp is predicted to produce a 539-amino acid protein. AtMHX is quite closely related to the SLC8 family at the amino acid level and shares a topology with eleven predicted TM domains (Figure A10.5). There is one major difference in the sequence, however, in that the long non-membranal loop (see Figure A10.5) is 148 amino acids in the AtMHX protein but 500 amino acids in the SLC8 proteins. However, this loop is not well conserved and is not required for transport function in the SLC8 family.

The AtMHX gene is expressed throughout the plant but most strongly in the vascular tissue. The authors suggest that the physiological role of the protein is to store Mg2+ in these tissues for later release on need. The protein localisation to the vacuolar membrane supports this suggestion (see also Chapter 1.5).

The protein transports Mg2+ into the vacuolar space and H+ out, as demonstrated by electrophysiological techniques. The transport is driven by the ΔpH maintained between the vacuolar space (pH 4.5 – 5.9) and the cytoplasm (pH 7.3 – 7.6) by an H+-ATPasecite journal| last=Kurkdjian| first= A. | coauthors=Guern, J.|year=1989| title= Intracellular pH: measurement and importance in cell activity| journal=Annual Review of Plant Physiology and Plant Molecular Biology | volume=40| pages=271–303| doi= 10.1146/annurev.pp.40.060189.001415] cite book| last=Marschner| first= H.|year=1995| title= Mineral Nutrition in Higher Plants. (San Diego: Academic Press)] . How the transport of Mg2+ by the protein is regulated was not determined. Currents were observed to pass through the protein in both directions, but the Mg2+ out current required a ‘cytoplasmic’ pH of 5.5, a condition not found in plant cells under normal circumstances. In addition to the transport of Mg2+, Shaul "et al." (1999) also showed that the protein could transport Zn2+ and Fe2+, but did not report on the capacity of the protein to transport other divalent cations (e.g. Co2+ and Ni2+) or its susceptibility to inhibition by cobalt (III) hexaammine.

The detailed kinetics of Mg2+ transport have not been determined for AtMHX. However, physiological effects have been demonstrated. When A. thaliana plants were transformed with overexpression constructs of the AtMHX gene driven by the CaMV 35S promoter, the plants over-accumulated the protein. The plants showed a phenotype of necrotic lesions in the leaves, and the authors suggest that this is caused by a disruption in the normal function of the vacuole, given their observation that the total Mg2+ (or Zn2+) content of the plants was not altered in the transgenic plants.

The image has been adapted from Shaul "et al." (1999) and Quednau et al. (2004), and combined with an analysis using HMMTOP, this figure shows the computer predicted membrane topology of the AtMHX protein in "Arabidopsis thaliana". At this time the topology shown should be considered a tentative hypothesis. The TM domains are shown in light blue. The orientation in the membrane and the positions of the N- and C-termini are indicated. Not drawn to scale. The α1 and α2 domains (shown in green) are both quite hydrophobic and may both be inserted into the membrane.


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