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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 protein^[1] and, where the CorA protein is absent, by the MgtE protein^[2]^[3]. In yeast the initial uptake is via the Alr1p and Alr2p proteins^[4], but at this stage the only internal Mg²⁺ distributing protein identified is Mrs2p^[5]. Within the protozoa only one Mg²⁺ transporter (XntAp) has been identified^[6]. In metazoa, Mrs2p^[7] and MgtE homologues^[8] have been identified, along with two novel Mg²⁺ transport systems TRPM6/TRPM7^[9]^[10] and PCLN-1^[11]. Finally, in plants, a family of Mrs2p homologues has been identified^[12]^[13] along with another novel protein, AtMHX^[14].

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, Borrelly^[15] 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 directly^[16].

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 Kolisek^[17] and Froschauer^[18] 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.

Bacteria

Early research

In 1968 Lusk^[19] 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 group^[20] and another group, Silver^[21], 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^[22] and in Aerobacter aerogenes and Bacillus megaterium^[23]. 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⁺ ^[24]^[25].

CorA

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^[24].

The gene was genetically identified in E. coli by Park et al.^[25], but wasn’t cloned until Hmiel et al.^[1] isolated the Salmonella enterica serovar Typhimurium (S. typhimurium) homologue. Later it would be shown by Smith and Maguire^[26] 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 Archaea^[27]. 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%^[27]. 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 (www.sanger.ac.uk/Software/Pfam/), 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⁺ concentrations^[28]. However, recent evidence suggests that the activity of the protein may be regulated by the PhoPQ two-component regulatory system^[29]. This sensor responds to low external Mg²⁺ during the infection process of S. typhimurium in humans^[30]. 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^[28]. 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⁺^[29]. For a conflicting report on the source of toxicity, see Papp and Maguire^[31].

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.^[32]. Evidence for CorA acting as a homotetramer was published by Warren et al. in 2004^[33]. 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. Follow this link to see the structure in 3D. 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 transporter^[34]. 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)^[34]^[35].

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 constructed^[36](see also below), and the uptake kinetics of the transporter were analysed^[37]. 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^[36].

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^[37](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^[18]. 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^[1]. Co²⁺ and Ni²⁺ have been shown to be transported by CorA by using radioactive tracer analysis^[1]^[38], 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^[1]. To date, the evidence for Mn²⁺ transport by CorA is limited to E. coli^[25].

	Mg²⁺	Co²⁺	Ni²⁺
Km (µM)	15	30	240
Vmax (pmol/min/10⁸ cells)	250	500	360
Ki (µM) - Mg	-	-	10
Ki (µM) - Co	50	-	20
Ki (µM) - Mn	30	-	-
Ki (µM) - Ni	300	-	300

The table lists the transport kinetics of the CorA Mg²⁺ transport system. This table has been compiled from the publications of Snavely et al. (1989b)^[37], Gibson et al. (1991)^[38] and Smith et al. (1998a)^[34] and summarises the kinetic data currently available for the CorA transport protein expressed from the wild type promoter in bacteria lacking MgtA and MgtB. Km and Vmax were determined at 20 °C as the uptake of Mg²⁺ at 37 °C was too rapid to measure accurately.

Recently the Mg²⁺-dependent fluorescence of mag-fura 2 was used to measure the free Mg²⁺ content of S. typhimurium cells in response to external Mg²⁺, and this again showed that CorA is the major uptake system for Mg²⁺ in bacteria^[18]. The authors also showed for the first time that the changes in the electric potential (ΔΨ) across the plasma membrane of the cell affected both the rate of Mg²⁺ uptake and the free Mg²⁺ content of the cell; depolarisation suppressed transport, while hyperpolarisation increased transport. The kinetics of transport were defined only by the rate of change of free Mg²⁺ inside the cells (250 μM s^-1). Because no quantification of the amount of CorA protein in the membrane was made, this value is difficult to compare with other experiments on Mg²⁺ transporters^[17].

The efflux of Mg²⁺ from bacterial cells was first observed by Lusk and Kennedy (1969)^[20] and is mediated by the CorA Mg²⁺ transport system in the presence of high extracellular concentrations of Mg²⁺^[37]. The efflux can also be triggered by Co²⁺, Mn²⁺ and Ni²⁺, although not to the same degree as Mg²⁺^[22]. No Co²⁺ efflux through the CorA transport system was observed. The process of Mg²⁺ efflux additionally requires one of the CorB, CorC or CorD genes^[38]. Interestingly, the mutation of any single one of these genes leads to a Co²⁺ resistance a little less than half of that provided by a corA mutant. Presumably this effect is due to the inhibition of Mg²⁺ loss that would otherwise occur in the presence of high levels of Co²⁺. An interesting, and currently unanswered, question is whether Mg²⁺ is more toxic when the CorBCD genes are deleted.

It has been speculated that the Mg²⁺ ion will initially interact with any transport protein through its hydration shell^[39]. Cobalt (III) hexaammine, Co(III)Hex, is a covalently bound (non-labile) analog for the first shell of hydration for several divalent cations, including Mg²⁺. The radius of the Co(III)Hex molecule is 244 pm, which is very similar to the 250 pm radius of the first hydration shell of Mg²⁺. This analog is a potent inhibitor of the CorA transport system, more so than Mg²⁺, Co²⁺ or Ni²⁺^[40]. The additional strength of the Co(III)Hex inhibition might come from the blocking of the transport pore due to the inability of the protein to ‘dehydrate’ the substrate. It was also shown that Co(III)Hex was not transported into the cells^[40], suggesting that at least partial dehydration would be required for the transport of the normal substrate (Mg²⁺). Nickel (II) hexaammine , with a radius of 255 pm, did not inhibit the CorA transport system, suggesting a maximum size limit exists for the binding of the CorA substrate ion^[40]. These results suggest that the important property involved in the recognition of Mg²⁺ by CorA is the size of the ion with its first shell of hydration. Hence, the volume change generally quoted for the bare to hydrated Mg²⁺ ion of greater than 500-fold, including the second sphere of hydration, may not be biologically relevant and perhaps the first sphere volume change of 56-fold should be more commonly used.

MgtA and MgtB

The presence of these two genes was first suspected when Nelson and Kennedy (1972)^[24] showed that there were Mg²⁺-repressible and Mg²⁺-non-repressible Mg²⁺ uptake systems in E. coli. The non-repressible uptake of Mg²⁺ is mediated by the CorA protein. In S. typhimurium the repressible Mg²⁺ uptake was eventually shown to be via the MgtA and MgtB proteins^[36].

Both MgtA and MgtB are regulated by the PhoPQ system and are actively transcribed during the process of infection of human patients by S. typhimurium^[41]^[42]^[30]. Although neither gene is required for pathogenicity the MgtB protein does enhance the long term survival of the pathogen in the cell^[43]. The genes are also upregulated in vitro when the Mg²⁺ concentration falls below 50 μM (Snavely et al., 1991a). Although the proteins have Km values similar to CorA and transport rates approximately 10-fold less the genes may be part of a Mg²⁺ scavenging system. Chamnongpol and Groisman (2002) presents evidence that the role of these proteins may be to compensate for the inactivation of the CorA protein by the PhoPQ regulon^[29]. The authors suggest that the CorA protein is inactivated to allow the avoidance of metal toxicity via the protein in the low Mg²⁺ environments S. typhimurium is subjected to by cells after infection.

The proteins are both P-type ATPases^[37] ^[44] and neither gene shows any similarity to CorA. The MgtA and MgtB proteins are 75% similar (50% identical), although it seems that MgtB may have been acquired by horizontal gene transfer as part of Salmonella Pathogenicity Island 3^[45]^[44]. The TM topology of the MgtB protein has been experimentally determined, showing that the protein has ten TM-spanning helices with the termini of the protein in the cytoplasm (see figure ). MgtA is present in widely divergent bacteria, but is not nearly as common as CorA, while MgtB appears to have a quite restricted distribution^[46]. No hypotheses for the unusual distribution have been suggested.

The figure, adapted from Smith et al. (1993b)^[47], shows the experimentally determined membrane topology of the MgtB protein in S. typhimurium. 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.

While the MgtA and MgtB proteins are very similar, they do show some minor differences in activity. MgtB is highly temperature sensitive, losing all activity (with regard to Mg²⁺ transport) at a temperature of 20°C^[37]. Additionally, MgtB and MgtA are inhibited by different ranges of cations (Table A10.1^[37]).

The table lists cation transport characteristics of the MgtA and MgtB proteins in S. typhimurium. The kinetic data for the MgtA and MgtB transport proteins at 37 °C are listed^[37]. The Vmax numbers listed in parentheses are those for uptake at 20 °C. The inhibition of Mg²⁺ transport by Mn²⁺ via MgtA showed unusual kinetics (see Figure 1 of Snavely et al., 1989b^[37])

	Mg²⁺		Co²⁺
	Km (μM)	Vmax (pmol/min/10⁸ cells)	Ki (µM)
			Co²⁺	Mn²⁺	Ni²⁺
MgtA	29	115(24)	40	x	30
MgtB	6	75(<2)	8	40	13

The MgtA and MgtB proteins are ATPases, using one molecule of ATP per transport cycle, whereas the Mg²⁺ uptake via CorA is simply electrochemically favourable. Chamnongpol and Groisman (2002) have suggested that the MgtA and MgtB proteins form part of a metal toxicity avoidance system^[29]. Alternatively as most P-type ATPases function as efflux mediating transporters it has been suggested that the MgtA and MgtB proteins act as efflux proteins for some as yet unidentified cation, and Mg²⁺ transport is either non-specific or exchanged to maintain the electro-neutrality of the transport process^[48]. Further experiments will be required to define the physiological function of these proteins.

MgtE

Two papers describe MgtE, a fourth Mg²⁺ uptake protein in bacteria, unrelated to MgtA/B or CorA^[3]^[2]. The gene has been sequenced and the protein, which is 312 amino acids in size, is predicted to contain either four or five TM spanning domains that are closely arranged in the C-terminal part of the protein (see figure). This region of the protein has been identified in the pFAM database as a conserved protein domain (PF01769) and species containing proteins that have this protein domain are roughly equally distributed amongst the Eubacteria and Archaea, although it is quite rare in comparison with the distribution of CorA. However, the diversity of the proteins containing the domain is significantly larger than that of the CorA domain. The pFAM database lists seven distinct groups of MgtE domain containing proteins, of which six contain an archebacterial or eubacterial member.

The figure (right), adapted from Smith et al. (1995)^[3] and the pFAM database entry, this shows the computer predicted membrane topology of the MgtE protein in Bacillus firmus OF4. The TM domains are shown in light blue. The CBS domains (named for the protein the were identified in, cystathionine-beta synthase), shown in orange, are identified in the pFAM database as regulatory domains, but the mechanism of action has not yet been described. They are found in several voltage-gated chloride channels^[49]. The orientation in the membrane and the positions of the N- and C-termini are indicated. Not drawn to scale. This transporter has recently had its structure solved by x-ray crystallography. Published in Nature

The MgtE gene was first identified by Smith et al. (1995) during a screen for CorA-like proteins in bacteria, and complements the Mg²⁺-uptake-deficient S. typhimurium strain MM281 (corA mgtA mgtB), restoring wild type growth on standard media^[3]. The kinetics of Mg²⁺ transport for the protein were not determined, as ²⁸Mg²⁺ was unavailable. As a substitute the uptake of ⁵⁷Co²⁺ was measured and shown to have a Km of 82 μM and a Vmax of 354 pmol min^-1 10⁸ cells^-1. Mg²⁺ was a competitive inhibitor with a Ki of 50 μM — the Ki of Mg²⁺ inhibition of ⁶⁰Co²⁺ uptake via CorA is 10 μM^[1]. A comparison of the available kinetic data for MgtA and CorA is shown in the table. Clearly MgtE does not transport Co²⁺ to the same degree as CorA and the inhibition of transport by Mg²⁺ is also less efficient, which suggests that the affinity of MgtE for Mg²⁺ is lower than that of CorA. The strongest inhibitor of Co²⁺ uptake was Zn²⁺ with a Ki of 20 μM^[3]. The transport of Zn²⁺ by this protein may be as important as that of Mg²⁺.

	Mg²⁺		Co²⁺
	Km (μM)	Vmax (pmol/min/10⁸ cells)	Km (μM)	Vmax (pmol/min/10⁸ cells)	Ki(Mg²⁺) (μM)
MgtE	-	-	82^[3] (at 37°C)	354^[3] (at 37°C)	50^[3] (at 37°C)
CorA	15^[37] (at 20°C)	250^[37] (at 20°C)	30^[1] (at 22°C)	500^[1] (at 22°C)	10^[1] (at 22°C)

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.

Yeast

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 Rothstein^[50]^[51] 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 system^[51]^[52]^[53]^[54] — 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^[4]. 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^[4]. 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)^[4] on the strength on sequence homology and more recently by Lee and Gardner (2006)^[55], 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^[55].

File:ALR1 topology.png

The TM topology of the ALR1 protein

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)^[55]. 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 membrane^[56]. 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^[56]. 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⁺^[4]) and Alr1p can support normal growth at Mg²⁺ concentrations as low as 30 μM^[56]. ⁵⁷Co²⁺ is taken up into yeast via the Alr1p protein with a Km of 77 – 105 μM (^[52]; 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^[4]. 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^[56]. Additionally, two electrophysiological studies where Alr1p was produced in yeast or Xenopus oocytes showed a Mg²⁺-dependent current in the presence of the protein^[57]; Salih et al., in prep.).

The kinetics of Mg²⁺ uptake by Alr1p have been investigated by electrophysiology techniques on whole yeast cells^[57]. 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⁺^[58] 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 mutation^[59], and was cloned and sequenced by Wiesenberger et al. (1992)^[60]. Mrs2p was not identified as a putative Mg²⁺ transporter until Bui et al. (1999)^[5]. 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 organelle^[61]^[62]. Mg²⁺ transport has been shown to be directly mediated by Mrs2p^[17], 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^[5]^[61] 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^[17].

The TM topology of the MRS2 and LPE10 proteins

The figure shows the experimentally determined topology of Mrs2p and Lpe10p as adapted from Bui et al. (1999)^[5] and Gregan et al. (2001a)^[61]. 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^[5] and Lpe10p to the mitochondria^[61]. 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^[17]. 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^[61].

The characteristics of Mg²⁺ uptake into isolated mitochondria by Mrs2p were quantified using mag-fura 2^[17]. 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^[17].

The kinetics of Mg²⁺ uptake by Mrs2p were determined as in the Froschauer et al. (2004) paper on CorA in bacteria^[18]. 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 papers^[63]^[64]^[65]^[66] before the gene was cloned by Haynes et al. (2002)^[6].

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^[6]. 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⁺ exchanger^[67]) and SLC24 (K+ dependent Na+/Ca²⁺ exchanger^[68]) 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^[6]. 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)^[6], this figure shows the computer predicted membrane topology of XntAp in Paramecium. The orientation in the membrane was determined using HMMTOP^[69]^[70]. 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^[66]. 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.

Metazoa

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 metabolism^[71].

MRS2

A bioinformatic search of the sequence databases identified one homologue of the MRS2 gene of yeast in a range of metazoans^[7]. 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^[7].

SLC41 (MgtE)

The identification of this gene family in the metazoa began with a signal sequence trap method for isolating secreted and membrane proteins^[8]. 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.

File:MGTE human.png

The predicted TM topology of MgtE from H. sapiens

Adapted from Wabakken et al. (2003)^[8] 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)^[8] 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^[8]. This will need to be verified using one of the now standard experiment systems for examining Mg²⁺ transport.

TRPM6/ TRPM7

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)^[72] have reviewed the research into the TRP genes, and a second review by Montell (2003)^[73] 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^[73].

The activity of these two proteins has been very difficult to quantify. TRPM7 by itself appears to be a Ca²⁺ channel^[74] but in the presence of TRPM6 the affinity series of transported cations places Mg²⁺ above Ca²⁺Cite error: A <ref> tag is missing the closing </ref> (see the help page).). 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 expression^[75]. An unfortunate choice of experimental system by Voets et al., (2004)^[76] led to the conclusion that TRPM6 is a functional Mg²⁺ transporter. However, later work by Chubanov et al. (2004)^[75] 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 TRPM6 and TRPM7 proteins

The predicted TM topology of the TPRM6 and TRPM7 proteins have been adapted from Nadler et al. (2001)^[9], Runnels et al. (2001)^[77] and Montell et al. (2002)^[72], 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)^[76] 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)^[11], again only after a series of reports describing the Mg²⁺ flux itself with no gene/protein^[78]^[79]^[80]. 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^[11]. 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 nephrocalcinosis^[81]^[82], strengthening the links between the gene and the uptake of Mg²⁺.

Plants

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^[14]^[12]^[13]. 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^[14]), 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^[12]. The authors also showed that the AtMRS2-1 gene could complement a Δmrs2 yeast mutant phenotype. Independently, Li et al. (2001)^[13] 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^[12]. 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^[13]. 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 inside^[83]. 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^[13]. 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^[13]. A similar experiment was attempted in the Schock et al. (2000) paper^[12], 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^[83]. 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.

AtMHX

The first magnesium transporter isolated in any multicellular organism, AtMHX shows no similarity to any previously isolated Mg²⁺ transport protein^[14]. 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^[14].

The AtMHX gene is expressed throughout the plant but most strongly in the vascular tissue^[14]. 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^[14]. 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⁺-ATPase^[84]^[85]. 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)^[14] 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 predicted TM topology of the AtMHX protein

The image has been adapted from Shaul et al. (1999)^[14] and Quednau et al. (2004)^[67], 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.

References

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^ ^a ^b Norris, P.R. (1977). "Accumulation of cadmium and cobalt by Saccharomyces cerevisiae". Journal of General Microbiology. 99: 317–324. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)
^ Okorokov, L.A. (1977). "Energy-dependent transport of manganese into yeast cells and distribtuion of accumulated ions". European Journal of Biochemistry. 75: 373–377. doi:10.1111/j.1432-1033.1977.tb11538.x. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)
^ Conklin, D.S. (1993). "The COT2 gene is required for glucose-dependent divalent cation transport in Saccharomyces cerevisiae". Molecular and Cellular Biology. 13: 2041–2049. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)
^ ^a ^b ^c Lee J, Gardner R (2006). "Residues of the yeast ALR1 protein that are critical for magnesium uptake". Curr Genet. 49 (1): 7–20. doi:10.1007/s00294-005-0037-y. PMID 16328501.
^ ^a ^b ^c ^d Graschopf, A. (2001). "The yeast plasma membrane protein Alr1 controls Mg²⁺ homeostasis and is subject to Mg²⁺-dependent control of its synthesis and degradation". Journal of Biological Chemistry. 276: 16216–16222. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)
^ ^a ^b Liu, G.J. (2002). "Large Mg²⁺-dependent currents are associated with the increased expression of ALR1 in Saccharomyces cerevisiae". FEMS Microbiology Letters. 213: 231–237. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)
^ Zhang, A. (1997). "Extracellular Mg²⁺ regulates intracellular Mg²⁺ and its subcellular compartmentation in fission yeast, Schizosaccharomyces pombe". Cellular and Molecular Life Sciences. 53: 69–72. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)
^ Koll, H. (1987). "Three nuclear genes suppress a yeast mitochondrial splice defect when present in high copy number". Current Genetics. 12: 503–510. doi:10.1007/BF00419559. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)
^ Wiesenberger, G. (1992). "The nuclear gene MRS2 is essential for the excision of group II introns from yeast mitochondrial transcripts in vivo". Journal of Biological Chemistry. 267: 6963–6969. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)
^ ^a ^b ^c ^d ^e Gregan, J. (2001a). "The mitochondrial inner membrane protein Lpe10p, a homologue of Mrs2p, is essential for magnesium homeostasis and group II intron splicing in yeast". Molecular Genome and Genetics. 264: 773–781. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)
^ Gregan, J. (2001b). "Mitochondrial Mg²⁺ homeostasis is critical for group II intron splicing in vivo". Genes & Development. 15: 2229–2237. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)
^ Preston, R.R. (1990). "A magnesium current in Paramecium". Science. 250: 285–288. doi:10.1126/science.2218533.
^ Preston, R.R. (1994a). "Inhibition of Mg²⁺ current by single-gene mutation in Paramecium". Journal of Membrane Biology. 137: 203–212. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)
^ Preston, R.R. (1994b). "Isolation and characterization of paramecium mutants defective in their response to magnesium". Genetics. 137: 759–769. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)
^ ^a ^b Preston, R.R. (1998). "Transmembrane Mg²⁺ currents and intracellular free Mg²⁺ concentration in Paramecium tetraurelia". Journal of Membrane Biology. 164: 11–24.
^ ^a ^b Quednau, B.D. (2004). "The sodium/calcium exchanger family—SLC8". Pflügers Archiv European Journal of Physiology. 447: 543–548. doi:10.1007/s00424-003-1065-4. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)
^ Schnetkamp, P.P.M. (2004). "The SLC24 Na+/Ca²⁺-K⁺ exchanger family: vision and beyond". Pflügers Archiv European Journal of Physiology. 447: 683–688.
^ Tusnady, G.E. (1998). "Principles governing amino acid composition of integral membrane proteins: application to topology prediction". Journal of Molecular Biology. 283: 489–506. doi:10.1006/jmbi.1998.2107. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)
^ Tusnady, G.E. (2001). "The HMMTOP transmembrane topology prediction server". Bioinformatics. 17: 849–850. doi:10.1093/bioinformatics/17.9.849. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)
^ Romani, A.M.P. (2002). "Hormonal regulation of Mg²⁺ transport and homeostasis in eukaryotic cells". BioMetals. 15: 271–283. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)
^ ^a ^b Montell, C. (2002). "The TRP Channels, a remarkably functional family". Cell. 108: 595–598. doi:10.1016/S0092-8674(02)00670-0. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)
^ ^a ^b Montell, C. (2003). "Mg²⁺ Homeostasis: The Mg²⁺nificent TRPM Chanzymes". Current Biology. 13: R799–R801.
^ Runnels, L.W. (2002). "The TRPM7 channel is inactivated by PIP2 hydrolysis". Nature Cell Biology. 4: 329–336. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)
^ ^a ^b Chubanov, V. (2004). "Disruption of TRPM6/TRPM7 complex formation by a mutation in the TRPM6 gene causes hypomagnesemia with secondary hypocalcemia". PNAS. 101: 2894–2899. doi:10.1073/pnas.0305252101. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)
^ ^a ^b Voets, T. (2004). "TRPM6 forms the Mg²⁺ influx channel involved in intestinal and renal Mg²⁺ absorption". Journal of Biological Chemistry. 279: 19–25. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)
^ Runnels, L.W. (2001). "TRP-PLIK, a bifunctional protein with kinase and ion channel activities". Science. 291: 1043–1047. doi:10.1126/science.1058519. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)
^ Shareghi, G.R. (1982). "Magnesium transport in the cortical thick ascending limb of Henle's loop of the rabbit". Journal of Clinical Investigation. 69: 759–769. doi:10.1172/JCI110514. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)
^ di Stefano, A. (1993). "Transepithelial Ca²⁺ and Mg²⁺ transport in the cortical thick ascending limb of Henle's loop of the mouse is a voltage-dependent process". Renal Physiology and Biochemistry. 16: 157–166. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)
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^ Weber, S. (2000). "Familial hypomagnesaemia with hypercalciuria and nephrocalcinosis maps to chromosome 3q27 and is associated with mutations in the PCLN-1 gene". European Journal of Human Genetics. 8: 414–422. doi:10.1038/sj.ejhg.5200475. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)
^ Weber, S. (2001). "Novel paracellin-1 mutations in 25 families with familial hypomagnesemia with hypercalciuria and nephrocalcinosis". Journal of the American Society of Nephrology. 12: 1872–1881. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)
^ ^a ^b Tutone, A.F. (2004). "Cloning and chararcterisation of the Mg²⁺ transport gene from A. thaliana. In School of Biological Sciences (Auckland: University of Auckland)". {{cite journal}}: Cite journal requires |journal= (help)
^ Kurkdjian, A. (1989). "Intracellular pH: measurement and importance in cell activity". Annual Review of Plant Physiology and Plant Molecular Biology. 40: 271–303. doi:10.1146/annurev.pp.40.060189.001415. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)
^ Marschner, H. (1995). Mineral Nutrition in Higher Plants. (San Diego: Academic Press).

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[Townsend_1995-2] Townsend, D.E. (1995). "Cloning of the mgtE Mg²⁺ transporter from Providencia stuartii and the distribution of mgtE in gram-negative and gram-positive bacteria". Journal of Bacteriology. 177: 5350–5354. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Smith_1995-3] ^ ^a ^b ^c ^d ^e ^f ^g ^h Smith, R.L. (1995). "Cloning and characterization of MgtE, a putative new class of Mg²⁺ transporter from Bacillus firmus OF4". Journal of Bacteriology. 177: 1233–1238. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

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[Bui_1999-5] Bui, D.M. (1999). "The bacterial magnesium transporter CorA can functionally substitute for its putative homologue Mrs2p in the yeast inner mitochondrial membrane". Journal of Biological Chemistry. 274: 20438–20443. doi:10.1074/jbc.274.29.20438. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)CS1 maint: unflagged free DOI (link)

[Haynes_2002-6] Haynes, W.J. (2002). "An exchanger-like protein underlies the large Mg²⁺ current in Paramecium". PNAS. 99: 15717–15722. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Zsurka_2001-7] Zsurka, G. (2001). "The human mitochondrial Mrs2 protein functionally substitutes for its yeast homologue, a candidate magnesium transporter". Genomics. 72: 158–168. doi:10.1006/geno.2000.6407. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Wabakken_2003-8] Wabakken, T. (2003). "The human solute carrier SLC41A1 belongs to a novel eukaryotic subfamily with homology to prokaryotic MgtE Mg²⁺ transporters". Biochemical and Biophysical Research Communications. 306: 718–724. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Nadler_2001-9] Nadler, M.J.S. (2001). "LTRPC7 is a Mg. ATP-regulated divalent cation channel required for cell viability". Nature. 411: 590–595. doi:10.1038/35079092. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Walder_2002-10] Walder, R.Y. (2002). "Mutation of TRPM6 causes familial hypomagnesemia with secondary hypocalcemia". Nature Genetics. 31: 171–174. doi:10.1038/ng901. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Simon_1999-11] Simon, D.B. (1999). "Paracellin-1, a renal tight junction protein required for paracellular Mg²⁺ resorption". Science. 285: 103–106. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Schock_2000-12] Schock, I. (2000). "A member of a novel Arabidopsis thaliana gene family of candidate Mg²⁺ ion transporters complements a yeast mitochondrial group II intron-splicing mutant". Plant Journal. 24: 489–501. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Li_2001-13] ^ ^a ^b ^c ^d ^e ^f Li, L. (2001). "A novel family of magnesium transport genes in Arabidopsis". Plant Cell. 13: 2761–2775. doi:10.1105/tpc.13.12.2761. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Shaul_1999-14] ^ ^a ^b ^c ^d ^e ^f ^g ^h ⁱ Shaul, O. (1999). "Cloning and characterization of a novel Mg²⁺/H⁺ exchanger". EMBO Journal. 18: 3973–3980. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Borelly_2001-15] Borrelly, G. (2001). "The yeast mutant vps5D affected in the recycling of Golgi membrane proteins displays an enhanced vacuolar Mg²⁺/H⁺ exchange activity". PNAS. 98: 9660–9665. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Tevelev_1995-16] Tevelev, A. (1995). "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)". {{cite journal}}: Cite journal requires |journal= (help); Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Kolisek_2003-17] ^ ^a ^b ^c ^d ^e ^f ^g Kolisek, M. (2003). "Mrs2p is an essential component of the major electrophoretic Mg²⁺ influx system in mitochondria". EMBO Journal. 22: 1235–1244. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Froschauer_2004-18] Froschauer, E.M. (2004). "Fluorescence measurements of free [Mg²⁺] by use of mag-fura 2 in Salmonella enterica". FEMS Microbiology Letters. 237: 49–55. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Lusk_1968-19] Lusk, J.E. (1968). "Magnesium and the growth of Escherichia coli". Journal of Biological Chemistry. 243: 2618–2624. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Lusk_1969-20] Lusk, J.E. (1969). "Magnesium transport in Escherichia coli". Journal of Biological Chemistry. 244: 1653–1655. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Silver_1969-21] Silver, S. (1969). "Active transport of magnesium in Escherichia coli". PNAS. 62: 764–771. doi:10.1073/pnas.62.3.764.

[Nelson_1971-22] Nelson, D.L. (1971). "Magnesium transport in Escherichia coli. Inhibition by cobaltous ion". Journal of Biological Chemistry. 246: 3042–3049. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Webb_1970-23] Webb, M. (1970). "Interrelationships between the utilization of magnesium and the uptake of other bivalent cations by bacteria". Biochima et Biophysica Acta. 222: 428–440.

[Nelson_1972-24] Nelson, D.L. (1972). "Transport of magnesium by a repressible and a nonrepressible system in Escherichia coli". PNAS. 69: 1091–1093. doi:10.1073/pnas.69.5.1091. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

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[Smith_1995a-26] Smith, R.L. (1995a). "Distribution of the CorA Mg²⁺ transport system in gram-negative bacteria". Journal of Bacteriology. 177: 1638–1640. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Kehres_1998-27] Kehres, D.G. (1998). "The CorA magnesium transporter gene family". Microbial and Comparative Genomics. 3: 151–169. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Snavely_1991a-28] Snavely, M.D. (1991a). "Magnesium transport in Salmonella typhimurium. Regulation of mgtA and mgtB expression". Journal of Biological Chemistry. 266: 824–829. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Chamnongpol_2002-29] Chamnongpol, S. (2002). "Mg²⁺ homeostasis and avoidance of metal toxicity". Molecular Microbiology. 44: 561–571. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Groisman_2001-30] Groisman, E.A. (2001). "The pleiotropic two-component regulatory system PhoP-PhoQ". Journal of Bacteriology. 183: 1835–1842. doi:10.1128/JB.183.6.1835-1842.2001.

[Papp_2004-31] Papp, K.M. (2004). "The CorA Mg²⁺ transporter does not transport Fe²⁺". Journal of Bacteriology. 186: 7653–7658. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Smith_1993a-32] Smith, R.L. (1993a). "Sequence and topology of the CorA magnesium transport systems of Salmonella typhimurium and Escherichia coli: Identification of a new class of transport protein". Journal of Biological Chemistry. 268: 14071–14080. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Warren_2004-33] Warren, M.A. (2004). "The CorA Mg²⁺ transporter is a homotetramer". Journal of Bacteriology. 186: 4605–4612. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Smith_1998a-34] Smith, R.L. (1998a). "The CorA Mg²⁺ transport protein of Salmonella typhimurium: Mutagenesis of conserved residues in the third membrane domain identifies a Mg²⁺ pore". Journal of Biological Chemistry. 273: 28663–28669. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Szegedy_1999-35] Szegedy, M.A. (1999). "The CorA Mg²⁺ transport protein of Salmonella typhimurium. Mutatgenesis of conserved residues in the second transmembrane domain". Journal of Biological Chemistry. 274: 36973–36979. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Hmiel_1989-36] Hmiel, S.P. (1989). "Magnesium transport in Salmonella typhimurium: genetic characterization and cloning of three magnesium transport loci". Journal of Bacteriology. 171: 4742–4751. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Snavely_1989b-37] ^ ^a ^b ^c ^d ^e ^f ^g ^h ⁱ ^j ^k Snavely, M.D. (1989b). "Magnesium transport in Salmonella typhimurium: ^{28/sup>Mg²⁺ transport by the CorA, MgtA, and MgtB systems". Journal of Bacteriology. 171: 4761–4766.} {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Gibson_1991-38] Gibson, M.M. (1991). "Magnesium transport in Salmonella typhimurium: the influence of new mutations conferring Co²⁺ resistance on the CorA Mg²⁺ transport system". Molecular Microbiology. 5: 2753–2762. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Smith_1995b-39] Smith, R.L. (1995b). "Genetics and molecular biology of magnesium transport systems. In The Biological Chemistry of Magnesium, J.A. Cowan, ed (New York: VCH), pp. 211-234". {{cite journal}}: Cite journal requires |journal= (help); Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Kucharski_2000-40] Kucharski, L.M. (2000). "Cation hexaammines are selective and potent inhibitors of the CorA magnesium transport system". Journal of Biological Chemistry. 22: 16767–16773. doi:10.1074/jbc.M001507200. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)CS1 maint: unflagged free DOI (link)

[Smith_1998b-41] Smith, R.L. (1998b). "Magnesium transport in Salmonella typhimurium: Regulation of mgtA and mgtCB during invasion of epithelial and macrophage cells". Microbiology. 144: 1835–1843. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Snavely_1989a-42] Snavely, M.D. (1989a). "Magnesium transport in Salmonella typhimurium: expression of cloned genes for three distinct Mg²⁺ transport systems". Journal of Bacteriology. 171: 4752–4760. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Moncrief_1999-43] Moncrief, M.B.C. (1999). "Magnesium transport in prokaryotes". Journal of Biological Inorganic Chemistry. 4: 523–527. doi:10.1007/s007750050374. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Tao_1995-44] Tao, T. (1995). "Magnesium transport in Salmonella typhimurium: mgtA encodes a P-type ATPase and is regulated by Mg²⁺ in a manner similar to that of the mgtB P-type ATPase". Journal of Bacteriology. 177: 2654–2662. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Snavely_1991b-45] Snavely, M.D. (1991b). "The mgtB Mg²⁺ transport locus of Salmonella typhimurium encodes a P- type ATPase". Journal of Biological Chemistry. 266: 815–823. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Blanc_1997-46] Blanc-Potard, A.-B. (1997). "The Salmonella selC locus contains a pathogenicity island mediating intramacrophage survival". EMBO Journal. 16: 5376–5385. doi:10.1093/emboj/16.17.5376. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Smith_1993b-47] Smith, D.L. (1993b). "Membrane topology of a P-type ATPase. The MgtB magnesium transport protein of Salmonella typhimurium". Journal of Biological Chemistry. 268: 22469–22479. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Kehres_2002-48] Kehres, D.G. (2002). "Structure, properties and regulation of magnesium transport proteins". BioMetals. 15: 261–270. doi:10.1023/A:1016078832697. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Ponting_1997-49] Ponting, C.P. (1997). "CBS domains in CIC chloride channels implicated in myotonia and nephrolithiasis (kidney stones)". Journal of Molecular Medicine. 75: 160–163.

[Rothstein_1958-50] Rothstein, A., Hayes (1958). "The active transport of Mg²⁺ and Mn²⁺ into the yeast cell". Journal of General Physiology. 41: 585–594. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)CS1 maint: multiple names: authors list (link)

[Fuhrmann_1968-51] Fuhrmann, G.-F. (1968). "The transport of Zn²⁺, Co²⁺ and Ni²⁺ into yeast cells". Biochimica et Biophysica Acta. 163: 325–330. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Norris_1977-52] Norris, P.R. (1977). "Accumulation of cadmium and cobalt by Saccharomyces cerevisiae". Journal of General Microbiology. 99: 317–324. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Okorokov_1977-53] Okorokov, L.A. (1977). "Energy-dependent transport of manganese into yeast cells and distribtuion of accumulated ions". European Journal of Biochemistry. 75: 373–377. doi:10.1111/j.1432-1033.1977.tb11538.x. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Conklin_1993-54] Conklin, D.S. (1993). "The COT2 gene is required for glucose-dependent divalent cation transport in Saccharomyces cerevisiae". Molecular and Cellular Biology. 13: 2041–2049. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Lee_2006-55] Lee J, Gardner R (2006). "Residues of the yeast ALR1 protein that are critical for magnesium uptake". Curr Genet. 49 (1): 7–20. doi:10.1007/s00294-005-0037-y. PMID 16328501.

[Graschopf_2001-56] Graschopf, A. (2001). "The yeast plasma membrane protein Alr1 controls Mg²⁺ homeostasis and is subject to Mg²⁺-dependent control of its synthesis and degradation". Journal of Biological Chemistry. 276: 16216–16222. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Liu_2002-57] Liu, G.J. (2002). "Large Mg²⁺-dependent currents are associated with the increased expression of ALR1 in Saccharomyces cerevisiae". FEMS Microbiology Letters. 213: 231–237. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Zhang_1997-58] Zhang, A. (1997). "Extracellular Mg²⁺ regulates intracellular Mg²⁺ and its subcellular compartmentation in fission yeast, Schizosaccharomyces pombe". Cellular and Molecular Life Sciences. 53: 69–72. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Koll_1987-59] Koll, H. (1987). "Three nuclear genes suppress a yeast mitochondrial splice defect when present in high copy number". Current Genetics. 12: 503–510. doi:10.1007/BF00419559. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Wiesenberger_1992-60] Wiesenberger, G. (1992). "The nuclear gene MRS2 is essential for the excision of group II introns from yeast mitochondrial transcripts in vivo". Journal of Biological Chemistry. 267: 6963–6969. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Gregan_2001a-61] Gregan, J. (2001a). "The mitochondrial inner membrane protein Lpe10p, a homologue of Mrs2p, is essential for magnesium homeostasis and group II intron splicing in yeast". Molecular Genome and Genetics. 264: 773–781. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Gregan_2001b-62] Gregan, J. (2001b). "Mitochondrial Mg²⁺ homeostasis is critical for group II intron splicing in vivo". Genes & Development. 15: 2229–2237. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Preston_1990-63] Preston, R.R. (1990). "A magnesium current in Paramecium". Science. 250: 285–288. doi:10.1126/science.2218533.

[Preston_1994a-64] Preston, R.R. (1994a). "Inhibition of Mg²⁺ current by single-gene mutation in Paramecium". Journal of Membrane Biology. 137: 203–212. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Preston_1994b-65] Preston, R.R. (1994b). "Isolation and characterization of paramecium mutants defective in their response to magnesium". Genetics. 137: 759–769. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Preston_1998-66] Preston, R.R. (1998). "Transmembrane Mg²⁺ currents and intracellular free Mg²⁺ concentration in Paramecium tetraurelia". Journal of Membrane Biology. 164: 11–24.

[Quednau_2004-67] Quednau, B.D. (2004). "The sodium/calcium exchanger family—SLC8". Pflügers Archiv European Journal of Physiology. 447: 543–548. doi:10.1007/s00424-003-1065-4. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Schnetkamp_2004-68] Schnetkamp, P.P.M. (2004). "The SLC24 Na+/Ca²⁺-K⁺ exchanger family: vision and beyond". Pflügers Archiv European Journal of Physiology. 447: 683–688.

[Tusnady_1998-69] Tusnady, G.E. (1998). "Principles governing amino acid composition of integral membrane proteins: application to topology prediction". Journal of Molecular Biology. 283: 489–506. doi:10.1006/jmbi.1998.2107. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Tusnady_2001-70] Tusnady, G.E. (2001). "The HMMTOP transmembrane topology prediction server". Bioinformatics. 17: 849–850. doi:10.1093/bioinformatics/17.9.849. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

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[Montell_2002-72] Montell, C. (2002). "The TRP Channels, a remarkably functional family". Cell. 108: 595–598. doi:10.1016/S0092-8674(02)00670-0. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

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[Chubanov_2004-75] Chubanov, V. (2004). "Disruption of TRPM6/TRPM7 complex formation by a mutation in the TRPM6 gene causes hypomagnesemia with secondary hypocalcemia". PNAS. 101: 2894–2899. doi:10.1073/pnas.0305252101. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Voets_2004-76] Voets, T. (2004). "TRPM6 forms the Mg²⁺ influx channel involved in intestinal and renal Mg²⁺ absorption". Journal of Biological Chemistry. 279: 19–25. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Runnels_2001-77] Runnels, L.W. (2001). "TRP-PLIK, a bifunctional protein with kinase and ion channel activities". Science. 291: 1043–1047. doi:10.1126/science.1058519. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[Shareghi_1982-78] Shareghi, G.R. (1982). "Magnesium transport in the cortical thick ascending limb of Henle's loop of the rabbit". Journal of Clinical Investigation. 69: 759–769. doi:10.1172/JCI110514. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

[di_Stefano_1993-79] Stefano, A. (1993). "Transepithelial Ca²⁺ and Mg²⁺ transport in the cortical thick ascending limb of Henle's loop of the mouse is a voltage-dependent process". Renal Physiology and Biochemistry. 16: 157–166. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help)

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@@ Line 2: / Line 2: @@
 :''This page links directly from the [[magnesium in biological systems]] page.''
-All forms of life require '''[[magnesium]]''', and yet the molecular mechanisms of Mg<sup>2+</sup> uptake from the environment and the distribution ('''transport''') of this vital element within the organism are only slowly being elucidated. In bacteria Mg<sup>2+</sup> is probably mainly supplied by [[CorA metal ion transporter|the CorA protein]]<ref name="Hmiel 1986">{{cite 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}}</ref> and, where the CorA protein is absent, by the MgtE protein<ref name="Townsend 1995">{{cite 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 Mg<sup>2+</sup> 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}}</ref><ref name="Smith 1995">{{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 Mg<sup>2+</sup> transporter from Bacillus firmus OF4| journal=Journal of Bacteriology | volume=177| pages=1233-1238}}</ref>. In yeast the initial uptake is via the Alr1p and Alr2p proteins<ref name="MacDiarmid 1998">{{cite 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}}</ref>, but at this stage the only internal Mg<sup>2+</sup> distributing protein identified is Mrs2p<ref name="Bui 1999">{{cite 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}}</ref>. Within the protozoa only one Mg<sup>2+</sup> transporter (XntAp) has been identified<ref name="Haynes 2002">{{cite 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 Mg<sup>2+</sup> current in Paramecium| journal=PNAS| volume= 99| pages=15717-15722}}</ref>. In metazoa, Mrs2p<ref name="Zsurka 2001">{{cite 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}}</ref> and MgtE homologues<ref name="Wabakken 2003">{{cite 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 Mg<sup>2+</sup> transporters| journal=Biochemical and Biophysical Research Communications | volume=306| pages=718-724}}</ref> have been identified, along with two novel Mg<sup>2+</sup> transport systems TRPM6/TRPM7<ref name="Nadler 2001">{{cite 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}}</ref><ref name="Walder 2002">{{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}}</ref> and PCLN-1<ref name="Simon 1999">{{cite 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 Mg<sup>2+</sup> resorption| journal=Science| volume= 285| pages=103-106}}</ref>. Finally, in plants, a family of Mrs2p homologues has been identified<ref name="Schock 2000">{{cite 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 Mg<sup>2+</sup> ion transporters complements a yeast mitochondrial group II intron-splicing mutant| journal=Plant Journal | volume=24| pages=489-501}}</ref><ref name="Li 2001">{{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}}</ref> along with another novel protein, AtMHX<ref name="Shaul 1999">{{cite 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 Mg<sup>2+</sup>/H<SUP>+</SUP> exchanger| journal=EMBO Journal | volume=18| pages=3973-3980}}</ref>.
+All forms of life require '''[[magnesium]]''', and yet the molecular mechanisms of Mg<sup>2+</sup> uptake from the environment and the distribution ('''transport''') of this vital element within the organism are only slowly being elucidated. In bacteria Mg<sup>2+</sup> is probably mainly supplied by [[CorA metal ion transporter|the CorA protein]]<ref name="Hmiel 1986">{{cite 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}}</ref> and, where the CorA protein is absent, by the MgtE protein<ref name="Townsend 1995">{{cite 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 Mg<sup>2+</sup> 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}}</ref><ref name="Smith 1995">{{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 Mg<sup>2+</sup> transporter from Bacillus firmus OF4| journal=Journal of Bacteriology | volume=177| pages=1233–1238}}</ref>. In yeast the initial uptake is via the Alr1p and Alr2p proteins<ref name="MacDiarmid 1998">{{cite 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}}</ref>, but at this stage the only internal Mg<sup>2+</sup> distributing protein identified is Mrs2p<ref name="Bui 1999">{{cite 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}}</ref>. Within the protozoa only one Mg<sup>2+</sup> transporter (XntAp) has been identified<ref name="Haynes 2002">{{cite 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 Mg<sup>2+</sup> current in Paramecium| journal=PNAS| volume= 99| pages=15717–15722}}</ref>. In metazoa, Mrs2p<ref name="Zsurka 2001">{{cite 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}}</ref> and MgtE homologues<ref name="Wabakken 2003">{{cite 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 Mg<sup>2+</sup> transporters| journal=Biochemical and Biophysical Research Communications | volume=306| pages=718–724}}</ref> have been identified, along with two novel Mg<sup>2+</sup> transport systems TRPM6/TRPM7<ref name="Nadler 2001">{{cite 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}}</ref><ref name="Walder 2002">{{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}}</ref> and PCLN-1<ref name="Simon 1999">{{cite 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 Mg<sup>2+</sup> resorption| journal=Science| volume= 285| pages=103–106}}</ref>. Finally, in plants, a family of Mrs2p homologues has been identified<ref name="Schock 2000">{{cite 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 Mg<sup>2+</sup> ion transporters complements a yeast mitochondrial group II intron-splicing mutant| journal=Plant Journal | volume=24| pages=489–501}}</ref><ref name="Li 2001">{{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}}</ref> along with another novel protein, AtMHX<ref name="Shaul 1999">{{cite 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 Mg<sup>2+</sup>/H<SUP>+</SUP> exchanger| journal=EMBO Journal | volume=18| pages=3973–3980}}</ref>.
 The evolution of Mg<sup>2+</sup> 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 Mg<sup>2+</sup> transport systems that are found only in the higher organisms.
-Clearly there are a large number of proteins yet to be identified that transport Mg<sup>2+</sup>. Even in the best studied eukaryote, yeast, Borrelly<ref name="Borelly 2001">{{cite 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 Mg<sup>2+</sup>/H<SUP>+</SUP> exchange activity| journal=PNAS| volume= 98| pages=9660-9665}}</ref> have reported a Mg<sup>2+</sup>/H<sup>+</sup> exchanger, without an associated protein, which is probably localised to the Golgi. At least one other major Mg<sup>2+</sup> transporter in yeast still unaccounted for — that effecting Mg<sup>2+</sup> transport into and out of the yeast vacuole. In higher, multicellular organisms it seems that many Mg<sup>2+</sup> transporting proteins await discovery.
+Clearly there are a large number of proteins yet to be identified that transport Mg<sup>2+</sup>. Even in the best studied eukaryote, yeast, Borrelly<ref name="Borelly 2001">{{cite 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 Mg<sup>2+</sup>/H<SUP>+</SUP> exchange activity| journal=PNAS| volume= 98| pages=9660–9665}}</ref> have reported a Mg<sup>2+</sup>/H<sup>+</sup> exchanger, without an associated protein, which is probably localised to the Golgi. At least one other major Mg<sup>2+</sup> transporter in yeast still unaccounted for — that effecting Mg<sup>2+</sup> transport into and out of the yeast vacuole. In higher, multicellular organisms it seems that many Mg<sup>2+</sup> transporting proteins await discovery.
 The CorA-domain-containing Mg<sup>2+</sup> 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 Mg<sup>2+</sup> transporters so far identified. This similarity suggests that the basic properties of Mg<sup>2+</sup> strongly influence the possible mechanisms of recognition and transport. However, this observation also suggests that using other metal ions as tracers for Mg<sup>2+</sup> uptake will not necessarily produce results comparable to the transporter’s ability to transport Mg<sup>2+</sup>. Ideally, Mg<sup>2+</sup> should be measured directly<ref name="Tevelev 1995">{{cite 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)}}</ref>.
-In a world where <sup>28</sup>Mg<sup>2+</sup> is practically unobtainable, much of the old data will need to be reinterpreted in terms of new tools for measuring Mg<sup>2+</sup> transport, if different transporters are to be compared directly. The pioneering work of Kolisek<ref name="Kolisek 2003">{{cite 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 Mg<sup>2+</sup> influx system in mitochondria| journal=EMBO Journal| volume= 22| pages=1235-1244}}</ref> and Froschauer<ref name="Froschauer 2004">{{cite journal| last=Froschauer| first= E.M. | coauthors=Kolisek, M., Dieterich, F., Schweigel, M., and Schweyen, R.J.|year=2004| title= Fluorescence measurements of free [Mg<sup>2+</sup>] by use of mag-fura 2 in Salmonella enterica| journal=FEMS Microbiology Letters| volume= 237| pages=49-55}}</ref> using mag-fura 2 has shown that free Mg<sup>2+</sup> 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 Mg<sup>2+</sup> 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 Mg<sup>2+</sup> transport proteins. However, the differences in biological systems between prokaryotes and eukaryotes will have to be considered as part of any experiment.
+In a world where <sup>28</sup>Mg<sup>2+</sup> is practically unobtainable, much of the old data will need to be reinterpreted in terms of new tools for measuring Mg<sup>2+</sup> transport, if different transporters are to be compared directly. The pioneering work of Kolisek<ref name="Kolisek 2003">{{cite 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 Mg<sup>2+</sup> influx system in mitochondria| journal=EMBO Journal| volume= 22| pages=1235–1244}}</ref> and Froschauer<ref name="Froschauer 2004">{{cite journal| last=Froschauer| first= E.M. | coauthors=Kolisek, M., Dieterich, F., Schweigel, M., and Schweyen, R.J.|year=2004| title= Fluorescence measurements of free [Mg<sup>2+</sup>] by use of mag-fura 2 in Salmonella enterica| journal=FEMS Microbiology Letters| volume= 237| pages=49–55}}</ref> using mag-fura 2 has shown that free Mg<sup>2+</sup> 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 Mg<sup>2+</sup> 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 Mg<sup>2+</sup> 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 Mg<sup>2+</sup> 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 Mg<sup>2+</sup> 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.
@@ Line 16: / Line 16: @@
 ==Bacteria==
 ===Early research===
-In 1968 Lusk<ref name="Lusk 1968">{{cite 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}}</ref> described the limitation of bacterial (''Escherichia coli'') growth on Mg<sup>2+</sup>-poor media, suggesting that bacteria required Mg<sup>2+</sup> and were likely to actively take up this ion from the environment. The following year the same group<ref name="Lusk 1969">{{cite 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}}</ref> and another group, Silver<ref name="Silver 1969">{{cite journal| last=Silver| first= S.|year=1969| title= Active transport of magnesium in Escherichia coli| journal=PNAS | volume=62| pages=764-771}}</ref>, independently described the uptake and efflux of Mg<sup>2+</sup> in metabolically active ''E. coli'' cells using <sup>28</sup>Mg<sup>2+</sup>. By the end of 1971 two papers had been published describing the interference of Co<sup>2+</sup>, Ni<sup>2+</sup> and Mn<sup>2+</sup> on the transport of Mg<sup>2+</sup> in ''E. coli''<ref name="Nelson 1971">{{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}}</ref> and in Aerobacter aerogenes and Bacillus megaterium<ref name="Webb 1970">{{cite 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}}</ref>. In a last major development prior to the cloning of the genes encoding the transporters, it was discovered that there was a second Mg<sup>2+</sup> 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 Mg<sup>2+</sup>
+In 1968 Lusk<ref name="Lusk 1968">{{cite 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}}</ref> described the limitation of bacterial (''Escherichia coli'') growth on Mg<sup>2+</sup>-poor media, suggesting that bacteria required Mg<sup>2+</sup> and were likely to actively take up this ion from the environment. The following year the same group<ref name="Lusk 1969">{{cite 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}}</ref> and another group, Silver<ref name="Silver 1969">{{cite 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}}</ref>, independently described the uptake and efflux of Mg<sup>2+</sup> in metabolically active ''E. coli'' cells using <sup>28</sup>Mg<sup>2+</sup>. By the end of 1971 two papers had been published describing the interference of Co<sup>2+</sup>, Ni<sup>2+</sup> and Mn<sup>2+</sup> on the transport of Mg<sup>2+</sup> in ''E. coli''<ref name="Nelson 1971">{{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}}</ref> and in Aerobacter aerogenes and Bacillus megaterium<ref name="Webb 1970">{{cite 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}}</ref>. In a last major development prior to the cloning of the genes encoding the transporters, it was discovered that there was a second Mg<sup>2+</sup> 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 Mg<sup>2+</sup>
-<ref name="Nelson 1972">{{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}}</ref><ref name="Park 1976">{{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}}</ref>.
+<ref name="Nelson 1972">{{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}}</ref><ref name="Park 1976">{{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}}</ref>.
 ===CorA===
 The CorA gene and its corresponding protein are the most exhaustively studied Mg<sup>2+</sup> 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 Mg<sup>2+</sup> transport by CorA. The gene was originally named after the cobalt-resistant phenotype in ''E. coli'' that was caused by the gene’s inactivation<ref name="Nelson 1972"/>.
-The gene was genetically identified in ''E. coli'' by Park ''et al.''<ref name="Park 1976"/>, but wasn’t cloned until Hmiel ''et al.''<ref name="Hmiel 1986"/> isolated the ''Salmonella enterica'' serovar Typhimurium (''S. typhimurium'') homologue. Later it would be shown by Smith and Maguire<ref name="Smith 1995a">{{cite journal| last=Smith| first= R.L. | coauthors=Maguire, M.E.|year=1995a| title= Distribution of the CorA Mg<sup>2+</sup> transport system in gram-negative bacteria| journal=Journal of Bacteriology | volume=177| pages=1638-1640}}</ref> 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 Archaea<ref name="Kehres 1998">{{cite 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}}</ref>. 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%<ref name="Kehres 1998"/>. 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 (www.sanger.ac.uk/Software/Pfam/), is additionally present in a wide range of higher organisms and these transporters will be reviewed below.
+The gene was genetically identified in ''E. coli'' by Park ''et al.''<ref name="Park 1976"/>, but wasn’t cloned until Hmiel ''et al.''<ref name="Hmiel 1986"/> isolated the ''Salmonella enterica'' serovar Typhimurium (''S. typhimurium'') homologue. Later it would be shown by Smith and Maguire<ref name="Smith 1995a">{{cite journal| last=Smith| first= R.L. | coauthors=Maguire, M.E.|year=1995a| title= Distribution of the CorA Mg<sup>2+</sup> transport system in gram-negative bacteria| journal=Journal of Bacteriology | volume=177| pages=1638–1640}}</ref> 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 Archaea<ref name="Kehres 1998">{{cite 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}}</ref>. 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%<ref name="Kehres 1998"/>. 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 (www.sanger.ac.uk/Software/Pfam/), 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 Mg<sup>2+</sup> concentrations<ref name="Snavely 1991a">{{cite 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}}</ref>. However, recent evidence suggests that the activity of the protein may be regulated by the PhoPQ two-component regulatory system<ref name="Chamnongpol 2002">{{cite journal| last=Chamnongpol| first= S. | coauthors=Groisman, E.A. | year=2002| title= Mg<sup>2+</sup> homeostasis and avoidance of metal toxicity| journal=Molecular Microbiology| volume= 44| pages=561-571}}</ref>. This sensor responds to low external Mg<sup>2+</sup> during the infection process of ''S. typhimurium'' in humans<ref name="Groisman 2001">{{cite 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}}</ref>. In low external Mg<sup>2+</sup> conditions the PhoPQ system was reported to suppress the function of CorA  and it has been previously shown that the transcription of the alternative Mg<sup>2+</sup> transporters MgtA and MgtB is activated in these conditions<ref name="Snavely 1991a"/>. 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 Mg<sup>2+</sup><ref name="Chamnongpol 2002"/>. For a conflicting report on the source of toxicity, see Papp and Maguire<ref name="Papp 2004">{{cite journal| last=Papp| first= K.M. | coauthors=Maguire, M.E.|year=2004| title= The CorA Mg<sup>2+</sup> transporter does not transport Fe<sup>2+</sup>| journal=Journal of Bacteriology | volume=186| pages=7653-7658}}</ref>.
+The CorA gene is constitutively expressed in ''S. typhimurium'' under a wide range of external Mg<sup>2+</sup> concentrations<ref name="Snavely 1991a">{{cite 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}}</ref>. However, recent evidence suggests that the activity of the protein may be regulated by the PhoPQ two-component regulatory system<ref name="Chamnongpol 2002">{{cite journal| last=Chamnongpol| first= S. | coauthors=Groisman, E.A. | year=2002| title= Mg<sup>2+</sup> homeostasis and avoidance of metal toxicity| journal=Molecular Microbiology| volume= 44| pages=561–571}}</ref>. This sensor responds to low external Mg<sup>2+</sup> during the infection process of ''S. typhimurium'' in humans<ref name="Groisman 2001">{{cite 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}}</ref>. In low external Mg<sup>2+</sup> conditions the PhoPQ system was reported to suppress the function of CorA  and it has been previously shown that the transcription of the alternative Mg<sup>2+</sup> transporters MgtA and MgtB is activated in these conditions<ref name="Snavely 1991a"/>. 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 Mg<sup>2+</sup><ref name="Chamnongpol 2002"/>. For a conflicting report on the source of toxicity, see Papp and Maguire<ref name="Papp 2004">{{cite journal| last=Papp| first= K.M. | coauthors=Maguire, M.E.|year=2004| title= The CorA Mg<sup>2+</sup> transporter does not transport Fe<sup>2+</sup>| journal=Journal of Bacteriology | volume=186| pages=7653–7658}}</ref>.
 [[Image:cora_mag.png|thumb|300px|right|The originally published TM topology of the CorA protein]]
-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.''<ref name="Smith 1993a">{{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}}</ref>. Evidence for CorA acting as a homotetramer was published by Warren ''et al.'' in 2004<ref name="Warren 2004">{{cite 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 Mg<sup>2+</sup> transporter is a homotetramer| journal=Journal of Bacteriology | volume=186| pages=4605-4612}}</ref>. 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. [http://www.rcsb.org/pdb/navbarsearch.do?newSearch=yes&isAuthorSearch=no&radioset=Structures&inputQuickSearch=2bbj&image.x=0&image.y=0&image=Search, Follow this link to see the structure in 3D]. 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 transporter<ref name="Smith 1998a">{{cite 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 Mg<sup>2+</sup> transport protein of Salmonella typhimurium: Mutagenesis of conserved residues in the third membrane domain identifies a Mg<sup>2+</sup> pore| journal=Journal of Biological Chemistry | volume=273| pages=28663-28669}}</ref>. From mutagenesis experiments it appears that the chemistry of Mg<sup>2+</sup> 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)<ref name="Smith 1998a"/><ref name="Szegedy 1999">{{cite journal| last=Szegedy| first= M.A. | coauthors=Maguire, M.E.|year=1999| title= The CorA Mg<sup>2+</sup> transport protein of Salmonella typhimurium. Mutatgenesis of conserved residues in the second transmembrane domain| journal=Journal of Biological Chemistry | volume=274| pages=36973-36979}}</ref>.
+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.''<ref name="Smith 1993a">{{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}}</ref>. Evidence for CorA acting as a homotetramer was published by Warren ''et al.'' in 2004<ref name="Warren 2004">{{cite 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 Mg<sup>2+</sup> transporter is a homotetramer| journal=Journal of Bacteriology | volume=186| pages=4605–4612}}</ref>. 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. [http://www.rcsb.org/pdb/navbarsearch.do?newSearch=yes&isAuthorSearch=no&radioset=Structures&inputQuickSearch=2bbj&image.x=0&image.y=0&image=Search, Follow this link to see the structure in 3D]. 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 transporter<ref name="Smith 1998a">{{cite 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 Mg<sup>2+</sup> transport protein of Salmonella typhimurium: Mutagenesis of conserved residues in the third membrane domain identifies a Mg<sup>2+</sup> pore| journal=Journal of Biological Chemistry | volume=273| pages=28663–28669}}</ref>. From mutagenesis experiments it appears that the chemistry of Mg<sup>2+</sup> 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)<ref name="Smith 1998a"/><ref name="Szegedy 1999">{{cite journal| last=Szegedy| first= M.A. | coauthors=Maguire, M.E.|year=1999| title= The CorA Mg<sup>2+</sup> transport protein of Salmonella typhimurium. Mutatgenesis of conserved residues in the second transmembrane domain| journal=Journal of Biological Chemistry | volume=274| pages=36973–36979}}</ref>.
 Before the activity of CorA could be studied ''in vivo'', any other Mg<sup>2+</sup> 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 constructed<ref name="Hmiel 1989">{{cite 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}}</ref>(see also below), and the uptake kinetics of the transporter were analysed<ref name="Snavely 1989b">{{cite 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: <sup>28/sup>Mg<sup>2+</sup> transport by the CorA, MgtA, and MgtB systems| journal=Journal of Bacteriology | volume=171| pages=4761–4766}}</ref>. This strain showed nearly normal growth rates on standard media (50 μM Mg<sup>2+</sup>), but the removal of all three genes created a bacterial strain requiring 100 mM external Mg<sup>2+</sup> for normal growth<ref name="Hmiel 1989"/>.
-Mg<sup>2+</sup> is taken up into cells containing only the CorA transport system with similar kinetics and cation sensitivities as the Mg<sup>2+</sup> uptake described in the earlier papers, and has additionally been quantified<ref name="Snavely 1989b"/>(see table). The uptake of Mg<sup>2+</sup> 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<ref name="Froschauer 2004"/>. Co<sup>2+</sup> and Ni<sup>2+</sup> are toxic to ''S. typhimurium'' cells containing a functional CorA protein and this toxicity stems from the blocking of Mg<sup>2+</sup> uptake (competitive inhibition) and the accumulation of these ions inside the cell<ref name="Hmiel 1986"/>. Co<sup>2+</sup> and Ni<sup>2+</sup> have been shown to be transported by CorA by using radioactive tracer analysis<ref name="Hmiel 1986"/><ref name="Gibson 1991">{{cite 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 Co<sup>2+</sup> resistance on the CorA Mg<sup>2+</sup> transport system| journal=Molecular Microbiology| volume= 5| pages=2753-2762}}</ref>, although with lower affinities (Km) and velocities (Vmax) than for Mg<sup>2+</sup> (see table). The Km values for Co<sup>2+</sup> and Ni<sup>2+</sup> 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<ref name="Hmiel 1986"/>. To date, the evidence for Mn<sup>2+</sup> transport by CorA is limited to ''E. coli''<ref name="Park 1976"/>.
+Mg<sup>2+</sup> is taken up into cells containing only the CorA transport system with similar kinetics and cation sensitivities as the Mg<sup>2+</sup> uptake described in the earlier papers, and has additionally been quantified<ref name="Snavely 1989b"/>(see table). The uptake of Mg<sup>2+</sup> 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<ref name="Froschauer 2004"/>. Co<sup>2+</sup> and Ni<sup>2+</sup> are toxic to ''S. typhimurium'' cells containing a functional CorA protein and this toxicity stems from the blocking of Mg<sup>2+</sup> uptake (competitive inhibition) and the accumulation of these ions inside the cell<ref name="Hmiel 1986"/>. Co<sup>2+</sup> and Ni<sup>2+</sup> have been shown to be transported by CorA by using radioactive tracer analysis<ref name="Hmiel 1986"/><ref name="Gibson 1991">{{cite 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 Co<sup>2+</sup> resistance on the CorA Mg<sup>2+</sup> transport system| journal=Molecular Microbiology| volume= 5| pages=2753–2762}}</ref>, although with lower affinities (Km) and velocities (Vmax) than for Mg<sup>2+</sup> (see table). The Km values for Co<sup>2+</sup> and Ni<sup>2+</sup> 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<ref name="Hmiel 1986"/>. To date, the evidence for Mn<sup>2+</sup> transport by CorA is limited to ''E. coli''<ref name="Park 1976"/>.
 {| class="wikitable"
@@ Line 77: / Line 77: @@
 The efflux of Mg<sup>2+</sup> from bacterial cells was first observed by Lusk and Kennedy (1969)<ref name="Lusk 1969"/> and is mediated by the CorA Mg<sup>2+</sup> transport system in the presence of high extracellular concentrations of Mg<sup>2+</sup><ref name="Snavely 1989b"/>. The efflux can also be triggered by Co<sup>2+</sup>, Mn<sup>2+</sup> and Ni<sup>2+</sup>, although not to the same degree as Mg<sup>2+</sup><ref name="Nelson 1971"/>. No Co<sup>2+</sup> efflux through the CorA transport system was observed. The process of Mg<sup>2+</sup> efflux additionally requires one of the CorB, CorC or CorD genes<ref name="Gibson 1991"/>. Interestingly, the mutation of any single one of these genes leads to a Co<sup>2+</sup> resistance a little less than half of that provided by a corA mutant. Presumably this effect is due to the inhibition of Mg<sup>2+</sup> loss that would otherwise occur in the presence of high levels of Co<sup>2+</sup>. An interesting, and currently unanswered, question is whether Mg<sup>2+</sup> is more toxic when the CorBCD genes are deleted.
-It has been speculated that the Mg<sup>2+</sup> ion will initially interact with any transport protein through its hydration shell<ref name="Smith 1995b">{{cite journal| last=Smith| first= R.L. | coauthors=Maguire, M.E.|year=1995b| title= Genetics and molecular biology of magnesium transport systems. In The Biological Chemistry of Magnesium, J.A. Cowan, ed (New York: VCH), pp. 211-234}}</ref>. Cobalt (III) hexaammine, Co(III)Hex, is a covalently bound (non-labile) analog for the first shell of hydration for several divalent cations, including Mg<sup>2+</sup>. The radius of the Co(III)Hex molecule is 244 pm, which is very similar to the 250 pm radius of the first hydration shell of Mg<sup>2+</sup>. This analog is a potent inhibitor of the CorA transport system, more so than Mg<sup>2+</sup>, Co<sup>2+</sup> or Ni<sup>2+</sup><ref name="Kucharski 2000">{{cite journal| last=Kucharski| first= L.M. | coauthors=Lubbe, W.J., and Maguire, M.E.|year=2000| title= Cation hexaammines are selective and potent inhibitors of the CorA magnesium transport system| journal=Journal of Biological Chemistry| volume= 22| pages=16767-16773}}</ref>. The additional strength of the Co(III)Hex inhibition might come from the blocking of the transport pore due to the inability of the protein to ‘dehydrate’ the substrate. It was also shown that Co(III)Hex was not transported into the cells<ref name="Kucharski 2000"/>, suggesting that at least partial dehydration would be required for the transport of the normal substrate (Mg<sup>2+</sup>). Nickel (II) hexaammine , with a radius of 255 pm, did not inhibit the CorA transport system, suggesting a maximum size limit exists for the binding of the CorA substrate ion<ref name="Kucharski 2000"/>. These results suggest that the important property involved in the recognition of Mg<sup>2+</sup> by CorA is the size of the ion with its first shell of hydration. Hence, the volume change generally quoted for the bare to hydrated Mg<sup>2+</sup> ion of greater than 500-fold, including the second sphere of hydration, may not be biologically relevant and perhaps the first sphere volume change of 56-fold should be more commonly used.
+It has been speculated that the Mg<sup>2+</sup> ion will initially interact with any transport protein through its hydration shell<ref name="Smith 1995b">{{cite journal| last=Smith| first= R.L. | coauthors=Maguire, M.E.|year=1995b| title= Genetics and molecular biology of magnesium transport systems. In The Biological Chemistry of Magnesium, J.A. Cowan, ed (New York: VCH), pp. 211-234}}</ref>. Cobalt (III) hexaammine, Co(III)Hex, is a covalently bound (non-labile) analog for the first shell of hydration for several divalent cations, including Mg<sup>2+</sup>. The radius of the Co(III)Hex molecule is 244 pm, which is very similar to the 250 pm radius of the first hydration shell of Mg<sup>2+</sup>. This analog is a potent inhibitor of the CorA transport system, more so than Mg<sup>2+</sup>, Co<sup>2+</sup> or Ni<sup>2+</sup><ref name="Kucharski 2000">{{cite journal| last=Kucharski| first= L.M. | coauthors=Lubbe, W.J., and Maguire, M.E.|year=2000| title= Cation hexaammines are selective and potent inhibitors of the CorA magnesium transport system| journal=Journal of Biological Chemistry| volume= 22| pages=16767–16773| doi= 10.1074/jbc.M001507200}}</ref>. The additional strength of the Co(III)Hex inhibition might come from the blocking of the transport pore due to the inability of the protein to ‘dehydrate’ the substrate. It was also shown that Co(III)Hex was not transported into the cells<ref name="Kucharski 2000"/>, suggesting that at least partial dehydration would be required for the transport of the normal substrate (Mg<sup>2+</sup>). Nickel (II) hexaammine , with a radius of 255 pm, did not inhibit the CorA transport system, suggesting a maximum size limit exists for the binding of the CorA substrate ion<ref name="Kucharski 2000"/>. These results suggest that the important property involved in the recognition of Mg<sup>2+</sup> by CorA is the size of the ion with its first shell of hydration. Hence, the volume change generally quoted for the bare to hydrated Mg<sup>2+</sup> ion of greater than 500-fold, including the second sphere of hydration, may not be biologically relevant and perhaps the first sphere volume change of 56-fold should be more commonly used.
 ===MgtA and MgtB===
 The presence of these two genes was first suspected when Nelson and Kennedy (1972)<ref name="Nelson 1972"/> showed that there were Mg<sup>2+</sup>-repressible and Mg<sup>2+</sup>-non-repressible Mg<sup>2+</sup> uptake systems in ''E. coli''. The non-repressible uptake of Mg<sup>2+</sup> is mediated by the CorA protein. In ''S. typhimurium'' the repressible Mg<sup>2+</sup> uptake was eventually shown to be via the MgtA and MgtB proteins<ref name="Hmiel 1989"/>.
-Both MgtA and MgtB are regulated by the PhoPQ system and are actively transcribed during the process of infection of human patients by ''S. typhimurium''<ref name="Smith 1998b">{{cite journal| last=Smith| first= R.L. | coauthors=Kaczmarek, M.T., Kucharski, L.M., and Maguire, M.E.|year=1998b| title= Magnesium transport in Salmonella typhimurium: Regulation of mgtA and mgtCB during invasion of epithelial and macrophage cells| journal=Microbiology | volume=144| pages=1835-1843}}</ref><ref name="Snavely 1989a">{{cite journal| last=Snavely| first= M.D. | coauthors=Florer, J.B., Miller, C.G., and Maguire, M.E.|year=1989a| title= Magnesium transport in Salmonella typhimurium: expression of cloned genes for three distinct Mg<sup>2+</sup> transport systems| journal=Journal of Bacteriology| volume= 171| pages=4752–4760}}</ref><ref name="Groisman 2001"/>. Although neither gene is required for pathogenicity the MgtB protein does enhance the long term survival of the pathogen in the cell<ref name="Moncrief 1999">{{cite journal| last=Moncrief| first= M.B.C. | coauthors=Maguire, M.E.|year=1999| title= Magnesium transport in prokaryotes| journal=Journal of Biological Inorganic Chemistry | volume=4| pages=523 - 527}}</ref>. The genes are also upregulated ''in vitro'' when the Mg<sup>2+</sup> concentration falls below 50 μM (Snavely ''et al.'', 1991a). Although the proteins have Km values similar to CorA and transport rates approximately 10-fold less the genes may be part of a Mg<sup>2+</sup> scavenging system. Chamnongpol and Groisman (2002) presents evidence that the role of these proteins may be to compensate for the inactivation of the CorA protein by the PhoPQ regulon<ref name="Chamnongpol 2002"/>. The authors suggest that the CorA protein is inactivated to allow the avoidance of metal toxicity via the protein in the low Mg<sup>2+</sup> environments ''S. typhimurium'' is subjected to by cells after infection.
+Both MgtA and MgtB are regulated by the PhoPQ system and are actively transcribed during the process of infection of human patients by ''S. typhimurium''<ref name="Smith 1998b">{{cite journal| last=Smith| first= R.L. | coauthors=Kaczmarek, M.T., Kucharski, L.M., and Maguire, M.E.|year=1998b| title= Magnesium transport in Salmonella typhimurium: Regulation of mgtA and mgtCB during invasion of epithelial and macrophage cells| journal=Microbiology | volume=144| pages=1835–1843}}</ref><ref name="Snavely 1989a">{{cite journal| last=Snavely| first= M.D. | coauthors=Florer, J.B., Miller, C.G., and Maguire, M.E.|year=1989a| title= Magnesium transport in Salmonella typhimurium: expression of cloned genes for three distinct Mg<sup>2+</sup> transport systems| journal=Journal of Bacteriology| volume= 171| pages=4752–4760}}</ref><ref name="Groisman 2001"/>. Although neither gene is required for pathogenicity the MgtB protein does enhance the long term survival of the pathogen in the cell<ref name="Moncrief 1999">{{cite journal| last=Moncrief| first= M.B.C. | coauthors=Maguire, M.E.|year=1999| title= Magnesium transport in prokaryotes| journal=Journal of Biological Inorganic Chemistry | volume=4| pages=523–527| doi= 10.1007/s007750050374}}</ref>. The genes are also upregulated ''in vitro'' when the Mg<sup>2+</sup> concentration falls below 50 μM (Snavely ''et al.'', 1991a). Although the proteins have Km values similar to CorA and transport rates approximately 10-fold less the genes may be part of a Mg<sup>2+</sup> scavenging system. Chamnongpol and Groisman (2002) presents evidence that the role of these proteins may be to compensate for the inactivation of the CorA protein by the PhoPQ regulon<ref name="Chamnongpol 2002"/>. The authors suggest that the CorA protein is inactivated to allow the avoidance of metal toxicity via the protein in the low Mg<sup>2+</sup> environments ''S. typhimurium'' is subjected to by cells after infection.
 The proteins are both P-type ATPases<ref name="Snavely 1989b"/>
-<ref name="Tao 1995">{{cite journal| last=Tao| first= T. | coauthors=Snavely, M.D., Farr, S.G., and Maguire, M.E.|year=1995| title= Magnesium transport in Salmonella typhimurium: mgtA encodes a P-type ATPase and is regulated by Mg<sup>2+</sup> in a manner similar to that of the mgtB P-type ATPase| journal=Journal of Bacteriology| volume= 177| pages=2654-2662}}</ref> and neither gene shows any similarity to CorA. The MgtA and MgtB proteins are 75% similar (50% identical), although it seems that MgtB may have been acquired by horizontal gene transfer as part of Salmonella Pathogenicity Island 3<ref name="Snavely 1991b">{{cite journal| last=Snavely| first= M.D. | coauthors=Miller, C.G., and Maguire, M.E.|year=1991b| title= The mgtB Mg<sup>2+</sup> transport locus of Salmonella typhimurium encodes a P- type ATPase| journal=Journal of Biological Chemistry | volume=266| pages=815-823}}</ref><ref name="Tao 1995"/>. The TM topology of the MgtB protein has been experimentally determined, showing that the protein has ten TM-spanning helices with the termini of the protein in the cytoplasm (see figure
+<ref name="Tao 1995">{{cite journal| last=Tao| first= T. | coauthors=Snavely, M.D., Farr, S.G., and Maguire, M.E.|year=1995| title= Magnesium transport in Salmonella typhimurium: mgtA encodes a P-type ATPase and is regulated by Mg<sup>2+</sup> in a manner similar to that of the mgtB P-type ATPase| journal=Journal of Bacteriology| volume= 177| pages=2654–2662}}</ref> and neither gene shows any similarity to CorA. The MgtA and MgtB proteins are 75% similar (50% identical), although it seems that MgtB may have been acquired by horizontal gene transfer as part of Salmonella Pathogenicity Island 3<ref name="Snavely 1991b">{{cite journal| last=Snavely| first= M.D. | coauthors=Miller, C.G., and Maguire, M.E.|year=1991b| title= The mgtB Mg<sup>2+</sup> transport locus of Salmonella typhimurium encodes a P- type ATPase| journal=Journal of Biological Chemistry | volume=266| pages=815–823}}</ref><ref name="Tao 1995"/>. The TM topology of the MgtB protein has been experimentally determined, showing that the protein has ten TM-spanning helices with the termini of the protein in the cytoplasm (see figure
-). MgtA is present in widely divergent bacteria, but is not nearly as common as CorA, while MgtB appears to have a quite restricted distribution<ref name="Blanc 1997">{{cite journal| last=Blanc-Potard| first= A.-B. | coauthors=Groisman, E.A. | year= 1997| title= The Salmonella selC locus contains a pathogenicity island mediating intramacrophage survival| journal=EMBO Journal | volume=16| pages=5376–5385}}</ref>. No hypotheses for the unusual distribution have been suggested.
+). MgtA is present in widely divergent bacteria, but is not nearly as common as CorA, while MgtB appears to have a quite restricted distribution<ref name="Blanc 1997">{{cite journal| last=Blanc-Potard| first= A.-B. | coauthors=Groisman, E.A. | year= 1997| title= The Salmonella selC locus contains a pathogenicity island mediating intramacrophage survival| journal=EMBO Journal | volume=16| pages=5376–5385| doi= 10.1093/emboj/16.17.5376}}</ref>. No hypotheses for the unusual distribution have been suggested.
 [[Image:mgta-b_mag.png|frame|right|The TM topology of the MgtB protein]]
-The figure, adapted from Smith ''et al.'' (1993b)<ref name="Smith 1993b">{{cite journal| last=Smith| first= D.L. | coauthors=Tao, T., and Maguire, M.E.|year=1993b| title= Membrane topology of a P-type ATPase. The MgtB magnesium transport protein of Salmonella typhimurium| journal= Journal of Biological Chemistry | volume=268| pages=22469-22479}}</ref>,  shows the experimentally determined membrane topology of the MgtB protein in ''S. typhimurium''. 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 figure, adapted from Smith ''et al.'' (1993b)<ref name="Smith 1993b">{{cite journal| last=Smith| first= D.L. | coauthors=Tao, T., and Maguire, M.E.|year=1993b| title= Membrane topology of a P-type ATPase. The MgtB magnesium transport protein of Salmonella typhimurium| journal= Journal of Biological Chemistry | volume=268| pages=22469–22479}}</ref>,  shows the experimentally determined membrane topology of the MgtB protein in ''S. typhimurium''. 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.
 While the MgtA and MgtB proteins are very similar, they do show some minor differences in activity. MgtB is highly temperature sensitive, losing all activity (with regard to Mg<sup>2+</sup> transport) at a temperature of 20°C<ref name="Snavely 1989b"/>. Additionally, MgtB and MgtA are inhibited by different ranges of cations (Table A10.1<ref name="Snavely 1989b"/>).
@@ Line 129: / Line 129: @@
 |}
-The MgtA and MgtB proteins are ATPases, using one molecule of ATP per transport cycle, whereas the Mg<sup>2+</sup> uptake via CorA is simply electrochemically favourable. Chamnongpol and Groisman (2002) have suggested that the MgtA and MgtB proteins form part of a metal toxicity avoidance system<ref name="Chamnongpol 2002"/>. Alternatively as most P-type ATPases function as efflux mediating transporters it has been suggested that the MgtA and MgtB proteins act as efflux proteins for some as yet unidentified cation, and Mg<sup>2+</sup> transport is either non-specific or exchanged to maintain the electro-neutrality of the transport process<ref name="Kehres 2002">{{cite journal| last=Kehres| first= D.G. | coauthors=Maguire, M.E.|year=2002| title= Structure, properties and regulation of magnesium transport proteins| journal=BioMetals | volume=15| pages=261-270}}</ref>. Further experiments will be required to define the physiological function of these proteins.
+The MgtA and MgtB proteins are ATPases, using one molecule of ATP per transport cycle, whereas the Mg<sup>2+</sup> uptake via CorA is simply electrochemically favourable. Chamnongpol and Groisman (2002) have suggested that the MgtA and MgtB proteins form part of a metal toxicity avoidance system<ref name="Chamnongpol 2002"/>. Alternatively as most P-type ATPases function as efflux mediating transporters it has been suggested that the MgtA and MgtB proteins act as efflux proteins for some as yet unidentified cation, and Mg<sup>2+</sup> transport is either non-specific or exchanged to maintain the electro-neutrality of the transport process<ref name="Kehres 2002">{{cite journal| last=Kehres| first= D.G. | coauthors=Maguire, M.E.|year=2002| title= Structure, properties and regulation of magnesium transport proteins| journal=BioMetals | volume=15| pages=261–270| doi= 10.1023/A:1016078832697}}</ref>. Further experiments will be required to define the physiological function of these proteins.
 ===MgtE===
@@ Line 135: / Line 135: @@
 [[Image:MGTE.png|frame|right|The predicted TM topology of the MgtE protein]]
-The figure (right), adapted from Smith ''et al.'' (1995)<ref name="Smith 1995"/> and the pFAM database entry, this shows the computer predicted membrane topology of the MgtE protein in ''Bacillus firmus'' OF4. The TM domains are shown in light blue. The CBS domains (named for the protein the were identified in, [[cystathionine beta synthase|cystathionine-beta synthase]]), shown in orange, are identified in the pFAM database as regulatory domains, but the mechanism of action has not yet been described. They are found in several voltage-gated chloride channels<ref name="Ponting 1997">{{cite journal| last=Ponting| first= C.P.|year=1997| title= CBS domains in CIC chloride channels implicated in myotonia and nephrolithiasis (kidney stones)| journal=Journal of Molecular Medicine| volume= 75| pages=160-163}}</ref>. The orientation in the membrane and the positions of the N- and C-termini are indicated. Not drawn to scale. This transporter has recently had its structure solved by x-ray crystallography. [http://www.nature.com/nature/journal/vaop/ncurrent/full/nature06093.html Published in Nature]
+The figure (right), adapted from Smith ''et al.'' (1995)<ref name="Smith 1995"/> and the pFAM database entry, this shows the computer predicted membrane topology of the MgtE protein in ''Bacillus firmus'' OF4. The TM domains are shown in light blue. The CBS domains (named for the protein the were identified in, [[cystathionine beta synthase|cystathionine-beta synthase]]), shown in orange, are identified in the pFAM database as regulatory domains, but the mechanism of action has not yet been described. They are found in several voltage-gated chloride channels<ref name="Ponting 1997">{{cite journal| last=Ponting| first= C.P.|year=1997| title= CBS domains in CIC chloride channels implicated in myotonia and nephrolithiasis (kidney stones)| journal=Journal of Molecular Medicine| volume= 75| pages=160–163}}</ref>. The orientation in the membrane and the positions of the N- and C-termini are indicated. Not drawn to scale. This transporter has recently had its structure solved by x-ray crystallography. [http://www.nature.com/nature/journal/vaop/ncurrent/full/nature06093.html Published in Nature]
 The MgtE gene was first identified by Smith ''et al.'' (1995) during a screen for CorA-like proteins in bacteria, and complements the Mg<sup>2+</sup>-uptake-deficient ''S. typhimurium'' strain MM281 (corA mgtA mgtB), restoring wild type growth on standard media<ref name="Smith 1995"/>. The kinetics of Mg<sup>2+</sup> transport for the protein were not determined, as <sup>28</sup>Mg<sup>2+</sup> was unavailable. As a substitute the uptake of <sup>57</sup>Co<sup>2+</sup> was measured and shown to have a Km of 82 μM and a Vmax of 354 pmol min<sup>-1</sup> 10<sup>8</sup> cells<sup>-1</sup>. Mg<sup>2+</sup> was a competitive inhibitor with a Ki of 50 μM — the Ki of Mg<sup>2+</sup> inhibition of <sup>60</sup>Co<sup>2+</sup> uptake via CorA is 10 μM<ref name="Hmiel 1986"/>. A comparison of the available kinetic data for MgtA and CorA is shown in the table. Clearly MgtE does not transport Co<sup>2+</sup> to the same degree as CorA and the inhibition of transport by Mg<sup>2+</sup> is also less efficient, which suggests that the affinity of MgtE for Mg<sup>2+</sup> is lower than that of CorA. The strongest inhibitor of Co<sup>2+</sup> uptake was Zn<sup>2+</sup> with a Ki of 20 μM<ref name="Smith 1995"/>. The transport of Zn<sup>2+</sup> by this protein may be as important as that of Mg<sup>2+</sup>.
@@ Line 173: / Line 173: @@
 ==Yeast==
 ===Early research===
-The earliest research showing that yeast take up Mg<sup>2+</sup> appears to be Schmidt ''et al.'' (1949). However, these authors only showed altered yeast Mg<sup>2+</sup> content in a table within the paper, and the report’s conclusions dealt entirely with the metabolism of phosphate. A series of experiments by Rothstein<ref name="Rothstein 1958">{{cite journal| last=Rothstein| first= A., Hayes| coauthors=A., Jennings, D., and Hooper, D.|year=1958| title= The active transport of Mg<sup>2+</sup> and Mn<sup>2+</sup> into the yeast cell| journal=Journal of General Physiology | volume=41| pages=585-594}}</ref><ref name="Fuhrmann 1968">{{cite journal| last=Fuhrmann| first= G.-F. | coauthors=Rothstein, A.|year=1968| title= The transport of Zn<sup>2+</sup>, Co<sup>2+</sup> and Ni<sup>2+</sup> into yeast cells| journal=Biochimica et Biophysica Acta | volume=163| pages=325-330}}</ref> shifted the focus more towards the uptake of the metal cations, showing that yeast take up cations with the following affinity series: Mg<sup>2+</sup>, Co<sup>2+</sup>, Zn<sup>2+</sup> > Mn<sup>2+</sup> > Ni<sup>2+</sup> > Ca<sup>2+</sup> > Sr<sup>2+</sup>. Additionally, it was suggested that the transport of the different cations is mediated by the same transport system<ref name="Fuhrmann 1968"/><ref name="Norris 1977">{{cite 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}}</ref><ref name="Okorokov 1977">{{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}}</ref><ref name="Conklin 1993">{{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}}</ref> — a situation very much like that in bacteria.
+The earliest research showing that yeast take up Mg<sup>2+</sup> appears to be Schmidt ''et al.'' (1949). However, these authors only showed altered yeast Mg<sup>2+</sup> content in a table within the paper, and the report’s conclusions dealt entirely with the metabolism of phosphate. A series of experiments by Rothstein<ref name="Rothstein 1958">{{cite journal| last=Rothstein| first= A., Hayes| coauthors=A., Jennings, D., and Hooper, D.|year=1958| title= The active transport of Mg<sup>2+</sup> and Mn<sup>2+</sup> into the yeast cell| journal=Journal of General Physiology | volume=41| pages=585–594}}</ref><ref name="Fuhrmann 1968">{{cite journal| last=Fuhrmann| first= G.-F. | coauthors=Rothstein, A.|year=1968| title= The transport of Zn<sup>2+</sup>, Co<sup>2+</sup> and Ni<sup>2+</sup> into yeast cells| journal=Biochimica et Biophysica Acta | volume=163| pages=325–330}}</ref> shifted the focus more towards the uptake of the metal cations, showing that yeast take up cations with the following affinity series: Mg<sup>2+</sup>, Co<sup>2+</sup>, Zn<sup>2+</sup> > Mn<sup>2+</sup> > Ni<sup>2+</sup> > Ca<sup>2+</sup> > Sr<sup>2+</sup>. Additionally, it was suggested that the transport of the different cations is mediated by the same transport system<ref name="Fuhrmann 1968"/><ref name="Norris 1977">{{cite 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}}</ref><ref name="Okorokov 1977">{{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}}</ref><ref name="Conklin 1993">{{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}}</ref> — 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''<ref name="MacDiarmid 1998"/>. The genes involved in this system and a second mitochondrial Mg<sup>2+</sup> transport system, functionally identified significantly after the gene was cloned, are described in the sections below.
@@ Line 180: / Line 180: @@
 Two genes, ALR1 and ALR2, were isolated in a screen for Al<sup>3+</sup> tolerance (resistance) in yeast<ref name="MacDiarmid 1998"/>. Overexpression constructs containing yeast genomic DNA were introduced into wild type yeast and the transformants screened for growth on toxic levels of Al<sup>3+</sup>. 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)<ref name="MacDiarmid 1998"/> on the strength on sequence homology and more recently by Lee and Gardner (2006)<ref name="Lee 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}}</ref>, 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<ref name="Lee 2006"/>.
+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)<ref name="MacDiarmid 1998"/> on the strength on sequence homology and more recently by Lee and Gardner (2006)<ref name="Lee 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}}</ref>, 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<ref name="Lee 2006"/>.
 [[Image:ALR1 topology.png|frame|right|The TM topology of the ALR1 protein]]
@@ Line 186: / Line 186: @@
 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 membrane<ref name="Graschopf 2001">{{cite 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 Mg<sup>2+</sup> homeostasis and is subject to Mg<sup>2+</sup>-dependent control of its synthesis and degradation| journal=Journal of Biological Chemistry| volume= 276| pages=16216-16222}}</ref>. Interestingly, the localisation of the Alr1p was observed to be internalised and degraded in the vacuole in response to extracellular cations. Mg<sup>2+</sup> at very low extracellular concentrations (100 μM; < 10% of the standard media Mg<sup>2+</sup> content) and Co<sup>2+</sup> and Mn<sup>2+</sup> 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<ref name="Graschopf 2001"/>. This mechanism allows a regulation of Mg<sup>2+</sup> 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.
+Membrane fractionation and green fluorescent protein (GFP) fusion studies established that Alr1p is localised to the plasma membrane<ref name="Graschopf 2001">{{cite 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 Mg<sup>2+</sup> homeostasis and is subject to Mg<sup>2+</sup>-dependent control of its synthesis and degradation| journal=Journal of Biological Chemistry| volume= 276| pages=16216–16222}}</ref>. Interestingly, the localisation of the Alr1p was observed to be internalised and degraded in the vacuole in response to extracellular cations. Mg<sup>2+</sup> at very low extracellular concentrations (100 μM; < 10% of the standard media Mg<sup>2+</sup> content) and Co<sup>2+</sup> and Mn<sup>2+</sup> 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<ref name="Graschopf 2001"/>. This mechanism allows a regulation of Mg<sup>2+</sup> 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 Mg<sup>2+</sup><ref name="MacDiarmid 1998"/>) and Alr1p can support normal growth at Mg<sup>2+</sup> concentrations as low as 30 μM<ref name="Graschopf 2001"/>. <sup>57</sup>Co<sup>2+</sup> is taken up into yeast via the Alr1p protein with a Km of 77 – 105 μM (<ref name="Norris 1977"/>; C. MacDiarmid and R. C. Gardner, unpublished data), but the Ki for Mg<sup>2+</sup> 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 Ca<sup>2+</sup>, Co<sup>2+</sup>, Cu<sup>2+</sup>, La<sup>3+</sup>, Mn<sup>2+</sup>, Ni<sup>2+</sup> and Zn<sup>2+</sup>, an array of cations similar to those shown to be transported into yeast by a CorA-like transport system<ref name="MacDiarmid 1998"/>. 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 Mg<sup>2+</sup> transporter is that the loss of Alr1p leads to a decreased total cell content of Mg<sup>2+</sup> but not of the other cations, and that the regulation of the transporter occurs at environmental concentrations of Mg<sup>2+</sup>, but not the other cations tested<ref name="Graschopf 2001"/>. Additionally, two electrophysiological studies where Alr1p was produced in yeast or ''Xenopus'' oocytes showed a Mg<sup>2+</sup>-dependent current in the presence of the protein<ref name="Liu 2002">{{cite journal| last=Liu| first= G.J. | coauthors=Martin, D.K., Gardner, R.C., and Ryan, P.R.|year=2002| title= Large Mg<sup>2+</sup>-dependent currents are associated with the increased expression of ALR1 in Saccharomyces cerevisiae| journal=FEMS Microbiology Letters | volume=213| pages=231-237}}</ref>; Salih ''et al.'', in prep.).
+The evidence that Alr1p is primarily a Mg<sup>2+</sup> transporter is that the loss of Alr1p leads to a decreased total cell content of Mg<sup>2+</sup> but not of the other cations, and that the regulation of the transporter occurs at environmental concentrations of Mg<sup>2+</sup>, but not the other cations tested<ref name="Graschopf 2001"/>. Additionally, two electrophysiological studies where Alr1p was produced in yeast or ''Xenopus'' oocytes showed a Mg<sup>2+</sup>-dependent current in the presence of the protein<ref name="Liu 2002">{{cite journal| last=Liu| first= G.J. | coauthors=Martin, D.K., Gardner, R.C., and Ryan, P.R.|year=2002| title= Large Mg<sup>2+</sup>-dependent currents are associated with the increased expression of ALR1 in Saccharomyces cerevisiae| journal=FEMS Microbiology Letters | volume=213| pages=231–237}}</ref>; Salih ''et al.'', in prep.).
 The kinetics of Mg<sup>2+</sup> uptake by Alr1p have been investigated by electrophysiology techniques on whole yeast cells<ref name="Liu 2002"/>. The results suggested that Alr1p is very likely to act as an ion-selective channel. In the same paper the authors reported that Mg<sup>2+</sup> 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 Mg<sup>2+</sup> transport proteins.
-The alternative techniques of <sup>28</sup>Mg<sup>2+</sup> radiotracer analysis and mag-fura 2 to measure Mg<sup>2+</sup> uptake have not yet been used with Alr1p. <sup>28</sup>Mg<sup>2+</sup> 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 Mg<sup>2+</sup><ref name="Zhang 1997">{{cite journal| last=Zhang| first= A. | coauthors=Cheng, T.P., Wu, X.Y., Altura, B.T., and Altura, B.M.|year=1997| title= Extracellular Mg<sup>2+</sup> regulates intracellular Mg<sup>2+</sup> and its subcellular compartmentation in fission yeast, Schizosaccharomyces pombe| journal=Cellular and Molecular Life Sciences | volume=53| pages=69-72}}</ref> suggesting that multiple systems inside the yeast are transporting Mg<sup>2+</sup> into storage compartments. This internal transport will very likely mask the uptake process. The expression of ALR1 in ''S. typhimurium'' without Mg<sup>2+</sup> uptake genes may be an alternative, but as stated earlier, the effects of a heterologous expression system would need to be taken into account.
+The alternative techniques of <sup>28</sup>Mg<sup>2+</sup> radiotracer analysis and mag-fura 2 to measure Mg<sup>2+</sup> uptake have not yet been used with Alr1p. <sup>28</sup>Mg<sup>2+</sup> 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 Mg<sup>2+</sup><ref name="Zhang 1997">{{cite journal| last=Zhang| first= A. | coauthors=Cheng, T.P., Wu, X.Y., Altura, B.T., and Altura, B.M.|year=1997| title= Extracellular Mg<sup>2+</sup> regulates intracellular Mg<sup>2+</sup> and its subcellular compartmentation in fission yeast, Schizosaccharomyces pombe| journal=Cellular and Molecular Life Sciences | volume=53| pages=69–72}}</ref> suggesting that multiple systems inside the yeast are transporting Mg<sup>2+</sup> into storage compartments. This internal transport will very likely mask the uptake process. The expression of ALR1 in ''S. typhimurium'' without Mg<sup>2+</sup> 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 Mg<sup>2+</sup> transporter. The MRS2 gene was identified in the nuclear genome of yeast in a screen for suppressors of a mitochondrial gene RNA splicing mutation<ref name="Koll 1987">{{cite 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}}</ref>, and was cloned and sequenced by Wiesenberger ''et al.'' (1992)<ref name="Wiesenberger 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}}</ref>. Mrs2p was not identified as a putative Mg<sup>2+</sup> transporter until Bui ''et al.'' (1999)<ref name="Bui 1999"/>. Gregan ''et al.'' (2001a) identified LPE10 by homology to MRS2 and showed that both LPE10 and MRS2 mutants altered the Mg<sup>2+</sup> content of yeast mitochondria and affected RNA splicing activity in the organelle<ref name="Gregan 2001a">{{cite 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}}</ref><ref name="Gregan 2001b">{{cite journal| last=Gregan| first= J. | coauthors=Kolisek, M., and Schweyen, R.J.|year=2001b| title= Mitochondrial Mg<sup>2+</sup> homeostasis is critical for group II intron splicing ''in vivo''| journal=Genes & Development | volume=15| pages=2229-2237}}</ref>. Mg<sup>2+</sup> transport has been shown to be directly mediated by Mrs2p<ref name="Kolisek 2003"/>, but not for Lpe10p.
+Like the ALR genes the MRS2 gene was cloned and sequenced before it was identified as a Mg<sup>2+</sup> transporter. The MRS2 gene was identified in the nuclear genome of yeast in a screen for suppressors of a mitochondrial gene RNA splicing mutation<ref name="Koll 1987">{{cite 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}}</ref>, and was cloned and sequenced by Wiesenberger ''et al.'' (1992)<ref name="Wiesenberger 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}}</ref>. Mrs2p was not identified as a putative Mg<sup>2+</sup> transporter until Bui ''et al.'' (1999)<ref name="Bui 1999"/>. Gregan ''et al.'' (2001a) identified LPE10 by homology to MRS2 and showed that both LPE10 and MRS2 mutants altered the Mg<sup>2+</sup> content of yeast mitochondria and affected RNA splicing activity in the organelle<ref name="Gregan 2001a">{{cite 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}}</ref><ref name="Gregan 2001b">{{cite journal| last=Gregan| first= J. | coauthors=Kolisek, M., and Schweyen, R.J.|year=2001b| title= Mitochondrial Mg<sup>2+</sup> homeostasis is critical for group II intron splicing ''in vivo''| journal=Genes & Development | volume=15| pages=2229–2237}}</ref>. Mg<sup>2+</sup> transport has been shown to be directly mediated by Mrs2p<ref name="Kolisek 2003"/>, 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<ref name="Bui 1999"/><ref name="Gregan 2001a"/> 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, Mg<sup>2+</sup> transport was strongly reduced<ref name="Kolisek 2003"/>.
@@ Line 210: / Line 210: @@
 ==Protozoan (''Paramecium'')==
-The transport of Mg<sup>2+</sup> into Paramecium has been characterised largely by R. R. Preston and coworkers. Electrophysiological techniques on whole Paramecium were used to identify and characterise Mg<sup>2+</sup> currents in a series of papers<ref name="Preston 1990">{{cite journal| last=Preston| first= R.R.|year=1990| title= A magnesium current in Paramecium| journal=Science | volume=250| pages=285-288}}</ref><ref name="Preston 1994a">{{cite journal| last=Preston| first= R.R. | coauthors=Kung, C.|year=1994a| title= Inhibition of Mg<sup>2+</sup> current by single-gene mutation in Paramecium| journal=Journal of Membrane Biology | volume=137| pages=203-212}}</ref><ref name="Preston 1994b">{{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}}</ref><ref name="Preston 1998">{{cite journal| last=Preston| first= R.R.|year=1998| title= Transmembrane Mg<sup>2+</sup> currents and intracellular free Mg<sup>2+</sup> concentration in Paramecium tetraurelia| journal=Journal of Membrane Biology| volume= 164| pages=11-24}}</ref> before the gene was cloned by Haynes ''et al.'' (2002)<ref name="Haynes 2002"/>.
+The transport of Mg<sup>2+</sup> into Paramecium has been characterised largely by R. R. Preston and coworkers. Electrophysiological techniques on whole Paramecium were used to identify and characterise Mg<sup>2+</sup> currents in a series of papers<ref name="Preston 1990">{{cite 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}}</ref><ref name="Preston 1994a">{{cite journal| last=Preston| first= R.R. | coauthors=Kung, C.|year=1994a| title= Inhibition of Mg<sup>2+</sup> current by single-gene mutation in Paramecium| journal=Journal of Membrane Biology | volume=137| pages=203–212}}</ref><ref name="Preston 1994b">{{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}}</ref><ref name="Preston 1998">{{cite journal| last=Preston| first= R.R.|year=1998| title= Transmembrane Mg<sup>2+</sup> currents and intracellular free Mg<sup>2+</sup> concentration in Paramecium tetraurelia| journal=Journal of Membrane Biology| volume= 164| pages=11–24}}</ref> before the gene was cloned by Haynes ''et al.'' (2002)<ref name="Haynes 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<ref name="Haynes 2002"/>. The protein has been predicted to contain 11 TM domains and also contains the α1 and α2 motifs (see figure) of the SLC8 (Na+/Ca<sup>2+</sup> exchanger<ref name="Quednau 2004">{{cite 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}}</ref>) and SLC24 (K+ dependent Na+/Ca<sup>2+</sup> exchanger<ref name="Schnetkamp 2004">{{cite journal| last=Schnetkamp| first= P.P.M.|year=2004| title= The SLC24 Na+/Ca<sup>2+</sup>-K<SUP>+</SUP> exchanger family: vision and beyond| journal=Pflügers Archiv European Journal of Physiology| volume= 447| pages=683 - 688}}</ref>) 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<ref name="Haynes 2002"/>. It is interesting to note that the AtMHX protein from plants also shares a distant relationship with the SLC8 proteins.
+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<ref name="Haynes 2002"/>. The protein has been predicted to contain 11 TM domains and also contains the α1 and α2 motifs (see figure) of the SLC8 (Na+/Ca<sup>2+</sup> exchanger<ref name="Quednau 2004">{{cite 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}}</ref>) and SLC24 (K+ dependent Na+/Ca<sup>2+</sup> exchanger<ref name="Schnetkamp 2004">{{cite journal| last=Schnetkamp| first= P.P.M.|year=2004| title= The SLC24 Na+/Ca<sup>2+</sup>-K<SUP>+</SUP> exchanger family: vision and beyond| journal=Pflügers Archiv European Journal of Physiology| volume= 447| pages=683–688}}</ref>) 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<ref name="Haynes 2002"/>. It is interesting to note that the AtMHX protein from plants also shares a distant relationship with the SLC8 proteins.
-[[Image:XNTA topology.png|frame|right|The TM topology of the XNTA protein]]The figure shows the predicted TM topology of XntAp. Adapted from Haynes ''et al.'' (2002)<ref name="Haynes 2002"/>, this figure shows the computer predicted membrane topology of XntAp in ''Paramecium''. The orientation in the membrane was determined using HMMTOP<ref name="Tusnady 1998">{{cite 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}}</ref><ref name="Tusnady 2001">{{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}}</ref>. 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.
+[[Image:XNTA topology.png|frame|right|The TM topology of the XNTA protein]]The figure shows the predicted TM topology of XntAp. Adapted from Haynes ''et al.'' (2002)<ref name="Haynes 2002"/>, this figure shows the computer predicted membrane topology of XntAp in ''Paramecium''. The orientation in the membrane was determined using HMMTOP<ref name="Tusnady 1998">{{cite 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}}</ref><ref name="Tusnady 2001">{{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}}</ref>. 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 Mg<sup>2+</sup>-dependent currents carried by XntAp are kinetically like that of a channel protein, and have an ion selectivity order of Mg<sup>2+</sup> > Co<sup>2+</sup>, Mn<sup>2+</sup> > Ca<sup>2+</sup> — a series again very similar to that of CorA<ref name="Preston 1998"/>. Unlike the other transport proteins reported so far, XntAp is dependent on intracellular Ca<sup>2+</sup>. The transport was also dependent on ΔΨ, but again Mg<sup>2+</sup> is not transported to equilibrium, being limited to approximately 0.4 mM free Mg<sup>2+</sup> in the cytoplasm. The existence of an intracellular compartment with a much higher free concentration of Mg<sup>2+</sup> (8 mM) was also supported by the results.
 ==Metazoa==
-The investigation of Mg<sup>2+</sup> 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 Mg<sup>2+</sup> 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 Mg<sup>2+</sup> content is maintained at levels where changes might influence cellular metabolism<ref name="Romani 2002">{{cite journal| last=Romani| first= A.M.P. | coauthors=Maguire, M.E.|year=2002| title= Hormonal regulation of Mg<sup>2+</sup> transport and homeostasis in eukaryotic cells| journal=BioMetals | volume=15| pages=271-283}}</ref>.
+The investigation of Mg<sup>2+</sup> 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 Mg<sup>2+</sup> 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 Mg<sup>2+</sup> content is maintained at levels where changes might influence cellular metabolism<ref name="Romani 2002">{{cite journal| last=Romani| first= A.M.P. | coauthors=Maguire, M.E.|year=2002| title= Hormonal regulation of Mg<sup>2+</sup> transport and homeostasis in eukaryotic cells| journal=BioMetals | volume=15| pages=271–283}}</ref>.
 ===MRS2===
@@ Line 237: / Line 237: @@
 ===TRPM6/ TRPM7===
-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)<ref name="Montell 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}}</ref> have reviewed the research into the TRP genes, and a second review by Montell (2003)<ref name="Montell 2003">{{cite journal| last=Montell| first= C.|year=2003| title= Mg<sup>2+</sup> Homeostasis: The Mg<sup>2+</sup>nificent TRPM Chanzymes| journal=Current Biology | volume=13| pages=R799-R801}}</ref> has reviewed the research into the TRPM genes.
+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)<ref name="Montell 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}}</ref> have reviewed the research into the TRP genes, and a second review by Montell (2003)<ref name="Montell 2003">{{cite journal| last=Montell| first= C.|year=2003| title= Mg<sup>2+</sup> Homeostasis: The Mg<sup>2+</sup>nificent TRPM Chanzymes| journal=Current Biology | volume=13| pages=R799–R801}}</ref> 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<ref name="Montell 2003"/>.
-The activity of these two proteins has been very difficult to quantify. TRPM7 by itself appears to be a Ca<sup>2+</sup> channel<ref name="Runnels 2002">{{cite 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}}</ref> but in the presence of TRPM6 the affinity series of transported cations places Mg<sup>2+</sup> above Ca<sup>2+</sup><ref name="Nadler 2001">; Monteilh-Zoller ''et al.'', 2003<ref name="Monteilh 2003">{{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}}</ref>). 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 expression<ref name="Chubanov 2004">{{cite 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}}</ref>. An unfortunate choice of experimental system by Voets ''et al.'', (2004)<ref name="Voets 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 Mg<sup>2+</sup> influx channel involved in intestinal and renal Mg<sup>2+</sup> absorption| journal=Journal of Biological Chemistry | volume=279| pages=19-25}}</ref> led to the conclusion that TRPM6 is a functional Mg<sup>2+</sup> transporter. However, later work by Chubanov ''et al.'' (2004)<ref name="Chubanov 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 activity of these two proteins has been very difficult to quantify. TRPM7 by itself appears to be a Ca<sup>2+</sup> channel<ref name="Runnels 2002">{{cite 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}}</ref> but in the presence of TRPM6 the affinity series of transported cations places Mg<sup>2+</sup> above Ca<sup>2+</sup><ref name="Nadler 2001">; Monteilh-Zoller ''et al.'', 2003<ref name="Monteilh 2003">{{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}}</ref>). 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 expression<ref name="Chubanov 2004">{{cite 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}}</ref>. An unfortunate choice of experimental system by Voets ''et al.'', (2004)<ref name="Voets 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 Mg<sup>2+</sup> influx channel involved in intestinal and renal Mg<sup>2+</sup> absorption| journal=Journal of Biological Chemistry | volume=279| pages=19–25}}</ref> led to the conclusion that TRPM6 is a functional Mg<sup>2+</sup> transporter. However, later work by Chubanov ''et al.'' (2004)<ref name="Chubanov 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.
 [[Image:TRPM6-7_topology.png|frame|right|The predicted TM topology of the TRPM6 and TRPM7 proteins]]
 The predicted TM topology of the TPRM6 and TRPM7 proteins
-have been adapted from Nadler ''et al.'' (2001)<ref name="Nadler 2001"/>, Runnels ''et al.'' (2001)<ref name="Runnels 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}}</ref> and Montell ''et al.'' (2002)<ref name="Montell 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.
+have been adapted from Nadler ''et al.'' (2001)<ref name="Nadler 2001"/>, Runnels ''et al.'' (2001)<ref name="Runnels 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}}</ref> and Montell ''et al.'' (2002)<ref name="Montell 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)<ref name="Voets 2004"/> paper are probably incorrect in attributing the Mg<sup>2+</sup> 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 Mg<sup>2+</sup>, based on both electrophysiological techniques and also mag-fura 2 to determine changes in cytoplasmic free Mg<sup>2+</sup>.
@@ Line 252: / Line 252: @@
 This gene is the only known example of Mg<sup>2+</sup> 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 Mg<sup>2+</sup> in the human kidney.
-The gene Claudin-16 was cloned by Simon ''et al.'' (1999)<ref name="Simon 1999"/>, again only after a series of reports describing the Mg<sup>2+</sup> flux itself with no gene/protein<ref name="Shareghi 1982">{{cite 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}}</ref><ref name="di Stefano 1993">{{cite journal| last=di Stefano| first= A. | coauthors=Roinel, N., de Rouffignac, C., and Wittner, M.|year=1993| title= Transepithelial Ca<sup>2+</sup> and Mg<sup>2+</sup> 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}}</ref><ref name="de Rouffignac 1994">{{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}}</ref>. 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<ref name="Simon 1999"/>. This localisation was consistent with the earlier reports for the location of Mg<sup>2+</sup> re-uptake by the kidney. Following the cloning, mutations in the gene were identified in patients with familial hypomagnesaemia with hypercalciuria and nephrocalcinosis<ref name="Weber 2000">{{cite 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}}</ref><ref name="Weber 2001">{{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}}</ref>, strengthening the links between the gene and the uptake of Mg<sup>2+</sup>.
+The gene Claudin-16 was cloned by Simon ''et al.'' (1999)<ref name="Simon 1999"/>, again only after a series of reports describing the Mg<sup>2+</sup> flux itself with no gene/protein<ref name="Shareghi 1982">{{cite 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}}</ref><ref name="di Stefano 1993">{{cite journal| last=di Stefano| first= A. | coauthors=Roinel, N., de Rouffignac, C., and Wittner, M.|year=1993| title= Transepithelial Ca<sup>2+</sup> and Mg<sup>2+</sup> 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}}</ref><ref name="de Rouffignac 1994">{{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}}</ref>. 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<ref name="Simon 1999"/>. This localisation was consistent with the earlier reports for the location of Mg<sup>2+</sup> re-uptake by the kidney. Following the cloning, mutations in the gene were identified in patients with familial hypomagnesaemia with hypercalciuria and nephrocalcinosis<ref name="Weber 2000">{{cite 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}}</ref><ref name="Weber 2001">{{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}}</ref>, strengthening the links between the gene and the uptake of Mg<sup>2+</sup>.
 ==Plants==
@@ Line 277: / Line 277: @@
 The AtMHX gene is expressed throughout the plant but most strongly in the vascular tissue<ref name="Shaul 1999"/>.  The authors suggest that the physiological role of the protein is to store Mg<sup>2+</sup> 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 Mg<sup>2+</sup> into the vacuolar space and H<sup>+</sup> out, as demonstrated by electrophysiological techniques<ref name="Shaul 1999"/>. 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<sup>+</sup>-ATPase<ref name="Kurkdjian 1989">{{cite 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}}</ref><ref name="Marschner 1995">{{cite book| last=Marschner| first= H.|year=1995| title= Mineral Nutrition in Higher Plants. (San Diego: Academic Press)}}</ref>. How the transport of Mg<sup>2+</sup> by the protein is regulated was not determined. Currents were observed to pass through the protein in both directions, but the Mg<sup>2+</sup> 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 Mg<sup>2+</sup>, Shaul ''et al.'' (1999)<ref name="Shaul 1999"/> also showed that the protein could transport Zn<sup>2+</sup> and Fe<sup>2+</sup>, but did not report on the capacity of the protein to transport other divalent cations (e.g. Co<sup>2+</sup> and Ni<sup>2+</sup>) or its susceptibility to inhibition by cobalt (III) hexaammine.
+The protein transports Mg<sup>2+</sup> into the vacuolar space and H<sup>+</sup> out, as demonstrated by electrophysiological techniques<ref name="Shaul 1999"/>. 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<sup>+</sup>-ATPase<ref name="Kurkdjian 1989">{{cite 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}}</ref><ref name="Marschner 1995">{{cite book| last=Marschner| first= H.|year=1995| title= Mineral Nutrition in Higher Plants. (San Diego: Academic Press)}}</ref>. How the transport of Mg<sup>2+</sup> by the protein is regulated was not determined. Currents were observed to pass through the protein in both directions, but the Mg<sup>2+</sup> 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 Mg<sup>2+</sup>, Shaul ''et al.'' (1999)<ref name="Shaul 1999"/> also showed that the protein could transport Zn<sup>2+</sup> and Fe<sup>2+</sup>, but did not report on the capacity of the protein to transport other divalent cations (e.g. Co<sup>2+</sup> and Ni<sup>2+</sup>) or its susceptibility to inhibition by cobalt (III) hexaammine.
 The detailed kinetics of Mg<sup>2+</sup> 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 Mg<sup>2+</sup> (or Zn<sup>2+</sup>) content of the plants was not altered in the transgenic plants.