Dissociation between reperfusion induced arrhythmias and increases in ventricular alpha 1 receptor density in the anaesthetised rat.

Using anaesthetised rats we have assessed (1) whether the density of alpha 1 adrenergic receptors increases during coronary artery occlusion, (2) whether any change in density can be associated with the onset of reperfusion induced ventricular fibrillation, and (3) whether alpha 1 blockade with prazosin modifies the incidence of reperfusion induced ventricular fibrillation. The incidence of fibrillation upon reperfusion after 3, 5, 10, 20 and 30 min occlusion was 20, 75, 50, 16 and 10% (n = 10-12 in each group) respectively. alpha 1 Receptor density was measured using [3H]-prazosin in non-ischaemic and ischaemic tissue obtained after 0, 5 and 30 min ischaemia. Receptor density was not significantly altered at the time of maximum incidence of reperfusion induced ventricular fibrillation (5 min occlusion) but did significantly increase in both non-ischaemic and ischaemic tissue after 30 min occlusion, when the incidence of fibrillation upon reperfusion was very low (8%). At this time the values were 17.0(SEM 2.3) and 18.4(0.6)fmol.mg-1 protein in non-ischaemic and ischaemic zones as compared to 10.7(0.6) and 12.8(1.0)fmol.mg-1 protein in sham operated control animals (p less than 0.05 in both cases). Prazosin (0.1 or 1.0 mg.kg-1 body wt intravenously, 5 min prior to coronary occlusion) did not alter the incidence of ventricular fibrillation, ventricular tachycardia or total number of premature ventricular complexes upon reperfusion. We conclude that ischaemia induced changes in alpha 1 receptor density do not parallel changes in vulnerability to reperfusion induced arrhythmias.(ABSTRACT TRUNCATED AT 250 WORDS)

Magnesium and cell proliferation.

Although studies in mammalian cells and yeast suggest that Mg2+ plays an important role in cell growth and hormone response, intracellular roles of Mg2+ are poorly understood. Thus, we are developing methods to study Mg2+ regulation of growth and hormonal response. Preliminary data using cell-permeable Mg2+ indicators based on tropolone suggest the feasibility of the dynamic and selective determination of intracellular free Mg2+ concentration. "Mg2+-deficient" cell lines have also been developed. Murine S49 lymphoma cells in normal 0.8 mM Mg2+ medium double in 17 hours, but die when placed in 0.2 mM Mg2+ medium. Two classes of S49 clones have been isolated which grow in 30 microM Mg2+ with doubling times of 22 and 60 hours. Although total cell Mg2+ is decreased by 50%, the decrease is selective since cytoplasmic Mg2+ is decreased 75% while particulate Mg2+ is unchanged. Hormonal response in the Mg2+ -deficient cells is defective. Cyclic AMP accumulation in response to beta-adrenergic receptor activation is decreased more than 95%. In contrast, the Mg2+ -deficient cells lose only about 50% of their response to PGE1 receptor activation, retain 50% of their beta-receptors, and accumulate cyclic AMP in response to cholera toxin at the wild-type rate. Mg2+ transport also occurs at the wild-type rate, but with a slightly higher affinity and is no longer hormone-sensitive. Ca2+ content is normal or slightly high. T-lymphocytes isolated from rats made Mg2+ -deficient for 8 weeks give similar results, indicating that the Mg2+ -deficient S49 lymphoma cell clones are a good model for Mg2+ -deficiency. The data suggest that lack of Mg2+ causes growth abnormalities and leads to markedly altered receptor-G-protein coupling, but may have less effect on G-protein-adenylate cylase interaction.

The MgtE Mg2+ transport protein is involved in Aeromonas hydrophila adherence.

Aeromonas hydrophila AH-3 strains carrying mutations in mgtE, which encodes a Mg2+ and Co2+ transport system, showed a 50% reduction of in vitro adherence to HEp-2 cells, a reduction in swarming in semisolid swarming agar, and decrease in biofilm formation of over 60% in comparison to the wild-type strain. The cloned A. hydrophila mgtE expressed from a plasmid complements a Salmonella typhimurium strain deleted for all Mg2+ transporters both phenotypically and by measurement of 57Co2+ uptake. Likewise, plasmid-borne mgtE was able to complement the changes observed in A. hydrophila mgtE mutants. We suggest that MgtE and thus Mg2+ and possibly Co2+ have a role in A. hydrophila related to their swarming ability and related consequences such as adherence and biofilm formation.

Hormonal regulation of magnesium uptake: differential coupling of membrane receptors to magnesium uptake.

Hormonal regulation of Mg2+ influx was examined in the neuroblastoma X glioma hybrid cell line NG108-15 and the skeletal muscle cell line G8 using 28Mg2+. Both cell lines express multiple classes of hormone receptors; in addition, G8 cells can be induced to differentiate from a single myoblast-like cell into fused myotube-like cells. In NG108-15 cells, 2-Cl-adenosine, an adenosine receptor agonist, stimulated Mg2+ influx by about 60%. This effect was not mimicked by norepinephrine or PGE1, agonists at alpha 2-adrenergic and prostaglandin receptors which NG108-15 cells also express. Carbachol, acting through a muscarinic receptor, gave minimal and variable stimulation of Mg2+ influx. The effect of 2-Cl-adenosine was not blocked by theophylline, an adenosine receptor antagonist, and was not mimicked by adenosine analogs selective for either A1 or A2 adenosine receptors, suggesting that a nonclassical adenosine receptor mediates the effect on Mg2+ influx. Theophylline slightly stimulated Mg2+ influx as did the permeable cyclic AMP analog, 8-Br-cyclic AMP. These results indicate that cyclic AMP may influence Mg2+ influx in NG108-15 cells unlike previous results in murine S49 lymphoma cells [Maguire and Erdos, J. biol. Chem. 255: 1030-1035, 1980] where receptor modulation of Mg2+ influx was independent of cyclic AMP. In G8 cells, the nicotinic cholinergic receptor agonist carbachol stimulated Mg2+ influx at the myoblast cell stage but had no effect on Mg2+ influx after cells had formed myotubes. The beta-adrenergic agonist isoproterenol had the opposite effect, stimulating Mg2+ influx in the myotube stage but not in the myoblast stage. Taken together, these results demonstrate that only a subset of receptors expressed by a cell may be coupled to Mg2+ influx, that the regulation of Mg2+ influx differs from cell type to cell type, and finally, that modulation of Mg2+ influx by hormone receptors may change with differentiation.

MgtA and MgtB: prokaryotic P-type ATPases that mediate Mg2+ influx.

The gram-negative bacterium Salmonella typhimurium possesses three distinct Mg2+ transport systems, encoded by the corA, mgtA, and mgtB loci. The CorA transport system is the constitutive Mg2+ influx system. It can also mediate Mg2+ efflux at very high extracellular Mg2+ concentrations. In contrast, the MgtA and MgtB Mg2+ transport systems are normally expressed only at low extracellular Mg2+ concentrations. A strain of S. typhimurium was constructed by mutagenesis which lacks Mg2+ transport and requires 100 mM Mg2+ for growth. Using this strain, both the MgtA and MgtB transport systems were cloned by complementation of the strains inability to grow without Mg2+ supplementation. After sequencing and further genetic analysis, the MgtB system appears to be an operon composed of the mgtC and mgtB genes (5' to 3'). The downstream mgtB gene encodes the 102 kDa MgtB protein which by sequence analysis is clearly a P-type ATPase. Interestingly, while MgtB has relatively poor homology to other known prokaryotic P-type ATPases, it is highly homologous to mammalian reticular Ca(2+)-ATPases. MgtC is a 22.5 kDa hydrophobic membrane protein that lacks homology to any known protein. Transposon insertions in this gene abolish uptake by the MgtB transport system. We hypothesize that MgtC is a subunit of the MgtB ATPase involved either in proper insertion of MgtB into the membrane or possibly in binding of extracellular Mg2+ for delivery to the ATPase subunit. The sequence of the MgtA gene has recently been completed, and it too is a P-type ATPase more similar to eukaryotic than prokaryotic P-type ATPases. Expression of both MgtA and MgtB are highly regulated by the concentration of extracellular Mg2+. Transcription of mgtB can be increased about 1000 fold by lowering Mg2+ from 1 mM to 1 microM. Likewise, when mgtB is expressed from a multicopy plasmid, a similar decrease in extracellular Mg2+ greatly increases transport. Under growth conditions of limiting Mg2+, MgtB becomes the dominant Mg2+ influx system in S. typhimurium. Even so, since MgtB (and MgtA) mediate only influx of Mg2+, it is unclear why the cell requires energy from ATP to mediate Mg2+ entry into the cell down a large electrochemical gradient. Further studies of the structure-function and energetics of these novel Mg2+ influx P-type ATPases should yield insights into the function of P-type ATPases in general as well as information about the regulation of cellular Mg2+ fluxes.

Magnesium regulation of the beta-receptor-adenylate cyclase complex. II. Sc3+ as a Mg2 antagonist.

Sc3+ bears the same relationship to Mg2+ as La3+ to Ca2+, a similar ionic radius but increased charge. Therefore, the possibility was investigated that Sc3+ would be a Mg2+ antagonist at Mg2+ sites on the beta-adrenergic receptor-adenylate cyclase complex of the murine S49 lymphoma cell. Sc3+ is consistently much more potent than La3+ in inhibiting adenylate cyclase regardless of the mode of activation. IC50 values for Sc3+ of 10-30 microM were observed, whereas those for La3+ were about 300 microM. However, Sc3+ does not block the ability of Mg2+ to increase beta-receptor affinity for agonist nor alter agonist affinity by itself. Furthermore, Sc3+ is a weak inhibitor of the beta-receptor-mediated inhibition of Mg2+ influx. In cyc- S49 membranes, in which the catalytic subunit of cyclase cannot interact with the nucleotide-coupling protein(s), Sc3+ is as potent as in wild-type S49 membranes and again more potent than La3+. Substrate kinetics show that Sc3+, like Mg2+, modulates adenylate cyclase activity by affecting the Vmax without altering the Km for substrate. The data suggest that Sc3+ is a specific antagonist of Mg2+ at the Mg2+ site on the catalytic subunit and support the suggestion that there are two distinct sites for Mg2+ with different functions, one site on the coupling protein(s) and one on the catalytic subunit. It was also found that an apparent complex of Sc3+ and F-, ScF4-, is a potent inhibitor of adenylate cyclase, with an IC50 of 3 microM.

Molecular aspects of Mg2+ transport systems.

The gram-negative bacterium Salmonella typhimurium possesses three distinct Mg2+ transport systems, encoded by the CorA, MgtA, and MgtB loci. The CorA transport system is the constitutive Mg2+ influx system but can also mediate efflux at high extracellular Mg2+ concentrations. The CorA protein lacks homology to any known protein, is an integral membrane protein containing 28% percent-charged amino acids, and has three carboxyl terminal membrane-spanning segments. Its properties indicate that it is a new class of membrane transport protein, likely found in all gram-negative bacteria and possibly other organisms. In contrast, the MgtA and MgtB Mg2+ transport systems are normally expressed only at low extracellular Mg2+ concentrations and can mediate only the influx of Mg2+. Both MgtA and MgtB system are P-type ATPases; they have relatively poor homology to other known prokaryotic P-type ATPases but are highly homologous to mammalian P-type ATPases, particularly reticular Ca(2+)-ATPases. Expression of both MgtA and MgtB is highly regulated by the concentration of extracellular Mg2+. Transcription of MgtB is increased about 1,000-fold by lowering Mg2+ from 1 mM to 1 microM, and, under growth conditions of limiting Mg2+, MgtB becomes the dominant Mg2+ influx system. However, it is unclear why the cells require the use of ATP to mediate the influx of Mg2+ down its electrochemical gradient. Study of these Mg2+ transport systems should lead to further understanding of cellular Mg2+ homeostasis and eventually to characterization of eukaryotic Mg2+ transport systems.

Magnesium but not calcium accumulation is inhibited by beta-adrenergic stimulation in S49 lymphoma cells.

The uptake of 28Mg and 45Ca was measured in S49 lymphoma cells. The beta-adrenergic agonist (-)-isoproterenol markedly inhibited the rate of 28Mg accumulation but had no effect on 45Ca accumulation. The effect of (-)-isoproterenol was blocked by (-)-propranolol. In variants of the S49 cell line deficient in adenylate cyclase activity (cyc-) or in hormone receptor-adenylate cyclase coupling (unc), (-)-isoproterenol had no effect on 28Mg accumulation. The S49 lymphoma cells also possess prostaglandin E1 receptors coupled to adenylate cyclase, and, like (-)-isoproterenol, prostaglandin E1 decreased the rate of 28Mg uptake. Experiments with the mouse erythroleukemia cell line GM86 also showed a beta-adrenergic-mediated decrease in the rate of accumulation of 28Mg. Previous work has shown that Mg2+ increases the affinity of agonists for the beta-adrenergic receptor (Bird, S.J., and Maguire, M.E. (1978) J. Biol. Chem. 253, in press). In view of these effects of Mg2+, it is suggested that Mg2+, but not Ca2+, may regulate the sensitivity of S49 cell adenylate cyclase to stimulation by catecholamines.

Microbial magnesium transport: unusual transporters searching for identity.

Mg2+ is unique among biological cations because of its charge density and solution chemistry. This is abundantly reflected in its transport systems, studied primarily in Salmonella typhimurium. The constitutively expressed CorA transport system is the primary Mg2+ influx pathway for the Bacteria and the Archaea. Its structure of a large N-terminal soluble periplasmic domain with three transmembrane segments at the C-terminus is unique among membrane carriers, and its protein sequence bears no resemblance to other known proteins. The MgtE transport system can also mediate Mg2+ uptake, but whether this is its primary function is not known. MgtE also lacks homology to other known proteins. In contrast, the MgtA and MgtB Mg2+ transport systems of enteric bacteria are P-type ATPases by sequence homology, mediating Mg2+ influx with, rather than against, the Mg2+ electrochemical gradient. They are closely related to mammalian Ca2+-ATPases. Expression of MgtA and MgtB is under the control of the PhoPQ two-component regulatory system, important in bacterial virulence. In S. typhimurium, MgtB is encoded by a two-gene operon mgtCB; the function of the MgtC protein is unknown, and it lacks close homologues. The ligand for the PhoQ membrane sensor kinase is Mg2+ and, at decreased extracellular Mg2+ concentrations, transcription of mgtA and mgtCB are enormously induced. All three genes are also induced upon S. typhimurium invasion of epithelial or macrophage cells. Mutation of these genes has no effect on invasion efficiency, but an insertion in mgtC renders S. typhimurium essentially avirulent in the mouse. The physiological roles of the known Mg2+ transport systems are not yet completely defined. Nonetheless, the singular sequence and apparent structure of the CorA and MgtE transport proteins, the complex regulation of MgtA, MgtB and MgtC and their involvement in pathogenesis suggests that further study will be rewarding.

Magnesium and the role of MgtC in growth of Salmonella typhimurium.

Salmonella typhimurium has three distinct transport systems for Mg2+: CorA, MgtA, and MgtB. The mgtCB operon encodes two proteins, MgtC, a hydrophobic protein with a predicted molecular mass of 22.5 kDa, and MgtB, a 102-kDa P-type ATPase Mg2+ transport protein. The mgtCB locus has been identified as part of a new Salmonella pathogenicity island, SPI-3. Transcription of mgtCB is regulated by extracellular Mg2+ via the two-component PhoPQ regulatory system important for virulence. To elucidate MgtC's role in a low-Mg2+ environment, we looked at growth and transport in strains lacking the CorA and MgtA Mg2+ transporters but expressing MgtB, MgtC, or both. mgtC mgtB+ and mgtC+ mgtB+ strains exhibited growth in N minimal medium without added Mg2+ with a 1- to 2-h lag phase. An mgtC+ mgtB strain was also able to grow in N minimal medium without added Mg2+ but only after a 24-h lag phase. In N minimal medium containing 10 mM Mg2+, all strains grew after a short lag phase; the mgtC+ mgtB strain grew to a higher optical density at 600 nm than an mgtC+ mgtB+ strain and was comparable to wild type. The lengthy lag phase before growth in an mgtC+ mgtB strain was not due to lack of expression of MgtC. Western blot analysis indicated that substantial MgtC protein is present by 2 h after suspension in N minimal medium. Surprisingly, in an mgtC+ mgtB+ strain, MgtC was undetectable during Mg2+ starvation, although large amounts of MgtB were observed. The lack of expression of MgtC is not dependent on functional MgtB, since a strain carrying a nonfunctional MgtB with a mutation (D379A) also did not make MgtC. Since, during invasion of eukaryotic cells, S. typhimurium appears to be exposed to a low-pH as well as a low-Mg2+ environment, the growth of an mgtC+ mgtB strain was tested at low pH with and without added Mg2+. While significant quantities of MgtC could be detected after suspension at pH 5.2, the mgtC+ mgtB strain was unable to grow at pH 5.2 whether or not Mg2+ was present. Finally, using 63Ni2+ and 57Co2+ as alternative substrates for the unavailable 28Mg2+, cation uptake could not be detected in an mgtC+ mgtB strain after Mg2+ starvation. We conclude that MgtC is not a Mg2+ transporter and that it does not have a primary role in the survival of S. typhimurium at low pH.

Regulation of magnesium but not calcium transport by phorbol ester.

Phorbol esters in the presence of Ca2+ apparently mimic diacylglycerol in activating protein kinase C. Resulting phosphorylations alter multiple cellular processes including inhibition of the action of Ca2+-mobilizing agonists. In contrast to this inhibition of Ca2+ mobilization, addition of 4 beta-phorbol 12-myristate 13-acetate (PMA) to murine S49 lymphoma cells stimulated Mg2+ influx severalfold without any detectable alteration of Mg2+ efflux or of Ca2+ influx or efflux. Stimulation of Mg2+ influx did not require extracellular Ca2+, was half-maximal at 10 nM PMA, and was characterized by a marked increase in the Vmax of Mg2+ influx without change in the Ka for Mg2+. Stimulation of Mg2+ influx was not mimicked by 4 alpha-phorbol didecanoate, which does not activate protein kinase C and was not the result of Na+/H+ exchange activity. The effect of PMA on Mg2+ influx was inhibited by the beta-adrenergic agonist (-)-isoproterenol, which we have previously shown to inhibit Mg2+ influx by a non-cyclic AMP-dependent mechanism (Maguire, M. E., and Erdos, J. J. (1980) J. Biol. Chem. 255, 1030-1035). Forskolin, a direct activator of adenylate cyclase, also inhibited PMA stimulation of Mg2+ influx, suggesting the presence of both cyclic AMP-dependent and -independent influences on Mg2+ influx. We have also previously demonstrated that Mg2+ influx occurs solely into a small subcytoplasmic pool (Grubbs, R. D., Collins, S. D., and Maguire, M. E. (1984) J. Biol. Chem. 259, 12184-12192). PMA did not alter this compartmentation; rather, it almost doubled the content of this cytosolic Mg2+ pool. These data indicate that, in addition to phorbol ester modulation of intracellular Ca2+ mobilization, substantial changes in Mg2+ flux and content occur. They further demonstrate that Mg2+ influx is regulated by a variety of stimuli. It seems likely that such alterations in Mg2+ flux and content would have physiological consequences.

Magnesium as a regulatory cation: criteria and evaluation.

Of the two major intracellular divalent cations, Ca2+ has been studied much more extensively than Mg2+ and is now well accepted as a major intracellular regulator. This review focuses instead on some recent advances in the understanding of the physiology and biochemistry of Mg2+. For purposes of discussion, four criteria have been developed that should be fulfilled if Mg2+ is to be accepted as an important intracellular regulatory cation: cellular processes must exist which are sensitive to free Mg2+ within the physiological concentration range; a (transport) mechanism(s) must exist which is capable of altering free Mg2+ concentration within a cell; if Mg2+ is compartmented within cells, any potentially regulated system or process and any change in intracellular free Mg2+ concentration must be shown to occur within the same compartment; and any change(s) in free Mg2+ concentration and any alteration(s) in a Mg2+-sensitive process must occur in a sequential manner. These criteria are largely but not completely met at the present time. Criteria 1 and probably 2 can be shown in at least some systems to be fully met. Criteria 3 and 4 are partially met but neither can be fully examined until methods for measuring intracellular free Mg2+ concentrations on an appropriate time scale are further developed. Thus, there exists strong but as yet incomplete evidence that Mg2+, like Ca2+, can play an active, regulatory role within cells. Finally, it is suggested that Ca2+ plays the specific role of the acute, transient regulatory element while Mg2+ plays the complementary role of a more long-term regulatory element which controls the set point or gain of a system or process.

Hormone-sensitive magnesium transport in murine S49 lymphoma cells: characterization and specificity for magnesium.

1. The hormone-sensitive transport of Mg(2+) into murine S49 lymphoma cells and its relationship to other divalent cation transport systems have been investigated.2. Mg(2+) influx, measured with (28)Mg(2+), is saturable with an apparent extracellular ion concentration at half-maximal influx (K(in)) for Mg(2+) of 330 muM and a maximal influx rate of 360 p-mole/min.10(7) cells (2.9 n-mole/min.mg cell protein or a flux rate of about 0.12 p-mole/sec.cm(2)). Efflux of Mg(2+) is biphasic with half-times of 55 and 240 min at 37 degrees C and is temperature-sensitive.3. beta-Adrenergic agonists inhibit influx but not efflux of Mg(2+) in S49 cells. Efflux of Mg(2+) is also unaffected by extracellular [Mg(2+)] or [Ca(2+)]. These results imply that the mechanism of the transport system does not involve Mg-Mg exchange.4. Mn(2+) is a non-competitive inhibitor of Mg(2+) influx with an inhibition constant, K(i), of about 200 muM. The weak inhibition exhibited by Ca(2+) (K(i) > 5 mM) is also non-competitive. La(3+) inhibits Mg(2+) transport half-maximally at about 100 muM; Ni(2+), Zn(2+), Co(2+) and Sc(3+) are all less effective than La(3+). The Ca(2+)-channel blockers cis-diltiazem, verapamil, and nifedipine and the monovalent cations Na(+) and K(+) also have no effect on Mg(2+) influx. However, increasing the extracellular pH stimulates Mg(2+) influx.5. Total cellular Mg(2+) is about 85 n-mole/10(7) cells; however, at apparent isotopic equilibrium with (28)Mg(2+) less than 3% of total cellular Mg(2+) has been exchanged. This indicates that cellular Mg(2+) is highly compartmented and that recently transported Mg(2+) exchanges very slowly with bulk intracellular Mg(2+).6. Ca(2+) influx has a K(in) of 80 muM and is much slower than Mg(2+) influx. V(max) varied in different experiments from 3 to 15 p-mole/min.10(7) cells (25-125 p-mole/min.mg cell protein). Efflux of Ca(2+) is biphasic with half-times of 22 and 200 min and is temperature-sensitive. Hormonal stimulation has no effect on either influx or efflux of Ca(2+). Mg(2+) is a competitive inhibitor of Ca(2+) influx (K(i) = 3 mM).7. Two kinetic components of Mn(2+) influx are present with apparent K(in)s of 4 muM and 100 muM. Maximal influx rates are 5 and 60 p-mole/min.10(7) cells (40 and 480 p-mole/min.mg cell protein), respectively. Influx of Mn(2+) is not altered by beta-adrenergic agonist.8. Uptake of Na(+) or K(+) is unaltered by beta-adrenergic stimulation. These data in the S49 lymphoma cell indicate that (a) Mg(2+) is translocated by a transport system independent of those that transport other divalent cations, (b) hormonal inhibition of divalent ion transport is specific for Mg(2+) and (c) cellular Mg(2+) is highly compartmented.

Magnesium transport systems: genetics and protein structure (a review).

Magnesium is unique among biological cations. Its volume change from hydrated cation to atomic ion is over an order of magnitude larger than that of any other biological cation. This volume change presents particular problems for a magnesium transport system and suggests that these systems may be significantly different from other classes of ion transport systems. Detailed study of Mg2+ transport in complex organisms is limited by severe technical problems. However, molecular genetic techniques have enabled the isolation of three Mg2+ transport systems from the Gram-negative bacterium Salmonella typhimurium. The MgtA and MgtB transport systems are members of the P-type ATPase superfamily of transporters but possess unique characteristics among members of this family. The CorA transport protein is the first member of an entirely new class of transport proteins. In addition, another completely new family of Mg2+ transport proteins have been identified that is present in both Gram-negative and Gram-positive bacteria. Characterization of these transporters should provide substantial insight into Mg2+ transport and cellular Mg2+ homeostasis.

The CorA Mg(2+) transport protein of Salmonella typhimurium. Mutagenesis of conserved residues in the second membrane domain.

Salmonella typhimurium CorA is the archetypal member of the largest family of Mg(2+) transporters of the Bacteria and Archaea. It contains three transmembrane segments. There are no conserved charged residues within these segments indicating electrostatic interactions are not used in Mg(2+) transport through CorA. Previous mutagenesis studies of CorA revealed a single face of the third transmembrane segment that is important for Mg(2+) transport. In this study, we mutated hydroxyl-bearing and other conserved residues in the second transmembrane segment to identify residues involved in transport. Residues Ser(260), Thr(270), and Ser(274) appear to be important for transport and are oriented such that they would also line a face of an alpha-helix. In addition, the sequence (276)YGMNF(280), found in virtually all CorA homologues, is critical for CorA function because even conservative mutations are not tolerated at these residues. Finally, mutations of residues in the second transmembrane segment, unlike those in the third transmembrane segment, revealed cooperative behavior for the influx of Mg(2+). We conclude that the second transmembrane segment forms a major part of the Mg(2+) pore with the third transmembrane segment of CorA.

Distribution of the CorA Mg2+ transport system in gram-negative bacteria.

The CorA Mg2+ transport system is the dominant constitutive uptake mechanism in Salmonella typhimurium and Escherichia coli. Southern blot hybridization and PCR techniques were used to screen a panel of 18 additional gram-negative bacterial species for corA homologs. Virtually all strains tested positive for the presence of corA. Thus, corA appears to be ubiquitous within gram-negative bacteria and is likely their major Mg2+ influx system.