Concentration, compartmentation and metabolic function of intracellular free Mg ²⁺

ARTICLE

Auteur(s) : T Günther

Charité–Universitätsmedizin Berlin, Campus Benjamin Franklin, Institut für Molekularbiologie und Biochemie, Arnimallee 22, 14195 Berlin, Germany

Compartmentation of intracellular Mg²⁺

In context with these facts, intracellular Mg²⁺ is compartmentalized. The percentage of Mg²⁺ localized in nuclei, mitochondria, microsomes (ribosomes) and cytosol is shown in table 1( Table 1 ). The differences found by various investigators are probably caused by a different intensity in tissue homogenisation. For a detailed discussion, see [1, 2]. These values are based on the total Mg²⁺ contents and include bound Mg²⁺ and free Mg²⁺. [Mg²⁺]_i in a cellular compartment depends on total Mg²⁺, on the concentration of Mg²⁺-binding substances, on their Mg²⁺ affinity, and on the Mg²⁺ transport activity of the membranes.
Table 1 Subcellular distribution of Mg²⁺ in rat liver. Values provided by 3 different investigators in % of total Mg²⁺ were taken from [1, 2].

Determination of [Mg²⁺]_i

Furaptra

R_min and R_max are the excitation wave length ratios at 335/370 nm for uncomplexed (excess EDTA) and Mg²⁺-saturated furaptra respectively. S_f and S_b are the fluorescence intensities measured at the 370 nm excitation wave length for furaptra with excess EDTA and excess Mg²⁺ respectively. R is the excitation ratio at 335/370 nm of the sample to be measured. For K_D of the Mg furaptra complex, usually a value of 1.5 mM was used. For uncertainty of K_D see below.

³¹P NMR

K_D^MgATP represents the dissociation constant of the MgATP complex. K_D^MgATP values have been measured for more than 50 years. A selection of these values is listed in table 2A( Table 2 ). More values for K_D^MgATP are shown in table 3( Table 3 ). Also, when measured by various investigators under identical conditions of temperature, pH and ionic strength, the values measured for K_D of MgATP show great variability.
Table 2 A) Various values of the dissociation constant (K_D) of the MgATP complex as published by various authors. Values for log K_A (association constant) from [12] were calculated as K_D for comparison with the values provided by other authors.B) Dissociation constant of MgATP (K_D) as a function of pH and temperature according to [14, 20].C) Dissociation constant of MgATP (K_D) as a function of ionic strength at 25 °C and pH 7.2 according to [14].

Mg²⁺-sensitive microelectrodes

[Mg²⁺]_i in cell organelles and cytosol

[Mg²⁺]_i in mitochondria is about the same as [Mg²⁺]_i in the cytosol [8]. Values of 0.67 mM [9], 0.5 mM and 0.8 - 1.5 mM and 0.82 mM, determined by furaptra in isolated beef heart mitochondria, have been reported [10]. Since there is a membrane potential of 200 mV inside negative across the mitochondrial membrane, there should be an outside directed Mg²⁺ transport mechanism. Mg²⁺ influx into mitochondria may be mediated by a protein (Mrs2p) that is inserted into the inner mitochondrial membrane [11].

Tables 3 and 4( Table 4 ) present a selection of the values of [Mg²⁺]_i in the cytosol taken from the enormous number of values reported by various investigators for different cell types and tissues. More values for [Mg²⁺]_i measured by various methods are listed in [14, 68]. As shown in table 3, very different K_Ds for Mg furaptra and MgATP were used. The K_Ds varied by a factor of up to 4. However, the variation of [Mg²⁺]_i within the same cell type is less expressed than was to be expected with respect to the different K_Ds e.g. in A7r5 cells, a rat embryonal aortic muscle derived cell line, or in brain.

When [Mg²⁺]_i was measured in the same cell type (lymphocytes) by different methods (furaptra, null-point, Mg²⁺ electrode), different values (0.15 mM (table 4), 0.24 mM, 0.9 mM and 1.35 mM (table 3)) were obtained. In erythrocytes, the null- point method yielded higher values than ³¹P NMR (0.4 mM versus 0.2 mM). See also [8]. Also, the same method used by various investigators yielded very different values of [Mg²⁺]_i in the same cell type: in resting platelets 0.266 mM to 0.644 mM, in VSMC 0.31 mM and 0.62 mM (table 4).

With Mg²⁺ microelectrodes, the first measurements of [Mg²⁺]_i yielded rather high values [49, 50]. Later measurements yielded values for [Mg²⁺]_i in the range as measured by other methods.
Table 4 Alteration of [Mg²⁺]_i by various effectors measured by means of furaptra.

Uncertainty of [Mg²⁺]_i measurements

There is another common uncertainty. Also K_D^{Mg furaptra} and K_D^MgATP must be determined in solutions identical to the ionic composition of the cytosol. However the solutions used are composed on the basis of chloride salts, and are thus different from the cytosolic anion composition. Due to its content of inorganic phosphate and phosphate esters, the Mg²⁺ activity coefficient in the cytosol is lower than in the chloride-containing solutions. Besides this uncertainty, the values for K_D^{Mg furaptra} used by different investigators varied from 1.0-6.8 mM [5, 7, 10]. K_D values estimated in situ yielded higher values (2.1 mM [9], 5.3 mM [27], 5.4 mM [27] and 6.8 mM [7]) than K_Ds determined under in vitro conditions (usually 1.5 mM).

For K_D^MgATP the situation is still more complex. ³¹P NMR measures the binding of Mg²⁺ to phosphoryl groups of ATP. Mg²⁺ binding is dependent on pH because the phosphate group dissociates at a pK value of about 6.8 [12], and is thus dependent on the intracellular pH. The degree by which the K_D of MgATP is dependent on pH is shown in table 2B. Besides H⁺, other cations compete with Mg²⁺ for binding to ATP. This encludes a weak binding of K⁺ and Na⁺ to ATP (log K_A = 0.99 and 0.98 M^-1[12]). To prevent the interaction of K⁺ and Na⁺ with ATP, 0.1 M tetraethylammonium salt was taken to adjust ionic strength. The effect of the ionic strength on the dissociation constant of MgATP is shown in table 2C.

A more realistic determination of K_D of MgATP was performed using an Mg²⁺-sensitive electrode and a background solution containing 140 mM K⁺ and 14.6 mM Na⁺. A value of 87.4 μM was obtained at pH 7.2 and 37° C [14, 20]. The same value (86 μM) was obtained by means of ³¹P NMR under approximately in vivo conditions [39].

When the more realistic K_D of MgATP was used to recalculate the values of [Mg²⁺]_i measured by other investigators, the values for [Mg²⁺]_i increased to a factor of 2.8 [14]. Values of 0.7 to 1.7 mM were obtained for 14 determinations of [Mg²⁺]_i in various tissues [14].

Microheterogeneity of [Mg²⁺]_i in the cytosol

Furaptra applied in cultured rat aortic smooth muscle cells and using a K_D of 1.5 mM yielded an [Mg²⁺]_i of 0.2 mM in the peripheral area, 0.6 mM in the perinuclear region and about 0.6 mM in the nuclear region [72]. A heterogeneous distribution of intracellular free Mg²⁺ in rat VSMC was also found by other investigators [73] using furaptra. Using a K_D of 1.45 mM, [Mg²⁺]_i in the nuclear region was 0.32 mM. [Mg²⁺]_i in the perinuclear region, which contains the highest density of intracellular organelles, amounted to 0.4-1.13 mM and the non- nuclear cytoplasmic area had an [Mg²⁺]_i of 0.77 mM. The peripheral region near the plasma membrane had the lowest [Mg²⁺]_i. By using mag-indo-1, other authors [74] found a mean [Mg²⁺]_i of 1.4 mM in human tracheal gland cells. Within a single cell, [Mg²⁺]_i was uniformly distributed within the nucleoplasm. Cytosolic [Mg²⁺]_i varied from 0.34 mM to 3 mM in another opposing region in the same cell.

Since positively charged Mg²⁺ ions are enriched at negatively charged membranes and macromolecules, it must be expected that the negatively charged furaptra is rejected by these structures, resulting in erroneous values of [Mg²⁺]_i. This may be a reason why a low [Mg²⁺]_i was measured with furaptra near the plasma membrane, although Mg²⁺ should be enriched at the inner side of the negatively charged plasma membrane.

Taken together, besides the uncertainty in the average values of [Mg²⁺]_i in the cytosol, there is an unequal distribution of free Mg²⁺ in the cytosol by a factor ranging between 3 and 10. The high [Mg²⁺]_i cannot be explained by enrichment of free Mg²⁺ in cell organelles (e.g. mitochondria, endoplasmic reticulum) because [Mg²⁺]_i in these cell organelles was about the same as the average [Mg²⁺]_i in the cytosol (see above). The mechanism causing the heterogeneous distribution of intracellular free Mg²⁺ is not defined.

Metabolic function of intracellular Mg²⁺

Since Mg²⁺ is enriched at negatively charged membranes, an exact knowledge of [Mg²⁺]_i at the inner surface of cell membranes would be essential to define the role of intracellular Mg²⁺ in the regulation of membrane-bound metabolic pathways and in the modulation of K⁺ and Ca²⁺ channels [77] as well as the role of intracellular Mg²⁺ in the feedback inhibition of Mg²⁺ influx via TRPM7 in the regulation of [Mg²⁺]_i[78, 79].

Generally, metabolic pathways are regulated by changing the activity of the rate-limiting enzymes. Usually these are allosteric enzymes with a complex regulation by activation and feedback inhibition by various metabolites, end products of biosynthesis and by effectors. Moreover, these enzymes can be regulated by phosphorylation-dephosphorylation. During evolution, the regulation of metabolic pathways was adapted to the requirements of the cell with respect to energy supply as well as intermediates and end products for biosynthesis.

What is the role of intracellular Mg²⁺ in these mechanisms? All reactions of Mg²⁺ with ligands such as low molecular substances (ATP, etc.), proteins, enzymes, and nucleic acids obey the law of mass action, yielding a sigmoidal curve when plotted as a function of pMg. With respect to Mg²⁺-dependent enzymes at high [Mg²⁺]_i, enzymes are usually inhibited by Mg²⁺. Activation and inhibition of an enzyme by Mg²⁺ as a function of pMg results in a bell-shaped curve [1]. Activation by Mg²⁺ occurs at the ascending part of the bell-shaped curve up to the pMg optimum. Alterations of [Mg²⁺]_i near or within the range of the pMg optimum of a Mg²⁺-dependent rate-limiting enzyme have no significant effect on the rate of a metabolic pathway.

Since the exact absolute value of [Mg²⁺]_i in the cytosol is uncertain (see above) and as there are considerable differences in the values of [Mg²⁺]_i for the same cell type (tables 3, 4), it is uncertain at which degree of activation (part of the bell-shaped pMg dependency) the Mg²⁺-dependent rate-limiting enzymes are operating.

For some glycolytic enzymes, the pMg optimum is at pMg 3 [80, 81]. However, the Mg²⁺ affinity and the pMg optimum of enzymes can change. This effect was shown for isolated glycolytic enzymes, when their Mg²⁺ dependency was determined at various constant Mg²⁺: ATP or Mg²⁺ : ADP ratios [81]. This effect may occur in intact cells. The synthesis of ATP and the utilization of ATP in the cytosol is compartmentalized [75, 76]. In organisms, there is an additional mechanism that can change the affinity of Mg²⁺ to enzymes. For example, the addition of glucagon to liver cells resulted in a dramatic decrease in the K_M of adenylyl cyclase for Mg²⁺[82].

In all Mg²⁺-dependent enzymes, which use MgATP as a substrate, an alteration of [Mg²⁺]_i has only a minor effect on the enzyme activity because ATP is nearly saturated with Mg²⁺ (to about 90%). Moreover, an alteration of MgATP affects enzyme activity according to Michaelis-Menten kinetics and depends on the K_M of MgATP and the MgATP concentration. Intracellular Mg²⁺ is buffered at a high level [20, 83]. Thus, intracellular Mg²⁺ is not suitable for a regulatory function. Its metabolic function can be tested by artificially changed [Mg²⁺]_i in experiments with intact cells. In such experiments it was found that [Mg²⁺]_i did not play a regulatory role in erythrocyte glycolysis [84].

When cells [85] or isolated mitochondria [86] were drastically Mg²⁺-depleted by means of A23187, respiration was reduced. In these experiments, [Mg²⁺]_i in mitochondria was not measured. Because of the complex effects under the experimental conditions, it cannot be decided whether [Mg²⁺]_i in mitochondria has a regulatory or a permissive function. [Ca²⁺]_i in mitochondria was also changed in these experiments and may have affected respiration. For a discussion of the interaction of H⁺, Ca²⁺, inorganic phosphate, spermine and various cofactors with Mg²⁺ in respiration and oxidative phosphorylation, see [87].

The synthesis of purine and pyrimidine precursors of nucleic acids depends on Mg²⁺, and at each step of DNA replication, RNA transcription and RNA translation Mg²⁺ is required for enzyme function. Mg²⁺ is a cofactor in all these processes. Regulation of enzyme activity through changes in [Mg²⁺]_i has not been observed [88].

Computer models have shown that Mg²⁺ does not regulate cardiac metabolism [89].

A drastic reduction in the cellular Mg²⁺ content by means of A23187 yielded a reduction in the biosynthesis of proteins, DNA and RNA, respiration and glycolysis [85]. Again these experiments did not prove that intracellular Mg²⁺ has a regulatory function. They may indicate a permissive function of intracellular Mg²⁺. Alternatively to conclusions about a permissive role of intracellular free Mg²⁺, other authors have suggested a regulatory function of intracellular free Mg²⁺[89-92].

Mg²⁺ content in malignant cells

When rapidly dividing HL60 cells, a promyelocytic leukemia cell line, were transformed to neutrophilic-like cells by incubation with retinoic acid, the total Mg²⁺ content of the cells was reduced by 19%. The total Mg²⁺ content in mitochondria and cytoplasm (cytoplasm was defined as the region excluding mitochondria and nuclei) were reduced by 18%, and the Mg²⁺ content of nuclei was unchanged [94]. The reduction of total Mg²⁺ content in cytoplasm may be caused by a reduction of ATP by 31% and ADP by 40% [94], and by an increase in Na⁺/Mg²⁺ antiport [95]. An alteration of Mg²⁺ bound to ribosomes was not investigated. Total cytoplasmic Ca²⁺ content in the transformed cells was reduced by 40%, and the K⁺/Na⁺ ratio in nuclei was reduced by about 28% [94]. The complex mechanisms by which the contents of the various ions were changed during the differentiation process of the cells and their metabolic significance are not defined.

Action of effectors on [Mg²⁺]_i and [Ca²⁺]_i

When intracellular Mg²⁺ has a regulatory function, [Mg²⁺]_i must be changed by effectors at a reasonable concentration within a reasonable time period and to a reasonable extent. The alterations of [Mg²⁺]_i after addition of various effectors are listed in table 4. Remarkably, different investigators measured different values of [Mg²⁺]_i in resting platelets and a different increase of [Mg²⁺]_i by insulin as well as controversial effects by thrombin in these cells. Thrombin decreased [Mg²⁺]_i in human platelets from 0.614 mM to 0.405 mM [59], whereas other authors found an increase in [Mg²⁺]_i in platelets from 0.54 mM to 1.33 mM by thrombin [96].

The increases in [Mg²⁺]_i induced by various effectors at the concentrations listed amounted maximally to a factor of 2, corresponding to an alteration in pMg of 0.3. A small part of the increase in [Mg²⁺]_i by some effectors may include an increase in [Ca²⁺]_i because of the unspecificity of furaptra. Due to the uncertainty of an absolute value of [Mg²⁺]_i, the alterations in [Mg²⁺]_i must be considered as relative.

Of the effectors listed in table 4, insulin had no effect on [Ca²⁺]_i[58, 59]. Insulin may increase Mg²⁺ uptake into platelets. In VSMC, vasopressin [65, 66] and endothelin-1 [65] induced rapid peaks of [Mg²⁺]_i and [Ca²⁺]_i followed by a decrease in [Mg²⁺]_i and [Ca²⁺]_i. The peak and sustained values of [Mg²⁺]_i are listed in table 4. The reduction in [Mg²⁺]_i may be caused by Mg²⁺ efflux via Na⁺/Mg²⁺ antiport [65]. Mg²⁺- and Ca²⁺-free medium decreased the vasopressin-mobilized [Ca²⁺]_i by 60.8% and prevented the increase in [Mg²⁺]_i[65]. Based on these results, the authors concluded that vasopressin and endothelin-1 mobilized Mg²⁺ in VSMC through the action of the intracellular second messenger Ca²⁺. However, in A7r5 cells, a VSMC line, 100 nM vasopressin had no effect on [Mg²⁺]_i[26].

In fibroblasts, bombesin induced an [Mg²⁺]_i and [Ca²⁺]_i peak [62, 63], followed by a sustained decrease in [Mg²⁺]_i and by oscillations of [Ca²⁺]_i. Endothelin-1 (0.5μM) had a similar effect on [Mg²⁺]_i as 3 nM bombesin. There was a considerable variation in the [Mg²⁺]_i response to bombesin, endothelin-1 [63] and EGF [97] among different individual cells of the same cell type.

Some effectors have a cell -specific action, e.g. endothelin-1 had no effect on [Mg²⁺]_i and [Ca²⁺]_i in platelets [60]. 8-Br-cGMP increased [Mg²⁺]_i in MDCK cells [57] and had no effect on [Mg²⁺]_i in platelets [59].

The increase in [Mg²⁺]_i by AVP and endothelin-1 [65] affected [Ca²⁺]_i and [Mg²⁺]_i simultaneously and in the same direction. In VSMC, AVP and endothelin [65] and in pancreatic acinar cells, acetylcholine and cholecystokinin induced short peaks of [Mg²⁺]_i followed by a decrease in [Mg²⁺]_i[67]. In a more detailed study with pancreatic acinar cells, 100 pM cholecystokinin and 100 μM acetylcholine decreased [Mg²⁺]_i from 0.58 mM to 0.47 mM. This may be caused by Mg²⁺ uptake into the endoplasmic reticulum [55] or by Mg²⁺ efflux via Na⁺/Mg²⁺ antiport [65, 67]. Cholecystokinin (10 pM) induced [Ca²⁺]_i oscillations. Frequency and amplitude of [Ca²⁺]_i oscillations were increased when [Mg²⁺]_i was decreased by preincubation at low extracellular Mg²⁺ concentration [55]. Thus, intracellular Mg²⁺ can modulate Ca²⁺ signaling.

The reviewed alterations in [Ca²⁺]_i reflect the general function of Ca²⁺ as a second messenger. Ca²⁺ signaling can occur as a single transition, as a sustained plateau or as repetitive oscillations. [Ca²⁺]_i oscillations are known to be involved in the control of a number of important cell processes, e.g. regulation of gene transcription, where the efficacy of the control varies with the amplitude and frequency of the oscillations [98, 99]. The different patterns of intracellular Ca²⁺ reveal a mechanism by which a multifunctional second messenger as Ca²⁺ can achieve specificity in signal transduction [98, 99].

Ca²⁺ versus Mg²⁺ as a second messenger

The effector-induced alterations of [Mg²⁺]_i were discussed as evidence that intracellular Mg²⁺ is a second messenger [94]. It merely indicates that intracellular Mg²⁺ can affect the action of Ca²⁺ as a second messenger. Additional evidence that intracellular Ca²⁺ but not Mg²⁺ is the second messenger was obtained with human lymphocytes stimulated by Con A. Con A increased [Ca ²⁺]_i and [Mg²⁺]_i. However, [Mg²⁺]_i was only elevated in cells with a high [Ca²⁺]_i[102]. This result indicates that a sufficient increase in [Ca²⁺]_i is necessary to induce the increase in [Mg²⁺]_i.

In the complex interactions between intracellular Mg²⁺ and Ca²⁺, [Mg²⁺]_i has a level at which Ca²⁺ release is almost maximally inhibited, and Ca²⁺ storage is almost maximally activated by intracellular Mg²⁺. Thus [Mg²⁺]_i provides a minimal [Ca²⁺]_i, so that effectors can induce pronounced changes in [Ca²⁺]_i[103].

These results are in agreement with the physico-chemical properties of Ca²⁺.

Because of the larger diameter of Ca²⁺ compared with Mg²⁺, in multidentate chelates with proteins there is less ligand-ligand repulsion by the negatively charged chelating groups. This yields 1000 times lower dissociation constants of Ca²⁺ with Ca²⁺-binding proteins compared with Mg²⁺ and to 1000 times lower [Ca²⁺]_i compared with [Mg²⁺]_i. Thus Ca²⁺ was favored against Mg²⁺ as a second messenger during evolution. For details see [99, 104].

Conclusion

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