The behaviour of inositol 1,3,4,5,6-pentakisphosphate in the presence of the major biological metal cations
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- Veiga, N., Torres, J., Godage, H.Y. et al. J Biol Inorg Chem (2009) 14: 1001. doi:10.1007/s00775-009-0510-z
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The inositol phosphates are ubiquitous metabolites in eukaryotes, of which the most abundant are inositol hexakisphosphate (InsP6) and inositol 1,3,4,5,6-pentakisphosphate [Ins(1,3,4,5,6)P5)]. These two compounds, poorly understood functionally, have complicated complexation and solid formation behaviours with multivalent cations. For InsP6, we have previously described this chemistry and its biological implications (Veiga et al. in J Inorg Biochem 100:1800, 2006; Torres et al. in J Inorg Biochem 99:828, 2005). We now cover similar ground for Ins(1,3,4,5,6)P5, describing its interactions in solution with Na+, K+, Mg2+, Ca2+, Cu2+, Fe2+ and Fe3+, and its solid-formation equilibria with Ca2+ and Mg2+. Ins(1,3,4,5,6)P5 forms soluble complexes of 1:1 stoichiometry with all multivalent cations studied. The affinity for Fe3+ is similar to that of InsP6 and inositol 1,2,3-trisphosphate, indicating that the 1,2,3-trisphosphate motif, which Ins(1,3,4,5,6)P5 lacks, is not absolutely necessary for high-affinity Fe3+ complexation by inositol phosphates, even if it is necessary for their prevention of the Fenton reaction. With excess Ca2+ and Mg2+, Ins(1,3,4,5,6)P5 also forms the polymetallic complexes [M4(H2L)] [where L is fully deprotonated Ins(1,3,4,5,6)P5]. However, unlike InsP6, Ins(1,3,4,5,6)P5 is predicted not to be fully associated with Mg2+ under simulated cytosolic/nuclear conditions. The neutral Mg2+ and Ca2+ complexes have significant windows of solubility, but they precipitate as [Mg4(H2L)]·23H2O or [Ca4(H2L)]·16H2O whenever they exceed 135 and 56 μM in concentration, respectively. Nonetheless, the low stability of the [M4(H2L)] complexes means that the 1:1 species contribute to the overall solubility of Ins(1,3,4,5,6)P5 even under significant Mg2+ or Ca2+ excesses. We summarize the solubility behaviour of Ins(1,3,4,5,6)P5 in straightforward plots.
Elucidation of the biological roles of the higher InsPs has been complicated by their intricate structural and metabolic interrelatedness. This has been aggravated by the unusual and often non-intuitive behaviour displayed by these highly charged compounds, in particular in the presence of multivalent cations. This behaviour can be the source of many artefacts (reviewed in ). For InsP6, we have over the past few years strived to make a rigorous and at the same time “biological-user-friendly” description of its chemistry with multivalent cations [13–15]. We have also studied the chemistry of inositol 1,2,3-trisphosphate [Ins(1,2,3)P3], focusing on the likelihood of its proposed interaction with Fe3+ in the cellular context . In the present paper we study Ins(1,3,4,5,6)P5, focusing on its interactions with Na+, K+, Ca2+, Mg2+, Cu2+, Fe2+ and Fe3+ in solution, and its solid-formation equilibria with Ca2+ and Mg2+. Our results predict that Ins(1,3,4,5,6)P5 does not have a high enough affinity for Mg2+ to be fully associated with this cation under cytosolic and nuclear conditions. They also predict that the compound would be fully soluble under those conditions, and that it even has a significant window of solubility under calcium-rich conditions such as those of the extracellular medium. We have summarized the non-intuitive solubility behaviour of Ins(1,3,4,5,6)P5 in straightforward plots that should be of help when planning experiments with this compound.
Materials and methods
All common laboratory chemicals were of reagent grade, purchased from commercial sources and used without further purification. NaCl, KCl, CaCl2·2H2O, MgCl2·6H2O, CuSO4·5H2O, (NH4)2Fe(SO4)2·6H2O, and Fe(ClO4)3·xH2O were used as metal sources. Solutions of the metals were standardized according to standard techniques . Ins(1,3,4,5,6)P5 was obtained from myo-inositol in 86% isolated overall yield over five steps. Briefly, transesterification of myo-inositol with trimethyl orthobenzoate in the presence of an acid catalyst followed by acid hydrolysis of the product, myo-inositol 1,3,5-orthobenzoate, gave 2-O-benzoyl myo-inositol, which was then phosphorylated. Deprotection of the fully protected pentakisphosphate followed by the removal of the benzoate ester in concentrated aqueous ammonia afforded Ins(1,3,4,5,6)P5 as the hexaammonium salt, (NH4)6H4L, which was verified by elemental analysis [Anal. calc. (%) for C6H35P5O21N6: C 10.6, H 5.2, N 12.3. Found (%): C 10.3, H 5.4, N 12.4.] to conform to the previous formula . Solutions of Ins(1,3,4,5,6)P5 were prepared by weighing this hexaammonium salt (NH4)6H4L. The standard HCl solutions were prepared from Merck standard ampoules. The titrant solution [0.1 M solution of Me4N(OH) in 0.15 M Me4NCl] was prepared by dissolving Me4N(OH)·5H2O (Fluka), and was standardized with potassium biphthalate.
IR spectroscopy, elemental analysis and thermal analysis
IR spectroscopy was carried out with a Bomen Fourier transform IR spectrophotometer, with samples present as 1% KBr pellets. Elemental analysis (C, H) was performed using a Carlo Erba EA 1108 instrument. The cacium content in the solid samples was determined gravimetrically as previously described . Magnesium was determined volumetrically according to standard techniques . Thermal analysis was performed with a Shimadzu DTA-50, TGA-50 instrument with a TA 50I interface, using a platinum cell and nitrogen atmosphere. Experimental conditions were 1 °C min−1 temperature ramp rate and 50 mL min−1 nitrogen flow rate.
All solutions were freed of carbon dioxide by argon bubbling. The protonation constants of Ins(1,3,4,5,6)P5 were determined at 37.0 °C, 0.15 M ionic strength in the non-interacting electrolyte Me4NCl. Five potentiometric titrations, comprising about 150 experimental points each, were carried out in the Ins(1,3,4,5,6)P5 concentration interval 0.5–3 mM, covering pH values between 2 and 11.
Since an ammonium salt was used as the Ins(1,3,4,5,6)P5 source, the (weak) acidity of the ammonium ion had to be taken into account when the desired constants were calculated. Thus, the acid dissociation constant of ammonium was determined de novo under the conditions of the study, through three potentiometric titrations using NH4Cl. The hydrolysis constants of Fe(III) under the same conditions were taken from data previously reported .
Then the behaviour of Ins(1,3,4,5,6)P5 in the presence of Na+, K+, Ca2+, Mg2+, Cu2+, Fe2+ and Fe3+ ions was analysed, also in 0.15 M Me4NCl and at 37.0 °C. Three to eight potentiometric titrations were carried out for each cation (about 150 experimental points for each titration), at metal ion concentrations ranging from 1 to 50 mM (for alkali metal ions) or 0.5 to 3 mM (for alkaline earth and transition metal ions), and Ins(1,3,4,5,6)P5 to metal molar ratios from 0.01 to 2 (for alkali metal ions) and from 0.2 to 3 (for alkaline earth and transition metal ions). Owing to the lower affinity towards Ins(1,3,4,5,6)P5 expected for M+ cations in comparison with M2+ or M3+, higher absolute concentrations of metal ions and lower Ins(1,3,4,5,6)P5 to metal molar ratios were used in the former cases. Potentiometric titrations were carried out as previously described , I being adjusted by addition of Me4NCl.
The cell constants, E°, and the liquid junction potentials were determined under the same conditions using the computer program GLEE . The data obtained were analysed using the HYPERQUAD program . In all cases, the fit of the values predicted by the model to the experimental data was estimated on the basis of the parameter σ corresponding to the scaled sum of square differences between predicted and experimental values. Then, the constants were used to produce species distribution diagrams using the HySS program .
Synthesis of [Mg4(H2L)]·23H2O and [Ca4(H2L)]·16H2O
An 8.8 mM aqueous solution of Ins(1,3,4,5,6)P5 was prepared, and the pH was adjusted to 10–11 by addition of 1 M LiOH. To 5 mL (0.044 mmol) of this solution, 36.0 mg of MgCl2·6H2O (0.18 mmol) dissolved in the minimum amount of water was added. A white solid immediately appeared, and was separated by centrifugation, washed with water (2 × 5 mL), and dried with ethanol (1 × 5 mL). The preparation of [Ca4(H2L)]·16H2O followed a similar procedure, starting from 26.0 mg of CaCl2·2H2O (0.18 mmol) as the metal source. The yield was 63% for [Mg4(H2L)]·23H2O and 56% for [Ca4(H2L)]·16H2O. Anal. Calc. for Mg4C6H55O44P5: C 6.7, H 5.1, Mg 9.0%. Found: C 6.3, H 5.0, Mg 9.5%. Anal. Calc. for Ca4C6H41O37P5: C 7.1, H 4.1, Ca 15.7%. Found: C 6.8, H 4.0, Ca 15.2%. The thermal analysis agreed with the proposed formula: 38.4% weight loss for [Mg4(H2L)]·23H2O and 28.1% weight loss for [Ca4(H2L)]·16H2O, corresponding to the elimination of water, compared with calculated values of 38.2% for [Mg4(H2L)]·23H2O and 28.2% for [Ca4(H2L)]·16H2O.
Solubility measurements were carried out at constant ionic strength, I = 0.15 M Me4NCl, and 37.0 °C. An amount of 10–40 mg of the compound—[Mg4(H2L)]·23H2O or [Ca4(H2L)]·16H2O)—was suspended in 10.0 mL of 0.15 M aqueous Me4NCl at 37.0 °C. Known amounts of HCl were added, so as to reach equilibrium points corresponding to measurable amounts of metal ion in solution. Each mixture was kept in a glass jacketed cell under continuous stirring until the measured pH was constant (about 1 week). After the equilibrium had been reached, the solid in excess was filtered out (Macherey-Nagel MN 640 m paper), and the total metal ion concentration was determined in the supernatant. Total calcium and magnesium contents were determined volumetrically according to standard techniques . With these M(II) concentration values, and with the assumption of a 4:1:2 stoichiometry [M(II)/Ins(1,3,4,5,6)P5/H+], total amounts of Ins(1,3,4,5,6)P5 in the solution were calculated. Then total concentrations of M(II), Ins(1,3,4,5,6)P5 and H+ were used as inputs for the HySS program  to determine the equilibrium concentrations of (free) M2+ and H2L8−, which define the Ks0. In this calculation, the complete set of solution equilibria previously measured were taken into account. At least three independent determinations were performed for each metal ion.
Results and discussion
Protonation equilibria of Ins(1,3,4,5,6)P5
Logarithms of the overall protonation constants of inositol 1,3,4,5,6-pentakisphosphate [Ins(1,3,4,5,6)P5] in 0.15 M Me4NCl at 37.0 °C; σ = 0.9
L10− + H+ → HL9−
L10− + 2H+ → H2L8−
L10− + 3H+ → H3L7−
L10− + 4H+ → H4L6−
L10− + 5H+ → H5L5−
L10− + 6H+ → H6L4−
L10− + 7H+ → H7L3−
Interactions between Ins(1,3,4,5,6)P5 and alkali metal ions
Logarithms of the overall formation constants for complexes between Ins(1,3,4,5,6)P5 and metal ions in 0.15 M Me4NCl at 37.0 °C
Interactions in solution between Ins(1,3,4,5,6)P5 and divalent ions and Fe3+
Comparative coordination ability of InsP6, Ins(1,3,4,5,6)P5 and Ins(1,2,3)P3
With respect to K+, Ins(1,2,3)P3 exhibits the strongest interaction at any pH value under the conditions studied (Fig. 6). This is possible because under 1:1 metal–ligand conditions, polymetallic species (which are formed by Ins(1,3,4,5,6)P5 and InsP6 but not by Ins(1,2,3)P3 except for [K2(H4L)] under acidic conditions) are quantitatively irrelevant. Under metal-excess conditions (100:1), which favour coordinative interactions in complexes between Ins(1,3,4,5,6)P5 or InsP6 and two to six K+ ions, the scenario changes, with InsP6 displaying the strongest interaction above pH 8 (Fig. S4).
The strongest interaction towards Mg2+ is clearly established by InsP6, as expected from the fact that it is the most highly charged of the three compounds at any pH value. InsP6 forms with Mg2+, in addition to 1:1 complexes, the neutral species [Mg5(H2L)], analogous to the neutral [Mg4(H2L)] complex now described for Ins(1,3,4,5,6)P5 (Table 2). Under conditions of metal excess, the formation of such polymetallic complexes can be expected to shift the relative strengths of interaction in favour of both InsP6 and Ins(1,3,4,5,6)P5 with respect to Ins(1,2,3)P3; however, the occurrence of precipitation in this range (see below) makes it difficult to draw meaningful plots analogous to that in Fig. 6.
The interaction with Fe3+ is very strong for the three InsPs. The log Q values are high and increase in very similar ways (from about 3 to about 7) between pH 4 and 7, while above pH 7, Ins(1,3,4,5,6)P5 becomes a more effective Fe3+ chelator than the other two InsPs, especially in comparison with InsP6. These results are unexpected, because, unlike Ins(1,2,3)P3 and InsP6, Ins(1,3,4,5,6)P5 lacks the 1,2,3-trisphosphate motif usually believed to be necessary and sufficient for high-affinity complexation of this cation by InsPs. In derivatives of myo-inositol, the substituent at C-2 is the only axial one (see Fig. 1), and therefore the 1,2,3-trisphosphate motif is unique in having three phosphates in a cis relationship to one another. This motif is thought to flip to the normally unfavourable axial–equatorial–axial disposition upon the complexation of Fe3+ . In contrast, Ins(1,3,4,5,6)P5 has all its phosphates in the equatorial form (and it is highly unlikely to adopt a thermodynamically unfavourable all-axial conformation). The evidence for the importance of the 1,2,3-trisphosphate motif comes from assays in which the InsPs prevent the iron-catalysed generation of hydroxyl radical through the Fenton reaction. All InsPs tested so far that contain the 1,2,3-trisphosphate motif, including InsP6 and Ins(1,2,3)P3, are highly effective at preventing hydroxyl radical formation, while those lacking this motif, including Ins(1,3,4,5,6)P5 in particular, are clearly less effective [24, 25]. Our results thus indicate that high-affinity complexation of Fe3+ by InsPs does not require the equatorial–axial–equatorial 1,2,3-trisphosphate motif, and is thus a separate property from their capacity to inhibit the iron-catalysed production of hydroxyl radical. Therefore all-equatorial vicinal trisphosphate groups, as present in Ins(1,3,4,5,6)P5, appear to support Fe3+ complexation of at least as high affinity as the 1,2,3-trisphosphate motif, but with the difference that iron is not prevented effectively from participating in the Fenton reaction.
Biological predictions for Ins(1,3,4,5,6)P5 under cytosolic/nuclear conditions of mammalian cells
Predictions for Ins(1,3,4,5,6)P5 under simulated cytosolic/nuclear conditions, in the absence and presence of Fe3+
Ins(1,3,4,5,6)P5 associated with Mg2+ (%)
Ins(1,3,4,5,6)P5 associated with K+ (%)
Unbound Ins(1,3,4,5,6)P5 (%)
Ins(1,3,4,5,6)P5 associated with Fe3+ (%)
Fe3+ associated with Ins(1,3,4,5,6)P5 (%)
We had previously shown that InsP6, because of its association with Mg2+, cannot bind Fe3+ under simulated cytosolic/nuclear conditions . In contrast, Ins(1,2,3)P3 associates much more weakly with Mg2+, and is able to bind fully Fe3+ present in equimolar amounts . The weak association with Mg2+ and the high cellular concentration of Ins(1,3,4,5,6)P5 mean that if a small concentration of Fe3+ (representing the “chelatable iron pool”, the size of which is unknown ) is included in the simulations, this iron associates completely with Ins(1,3,4,5,6)P5 (Table 3). However, we feel unsure about the biological significance of this result since cellular iron ligands are expected to prevent the participation of iron in the Fenton reaction, a property that Ins(1,3,4,5,6)P5 lacks.
Ins(1,3,4,5,6)P5 solids with calcium and magnesium: synthesis and IR spectra
The interaction of M2+ ions with Ins(1,3,4,5,6)P5 under metal excess gives rise to the formation of fairly insoluble compounds. We prepared and analysed the solids with Ca2+ and Mg2+. The elemental and thermogravimetric analyses agreed with the general formula [M4(H2L)]·xH2O [x = 23 (Mg), 16 (Ca)]. Therefore, the calcium and magnesium solids have the same metal-to-ligand stoichiometry as the neutral tetrametallic complexes formed with these cations in solution. The solids, as expected, also include a number of water molecules, which are lost during thermogravimetric analysis across a wide temperature range, namely between 50 and 210 °C.
IR bands and assignments for Ins(1,3,4,5,6)P5 and its solid complexes with magnesium and calcium
The IR spectra of the magnesium and calcium solids are very similar, indicative of isostructural compounds, differing only in the number of water molecules. A similar behaviour was observed for the magnesium and calcium solids of InsP6 . The δ(H–N–H) peak is absent from the spectra of the magnesium and calcium solids of Ins(1,3,4,5,6)P5, attesting to a complete NH4+–Mg2+/Ca2+ exchange during the syntheses. Complexation with the M(II) ions introduces several changes in the IR spectra (in comparison with the spectrum of the ammonium salt), the normal modes associated with phosphate groups being the most affected. The stretching signals of the PO2– group (five bands in the ligand) appear now as two intense peaks at about 990 and 1,120 cm−1, which are probably associated with νs(PO2−) and νas(PO2−), respectively. This fact (only two sharp and broad bands, with a small difference between them) was already reported for Ca2+ and Mg2+ solids of InsP6 [13, 38]. Besides, the frequency for the O–P–O bending mode changes upon coordination to higher wavenumber values. These two facts suggest that the metal cations are bound to the phosphate groups, possibly by means of a direct and bidentate M–O–P coordination .
Solubility of Ins(1,3,4,5,6)P5 solids with calcium and magnesium
Complete description of the behaviour of Ins(1,3,4,5,6)P5 in the presence of Ca2+ and/or Mg2+
The protonation constants of Ins(1,3,4,5,6)P5, together with the Ca2+ and Mg2+ complexation constants and the Ks0 values, allow a complete description of the speciation of Ins(1,3,4,5,6)P5 in the presence of Ca2+/Mg2+. Broadly, the behaviour is characterized by the predominance of soluble 1:1 species under Ins(1,3,4,5,6)P5 excess and the predominance of the tetrametallic species [M4(H2L)] under metal excess. The [M4(H2L)] complexes exist in solution up to fixed concentration limits, and any amount of them that forms in excess of those limits undergoes precipitation. Such concentration limits are given by the product of the value of each stability constant (4M2+ + H2L8− ↔ [M4(H2L)]) and the corresponding value of Ks0 , and they thus are 135 μM for [Mg4(H2L)] and 56 μM for [Ca4(H2L)]. Therefore, under conditions of predominance of the [M4(H2L)] complexes [large excess of M2+ with respect to Ins(1,3,4,5,6)P5, neutral or alkaline pH], these fixed values correspond in practice to the total solubility of Ins(1,3,4,5,6)P5.
The differences between the plots for Mg2+ and Ca2+ are slight, except for the fact that the lower solubility of [Ca4(H2L)] with respect to [Mg4(H2L)] causes the solid to be present across a wider range of conditions in the case of Ca2+. When both Ca2+ and Mg2+ are present, the system behaves in a way similar to what has been described, as long as equal total divalent cation concentrations are considered. Under conditions of total cation excess over Ins(1,3,4,5,6)P5, precipitation of the more insoluble Ca2+ complex will be favoured over that of the Mg2+ one.
Overall, this behaviour of Ins(1,3,4,5,6)P5 is similar that of InsP6 that we reported previously  except for the facts that in the InsP6 system (1) the stoichiometry of the neutral polymetallic InsP6 complex is 5:1 and (2) the solubility limit of the Mg2+ complex is 49 μM and that of the Ca2+ one is too low to be measured. An additional finer difference between the systems is that the size of the cation excess needed for complete predominance of the neutral polymetallic species over the anionic 1:1 complexes is smaller for InsP6 than for Ins(1,3,4,5,6)P5: while systems with InsP6 and either Mg2+ or Ca2+ at 5:1 metal-to-ligand ratios display [Mg5(H2L)] as practically the sole species, similar systems for Ins(1,3,4,5,6)P5 (with 4:1 metal-to-ligand ratios) display a mixture of [Mg4(H2L)] and 1:1 species.
Biological predictions for Ins(1,3,4,5,6)P5 present at high concentrations in non-mammalian erythrocytes
Avian and turtle erythrocytes contain very high (millimolar) concentrations of Ins(1,3,4,5,6)P5 (reviewed in ). Even if the compound has been proposed to interact with (and modulate the oxygen affinity of) haemoglobin, it is unlikely that the whole of the Ins(1,3,4,5,6)P5 present in these cells is bound to haemoglobin. We ran calculations to predict the physicochemical status of non-haemoglobin-bound Ins(1,3,4,5,6)P5 in red blood cells, using as conditions 150 mM K+ and pH 7.4. We explored Ins(1,3,4,5,6)P5 concentrations of 1, 3 and 7 mM: this spans the concentrations reported for avian and turtle erythrocytes [39, 40], and takes into account the possibility that the pool of haemoglobin-free Ins(1,3,4,5,6)P5 is smaller than the total Ins(1,3,4,5,6)P5 one. Also, the 7 mM figure in particular covers the extremely high Ins(1,3,4,5,6)P5 concentrations surprisingly found in the erythrocytes of the Amazonian fish pirarucu during its air-breathing phase (reviewed in ). For each Ins(1,3,4,5,6)P5 concentration we explored different figures for the total concentration of Mg2+, so as to obtain free Mg2+ in the 0.2-mM range reported for (mammalian) erythrocytes . The calculations predicted the whole of Ins(1,3,4,5,6)P5 to be soluble (as a mixture of non-complexed anion, K+ complexes, and 1:1 Mg2+ complexes, plus a small proportion of [Mg4(H2L)]). Precipitation was predicted to start only at higher free Mg2+ concentrations, i.e. approximately 0.32 mM free Mg2+ for 7 mM Ins(1,3,4,5,6)P5 and approximately 0.41 mM free Mg2+ for 3 mM Ins(1,3,4,5,6)P5. Interestingly, InsP6 was predicted (on the basis of the data in ) to be fully precipitated at approximately 0.2 mM free Mg2+, hinting that Ins(1,3,4,5,6)P5 might have been selected for its function in erythrocytes partly as a consequence of its solubility in the presence of Mg2+.
Biological predictions for Ins(1,3,4,5,6)P5 under extracellular conditions
It is relevant to predict the speciation of Ins(1,3,4,5,6)P5 under high-Ca2+, high-Mg2+ conditions such as those prevalent in the extracellular medium of mammals because (1) such conditions correspond to those experiments in which the compound is added to culture cells in physiological media and (2) concentrations of inositol pentakisphosphates in the 10–20-nM range have been reported for rat plasma  (although the accompanying measurements for InsP6 have been questioned ). We therefore chose 150 mM Na+, pH 7.5, 2 mM total Ca2+ and 2 mM total Mg2+ to simulate extracellular-like conditions. We first ran a simulation with Ins(1,3,4,5,6)P5 at 15 nM, i.e. a concentration similar to that reported by Grases et al. : the result shows that plasma Ins(1,3,4,5,6)P5, if present in the reported concentration range and not bound to proteins, would exist predominantly as a soluble mixture of soluble [Ca4(H2L)] (93%) and [Mg4(H2L)] (6%). We then ran simulations with increasing amounts of Ins(1,3,4,5,6)P5, so as to determine its maximum solubility in plasma: under the conditions detailed above, 60–65 μM Ins(1,3,4,5,6)P5 can exist in solution, mostly as the mixture [Mg4(H2L)]/[Ca4(H2L)]. The solubility in intracellular vesicular compartments, similarly rich in Ca2+ and Mg2+ as the extracellular medium but more acidic, will be in excess of the value given above. Therefore, the physicochemical properties of Ins(1,3,4,5,6)P5 would allow the existence of a significant pool of soluble, protein-free compound in the extracellular medium as well as in intracellular vesicular compartments.
Practical data for the experimentation with Ins(1,3,4,5,6)P5
Our data provide a few simple guidelines to keep experiments using added Ins(1,3,4,5,6)P5 within reasonably physiological conditions. When mimicking cytosolic/nuclear conditions, one must reason that Ins(1,3,4,5,6)P5 can complex up to 4 mol of Mg2+ per mole, although under most conditions except for very large Mg2+ excesses and/or very high pH, this will be less than 1 mol per mole (see, e.g., Table 3). Since cytosol and nucleus of mammalian cells contain 0.25–1 mM free Mg2+ , the total concentration of Mg2+ included in the experiments must be in excess of the molar concentration of Ins(1,3,4,5,6)P5 (plus the Mg2+-complexating capacity of any other chelators such as ATP that are present). Under these conditions Ins(1,3,4,5,6)P5 will remain in solution up to 135 μM (or higher, but this only for a very restricted subset of conditions).
N.V. is indebted to PEDECIBA-Química and ANII for a scholarship. We acknowledge support from the Wellcome Trust (programme grant 082837 to A.M.R. and B.V.L.P.). Thermal analyses were carried out by Jorge Castiglioni, LAFIDESU, DETEMA, Facultad de Química (Uruguay). A.D. is grateful to the Biochemical Society for a bursary to attend the Harden conference on inositol phosphates and lipids.