Solution behaviour of myo-inositol hexakisphosphate in the presence of multivalent cations. Prediction of a neutral pentamagnesium species under cytosolic/nuclear conditions

Abstract

myo-Inositol hexakisphosphate (InsP₆) is an ubiquitous and abundant molecule in the cytosol and nucleus of eukaryotic cells whose biological functions are incompletely known. A major hurdle for studying the biology of InsP₆ has been a deficiency of a full understanding of the chemistry of its interaction with divalent and trivalent cations. This deficiency has limited our appreciation of how it remains in solution within cells, and the likely degree to which it might interact in vivo with physiologically important cations such as Ca²⁺ and Fe³⁺. We report here the initial part of the description of the InsP₆-multivalent cation chemistry, including its solution equilibria studied by high resolution potentiometry and (for the Fe(III)/Fe(II) couple) cyclic voltammetry. InsP₆ forms anionic complexes of high affinities and 1:1 stoichiometry with Mg(II), Ca(II), Mn(II), Fe(II), Co(II), Ni(II), Cu(II), Zn(II) and Cd(II). Of particular importance is the observation that, in the exceptional case of Mg(II), InsP₆ forms the species [Mg₅(H₂L)] (L representing fully deprotonated InsP₆); this soluble neutral species is predicted to be the predominant form of InsP₆ under nuclear or cytosolic conditions in animal cells. Contrary to previous suggestions, InsP₆ is predicted not to interact with cytosolic calcium even when calcium is increased during signalling events. In vitro, InsP₆ also forms high affinity 1:1 complexes with Fe(III) and Al(III). However, our data predict that in the biological context of excess free Mg(II), neither Fe(III) nor Fe(II) are complexed by InsP₆.

Introduction

myo-Inositol hexakisphosphate or phytate (InsP₆; its different protonated forms are denoted in this work by H₁₂L, H₁₁L⁻, …, L¹²⁻) is an ubiquitous and abundant molecule in eukaryotic cells (reviewed in [1], [2], [3]). InsP₆ can act as a precursor of diphosphoinositol pentakisphosphates (PPInsP₅) and bis(diphospho)inositol tetrakisphosphates ((PP)₂InsP₄) (reviewed in [4]). The rate of metabolic turnover between InsP₆ and the derived pyrophosphates is very high [5], [6], [7], especially when considered in relation to the lower concentrations of the latter. It is thus easy to envisage the pyrophosphates as molecular switches regulating cellular processes through phosphorylation–dephosphorylation cycles, and indeed there is evidence some may regulate endocytosis [8], [9] and chemotaxis [7]. In contrast, it has not been so easy to class the biological functions of InsP₆ (thought not to be restricted to its role as precursor for the pyrophosphates) to broadly defined categories.

InsP₆ interacts strongly with divalent and trivalent cations in a complex, but poorly described, chemistry encompassing solution complexation and precipitation reactions. The lack of a rigorous description of these reactions is a significant hurdle for InsP₆ biology. First, it has been a widespread cause of artefactual results in in vitro experiments involving the compound. As pointed out by Shears [1], depletion of Ca²⁺ and Mg²⁺ through complexation is one problem. Another problem is unnoticed precipitation (when working with small volumes): this can both deplete the solution of the cation, and generate particulate species that interfere with experimental parameters. Second, it is difficult to extrapolate to the state of InsP₆ in vivo, since the physicochemical form of InsP₆ in cells is unknown. Most or all of the cellular InsP₆ is located in the cytosolic and nuclear compartments either free or bound to cellular components including membranes (see below), and requires chelating agents for its extraction [10]. If it is in free solution it is not entirely clear how InsP₆ can exist as such under cytosolic conditions [2]. Shears has suggested that InsP₆ may be “wallpapering” the cytosolic face of cell membranes [11], possibly through cation-mediated interactions [12]. Whether soluble or bound to fixed components, it is expected that nuclear and cytosolic InsP₆ exist as complexes with Mg(II) and/or Ca(II), but such species have not been defined. InsP₆ is not confined to the nuclear/cytosolic compartments in all eukaryotes [13], [14], an observation that extends the possibilities of interactions with Ca(II) in particular. Complexation of Fe(III) by InsP₆ has received particular attention, because it is surprisingly effective at preventing the participation of iron in the redox cycles catalysing the formation of the hydroxyl radical [15], [16], [17]. Whether InsP₆ can interact with Fe(III) in cells will depend on the relative affinities for Fe(III) and the much more abundant Mg(II).

Qualitative or semi-quantitative data on the InsP₆-multivalent cations chemistry have been previously reported (e.g., [18] and references therein, and [19]). The quantitative studies of Bebot-Brigaud et al. [20] and Vasca et al. [21], encompassed a range of divalent cations, but excluded Ca(II) and Mg(II), and employed conditions of temperature and ionic strength deviating from the physiologically most relevant. Two main conclusions stem from these two reports. The first is that, as mentioned, InsP₆-metal ion systems comprise two different aspects, namely solution complexation and solid formation. In the presence of excess InsP₆, formation of soluble complexes displaying 1:1 stoichiometries predominates. In contrast, when the metal ion is the component in excess, insoluble solids are formed, the compositions of which are mostly unknown. The second conclusion is that the stability constants of the soluble species are strongly influenced by ionic strength, apparently as a result of the formation of stable complexes with the small monovalent cations.

A complete description of the chemistry of InsP₆ with multivalent cations would constitute a significant tool for InsP₆ biology. We have begun such a study, the initial part of which is reported in this paper. This part comprises the potentiometric determination of the stability constants, at 37.0 °C and I = 0.15 M NaClO₄, of the complexes formed by InsP₆ and several divalent cations (Mg, Ca, Mn, Fe, Co, Ni, Cu, Zn, Cd), as well as Fe(III) and Al(III). It also comprises the determination of the stability constants for complexes formed between InsP₆ and Na(I) at 37.0 °C and I = 0.15 M (Me₄N)Cl. In addition, the work includes the isolation and characterization of an unexpected species ([Mg₅(H₂L)]), predicted to hold particular biological relevance. Finally, the work comprises the study by cyclic voltammetry of the redox exchange between InsP₆–Fe(III) and InsP₆–Fe(II). These data are of value in themselves as a part of our understanding of InsP₆-cation interaction, but more importantly they have allowed us to predict the degrees of association of InsP₆ with Mg(II) and Ca(II) in the cytosol of cells, and make inferences on the possibility of its interaction with Fe(III).