Insight into the protonation and K(I)-interaction of the inositol 1,2,3-trisphosphate as provided by 31P NMR and theoretical calculations

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.

Progress in the biology of myo-inositol hexakisphosphate (InsP 6) has been delayed by the lack of a quantitative description of its multiple interactions with divalent cations. Our recent initial description of these [J. Torres, S. Dominguez, M.F. Cerda, G. Obal, A. Mederos, R.F. Irvine, A. Diaz, C. Kremer, J. Inorg. Biochem. 99 (2005) 828–840] predicted that under cytosolic/nuclear conditions, protein-free soluble InsP 6 occurs as Mg5(H2L), a neutral complex that exists thanks to a significant, but undefined, window of solubility displayed by solid Mg5(H2L) · 22H2O (L is fully deprotonated InsP 6). Here we complete the description of the InsP 6–Mg2+–Ca2+ system, defining the solubilities of the Mg2+ and Ca2+ (Ca5(H2L) · 16H2O) solids in terms of K s0 = [M2+]5[H2L10−], with pK s0 = 32.93 for M = Mg and pK s0 = 39.3 for M = Ca. The concentration of soluble Mg5(H2L) at 37 °C and I = 0.15 M NaClO4 is limited to 49 μM, yet InsP 6 in mammalian cells may reach 100 μM. Any cytosolic/nuclear InsP 6 in excess of 49 μM must be protein- or membrane-bound, or as solid Mg5(H2L) · 22H2O, and any extracellular InsP 6 (e.g. in plasma) is surely protein-bound.

The discovery of the second messenger role of myo-inositol 1,4,5-trisdihydrogenphosphate [Ins(1,4,5)P3] has triggered tremendous interest in investigating the structure, metabolism, and biological roles of inositol phosphates. Although the conformation of phytic acid [(myo-inositol hexakisdihydrogenphosphate), Ins P6] has been the subject of much study, the conformations of lower inositol phosphates such as inositol-pentakis-, tetrakis-, and tris-dihydrogenphosphates have not been investigated. We investigated, by 1H NMR spectroscopy, the conformations of inositol phosphates (Ins P5, Ins P4, Ins P3, Ins P2, and Ins P1) and monitored the influence of pH on conformational preferences. Ins P6 adopts the sterically stable 1ax/5eq (one phosphate in the axial position and five phosphates in the equatorial position) conformation in the pH range 0.5-9.0, and the sterically hindered 5ax/1eq (five phosphates in the axial position and one phosphate in the equatorial position) conformation above pH 9.5. At pH 9.5, both conformations are in dynamic equilibrium. Ins(1,2,3,4,6)P5 and Ins(1,2,3,5,6)P5 adopt the 1ax/5eq form in the pH range 1.0-9.0; in the pH range 9.5-13.0, the 1ax/5eq and 5ax/1eq conformations are in dynamic equilibrium. In contrast to Ins P6 and Ins P5, all the lower inositol phosphates (Ins P4 to Ins P1) investigated adopt the 1ax/5eq conformation over the entire pH range, 1.0-13.0. Preference for the 5ax/1eq conformation by Ins P6 and Ins P5 is probably due to decreased electrostatic repulsion between negatively charged vicinal equatorial phosphates in the 1ax/5eq conformation and stabilization of the sterically hindered 5ax/1eq conformation by hydrogen bonding and/or sodium counter-ions bonding between the syn-oriented phosphates. On the basis of conformations adopted by the inositol phosphates (Ins P6 to Ins P1) at different pH, we conclude that the presence of four or five equatorial phosphates on the inositol ring induces a change in the conformation from the sterically unhindered 1ax/5eq structure to the sterically hindered 5ax/1eq conformation, at high pH. This investigation illustrates that the conformational preferences of inositol phosphates at different pH is unique to the particular isomer and does not parallel the behaviour of phytic acid.