The degree and position of phosphorylation determine the impact of toxic and trace metals on phosphoinositide containing model membranes

Abstract

This work assessed effects of metal binding on membrane fluidity, liposome size, and lateral organization in biomimetic membranes composed of 1 mol% of selected phosphorylated phosphoinositides in each system. Representative examples of phosphoinositide phosphate, bisphosphate and triphosphate were investigated. These include phosphatidylinositol-(4,5)-bisphosphate, an important signaling lipid constituting a minor component in plasma membranes whereas phosphatidylinositol-(4,5)-bisphosphate clusters support the propagation of secondary messengers in numerous signaling pathways. The high negative charge of phosphoinositides facilitates electrostatic interactions with metals. Lipids are increasingly identified as toxicological targets for divalent metals, which potentially alter lipid packing and domain formation. Exposure to heavy metals, such as lead and cadmium or elevated levels of essential metals, like cobalt, nickel, and manganese, implicated with various toxic effects were investigated. Phosphatidylinositol-(4)-phosphate and phosphatidylinositol-(3,4,5)-triphosphate containing membranes are rigidified by lead, cobalt, and manganese whilst cadmium and nickel enhanced fluidity of membranes containing phosphatidylinositol-(4,5)-bisphosphate. Only cobalt induced liposome aggregation. All metals enhanced lipid clustering in phosphatidylinositol-(3,4,5)-triphosphate systems, cobalt in phosphatidylinositol-(4,5)-bisphosphate systems, while all metals showed limited changes in lateral film organization in phosphatidylinositol-(4)-phosphate matrices. These observed changes are relevant from the biophysical perspective as interference with the spatiotemporal formation of intricate domains composed of important signaling lipids may contribute to metal toxicity.

Keywords: Lipid domains; Lipid-metal interactions; Liposomes; Membrane fluidity; Model membranes; Phosphatidylinositol.

Graphical abstract

Fig. 1

Schematic illustrations of PI lipids along with potential localization of metal ions in the bulk or bound to the phosphate groups of the PPIs. Panel A) A schematic of the lipid structures used and the fluorescent molecule laurdan. Panel B) Schematic of the proposed metal localization and interaction with PI containing liposomes. R₁ is palmitoyl acyl chain (16:0) and R₂ is oleoyl acyl chain (18:1).

Fig. 2

Change in the generalized polarization (A) and size (B) of PI lipids containing liposomes due to various metals. Panel A) The change in laurdan GP of POPC controls and liposomes containing 1% of either PIP, PIP₂, and PIP₃ upon addition of 1200 μM Pb²⁺ (red), Cd²⁺ (green), Co²⁺ (purple), Ni²⁺ (blue), and 900 μM Mn²⁺ (orange). Panel B) The change in liposome diameter size DLS of POPC controls liposomes containing 1% PIP, PIP₂, and PIP₃ liposomes assessed by DLS at 35°C upon addition of 1200 μM Pb²⁺ (red), Cd²⁺ (green), Co²⁺ (purple), Ni²⁺ (blue), and 900 μM Mn²⁺ (orange). Error bars note standard deviation of triplicates with red asterisks for statistical significance with a 95% confidence interval from the control non-metal reading.

Fig. 3

Effect of some of the metal ions on PI containing monolayers analyzed using BAM. Two images of the most significant effects for each system are displayed. Panel A) Primary metal-induced changes to BAM images of 1% PIP, 1% PIP₂, and 1% PIP₃ monolayers after treatment of 86.2 µM metals (500:1 metal: lipid mol ratio) in 100 mM NaCl at pH 7.4 subphase at room temperature. Inserts indicate zoomed in regions. Panel B) 3D image analysis of zoomed in regions in Panel A. The scale bar represents a qualitative representation of the intensity of domain clusters in height between monolayer and camera. Scale bars correspond to 50 μm.

Fig. 4

BAM images of POPC ± 1% PIP3 at 30 mN/m. 3D analysis of Pb²⁺ and Ni²⁺ are shown in Figure 3 above. Each system had a metal lipid mol ratio of 500:1 respectively.