Calcium Directly Regulates Phosphatidylinositol 4,5-Bisphosphate Headgroup Conformation and Recognition

Abstract

The orchestrated recognition of phosphoinositides and concomitant intracellular release of Ca²⁺ is pivotal to almost every aspect of cellular processes, including membrane homeostasis, cell division and growth, vesicle trafficking, as well as secretion. Although Ca²⁺ is known to directly impact phosphoinositide clustering, little is known about the molecular basis for this or its significance in cellular signaling. Here, we study the direct interaction of Ca²⁺ with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂), the main lipid marker of the plasma membrane. Electrokinetic potential measurements of PI(4,5)P₂ containing liposomes reveal that Ca²⁺ as well as Mg²⁺ reduce the zeta potential of liposomes to nearly background levels of pure phosphatidylcholine membranes. Strikingly, lipid recognition by the default PI(4,5)P₂ lipid sensor, phospholipase C delta 1 pleckstrin homology domain (PLC δ1-PH), is completely inhibited in the presence of Ca²⁺, while Mg²⁺ has no effect with 100 nm liposomes and modest effect with giant unilamellar vesicles. Consistent with biochemical data, vibrational sum frequency spectroscopy and atomistic molecular dynamics simulations reveal how Ca²⁺ binding to the PI(4,5)P₂ headgroup and carbonyl regions leads to confined lipid headgroup tilting and conformational rearrangements. We rationalize these findings by the ability of calcium to block a highly specific interaction between PLC δ1-PH and PI(4,5)P₂, encoded within the conformational properties of the lipid itself. Our studies demonstrate the possibility that switchable phosphoinositide conformational states can serve as lipid recognition and controlled cell signaling mechanisms.

Figure 1

(a) Setup of the LUV flotation assay. (b) PLC δ1-PH binding to LUVs with POPC/PI(4,5P)₂ (95/5 mol %). Error bars are standard deviations of three independent experiments. (c) GUVs after ECFP-PLC δ1-PH addition (green) and Dil as membrane marker (red). The scale bar corresponds to 10 μm. (d) The distribution of median ECFP-PLC δ1-PH intensity per pixel of individual GUVs and different sizes of control (blue) and after preincubation with 1 mM Mg²⁺ (green) or Ca²⁺ (red) (data from two additional independent experiments are provided in Figure S5). Each dot represents a single GUV. The number of analyzed GUVs is indicated in the respective color. The median intensity values with mean and standard deviation are depicted in the inset. The Mann–Whitney test was used as significance test (p value <0.0001 for all cases).

Figure 2

VSFS spectra of (a) the inositol ring and phosphate regions and (b) the carbonyl C=O symmetric stretch region of PI(4,5)P₂ on a buffer subphase (black spectra) containing 1 mM MgCl₂ (blue spectra) or 1 mM CaCl₂ (red spectrum) at a surface pressure of 17 mN/m. The open circles represent VSFS data points, and the solid lines are fits to the data. All spectra were taken with the ssp polarization combination. Spectra of the same data offset along the y-axis are provided in Figure S7. Details of monolayer preparation and images are provided in Figure S14.

Figure 3

Snapshots from MD simulations of the lipid bilayer taken at 1 μs (a) without and (b) with Ca²⁺. (c) Tilt angle distribution of the PI(4,5)P₂ headgroup and (d) density profiles of lipid headgroups without (blue) and with Mg²⁺ (green) or Ca²⁺ (red). Numbers in (c) represent mean tilt angles for each system. Here, only the results of the Berger force field simulations are presented. Additional force field simulations with similar outcomes can be found in the Supporting Information.