Zn²⁺binds to phosphatidylserine and induces membrane blebbing

Herein, we show that Zn2+ binds to phosphatidylserine (PS) lipids in supported lipid bilayers (SLBs), forming a PS-Zn2+ complex with an equilibrium dissociation constant of ∼100 μM. Significantly, Zn2+ binding to SLBs containing more than 10 mol % PS induces extensive reordering of the bilayer. This reordering is manifest through bright spots of high fluorescence intensity that can be observed when the bilayer contains a dye-labeled lipid. Measurements using atomic force microscopy (AFM) reveal that these spots represent three-dimensional unilamellar blebs. Bleb formation is ion specific, inducible by exposing the bilayer to μM concentrations of Zn2+ but not Mg2+, Cu2+, Co2+, or Mn2+. Moreover, Ca2+ can induce some blebbing at mM concentrations but not nearly as effectively as Zn2+. The interactions of divalent metal cations with PS lipids were further investigated by a combination of vibrational sum frequency spectroscopy (VSFS) and surface pressure.area isotherm measurements. VSFS revealed that Zn2+ and Ca2+ were bound to the phosphate and carboxylate moieties on PS via contact ion pairing, dehydrating the lipid headgroup, whereas Mg2+ and Cu2+ were bound without perturbing the hydration of these functional groups. Additionally, Zn2+ was found to dramatically reduce the area per lipid in lipid monolayers, while Mg2+ and Cu2+ did not. Ca2+ could also reduce the area per lipid but only when significantly higher surface pressures were applied. These measurements suggest that Zn2+ caused lipid blebbing by decreasing the area per lipid on the side of the bilayer to which the salt was exposed. Such findings have implications for blebbing, fusion, oxidation, and related properties of PS-rich membranes in biological systems where Zn2+ concentrations are asymmetrically distributed.