Ion-Induced Transient Potential Fluctuations Facilitate Pore Formation and Cation Transport through Lipid Membranes

Abstract

Unassisted ion transport through lipid membranes plays a crucial role in many cell functions without which life would not be possible, yet the precise mechanism behind the process remains unknown due to its molecular complexity. Here, we demonstrate a direct link between membrane potential fluctuations and divalent ion transport. High-throughput wide-field non-resonant second harmonic (SH) microscopy of membrane water shows that membrane potential fluctuations are universally found in lipid bilayer systems. Molecular dynamics simulations reveal that such variations in membrane potential reduce the free energy cost of transient pore formation and increase the ion flux across an open pore. These transient pores can act as conduits for ion transport, which we SH image for a series of divalent cations (Cu²⁺, Ca²⁺, Ba²⁺, Mg²⁺) passing through giant unilamellar vesicle (GUV) membranes. Combining the experimental and computational results, we show that permeation through pores formed via an ion-induced electrostatic field is a viable mechanism for unassisted ion transport.

Figure 1

Transmembrane potential fluctuations. (A) Top: schematic of divalent cations binding to anionic lipids, which disturbs the water orientation around one leaflet of the membrane, thus making the interface SH active. Bottom: divalent cation translocation disturbs the water orientation on both sides making the interface SH inactive. (B) Transmembrane potential ΔΦ₀ snapshots obtained from FLMs and GUVs with different sources of asymmetry: (i) asymmetric lipid composition (FLM, DPhPC:DPhPA 70:30/DPhPC in 150 μM KCl), (ii) asymmetric solutions (FLM, DPhPC:DPhPA 70:30 in 50 μM CaCl₂/150 μM KCl), (iii) symmetric lipid composition and solutions (FLM, DPhPC:DPhPA 70:30 in 50 μM CaCl₂), and (iv) asymmetric solutions (GUV, DPhPC:DPhPA 1:1, 5 mM CaCl₂ outside/0 mM CaCl₂ inside). Scale bars, 5 μm. (C) Potentials of mean force (PMFs) of pore formation over POPC membranes at transmembrane potentials between 0 and 600 mV, computed with the Charmm36-ECC force field (see legend).

Figure 2

Divalent cation translocation mechanism. (A) Translocation rate of divalent cations through the 1:1 DOPC:DOPA membrane extracted from intensity decay in SH images. (B) Transmembrane potential values induced by a 5 mM divalent cation solution on one side of 1:1 symmetric DPhPC:DPhPA GUVs. Each dot represents the average potential value of a single GUV and the colored lines represent the domain-wise spread in membrane potential. (C) Computed free energy of pore formation in the POPC lipid membrane (black curve - simulation) together with experimental values for Ba²⁺, Ca²⁺, and Cu²⁺ ions (colored squares - experiment) extracted from measured translocation rates. (D) MD snapshot of an open pore at a transmembrane potential magnitude of 600 mV in a POPC:POPS membrane. Lipid phosphorus atoms are rendered as brown spheres, lipid tails as gray lines, and Ca²⁺ ions as yellow spheres. Water molecules inside the membrane are represented as sticks and bulk water as a transparent surface. (E) Schematic of the voltage-induced ion transport mechanism. Transmembrane potential induced by divalent cation binding opens a transient pore through which divalent cations can translocate from one side of the membrane to the other.