Giant unilamellar vesicles as a model system for studying ion transport

Abstract

Cellular activity depends on constant flux of ions across biological membranes. Artificial membrane models like planar lipid bilayers and liposomes are ideal for studying membrane transport phenomena as they are free of the structural complexity of cells and allow examination of transport processes under tightly controlled conditions. Over the last decades, artificial membrane-based techniques like single-channel recording and fluorescent monitoring of transport through bulk lipid vesicle suspensions have revealed many molecular mechanisms of transport. Recently, giant unilamellar vesicles (GUVs), cell-sized liposomes, have emerged as an important tool for studying cellular processes, including ion transport. The principal advantage of GUVs derives from their micron scale, which enables ease of visualisation and manipulation using microscopy and microhandling. For that reason, GUVs have also become the state-of-the-art for recapitulating a host of cell structures and functions for the purpose of developing artificial cells. Taken together, GUVs represent a promising biomimetic system to elucidate ion transport mechanisms and unravel the association between ion fluxes and various cellular processes such as neuronal transduction, nutrient uptake, electrochemical gradient development. Nevertheless, despite their great potential as a model system, the use of GUVs in ion transport studies is still limited. The aim of this review is to outline recent GUV-based ion transport studies, describe the current techniques for measuring ion transport in GUVs, compare the utility of GUVs relative to other available techniques such as single-channel current recording, and explore the potential of using GUVs to investigate complex ion transport processes.

Keywords: Artificial cell membrane; Giant unilamellar vesicles; Ion transport.

Fig. 1

Ion transport pathways in living cells and artificial cell mimics. A Schematic illustrating different types of transport pathways by which ions can cross the membrane. B Ion transport pathways in living cells (left) and in artificial cell mimics (right). The measured net flux density, $J$, of an ion is the sum of the fluxes of each individual pathway $J_i$ across the membrane. To model ion transport pathways, we aim to measure the dependence of $J_i$ on ionic concentration gradients and the membrane voltage, $V_{\text{mem}}.$ In artificial cells, individual pathways can be reconstituted and studied under controlled membrane and solution compositions. However, in living cells, ion transport measurements only produce the net result of many coupled pathways both across the lipid bilayer and transport proteins. Cartoons of lipids adapted from lipid-yellowgray icon by Servier https://smart.servier.com/ licensed without endorsement under CC-BY 3.0 Unported https://creativecommons.org/licenses/by/3.0/

Fig. 2

Recent advances in quantifying ion transport in artificial cell models. A Transport rates of ions across giant unilamellar vesicles (GUVs) have been measured using electrical methods inspired by the cell-based patch clamp technique. A GUV is aspirated using a micropipette to form a high (electrical) resistance seal. Ionic transport properties of membrane systems can be extracted from I-V relationships for individual GUVs. B Fluorophores whose fluorescent properties are dependent on ionic concentrations of a specific ion can be used to measure ion transport rates by monitoring GUV fluorescence intensity. C Fluorescent constructs whose fluorescence intensity is dependent on membrane potential. Di Garten et al. measured I-V curves across GUV membranes before (left) and after (right) inducing salt asymmetry using perfusion (Inside GUV: 95mM Na⁺, 5mM Chol⁺, perfusion solution: 5mM Na⁺, 95mM Chol.⁺). Note, however, there is significant uncertainty in external solution concentration as the perfusion does not completely displace the external solution. Ii Using this method, the authors additionally measured reversal potentials (right) across GUVs with (red, solid) and without (black, solid) gramicidin A after exchange of potassium in the external solution for sodium. With symmetric ion conditions gramicidin A GUVs have a zero reversal potential (red, dotted). Reproduced with permission from (Garten et al. 2017) (Copyright (2016) National Academy of Sciences). E Tivony et al. determined the passive proton flux across lipid bilayers vs. concentration difference characteristic from monitoring proton concentrations both within and outside single GUVs (left). The flux vs. proton concentration difference characteristic is nonlinear due to the build up of membrane potential, ΔΨ, associated with different transport rates of protons and chloride, following the addition of hydrochloric acid (HCl). The authors identified linear regions in the flux vs. proton concentration gradient profiles for the early period of transport (left plot), corresponding to periods of low membrane potential. From these linear regions the authors estimated the proton permeability coefficient, $P_{H^{+}}$, for populations of GUVs. Using Eq. (4), the authors compared permeability values for lipid vesicles formed by distinct methods: electroformation (EF) and octanol-assisted liposome assembly (OLA), for two lipid compositions, DOPC:DOPG 3:1 and DOPC (middle plot). Using mean population values for $P_{H^{+}}$, the membrane potential, ΔΨ, variation during transport (right plot, inset) could be evaluated from relationships in flux vs concentration gradient plots, using Eq. (1). Taken with permission from reference (Tivony et al. 2022). Cartoons of lipids adapted from lipid-yellowgray icon by Servier https://smart.servier.com/ licensed without endorsement under CC-BY 3.0 Unported https://creativecommons.org/licenses/by/3.0/

Fig. 3

Experimental strategies for generating different facilitated ion transport pathways across GUV membranes A.i Ionophores (orange)—molecular carriers of ions across membranes like valinomycin and CCCP, and membrane active peptides (blue), such as Gramicidin A, which spontaneously embed in the lipid bilayer, are commonly used as model ion channels. These pores can display cell membrane protein reminiscent properties like ion selectivity(Finkelstein et al. 1981), Roux 1996). ii. Ruppelt et al. recently studied the proton transport characteristics of the pore forming ion channel gramicidin A (left)(Ruppelt et al. 2024). Then, the authors used their assay to establish archetypal proton transport characteristics for both pore forming peptides (gramicidin) and ionophores (valinomycin/CCCP) (right). Reproduced with permission from reference.(Ruppelt et al. 2024) B Membrane protein channels have also been used to study ion transport across GUVs. i. Aimon et al. reconstituted the voltage-sensitive ion channel KvAP (fluorescent labelled, shown in green, right) in GUV membranes. Using patch clamping of GUVs the authors determined the conductance for different applied voltages (bottom plot), indicating a gating behaviour. Reproduced with permission from reference (Aimon et al. 2011). ii Fletcher et al. reconstituted the outer membrane protein F from E. coli in GUVs and characterized its selectivity using an optofluidic potassium transport assay (Fletcher et al. 2022). The authors devised a novel potassium ion indicator using G-quadruplex forming DNA oligonucleotides, labelled with a fluorophore and quencher at each end of the DNA single strand. Presence of K + ions causes a transition in DNA structure from random coil to a folded G-quadruplex, resulting in a reduction in mean separation between fluorophore and quencher and an associated decrease in fluorophore fluorescence (left). Using microfluidic immobilisation of GUVs containing the DNA sensor (centre), the authors could develop K + ion gradients across GUV membranes and monitor the transmembrane transport of K + , for GUVs embedded with different model ion channels. The authors determined K + flux vs K + concentration difference relationships from K + transport dynamics for GUVs embedded with OmpF (blue) and gramicidin A (red). By analysing the non-linear characteristics of each profile using the GHK flux equation for K + influx and H + counter flux, the authors inferred a significantly lower selectivity (defined as) for OmpF than gramicidin A which reflects their differing pore sizes. Reproduced with permission from reference (Fletcher et al. 2022). Cartoons of lipids adapted from lipid-yellowgray icon by Servier https://smart.servier.com/ licensed without endorsement under CC-BY 3.0 Unported https://creativecommons.org/licenses/by/3.0/

Fig. 4

Coupling ion transport to function in artificial cells. A The photoresponsive proton pump, bacteriorhodopsin, was reconstituted into GUV membranes. i The fluorescent pH-sensitive dye, pyranine, was used to show accumulation of protons in GUV lumens due to proton pumping. Valinomycin was used to mediate membrane potential dissipation to maintain proton influx by bacteriorhodopsin. Note, proton transport into the GUV implies an excess of bacteriorhodopsins orientated such that proton pumping action transports protons inwards. ii Fluorescence intensity reduction of pyranine due to proton pumping activity of bacteriorhodopsin pores (Dezi et al. 2013).Reproduced with permission from (Dezi et al. 2013) (Copyright (2016) National Academy of Sciences. B Reconstitution and activity of Arabidopsis thaliana H⁺-ATPase isoform 2 (AHA2) in GUVs. i Schematic illustration of active transport of protons into pyranine loaded GUVs following activation of AHA2 by ATP. ii Pyranine fluorescence intensity of AHA2 reconstituted GUVs and GUVs following the addition of ATP. Fluorescence intensity reduction indicates that protons are actively pumped into the GUV lumen. Reproduced with permission from Uzun et al. (2025). Reproduced with permission from Fig. 4C in Uzun et al. under the CC licence (http://creativecommons.org/licenses/by/4.0/.) C. Illustrations of ion transport associated processes occurring in native cells which are yet, to our knowledge, to be demonstrated in artificial systems. i Action potential like travelling waves of membrane potential. ii Electrophoretic transport of charged molecules between cell compartments, used by cells as a form of signalling. iii Illustration of the steady state process of maintaining electrical potential by the coupling of passive and active transport processes. Cartoons of lipids adapted from lipid-yellowgray icon by Servier https://smart.servier.com/ licensed without endorsement under CC-BY 3.0 Unported https://creativecommons.org/licenses/by/3.0/