title: Whole-GUV patch-clamping.
abstract: Studying how the membrane modulates ion channel and transporter activity is challenging because cells actively regulate membrane properties, whereas existing in vitro systems have limitations, such as residual solvent and unphysiologically high membrane tension. Cell-sized giant unilamellar vesicles (GUVs) would be ideal for in vitro electrophysiology, but efforts to measure the membrane current of intact GUVs have been unsuccessful. In this work, two challenges for obtaining the "whole-GUV" patch-clamp configuration were identified and resolved. First, unless the patch pipette and GUV pressures are precisely matched in the GUV-attached configuration, breaking the patch membrane also ruptures the GUV. Second, GUVs shrink irreversibly because the membrane/glass adhesion creating the high-resistance seal (>1 GΩ) continuously pulls membrane into the pipette. In contrast, for cell-derived giant plasma membrane vesicles (GPMVs), breaking the patch membrane allows the GPMV contents to passivate the pipette surface, thereby dynamically blocking membrane spreading in the whole-GMPV mode. To mimic this dynamic passivation mechanism, beta-casein was encapsulated into GUVs, yielding a stable, high-resistance, whole-GUV configuration for a range of membrane compositions. Specific membrane capacitance measurements confirmed that the membranes were truly solvent-free and that membrane tension could be controlled over a physiological range. Finally, the potential for ion transport studies was tested using the model ion channel, gramicidin, and voltage-clamp fluorometry measurements were performed with a voltage-dependent fluorophore/quencher pair. Whole-GUV patch-clamping allows ion transport and other voltage-dependent processes to be studied while controlling membrane composition, tension, and shape.
authors.affiliation: Laboratoire Physico Chimie Curie, Institut Curie, PSL Research University, CNRS UMR168, 75005 Paris, France.|Sorbonne Universités, Université Pierre et Marie Curie, Université Paris 6, F-75005, Paris, France.|Section on Integrative Biophysics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892.
authors.forename: Matthias
authors.initials: M
authors.lastname: Garten
authors.affiliation: Niels Bohr Institute, University of Copenhagen, Copenhagen 2100, Denmark.
authors.forename: Lars D
authors.initials: LD
authors.lastname: Mosgaard
authors.affiliation: Laboratoire Physico Chimie Curie, Institut Curie, PSL Research University, CNRS UMR168, 75005 Paris, France.|Sorbonne Universités, Université Pierre et Marie Curie, Université Paris 6, F-75005, Paris, France.
authors.forename: Thomas
authors.initials: T
authors.lastname: Bornschlögl
authors.affiliation: Institut de Biologie de l'École Normale Supérieure, INSERM U1024 and CNRS UMR 8197, 75005 Paris, France.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Dieudonné
authors.affiliation: Laboratoire Physico Chimie Curie, Institut Curie, PSL Research University, CNRS UMR168, 75005 Paris, France.|Sorbonne Universités, Université Pierre et Marie Curie, Université Paris 6, F-75005, Paris, France.
authors.forename: Patricia
authors.initials: P
authors.lastname: Bassereau
authors.affiliation: Molecular Physiology and Biophysics Section, National Institute for Neurological Disorders and Stroke, National Institutes of Health, Porter Neuroscience Research Center, Bethesda, MD 20892 gilman.toombes@nih.gov.
authors.forename: Gilman E S
authors.initials: GE
authors.lastname: Toombes
keywords: biomimetic system; electrophysiology; giant unilamellar vesicle; lipid–glass interaction; patch clamp