title: Functional reconstitution of a voltage-gated potassium channel in giant unilamellar vesicles.
abstract: Voltage-gated ion channels are key players in cellular excitability. Recent studies suggest that their behavior can depend strongly on the membrane lipid composition and physical state. In vivo studies of membrane/channel and channel/channel interactions are challenging as membrane properties are actively regulated in living cells, and are difficult to control in experimental settings. We developed a method to reconstitute functional voltage-gated ion channels into cell-sized Giant Unilamellar Vesicles (GUVs) in which membrane composition, tension and geometry can be controlled. First, a voltage-gated potassium channel, KvAP, was purified, fluorescently labeled and reconstituted into small proteoliposomes. Small proteoliposomes were then converted into GUVs via electroformation. GUVs could be formed using different lipid compositions and buffers containing low (5 mM) or near-physiological (100 mM) salt concentrations. Protein incorporation into GUVs was characterized with quantitative confocal microscopy, and the protein density of GUVs was comparable to the small proteoliposomes from which they were formed. Furthermore, patch-clamp measurements confirmed that the reconstituted channels retained potassium selectivity and voltage-gated activation. GUVs containing functional voltage-gated ion channels will allow the study of channel activity, distribution and diffusion while controlling membrane state, and should prove a powerful tool for understanding how the membrane modulates cellular excitability.
authors: Unité Mixte de Recherche 168, Physico-Chimie Curie, Centre National de la Recherche Scientifique, Institut Curie, Centre de Recherche, Université Pierre et Marie Curie, Paris, France.
authors: Sophie
authors: S
authors: Aimon
authors: John
authors: J
authors: Manzi
authors: Daniel
authors: D
authors: Schmidt
authors: Jose Antonio
authors: JA
authors: Poveda Larrosa
authors: Patricia
authors: P
authors: Bassereau
authors: Gilman E S
authors: GE
authors: Toombes
mesh_terms: D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D024642:Potassium Channels, Voltage-Gated / Q000737:chemistry / Q000378:metabolism*; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D053835:Unilamellar Liposomes / Q000737:chemistry / Q000378:metabolism*
chemical_list: D024642:Potassium Channels, Voltage-Gated; D053835:Unilamellar Liposomes