Kinetic Study on Giant Vesicle Formation with Electroformation Method

Shimanouchi, Toshinori; Umakoshi, Hiroshi; Kuboi, Ryoichi

doi:10.1021/la8040488

Cited by 57 publications

(28 citation statements)

References 33 publications

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“…Buffer is then added, and in the presence of an applied alternating voltage, the membranes detach from the stack to form GUVs. Although the precise mechanism of electroformation is not yet fully understood [39], [40], Figure S3 summarizes the key steps in the fusion/electroformation approach. Experimentally, the challenge is to efficiently transform proteo-SUVs into GUVs while still maintaining the protein in a functional state.…”

Section: Resultsmentioning

confidence: 99%

Functional Reconstitution of a Voltage-Gated Potassium Channel in Giant Unilamellar Vesicles

et al. 2011

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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.

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Section: Resultsmentioning

confidence: 99%

Functional Reconstitution of a Voltage-Gated Potassium Channel in Giant Unilamellar Vesicles

et al. 2011

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“…Despite electroformation being first reported 30 years ago and extensively reused since then, the exact underlying principles and mechanisms which induce the self‐assembly of amphiphiles are not well understood. The assumption is that the electrical field, particularly the alternating current, facilitates bilayer separation and bending of the film by electro‐osmotic effects …”

Section: Methodsmentioning

confidence: 99%

Self‐Assembly of Giant Unilamellar Vesicles by Film Hydration Methodologies

2019

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Self‐assembly of lipids or polymeric amphiphiles into vesicular structures has been achieved by various methods since the first generation of liposomes in the 1960s. Vesicles can be obtained with diameters from the nanometer to the micrometer regime. From the perspective of cell mimicking, vesicles with diameters of several micrometers are most relevant. These vesicles are called giant unilamellar vesicles (GUVs). Commonly used methods to form GUVs are solvent‐displacement techniques, especially since the development of microfluidics. These methodologies however, trap undesirable organic solvents in their membrane as well as other potentially undesired additives (surfactants, polyelectrolytes, polymers, etc.). In contrast to those strategies, summarized herein are solvent‐free approaches as suitable clean alternatives. The vesicles are formed from a dry thin layer of the lipid or amphiphilic polymers and are hydrated in aqueous media using the entropically favored self‐assembly of amphiphiles into GUVs. The rearrangement of the amphiphilic films into vesicular structures is usually aided by shear forces such as an alternative current (electroformation) or the swelling of water‐soluble polymeric supports (gel‐assisted hydration).

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“…The vesicles were formed on a platinum wire by electroformation 2. 13 The fluorescence micrographs shown were recorded immediately after microinjection and after 40 min; scale bar: 100 μm. B) Changes in relative fluorescence intensity as a function of time for 2 (○) and for 1 (•) as obtained from ≥4 independent measurements.…”

Section: Methodsmentioning

confidence: 99%