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.2015 Feb 9;54(7):2137-41.
doi: 10.1002/anie.201410200. Epub 2014 Dec 30.

Visualization and quantification of transmembrane ion transport into giant unilamellar vesicles

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Visualization and quantification of transmembrane ion transport into giant unilamellar vesicles

Hennie Valkenier et al. Angew Chem Int Ed Engl..

Abstract

Transmembrane ion transporters (ionophores) are widely investigated as supramolecular agents with potential for biological activity. Tests are usually performed in synthetic membranes that are assembled into large unilamellar vesicles (LUVs). However transport must be followed through bulk properties of the vesicle suspension, because LUVs are too small for individual study. An alternative approach is described whereby ion transport can be revealed and quantified through direct observation. The method employs giant unilamellar vesicles (GUVs), which are 20-60 μm in diameter and readily imaged by light microscopy. This allows characterization of individual GUVs containing transporter molecules, followed by studies of transport through fluorescence emission from encapsulated indicators. The method provides new levels of certainty and relevance, given that the GUVs are similar in size to living cells. It has been demonstrated using a highly active anion carrier, and should aid the development of compounds for treating channelopathies such as cystic fibrosis.

Keywords: anions; giant unilamellar vesicles; ion transport; membranes; supramolecular chemistry.

© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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Figures

Figure 1
Figure 1
a) Representation of the transport of chloride by transporter 1 into giant unilamellar vesicles (GUVs). Upon addition of a solution of NaCl, the transporter exchanges exterior chloride for interior nitrate. The chloride that is transported into the GUVs quenches the fluorescence of lucigenin 2 present in the interior of the vesicles. b,c) Bright-field (left) and confocal fluorescence microscopy images (right) show three giant vesicles with transporter 1 (0.1 mol % of total lipid) preincorporated in the membrane before (b) and after (c) addition of NaCl.
Figure 2
Figure 2
Confocal fluorescence microscopy images of lucigenin-containing GUVs, incorporating varying amounts of transporter 1, before (a–d) and about 5 min after addition of NaCl (e–h).
Figure 3
Figure 3
Average traces of the normalized lucigenin emission intensity after addition of 50 mm NaCl to GUVs without transporter (purple), with 0.01 % transporter (green), with 0.04 % transporter (blue), and with 0.1 % transporter present (red) or without NaCl added (black).
Figure 4
Figure 4
a) Fluorescence microscopy images of GUVs containing lucigenin and incorporating rhodamine-labeled lipid +0.01 % transporter 1. Left: Illumination at 532 nm visualizes the rhodamine in the bilayer. Right: Illumination at 488 nm excites the lucigenin. b) As above, 20 min after addition of NaCl. c) Traces of the lucigenin fluorescence intensity of individual vesicles over time after addition of 50 mm NaCl. The four vesicles that display stronger rhodamine fluorescence, and their corresponding traces, are labeled A–D.
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