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Study of a voltage-gated ion channel reconstituted in Giant Unilamellar Vesicles

Sophie Aimon 1
1 Patricia Bassereau
PCC - Physico-Chimie-Curie
Abstract : Studying the role of the membrane in cell excitability is challenging using cells because of the homeostatic feedbacks preventing the external control of the relevant parameters. To circumvent these difficulties, I developed a model system in which a voltage-gated channel was reconstituted in a membrane where the composition, tension and geometry can be controlled. I thus expressed, purified and fluorescently labeled KvAP, a voltage-gated potassium channel. I then adapted a method to reconstitute the channel in Giant Unilamellar Vesicles, which are liposomes the same size as cells. I used quantitative confocal microscopy to measure the channel density in these GUVs (it can be controlled between few to thousands proteins per square micron). Furthermore, electrophysiology measurements showed that the protein is still functional after reconstitution. With these GUVs containing reconstituted KvAP, I have studied the affinity of the protein for curved membranes. I have pulled membrane nanotubes of controlled diameter from GUVs and measured the density of the protein in the tube as compared to the vesicle using confocal microscopy. I found that the channel is enriched in the nanotube and that this enrichment increases as a function of curvature, in agreement with an elasticity based theory. The second application was the study of the effect of membrane confinement on KvAP diffusion. Using single particle tracking, we found that the diffusion coefficient decreases by a factor 3 when the tube radius decreases from 250 to 10 nm. This result is in agreement with a hydrodynamic theory that extends the work of Saffman and Delbrück to cylindrical geometries.
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https://tel.archives-ouvertes.fr/tel-00736743
Contributor : Sophie Aimon <>
Submitted on : Thursday, November 1, 2012 - 11:21:18 PM
Last modification on : Thursday, December 10, 2020 - 10:50:38 AM
Long-term archiving on: : Saturday, February 2, 2013 - 2:50:18 AM

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  • HAL Id : tel-00736743, version 1

Citation

Sophie Aimon. Study of a voltage-gated ion channel reconstituted in Giant Unilamellar Vesicles. Biological Physics [physics.bio-ph]. Université Pierre et Marie Curie - Paris VI, 2011. English. ⟨tel-00736743⟩

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