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Reconstitution and functional characterization of ion channels from nanodiscs in lipid bilayers

Winterstein, Laura-Marie ; Kukovetz, Kerri ; Rauh, Oliver ; Turman, Daniel L. ; Braun, Christian ; Moroni, Anna ; Schroeder, Indra ; Thiel, Gerhard (2022):
Reconstitution and functional characterization of ion channels from nanodiscs in lipid bilayers. (Publisher's Version)
In: The Journal of General Physiology, 150 (4), pp. 637-646. Rockefeller Univ. Press, ISSN 0022-1295, e-ISSN 1540-7748,
DOI: 10.26083/tuprints-00013429,
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Item Type: Article
Origin: Secondary publication
Status: Publisher's Version
Title: Reconstitution and functional characterization of ion channels from nanodiscs in lipid bilayers
Language: English
Abstract:

Recent studies have shown that membrane proteins can be efficiently synthesized in vitro before spontaneously inserting into soluble nanoscale lipid bilayers called nanodiscs (NDs). In this paper, we present experimental details that allow a combination of in vitro translation of ion channels into commercially available NDs followed by their direct reconstitution from these nanobilayers into standard bilayer setups for electrophysiological characterization. We present data showing that two model K+ channels, Kcv and KcsA, as well as a recently discovered dual-topology F− channel, Fluc, can be reliably reconstituted from different types of NDs into bilayers without contamination from the in vitro translation cocktail. The functional properties of Kcv and KcsA were characterized electrophysiologically and exhibited sensitivity to the lipid composition of the target DPhPC bilayer, suggesting that the channel proteins were fully exposed to the target membrane and were no longer surrounded by the lipid/protein scaffold. The single-channel properties of the three tested channels are compatible with studies from recordings of the same proteins in other expression systems. Altogether, the data show that synthesis of ion channels into NDs and their subsequent reconstitution into conventional bilayers provide a fast and reliable method for functional analysis of ion channels.

Journal or Publication Title: The Journal of General Physiology
Volume of the journal: 150
Issue Number: 4
Publisher: Rockefeller Univ. Press
Classification DDC: 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin, Gesundheit
Divisions: 10 Department of Biology > Plant Membrane Biophysics
Date Deposited: 01 Mar 2022 13:19
Last Modified: 10 Mar 2022 09:25
DOI: 10.26083/tuprints-00013429
Corresponding Links:
URN: urn:nbn:de:tuda-tuprints-134292
URI: https://tuprints.ulb.tu-darmstadt.de/id/eprint/13429
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