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

The Journal of General Physiology
Laura-Marie WintersteinGerhard Thiel

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 N...Continue Reading

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Citations

Jan 9, 2020·Proceedings of the National Academy of Sciences of the United States of America·Pramod KumarClaudio Grosman
Apr 24, 2019·Channels·Denise EckertGerhard Thiel
Mar 22, 2020·BioDrugs : Clinical Immunotherapeutics, Biopharmaceuticals and Gene Therapy·Srujan Kumar DondapatiStefan Kubick
Nov 25, 2020·Cells·Anja J EngelGerhard Thiel
Jan 14, 2021·The Journal of General Physiology·Laura-Marie WintersteinOliver Rauh

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BETA
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Software Mentioned

IonoVation
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