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.
In: The Journal of General Physiology, 2018, 150 (4)
doi: 10.26083/tuprints-00013429
Artikel, Zweitveröffentlichung, Verlagsversion
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Kurzbeschreibung (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.
| Typ des Eintrags: | Artikel |
|---|---|
| Erschienen: | 2022 |
| Autor(en): | Winterstein, Laura-Marie ; Kukovetz, Kerri ; Rauh, Oliver ; Turman, Daniel L. ; Braun, Christian ; Moroni, Anna ; Schroeder, Indra ; Thiel, Gerhard |
| Art des Eintrags: | Zweitveröffentlichung |
| Titel: | Reconstitution and functional characterization of ion channels from nanodiscs in lipid bilayers |
| Sprache: | Englisch |
| Publikationsjahr: | 2022 |
| Publikationsdatum der Erstveröffentlichung: | 2018 |
| Verlag: | Rockefeller Univ. Press |
| Titel der Zeitschrift, Zeitung oder Schriftenreihe: | The Journal of General Physiology |
| Jahrgang/Volume einer Zeitschrift: | 150 |
| (Heft-)Nummer: | 4 |
| DOI: | 10.26083/tuprints-00013429 |
| URL / URN: | https://tuprints.ulb.tu-darmstadt.de/13429 |
| Zugehörige Links: | |
| Herkunft: | Zweitveröffentlichung |
| Kurzbeschreibung (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. |
| Status: | Verlagsversion |
| URN: | urn:nbn:de:tuda-tuprints-134292 |
| Sachgruppe der Dewey Dezimalklassifikatin (DDC): | 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin, Gesundheit |
| Fachbereich(e)/-gebiet(e): | 10 Fachbereich Biologie 10 Fachbereich Biologie > Plant Membrane Biophyscis (am 20.12.23 umbenannt in Biologie der Algen und Protozoen) |
| Hinterlegungsdatum: | 01 Mär 2022 13:19 |
| Letzte Änderung: | 02 Mär 2022 07:05 |
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| Suche nach Titel in: | TUfind oder in Google |
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- Reconstitution and functional characterization of ion channels from nanodiscs in lipid bilayers. (deposited 01 Mär 2022 13:19) [Gegenwärtig angezeigt]
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