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.

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