2018 - Reconstitution and Electrophysiological Characterization of Ion Channels in Lipid Bilayers
Port-a-Patch and Vesicle Prep Pro publication in Current Protocols in Pharmacology (2018)
Klaerke D.A., de los Angeles Tejada M., Grøsfjeld Christensen V., Lassen M., Amstrup Pedersen P., Calloe K.
Current Protools in Pharmacology (2018) e37:1–17. doi: 10.1002/cpph.37
Detergent‐solubilized purified ion channels can be reconstituted into lipid bilayers for electrophysiological analysis. Traditionally, ion channels were inserted into vesicles and subsequently fused with planar “black lipid membranes” formed from lipids dissolved in a hydrophobic solvent such as decane. Provided in this article is a step‐by‐step guide to reconstitute purified ion channel proteins into giant unilamellar vesicles (GUVs). This procedure results in the formation of proteoliposomes that can be used for planar bilayer formation and electrophysiological characterization of single‐channel currents. By using preformed GUVs it is possible to omit the membrane solvent. Compared to traditional preparations, the lipid bilayers formed from GUVs provide an environment that more closely resembles the native cell membrane. Also described is an alternate protocol that entails the production of planar lipid bilayers from GUVs onto which proteins in detergent are added.