Liposome-Based in Vitro Biosynthesis of Two-Pore Domain Potassium Channels for Functional Studies

Abstract

The Two-pore domain potassium channels (K2P) function as regulators of membrane resting potential and cellular electrical excitability. The family has 15 homo- and heterodimeric members, most of which can be characterized by electrophysiological recordings of transfected cells and RNA-injected Xenopus oocytes.However, drawbacks of heterologuous expression arise from ungovernable cellular regulation events, unknown membrane composition, or lack of wildtype currents at the plasma membrane. Alternative methods demand recombinant membrane protein production, which is still difficult.We report the in vitro biosynthesis of several K2P channels as well as mutant and GFP-fusion constructs in a lipid-containing wheat-germ cell-free system.We show that Giant Unilamellar Vesicles (GUV) as well as Small Unilamellar Vesicles (SUV) of defined lipid composition can be included in the cell-free reaction to promote production of full-length channel proteins without addition of detergents. Channel insertion and orientation in the GUV lipid membrane was visualized by fluorescence microscopy of GFP-tagged fusion proteins and a proteolytic assay. Channel containing GUV were probed by patch-clamp methods, while SUV were preferred for planar lipid bilayer experiments. Our results indicate that K2P channels are formed in a functional state.Liposome-based cell-free synthesis provides the opportunity of obtaining ion channels in artificial membrane environment to study lipid regulation effects or mechanosensitivity exclusive of endogenuous regulatory or accessory proteins. and coexpression experiments could reveal interaction partners. Furthermore, patch clamp techniques can be extended to channels of possibly intracellular location and could help to understand the role of K2P family members so far referred to as “silent”. As synthesis is accessible for reagent addition, site-specific labeling by fluorophores, cysteine reactive agents or non-natural amino acids can readily be achieved and might prove beneficial in studying structural changes in gating events.