Cell-free expression of functional Shaker potassium channels

Abstract

THE functional activity of ion channels and other membrane proteins requires that the proteins be correctly assembled in a transmembrane configuration. Thus, the functional expression of ion channels, neurotransmitter receptors and complex membrane-limited signalling mechanisms from complementary DNA has required the injection of messenger RNA or transfection of DNA into Xenopus oocytes or other target cells that are capable of processing newly translated protein into the surface membrane^1–4. These approaches, combined with voltage-clamp analysis of ion channel currents, have been especially powerful in the identification of structure–function relationships in ion channels^5–7. But oocytes express endogenous ion channels^8,9, neurotransmitter receptors¹⁰ and receptor–channel subunits¹¹, complicating the interpretation of results in mRNA-injected eggs. Furthermore, it is difficult to control experimentally the membrane lipids and post-translational modifications that underlie the regulation and modulation of ion channels in intact cells. A cell-free system for ion channel expression is ideal for good experimental control of protein expression and modulatory processes. Here we combine cell-free protein translation, microsomal membrane processing^12–14 of nascent channel proteins, and reconstitution of newly synthesized ion channels into planar lipid bilayers¹⁵ to synthesize, glycosylate, process into membranes, and record in vitro the activity of functional Shaker potassium channels.