Cell-free Expression and Functional Reconstitution of Homo-oligomeric α7 Nicotinic Acetylcholine Receptors into Planar Lipid Bilayers

Abstract The α7 nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel that modulates neurotransmitter release in the central nervous system. We show here that functional, homo-oligomeric α7 nAChRs can be synthesized in vitrowith a rabbit reticulocyte lysate translation system supplemented with endoplasmic reticulum microsomes, reconstituted into planar lipid bilayers, and evaluated using single-channel recording techniques. Because wild-type α7 nAChRs desensitize rapidly, we used a nondesensitizing form of the α7 receptor with mutations in the second transmembrane domain (S2′T and L9′T) to record channel activity in the continuous presence of agonist. Endoglycosidase H treatment of microsomes containing nascent α7 S2′T/L9′T nAChRs indicated that the receptors were glycosylated. A proteinase K protection assay revealed a 36-kDa fragment in the ER lumen, consistent with a large extracellular domain predicted by most topological models, indicating that the protein was folded integrally through the ER membrane. α7 S2′T/L9′T receptors reconstituted into planar lipid bilayers had a unitary conductance of ∼50 pS, were highly selective for monovalent cations over Cl−, were nonselective between K+ and Na+, and were blocked by α-bungarotoxin. This is the first demonstration that a functional ligand-gated ion channel can be synthesized using an in vitro expression system.