Cell-free expression and functional reconstitution of homo-oligomeric alpha 7 nicotinic acetylcholine receptors into planar lipid bilayers

The alpha 7 nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel that modulates neurotransmitter release in the central nervous syst em. We show here that functional, homo-oligomeric alpha 7 nAChRs can be syn thesized in vitro with a rabbit reticulocyte lysate translation system supp lemented with endoplasmic reticulum microsomes, reconstituted into planar l ipid bilayers, and evaluated using single-channel recording techniques. Bec ause wild-type alpha 7 nAChRs desensitize rapidly, we used a nondesensitizi ng form of the alpha 7 receptor with mutations in the second transmembrane domain (S2'T and L9'T) to record channel activity in the continuous presenc e of agonist. Endoglycosidase H treatment of microsomes containing nascent alpha 7 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, c onsistent with a large extracellular domain predicted by most topological m odels, indicating that the protein was folded integrally through the ER mem brane. alpha 7 S2'T/L9'T receptors reconstituted into planar lipid bilayers had a unitary conductance of similar to 50 pS, were highly selective for m onovalent cations over Cl-, were nonselective between K+ and Na+, and were blocked by alpha-bungarotoxin, This is the first demonstration that a funct ional ligand-gated ion channel can be synthesized using an in vitro express ion system.