Cell-free expression and functional reconstitution of homo-oligomeric alpha7 nicotinic acetylcholine receptors into planar lipid bilayers.
The alpha7 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 alpha7 nAChRs can be synthesized in vitro with 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 alpha7 nAChRs desensitize rapidly, we used a nondesensitizing form of the alpha7 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 alpha7 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. alpha7 S2'T/L9'T receptors reconstituted into planar lipid bilayers had a unitary conductance of approximately 50 pS, were highly selective for monovalent cations over Cl(-), were nonselective between K(+) and Na(+), and were blocked by alpha-bungarotoxin. This is the first demonstration that a functional ligand-gated ion channel can be synthesized using an in vitro expression system.
Lyford
L K
LK
Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599-7365, USA.
Rosenberg
R L
RL
D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D002474:Cell-Free System; D019281:Dimerization; D008051:Lipid Bilayers; D009154:Mutation; D018408:Patch-Clamp Techniques; D017510:Protein Folding; D011817:Rabbits; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000502:physiology*; D014981:Xenopus; D064569:alpha7 Nicotinic Acetylcholine Receptor
D008051:Lipid Bilayers; D011978:Receptors, Nicotinic; D064569:alpha7 Nicotinic Acetylcholine Receptor; D000109:Acetylcholine