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

Abstract

The ␣7 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 ␣7 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 ␣7 nAChRs desensitize rapidly, we used a nondesensitizing form of the ␣7 receptor with mutations in the second transmembrane domain (S2T and L9T) to record channel activity in the continuous presence of agonist. Endoglycosidase H treatment of microsomes containing nascent ␣7 S2T/L9T 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. ␣7 S2T/L9T receptors reconstituted into planar lipid bilayers had a unitary conductance of ϳ50 pS, were highly selective for monovalent cations over Cl ؊ , were nonselective between K ؉ and Na ؉ , and were blocked by ␣-bungarotoxin. This is the first demonstration that a functional ligand-gated ion channel can be synthesized using an in vitro expression system. The nicotinic acetylcholine receptor (nAChR)1 is a member of a superfamily of ligand-gated ion channels that also includes GABA A receptors, serotonin (5-HT 3 ) receptors, glycine receptors, and an invertebrate glutamate-gated chloride channel (1). Nicotinic receptors are located at the neuromuscular junction and in the central and peripheral nervous systems. Muscletype nAChRs are pentamers of homologous subunits in the stoichiometry of ␣ 2 ␤␥␦ (or ␣ 2 ␤␦⑀) arranged around a central pore (2). Neuronal nAChRs also form pentameric complexes (3, 4) from various combinations of the 11 neuronal nAChR genes (␣2-␣9 and ␤2-␤4) that have been identified to date (5). ␣7, ␣8, and ␣9 nAChRs are blocked by the snake peptide toxin ␣-bungarotoxin (␣-BTX), which also blocks muscle and Torpedo nAChRs but not other subtypes of neuronal nAChRs (5). ␣7 nAChRs are the most abundantly expressed nicotinic receptor subunit in the central nervous system (6) and are important in neuronal development, hippocampal function, and the modulation of fast neurotransmission (7,8). ␣7 nAChRs are highly calcium-permeable (9, 10), and calcium influx through presynaptic ␣7 nAChRs modulates the release of excitatory neurotransmitters (11,12).During biosynthesis of nAChRs, the polypeptide is translocated into the endoplasmic reticulum (ER) membrane. The ER contains enzymes necessary for signal sequence cleavage (13) and other post-translational modifications required for correct subunit folding, assembly, and ligand-binding site formation. These modifications include core glycosylation (13) and disulfide bond formation (14, 15). In addition, ER and cytoplasmic chaperone proteins are tho...

Functional Cell-free Synthesis of a Seven Helix Membrane Protein: In situ Insertion of Bacteriorhodopsin into Liposomes

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Advances in cell-free protein synthesis for the functional and structural analysis of membrane proteins

“…well as by addition of microsomes [54,55] or crude inner membrane vesicles [27,56]. Added chaperones did not increase the integration rate of functional aquaporin Z into synthetic liposomes, although a higher association of the MP to the lipid bilayer was noted [24].…”

Cell-Free Protein Synthesis: A Promising Option for Future Drug Development

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