Surface expression of the nicotinic acetylcholine receptor (AChR) requires the assembly of multiple subunits in the endoplasmic reticulum (ER). Little is known, however, about the mechanism by which assembled receptor pentamers are transported to the cell membrane while unassembled subunits are retained in the ER. Here we report that a motif conserved in the transmembrane domain of AChR subunits is critically involved in this process. In COS cells, mutation within this signal allowed surface expression of unassembled subunits. Conversely, insertion of the sequence to unrelated proteins that are normally transported to the surface resulted in ER retention. The signal is buried in AChR pentamers, but is exposed on unassembled subunits in the ER, where it promotes protein degradation. We therefore conclude that this signal ensures surface trafficking of only functional AChRs.

The ␣ subunit of the nicotinic acetylcholine receptor (AChR) from Torpedo electric organ and mammalian muscle contains high affinity binding sites for ␣-bungarotoxin and for autoimmune antibodies in sera of patients with myasthenia gravis. To obtain sufficient materials for structural studies of the receptor-ligand complexes, we have expressed part of the mouse muscle ␣ subunit as a soluble, secretory protein using the yeast Pichia pastoris. By testing a series of truncated fragments of the receptor protein, we show that ␣211, the entire amino-terminal extracellular domain of AChR ␣ subunit (amino acids 1-211), is the minimal segment that could fold properly in yeast. The ␣211 protein was secreted into the culture medium at a concentration of >3 mg/liter. It migrated as a 31-kDa polypeptide with Nlinked glycosylation on SDS-polyacrylamide gel. The protein was purified to homogeneity by isoelectric focusing electrophoresis (pI 5.8), and it appeared as a 4.5 S monomer on sucrose gradient at concentrations up to 1 mM (ϳ30 mg/ml). The receptor domain bound monoclonal antibody mAb35, a conformation-specific antibody against the main immunogenic region of the AChR. In addition, it formed a high affinity complex with ␣-bungarotoxin (k D 0.2 nM) but showed relatively low affinity to the small cholinergic ligand acetylcholine. Circular dichroism spectroscopy of ␣211 revealed a composition of secondary structure corresponding to a folded protein. Furthermore, the receptor fragment was efficiently 15 N-labeled in P. pastoris, and proton crosspeaks were well dispersed in nuclear Overhauser effect and heteronuclear single quantum coherence spectra as measured by NMR spectroscopy. We conclude that the soluble AChR protein is useful for high resolution structural studies.The nicotinic AChRs 1 are members of the superfamily of ligand-gated ion channels that mediate fast ion flow across postsynaptic membranes in neural and muscle cells. This family of proteins includes the receptors for glycine, ␥-aminobutyric acid, glutamate, and serotonin (1, 2). The AChR from Torpedo electric organ and skeletal muscle is a large (ϳ290 kDa) heteropentameric complex consisting of four homologous subunits in the stoichiometry of ␣ 2 ␤␦␥ (Torpedo and embryonic muscle) or ␣ 2 ␤␦⑀ (adult muscle (3, 4)). The subunits are arranged in the order ␣-␥-␣-␦-␤ to create a cylindrical complex around the ion channel (5-7). Each subunit is a single polypeptide chain that has a large extracellular amino-terminal domain followed by three transmembrane domains, a long intracellular loop, a fourth transmembrane domain, and a short extracellular carboxyl-terminal tail (8 -10). The large extracellular domain of the AChR confers high affinity binding activity for major agonists and competitive antagonists. The neurotransmitter ACh interacts with two nonequivalent sites near the interface of the ␣␦ and ␣␥ subunits (2, 11, 12). The binding site for ␣-bungarotoxin (␣-BuTx) is located largely on the amino-terminal extracellular domain of the ␣ subunit (13-16). In additio...

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