The adult muscle-type nicotinic acetylcholine receptor (AChR) is essential for neuromuscular transmission but is difficult to produce due to the requirement for coordinated subunit assembly. Here, we present a protocol for generating doxycycline-inducible stable cell lines that co-express all four subunits. We describe steps for lentivirus infection, puromycin selection, fluorescence-activated cell sorting, clonal expansion, and protein expression test. This protocol enables AChR production at quantities sufficient for single-particle cryoelectron microscopy (cryo-EM) and may be applicable to other hetero-multimeric protein complexes. For complete details on the use and execution of this protocol, please refer to Li et al. 1 • Guidance on designing lentivirus constructs to express human muscle-type AChR subunits • Steps for lentiviral co-infection and antibiotic selection for stably integrated cells • Instructions for FACS isolation of clones expressing all AChR subunits • Procedures for AChR expression test by 125 I-α-bungarotoxin binding assay Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. The adult muscle-type nicotinic acetylcholine receptor (AChR) is essential for neuromuscular transmission but is difficult to produce due to the requirement for coordinated subunit assembly. Here, we present a protocol for generating doxycycline-inducible stable cell lines that co-express all four subunits. We describe steps for lentivirus infection, puromycin selection, fluorescence-activated cell sorting, clonal expansion, and protein expression test. This protocol enables AChR production at quantities sufficient for single-particle cryoelectron microscopy (cryo-EM) and may be applicable to other hetero-multimeric protein complexes.
Li et al. (2026) studied this question.