Stable expression of the mouse nicotinic acetylcholine receptor in mouse fibroblasts comparison of receptors in native and transfected cells

A stable cell line expressing mouse acetylcholine receptors (AChRs), named AM4, was established by co-transfecting into NIH 3T3 fibroblasts, α-, β-, γ-, and δ-subunit cDNAs plus the neo^r gene by calcium phosphate precipitation. Surface AChRs on AM4 cells contain all four subunits, sediment as a single ∼9 S peak on sucrose gradients, and have the same ratio of α- to β-subunits as surface AChRs from mouse BC3H-1 cells. The surface AChRs exhibit pharmacological properties identical to those obtained for BC3H-1 cells, including the association and dissociation rates of α-bungarotoxin, a low affinity and cooperative instantaneous dose-response curve, cooperative steady state agonist binding and desensitization, cooperative enhancement of agonist binding affinity by local anesthetics, and distinct affinities for curariform antagonists. Patch clamp measurements on AM4 cells reveal AChR single channel properties identical to those obtained from BC3H-1 cells, including a single class of channels with a conductance of 56 pS, short and long duration openings at low and high agonist concentrations, brief and intermediate closed duration components at low agonist concentrations, and six distinct closed duration components at high agonist concentrations. The biochemical, pharmacological, and single channel measurements indicate at least 95% of the surface AChRs on AM4 cells are α₂βγδ pentamers.