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J. Biol. Chem., Vol. 266, Issue 21, 13679-13689, 07, 1991
SM Sine and T Claudio
A stable cell line expressing mouse acetylcholine receptors (AChRs), named
AM4, was established by cotransfecting into NIH 3T3 fibroblasts, alpha-,
beta-, gamma-, and delta-subunit cDNAs plus the neor gene by calcium
phosphate precipitation. Surface AChRs on AM4 cells contain all four
subunits, sediment as a single approximately 9 S peak on sucrose gradients,
and have the same ratio of alpha- to beta-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 alpha- 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 alpha 2 beta gamma delta
pentamers.
Stable expression of the mouse nicotinic acetylcholine receptor in mouse fibroblasts. Comparison of receptors in native and transfected cells
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510.
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