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Vol. 54, Issue 2, 322-333, August 1998
3/
4 Subtype of Neuronal Nicotinic Acetylcholine
Receptor Stably Expressed in a Transfected Cell Line: Pharmacology of
Ligand Binding and Function
Department of Pharmacology, Georgetown University School of
Medicine, Washington, DC 20007
We stably transfected human kidney embryonic 293 cells with the
rat neuronal nicotinic acetylcholine receptor (nAChR) 
3 and 
4
subunit genes. This new cell line, KX34R2, expresses a high level
of the 3/4 receptor subtype, which binds (±)-
[3H]epibatidine with a
Kd value of 304±16
pM and a Bmax value of
8942 ± 115 fmol/mg protein. Comparison of nicotinic drugs in
competing for 3/4 receptor binding sites in this cell line and
the binding sites in rat forebrain (predominantly 4/2 receptors) revealed marked differences in their
Ki values, but similar rank
orders of potency for agonists were observed, with the exception of
anatoxin-A. The affinity of the competitive antagonist
dihydro--erythroidine is >7000 times higher at 4/2 receptors
in rat forebrain than at the 3/4 receptors in these cells. The
3/4 nAChRs expressed in this cell line are functional, and in
response to nicotinic agonists, 86Rb+ efflux
was increased to levels 8-10 times the basal levels. Acetylcholine, (
)-nicotine, cytisine, carbachol, and (±)-epibatidine all stimulated 86Rb+ efflux, which was blocked by
mecamylamine. The EC50 values for acetylcholine and
()-nicotine to stimulate 86Rb+ effluxes were
114 ± 24 and 28 ± 4 µM, respectively.
The rank order of potency of nicotinic antagonists in blocking the
function of this 3/4 receptor was mecamylamine > d-tubocurarine > dihydro--erythroidine > hexamethonium. Mecamylamine, d-tubocurarine, and
hexamethonium blocked the function by a noncompetitive mechanism,
whereas dihydro--erythroidine blocked the function competitively.
The KX34R2 cell line should prove to be a very useful model for
studying this subtype of nAChRs.
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