Neuronal Nicotinic Receptor 2 and 4 Subunits Confer Large
Differences in Agonist Binding Affinity
MICHAEL J. PARKER, AVI BECK, and CHARLES W. LUETJE
Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, Florida 33101
Received July 10, 1998; Accepted September 14, 1998 This paper is available online at http://www.molpharm.org
ABSTRACT
We used equilibrium binding analysis to characterize the ago-
nist binding properties of six different rat neuronal nicotinic
receptor subunit combinations expressed in Xenopus laevis
oocytes. The 42 receptor bound [
3
H]cytisine with a K
dapp
of
0.74 0.14 nM. The rank order of K
iapp
values of additional
nicotinic ligands, determined in competition assays, was cy-
tisine nicotine acetylcholine carbachol curare. These
pharmacological properties of 42 expressed in oocytes are
comparable to published values for the high affinity cytisine
binding site in rat brain (42), demonstrating that rat neuronal
nicotinic receptors expressed in X. laevis oocytes display ap-
propriate pharmacological properties. Use of [
3
H]epibatidine
allowed detailed characterization of multiple neuronal nicotinic
receptor subunit combinations. K
dapp
values for [
3
H]epibatidine
binding were 10 pM for 22, 87 pM for 24, 14 pM for 32,
300 pM for 34, 30 pM for 42, and 85 pM for 44. Affinities
for six additional agonists (acetylcholine, anabasine, cytisine,
1,1-dimethyl-4-phenylpiperazinium, lobeline, and nicotine)
were determined in competition assays. The 2-containing re-
ceptors had consistently higher affinities for these agonists
than did 4-containing receptors. Particularly striking examples
are the affinities displayed by 22 and 24, which differ in
1,1-dimethyl-4-phenylpiperazinium, nicotine, lobeline, and ace-
tylcholine affinity by 120-, 86-, 85-, and 61-fold, respectively.
Although smaller differences in affinity could be ascribed to
different subunits, the major factor in determining agonist
affinity was the nature of the subunit.
Neuronal nAChRs form as pentameric assemblies of sub-
units, similar to muscle nAChRs (Anand et al., 1991; Cooper
et al., 1991). There are 11 known neuronal nAChR subunits,
2–9 and 2– 4 (Sargent, 1993; Elgoyhen et al., 1994). Many
different combinations of these subunits can assemble to
form functional nAChRs when expressed in Xenopus laevis
oocytes or mammalian cell lines, with each functional sub-
unit combination displaying a distinct array of biophysical
and pharmacological properties (Role, 1992; Patrick et al.,
1993; Sargent, 1993). Thus, differential subunit assembly is
likely to underlie biophysical and pharmacological observa-
tions of multiple subtypes of neuronal nAChRs in the ner-
vous system.
Nicotinic ligands are potentially useful as anxiolytics and
analgesics and are potentially useful in the treatment of
neurological disorders such as schizophrenia, Parkinson’s
disease, and Alzheimer’s disease (Brioni et al., 1997). Neuro-
nal nAChRs also are the sites at which nicotine exerts its
psychoactive and addictive effects (Dani and Heinemann,
1996). Thus, pharmacological intervention at neuronal
nAChRs holds promise for treating the effects of diseases of
the central nervous system and for understanding and treat-
ing addictive processes. Critical to the realization of this
potential is the development of subtype-selective nAChR li-
gands. Pursuit of this goal requires an understanding of the
molecular structure of the ligand binding sites of neuronal
nAChRs. In particular, the features of nicotinic binding sites
that are responsible for nAChR subtype selectivity must be
identified.
Affinity labeling and mutagenesis techniques have been
used to identify a series of residues on the , /, and
subunits that participate in the structure of the neurotrans-
mitter binding sites of muscle-type nAChRs (Karlin and Aka-
bas, 1995). The identification of critical residues on the /
and subunits, together with the repeated demonstration
that the two binding sites on muscle nAChRs are pharmaco-
logically distinct, has led to the concept that the neurotrans-
mitter binding sites are located at the interface between
and non- (/ and ) subunits (Blount and Merlie, 1989;
Galzi and Changeux, 1995). The neurotransmitter binding
sites on neuronal nAChRs seem to be formed in a similar
manner, because both and subunits make contributions
to the pharmacological properties of these receptors (Luetje
This work was supported by a grant to C.W.L. from the National Institute
on Drug Abuse (DA08102). M.J.P. was supported in part by T32-HL07188.
Portions of this work have been presented in preliminary form [Parker MJ and
Luetje CW (1996) Soc Neurosci Abstr 22:1271; Parker MJ and Luetje CW
(1997) Soc Neurosci Abstr 23:385].
ABBREVIATIONS: nAChR, nicotinic acetylcholine receptor; ACh, acetylcholine; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;
DMPP, 1,1-dimethyl-4-phenylpiperizinium.
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