Sazetidine-A, a novel ligand that desensitizes α4β2 nicotinic acetylcholine receptors without activating them

Article abstract of DOI:10.1124/mol.106.027318

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels found throughout the central and peripheral nervous systems. They are crucial to normal physiology and have been clearly implicated in nicotine addiction. In addition, they are possible therapeutic targets in a wide range of pathological conditions, including cognitive disorders, Parkinson's disease, and neuropathic pain. Nicotinic ligands are usually classified as agonists (or partial agonists), competitive antagonists, or noncompetitive antagonists. Sazetidine-A is a new nicotinic ligand that shows a different pharmacological profile from any of these known classes of ligands. Sazetidine-A competes with very high binding affinity (K_i ≈ 0.5 nM) and selectivity for the α4β2 nAChR subtype (K_i ratio α3β4/ α4β2 ～ 24,000). Despite its high affinity, sazetidine-A neither activates nAChR channel function nor prevents channel activation when it is applied simultaneously with nicotine. However, when it is pre-incubated for 10 min with the receptors, it potently blocks nicotine-stimulated α4β2 nAChR function (IC₅₀ ≈ 30 nM). The action of sazetidine-A may be explained by its very low affinity for the resting conformation of the α4β2 nAChRs, and its very high affinity for the desensitized state of the receptor. We propose that sazetidine-A is a "silent desensitizer" of nAChRs, meaning that it desensitizes the receptor without first activating it. Furthermore, comparison of the effects of sazetidine-A and nicotine at α4β2 nAChRs suggests that the predominant effects of nicotine and other nicotinic agonists are related to desensitization of the receptors and that sazetidine-A potently mimics these effects. Copyright

Full text of DOI:10.1124/mol.106.027318

0026-895X/06/7004-1454–1460$20.00
MOLECULAR PHARMACOLOGY
Copyright © 2006 The American Society for Pharmacology and Experimental Therapeutics
Mol Pharmacol 70:1454–1460, 2006
Vol. 70, No. 4
27318/3142740
Printed in U.S.A.
Sazetidine-A, A Novel Ligand That Desensitizes ␣4␤2 Nicotinic
Acetylcholine Receptors without Activating Them
Yingxian Xiao, Hong Fan, John L. Musachio,1 Zhi-Liang Wei,2 Sheela K. Chellappan,
Alan P. Kozikowski, and Kenneth J. Kellar
Department of Pharmacology, Georgetown University School of Medicine, Washington DC (Y.X., K.J.K.); Department of
Radiology, Johns Hopkins University School of Medicine, Baltimore, Maryland (H.F., J.L.M.); and Department of Medicinal
Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois (Z.-L.W., S.K.C.,
A.P.K.).
Received May 31, 2006; accepted July 14, 2006
ABSTRACT
Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand- channel function nor prevents channel activation when it is
gated ion channels found throughout the central and peripheral applied simultaneously with nicotine. However, when it is pre-
nervous systems. They are crucial to normal physiology and incubated for 10 min with the receptors, it potently blocks
have been clearly implicated in nicotine addiction. In addition, nicotine-stimulated ␣4␤2 nAChR function (IC50 Ϸ 30 nM). The
they are possible therapeutic targets in a wide range of patho- action of sazetidine-A may be explained by its very low affinity
logical conditions, including cognitive disorders, Parkinson’s for the resting conformation of the ␣4␤2 nAChRs, and its very
disease, and neuropathic pain. Nicotinic ligands are usually high affinity for the desensitized state of the receptor. We
classified as agonists (or partial agonists), competitive antago- propose that sazetidine-A is a “silent desensitizer” of nAChRs,
nists, or noncompetitive antagonists. Sazetidine-A is a new meaning that it desensitizes the receptor without first activating
nicotinic ligand that shows a different pharmacological profile it. Furthermore, comparison of the effects of sazetidine-A and
from any of these known classes of ligands. Sazetidine-A com- nicotine at ␣4␤2 nAChRs suggests that the predominant effects
petes with very high binding affinity (Ki Ϸ 0.5 nM) and selectivity of nicotine and other nicotinic agonists are related to desensi-
for the ␣4␤2 nAChR subtype (Ki ratio ␣3␤4/␣4␤2 ϳ 24,000). tization of the receptors and that sazetidine-A potently mimics
Despite its high affinity, sazetidine-A neither activates nAChR these effects.
nAChRs are members of the Cys-loop family of neurotrans- changes, which are translated to the pore through a pathway
mitter-gated ion channels (Karlin, 2002; Le Novere et al.,
2002; Lindstrom, 2002). High-resolution structural models
have greatly advanced our understanding of these receptors
(Brejc et al., 2001; Unwin, 2005). They are composed of five
subunits assembled around a central ion channel. A large
extracellular domain, composed of N-terminal portions of all
five subunits, contains two to five ligand binding sites formed
between an ␣ subunit and an adjacent subunit. Each subunit
has four transmembrane ␣-helices, M1–M4. The M2 helices
from the five subunits form a long narrow ion-conducting
pore. The binding of the endogenous agonist acetylcholine or
a drug such as nicotine initiates a series of conformational
in the ␣ subunits formed between three residues of the ex-
tracellular portion and two residues that link the M2 and M3
transmembrane helices (Lee and Sine, 2005).
In addition to acetylcholine and nicotine, many other nat-
ural products and synthetic drugs act on nAChRs (Daly,
2005; Jensen et al., 2005). These nicotinic compounds have
been classified into three groups: agonists (or partial ago-
nists), which act on acetylcholine binding sites and lead to
the opening of nAChR channels; competitive antagonists,
which bind to the agonist sites without stimulating the open-
ing of nAChR channels and so block access by agonists; and
noncompetitive antagonists, which act on sites of nAChRs
other than acetylcholine binding sites (e.g., within the ion
channel) to block functions of agonists. In addition, some
drugs have been proposed to be allosteric potentiating li-
gands, which may enhance the effects of agonists by acting at
a site close to but distinct from the acetylcholine binding sites
to increase the probability of channel opening in the presence
of an agonist (Pereira et al., 2002).
This work was supported by National Institutes of Health grants
DA017980, DA12976, and DA13199.
1 Current affiliation: National Institute of Mental Health, National Insti-
tutes of Health, Bethesda, Maryland.
2 Current affiliation: Renovis, Inc., South San Francisco, California.
Article, publication date, and citation information can be found at
http://molpharm.aspetjournals.org.
doi:10.1124/mol.106.027318.
ABBREVIATIONS: nAChR, nicotinic acetylcholine receptor; A-85380, 3-(2(S)-azetidinylmethoxy) pyridine; EB, (Ϯ)epibatidine; DH␤E, dihydro-␤-
erythroidine.
1454

A Silent Desensitizer of ␣4␤2 nAChRs
1455
nAChRs have at least four discrete conformations: a rest- Co. (St. Louis, MO), unless otherwise stated. Varenicline was syn-
thesized using methods described previously (O’Donnell et al., 2004;
Coe et al., 2005).
ing state, an open state, and two or more desensitized states
(Katz and Thesleff, 1957; Karlin, 1967; Changeux and Edel-
stein, 1998). Agonists have dual effects at nAChRs. They
initially open the channel, allowing conduction of Naϩ, Kϩ,
and Ca2ϩ ions, which can lead to membrane depolarization
and altered cell function, but agonists then drive the receptor
into a desensitized state, temporarily rendering it unrespon-
sive to further agonist stimulation. The desensitized state
may be brief, as is usually the case with the endogenous
agonist acetylcholine, or it may be prolonged, as seems to
occur when nicotine is the agonist, especially with repeated
exposure (Quick and Lester, 2002). In addition to these two
immediate effects, long-term exposure to nicotine or other
agonists increases the density of nAChRs in brain (Marks et
al., 1983; Schwartz and Kellar, 1983).
In this report, we have characterized the pharmacological
actions of sazetidine-A (Fig. 1), a new nicotinic ligand with
very high affinity and selectivity for nAChRs containing ␤2
subunits. Sazetidine-A is an analog of A-85380, which itself
has high affinity and selectivity for nAChRs containing ␤2
subunits. Because the 5-iodo analog of A-85380 retains high
affinity and displays even higher selectivity for ␣4␤2
nAChRs (Mukhin et al., 2000; Xiao and Kellar, 2004), we
examined other substituents at this position to determine the
possibility of creating high-affinity nAChR ligands that could
be used for affinity isolation and/or fluorescent labeling,
among other purposes. In the course of those studies, we
synthesized sazetidine-A. As described here, we found that it
retains high affinity for ␣4␤2 nAChRs and is more selective.
It is noteworthy that despite its high affinity for ␣4␤2 recep-
tors, sazetidine-A did not activate these receptors, and it did not
inhibit activation when added simultaneously with nicotine.
But after a 10 min preincubation, sazetidine-A very potently
inhibited nicotine-stimulated ␣4␤2 nAChR activation. These
results suggest that although sazetidine-A does not have mea-
surable agonist activity, it potently drives the receptors into a
Synthesis of Sazetidine-A
Sazetidine-A was synthesized from a precursor, (s)-5-bromo-3-{[1-
(tert-butoxycarbonyl)-2-azetinyl]-methoxy}pyridine(Musachio et al.,
1999). This precursor was coupled with 5-hexyn-1-ol via palladium-
catalyzed Sonogashira reaction at 80°C, in the presence of P(t-Bu)3,
to afford (s)-5-(5-hexyn-1-ol)-3-{[1-(tert-butoxycarbonyl)-2-azetidi-
nyl]-methoxy}pyridine. The coupling product was then de-protected
with 1 M HCl in diethyl ether to give 6-(5-(((S)-azetidin-2-yl)me-
thoxy)pyridin-3-yl)hex-5-yn-l-ol (sazetidine-A).
(s)-5-(5-Hexyn-1-ol)-3-{[1-(tert-butoxycarbonyl)-2-azetidi-
nyl]-methoxy}pyridine. To a v-vial containing of (s)-5-bromo-3-{[1-
(tert-butoxycarbonyl)-2-azetinyl]-methoxy}pyridine (60 mg, 0.17
mmol) was added a solution of 5-hexyn-1-ol (0.022 ml, 0.2 mmol) in
isopropylamine (1 ml), followed by charging with copper(I) iodide (3.3
mg, 0.017 mmol), (PPh3)2PdCl2 (3.7 mg, 0.0085 mmol), and tri-tert-
butly phosphine (10% in hexane, 22.5 mg, 0.011 mmol). The mixture
was heated at 80°C for 36 h. The solution was then cooled, diluted
with EtOAc (5 ml), filtered through a small pad of silica gel, and
concentrated. The residue was purified by flush chromatography
using ethyl acetate/dichloromethane (2:3) to give 60 mg (81%) of
desired product. 1H NMR (CDCl3, ␦) 1.42 (s, 9H), 1.68–1.78 (m, 4H),
2.22–2.40 (m, 2H), 2.47 (t, J ϭ 6 Hz, 2H), 3.70 (t, J ϭ 5.6 Hz, 2H),
3.88 (t, J ϭ 7.2 Hz, 2H), 4.11 (dd, J1 ϭ 10, J2 ϭ 2.8 Hz, 2H), 4.41 (m,
1H), 7.48 (m, 1H), 7.62 (m, 2H). FAM-HRMS: calculated for
C
20H28N2O4: 360.4473; found: 361.2132 (MϩH)ϩ.
(s)-5-(5-Hexyn-1-ol)-3-(2-azetidinylmethoxy)pyridine. To an
ethanolic solution (0.2 ml) of (s)-5-(5-Hexyn-1-ol)-3-{[1-(tert-butoxycar-
bonyl)-2-azetidinyl]-methoxy}pyridine (20 mg, 56 ␮mol), was added 1
ml of 1 M HCl in anhydrous diethyl ether. The reaction was allowed to
proceed for 12 h before collection of the resulting white precipitate via
suction filtration and washing with ice-cold diethyl ether. Desired prod-
uct (15 mg, 78%) was obtained. 1H NMR (CD3OD, ␦) 1.71 (m, 4H), 2.56
(m, 2H), 2.69(t, J ϭ 8.0 Hz, 2H), 3.60 (t, J ϭ 2.8 Hz, 2H), 4.10 (m, 2H),
4.55 (m, 2H), 4.82 (m, 1H), 8.21 (m, 1H), 8.59 (m, 2H). FAM-HRMS:
calculated for C15H20N2O2: 260.1525; found: 261.1613 (MϩH)ϩ.
desensitized state. Taken together, these data imply that saze- Cell Lines and Cell Culture
tidine-A may represent a new class of nicotinic cholinergic
Tissue culture medium and antibiotics were obtained from In-
drugs with a novel mechanism of action. These drugs may have
important potential for treatment of nicotine addiction, as well
as other conditions involving nAChRs.
vitrogen Corporation (Carlsbad, CA), unless otherwise stated. Fetal
bovine serum and horse serum were provided by Gemini Bio-Prod-
ucts (Woodland, CA). The cell lines KX␣3␤4R2, expressing rat ␣3␤4
nAChRs, and KX␣4␤2, expressing rat ␣4␤2 nAChRs, were estab-
lished previously by stably transfecting human embryonic kidney
293 cells with combinations of rat nAChR subunit genes (Xiao et al.,
1998; Xiao and Kellar, 2004). These cell lines were maintained in
minimum essential medium supplemented with 10% fetal bovine
serum, 100 units/ml penicillin G, 100 mg/ml streptomycin, and 0.7
mg/ml G418 (Geneticin) at 37°C with 5% CO2 in a humidified incu-
bator. The cell line SH-EP1-pcDNA-h␣4␤2, expressing human ␣4␤2
nAChRs, was described previously (Eaton et al., 2003) and was
generously provided by Dr. Ronald J. Lukas (Barrow Neurological
Institute, Phoenix, AZ). The SH-EP1-pcDNA-h␣4␤2 cells were grown
in Dulbecco’s modified Eagle’s medium supplemented with 5% fetal
bovine serum, 10% horse serum, 100 units/ml penicillin G, 100 ␮g/ml
streptomycin, 320 ␮g/ml hygromycin B (Calbiochem, La Jolla, CA),
400 units/ml Zeocin at 37°C with 5% CO2 in a humidified incubator.
Radioligand Binding Assay. Binding of [3H]EB to nAChRs was
measured as described previously (Xiao et al., 1998). In brief, cul-
tured cells at Ͼ80% confluence were removed from their flasks (80
cm2) with a disposable cell scraper and placed in 10 ml of 50 mM
Tris-HCl buffer, pH 7.4, 4°C. The cell suspension was centrifuged at
10,000g for 5 min and the pellet was collected. For cells undergoing
prolonged treatment with nicotinic agonists during culturing, the
Materials and Methods
Materials and Chemicals
[3H](Ϯ)Epibatidine ([3H]EB) and [86Rb]rubidium chloride (86RbCl)
were purchased from PerkinElmer Life and Analytical Sciences (Bos-
ton, MA). All other chemicals were purchased from Sigma Chemical
Fig. 1. Structure of sazetidine-A, 6-(5-(((S)-azetidin-2-yl)methoxy)pyri-
dine-3-yl)hex-5-yn-1-ol.

1456
Xiao et al.
pellet was washed two more times by centrifugation in fresh buffer to lysed by adding 1 ml of 100 mM NaOH to each well, and the lysate
remove residual drug. The cell pellet was then homogenized in 10 ml
was collected for determination of the amount of 86Rbϩ that was in
buffer with a Polytron homogenizer (12-mm aggregate, 26,000 rpm, the cells at the end of the efflux assay. Radioactivity of assay samples
20 s; model PT2100; Kinematica, Basel, Switzerland) and centri- and lysates was measured by liquid scintillation counting. Total
fuged at 36,000g for 10 min at 4°C. The membrane pellet was
loading (cpm) was calculated as the sum of the assay sample and the
resuspended in fresh buffer, and aliquots of the membrane prepara- lysate of each well. The amount of 86Rbϩ efflux was expressed as a
tion equivalent to 30 to 200 ␮g of protein were used for binding percentage of 86Rbϩ loaded. Stimulated 86Rbϩ efflux was defined as
assays. The concentration of [3H]EB used was ϳ100 pM for compe- the difference between efflux in the presence and absence of nicotine.
tition binding assays and ϳ2.4 nM, which is a saturating concentra- For obtaining an IC50 value, inhibition curves were constructed in
tion for the ␣4␤2 receptor subtype, for measuring receptor density. which different concentrations of an antagonist were included in the
Nonspecific binding was assessed in parallel incubations in the pres- assay to inhibit efflux stimulated by 100 ␮M nicotine. IC50 values
ence of 300 ␮M nicotine. Bound and free ligands were separated by were determined by nonlinear least-squares regression analyses
vacuum filtration through Whatman GF/C filters treated with 0.5% (GraphPad Software).
polyethylenimine. The filter-retained radioactivity was measured by
liquid scintillation counting. Specific binding was defined as the
difference between total binding and nonspecific binding. Data from
saturation and competition binding assays were analyzed using
Results
Binding Affinities of Sazetidine-A at nAChR Sub-
types. The structure of sazetidine-A is shown in Fig. 1. All
ligand binding assays were carried out using rat or human
nAChRs heterologously expressed in human cells or in native
rat brain tissues. As shown in Fig. 2 and summarized in
Table 1, sazetidine-A competes with very high affinity for rat
and human ␣4␤2 nAChRs heterologously expressed in cells
or from rat brain; moreover, its binding affinity for the het-
erologously expressed rat ␣4␤2 receptors is 24,000 times
higher than for rat ␣3␤4 receptors. As shown in Table 1, the
selectivity of sazetidine-A for ␣4␤2 receptors over ␣3␤4 re-
ceptors (Ki ratio) was much greater than that of nicotine or
dihydro-␤-erythroidine (DH␤E), both of which are relatively
selective ligands for ␣4␤2 nAChRs in binding assays (Xiao
and Kellar, 2004). The affinity of sazetidine-A for heterolo-
gously expressed rat and human ␣4␤2 subtype is comparable
with its affinity for native nAChRs from rat forebrain, which
expresses predominantly the ␣4␤2 subtype (Whiting and
Lindstrom, 1987; Flores et al., 1992).
Prism 4 (GraphPad Software, San Diego, CA). Additional statistical
analyses are indicated in the figure legends.
86Rb؉ Efflux Assay. Functional properties of compounds at
nAChRs expressed in the transfected cells were measured using
86Rbϩ efflux assays as described previously (Xiao et al., 1998). In
brief, cells were plated into 24-well plates coated with poly-D-lysine.
The plated cells were grown at 37°C for 18 to 24 h to reach 85 to 95%
confluence. The cells were then incubated in growth medium (0.5
ml/well) containing 86Rbϩ (2 ␮Ci/ml) for 4 h at 37°C. The loading
mixture was then aspirated, and the cells were washed four times
with 1 ml of buffer (15 mM HEPES, 140 mM NaCl, 2 mM KCl, 1 mM
MgSO4, 1.8 mM CaCl2, and 11 mM glucose, pH 7.4). One milliliter of
buffer with or without compounds to be tested was then added to
each well. After incubation for 2 min, the assay buffer was collected
for measurements of 86Rbϩ released from the cells. Cells were then
Effects of Sazetidine-A on nAChR Function. The func-
tional effects of sazetidine-A on nAChRs were assessed by
measuring agonist-stimulated 86Rbϩ efflux from stably
transfected cells. Consistent with previous reports, nicotine
stimulates 86Rbϩ release mediated by both ␣4␤2 (Eaton et
al., 2003) and ␣3␤4 (Xiao et al., 1998) nAChR subtypes in a
concentration dependent manner (Fig. 3). In contrast, saze-
tidine-A showed no agonist activity at either nAChR subtype
over a wide concentration range (Fig. 3). This indicates that
sazetidine-A does not activate nAChR channel function, or
that its action is so weak, and/or that the duration of its
action is so transient that no measurable channel function
can be detected by the 86Rbϩ efflux method.
Fig. 2. Competition by sazetidine-A for nAChR binding sites labeled by
[3H]epibatidine. Competition binding assays were carried out in SH-EP1
cells expressing human ␣4␤2 nAChRs (h␣4␤2), or HEK cells expressing
rat ␣4␤2 nAChRs (r␣4␤2) or rat ␣3␤4 nAChRs (r␣3␤4), as described
under Materials and Methods. The concentration of [3H]EB used was 100
pM. Data shown are representative of three to five independent experi-
ments. See Table 1 for a summary of the Ki values for sazetidine-A
binding at these receptors.
The antagonist activity of sazetidine-A was evaluated by
measuring its blockade of nicotine-stimulated 86Rbϩ efflux.
It is interesting that when sazetidine-A was added simulta-
TABLE 1
Comparison of binding affinities of Sazetidine-A, nicotine, and DH␤E at nAChR subtypes
Binding was measured using 100 pM ͓3H͔EB as described under Materials and Methods. Values are the mean Ϯ S.E.M. of three to five independent experiments. The Ki
values of nicotine and DH␤E at rat nAChRs were published previously (Xiao and Kellar, 2004) and are shown here for comparison. Approximately 90% of the nAChRs in
rat forebrain are the ␣4␤2 subtype.
Ki
Selectivity
(Ki ratio: rat ␣3␤4/rat ␣4␤2)
Ligand
␣4␤2 (rat)
␣4␤2 (human)
Forebrain (rat)
␣3␤4 (rat)
nM
Sazetidine-A
(Ϫ)-Nicotine
DH␤E
0.41 Ϯ 0.16
10 Ϯ 2
600 Ϯ 70
0.64 Ϯ 0.32
8.2 Ϯ 0.2
410 Ϯ 30
0.94 Ϯ 0.48
12 Ϯ 2
130 Ϯ 50
10,000 Ϯ 3000
440 Ϯ 60
110,000 Ϯ 10,000
24,000
44
180

A Silent Desensitizer of ␣4␤2 nAChRs
1457
neously with nicotine no antagonist activity in cells express- on dopamine turnover and release in rat brain (Coe et al., 2005),
ing either ␣4␤2 or ␣3␤4 nAChRs was detected (Fig. 4 and it potently and completely inhibited the ␣4␤2 receptor-medi-
Table 2). However, when preincubated with the cells for 10 ated channel response when it was preincubated with the cells
min, sazetidine-A potently inhibited nicotine activation of for 10 min (Table 2). Under these conditions, the inhibitory
the ␣4␤2 receptors with an IC50 value of ϳ30 nM, and it potency of varenicline was comparable with that of sazeti-
nearly completely inhibited activation at ϳ200 nM. In con- dine-A. Nicotine itself also potently inhibited channel function
trast, even with the 10 min preincubation, sazetidine-A was of the ␣4␤2 receptors after the 10-min pretreatment (Table 2);
still ineffective in blocking ␣3␤4 nAChRs (Fig. 4 and Table 2). in fact, in these studies, nicotine was ϳ50 times more potent at
For comparison, we also examined three other nicotinic li- inhibiting ␣4␤2 nAChRs than at activating them (compare Ta-
gands in the same manner. DH␤E, a relatively potent and ble 2 and Fig. 3).
selective competitive antagonist of ␣4␤2 nAChRs, blocked nic-
Recovery of Function after Sazetidine-A. We next ex-
otine activation of the ␣4␤2 subtype with an IC50 value of ϳ1.4 amined recovery of channel function after the ␣4␤2 nAChRs
␮M (Table 2). In contrast to sazetidine-A, however, the potency were pretreated with 1 ␮M sazetidine-A or 10 ␮M nicotine for
of DH␤E was similar with or without the 10 min preincubation. 10 min. When nicotine stimulation was measured immedi-
DH␤E also blocked nicotine activation of the ␣3␤4 subtype but ately after removal of buffer containing sazetidine-A (the no
with ϳ100-fold lower potency than it did the ␣4␤2 subtype recovery group), ϳ10% of channel function was observed
(Table 2). Varenicline, a new drug recently approved for smok- (Fig. 5). In cells pretreated with sazetidine-A and then
ing cessation, like sazetidine-A, has high affinity and selectivity washed and incubated in fresh medium for 1 h, channel
for ␣4␤2 nAChRs (Coe et al., 2005). However, whereas vareni-
cline was found to be a partial agonist at ␣4␤2 nAChRs ex-
pressed in frog oocytes or at receptors mediating nicotine effects
Fig. 4. Comparison of inhibitory effects of sazetidine-A on nAChR chan-
nel function with or without a 10 min pretreatment. The 86Rbϩ efflux
assays in cells expressing human ␣4␤2 and rat ␣3␤4 nAChRs were
Fig. 3. Comparison of agonist activity of sazetidine-A and nicotine. The
agonist activity of sazetidine-A and nicotine at nAChRs was compared by
measuring the stimulation of 86Rbϩ efflux by each compound in cells
expressing human ␣4␤2 and rat ␣3␤4 nAChRs, as described under Ma-
terials and Methods. Data shown are representative of four to seven
independent experiments. The EC50 values (mean Ϯ S.E.M.) for nicotine
activation of channel function were 10 Ϯ 1 ␮M at the human ␣4␤2
receptors and 34 Ϯ 5 ␮M at the rat ␣3␤4 receptors. EC50 values for
sazetidine-A could not be calculated.
carried out as described under Materials and Methods. Cells were either
preincubated with buffer alone and then exposed simultaneously to 100
␮M nicotine and the indicated concentration of sazetidine-A (0 min, no
pretreatment group), or the cells were preincubated for 10 min with the
indicated concentration of sazetidine-A alone and then exposed to 100 ␮M
nicotine plus sazetidine-A (10 min, pretreatment group). Concentration-
effect curves are representative of three to seven independent experi-
ments. See Table 2 for a summary of IC50 values for sazetidine-A from all
experiments.

1458
Xiao et al.
function recovered to ϳ35%; after incubation in fresh me- 1983; Schwartz and Kellar, 1983; Flores et al., 1992). This
dium for 20 h, recovery of function was nearly complete (Fig. effect is also seen in cells in culture and with other nicotinic
5). For comparison, after a 10-min pretreatment with nico- agonists (Peng et al., 1994; Gopalakrishnan et al., 1997). To
tine, channel function was ϳ15% of control when no recovery determine whether sazetidine-A can up-regulate ␣4␤2
period was included, but it was nearly fully recovered within nAChRs, cells were grown in the presence of 1 ␮M sazeti-
1 h after incubation in fresh medium (Fig. 5).
dine-A for 5 days before the binding sites in membrane ho-
Up-Regulation of ␣4␤2 nAChR Binding Sites by Saze- mogenates were measured with a saturating concentration of
tidine-A. Long-term administration of nicotine increases [3H]EB, which closely approximates the binding site density.
␣4␤2 nAChR density in mouse and rat brain (Marks et al., As shown in Fig. 6, the binding site density was ϳ2 times
higher in membrane homogenates from cells exposed to saze-
tidine-A than in control cells. As a comparison, in cells grown
in the presence of 10 ␮M nicotine, binding site density was
increased ϳ5-fold (Fig. 6). The concentrations of sazetidine-A
and nicotine that were used in these experiments are ϳ1500
and ϳ1200 times their respective Ki values.
TABLE 2
Inhibition of nAChR function by sazetidine-A, DH␤E, varenicline, and
nicotine
nAChR function in cells expressing human ␣4␤2 nAChRs and rat ␣3␤4 nAChR was
measured in the nicotine-stimulated 86Rbϩ efflux assays, as described under Mate-
rials and Methods. The IC50 values were derived from inhibition curves as shown in
Fig. 4 and are the mean Ϯ S.E.M. of three to seven independent experiments.
IC50
Discussion
Compound
␣4␤2 nAChRs
␣3␤4 nAChRs
The present study demonstrates that the main pharmaco-
logical action of sazetidine-A at ␣4␤2 nAChRs is different
from that of other known nicotinic ligands; in fact, it may
represent a new type of drug action at these receptors. Two
lines of evidence lead to this conclusion. First, sazetidine-A
has very high affinity (Ki value less than 1 nM) for ␣4␤2
nAChRs in equilibrium binding assays, in which it presum-
ably interacts with the desensitized form of the receptors
(Fig. 2, Table 1); despite its high affinity for the desensitized
receptor, however, sazetidine-A does not activate the channel
function of the receptors in their resting state when it is
applied alone (Fig. 3), and it does not potentiate or block
channel function when it is applied simultaneously with nic-
otine (Fig. 4). Thus, it is neither an agonist nor a classic
competitive antagonist. Second, it does, however, potently
block ␣4␤2 nAChR function when it is preincubated for 10
min with the cells expressing these receptors; in fact, under
these conditions, sazetidine-A is one of the most potent in-
hibitors of ␣4␤2 nAChR function ever reported, 50 times
more potent than DH␤E (Fig. 4, Table 2).
nM
Sazetidine-A
0-min
Pretreatment
10-min
Pretreatment
DH␤E
0-min
Pretreatment
10-min
Pretreatment
Varenicline
0-min
Pretreatment
10-min
Pretreatment
(Ϫ)-Nicotine
0-min
Ͼ10,000
26 Ϯ 7
Ͼ10,000
Ͼ10,000
1400 Ϯ 200
1400 Ϯ 300
160,000 Ϯ 20,000
130,000 Ϯ 58,000
Ͼ5,000
50 Ϯ 14
Not Tested
Not Tested
a
—
—
Pretreatment
10-min
Pretreatment
180 Ϯ 70
Not Tested
a Dashes indicate that nicotine is a full agonist in those conditions.
We term this inhibitory action “silent desensitization”—
i.e., desensitization of the receptors without first activating
them. The explanation for this action may derive from the
Fig. 5. Recovery of ␣4␤2 nAChR channel function after desensitization by
a 10-min treatment with sazetidine-A or nicotine. The 86Rbϩ efflux assays
in cells expressing human ␣4␤2 nAChRs were carried out as described
under Materials and Methods. Cells were incubated with buffer contain-
ing 1 ␮M sazetidine-A or 10 ␮M nicotine for 10 min. The buffer containing
the drug was then removed, and 86Rbϩ efflux stimulated by 100 ␮M
nicotine was either measured immediately (no recovery group), or the
cells were washed thoroughly and incubated in fresh medium for the
recovery period indicated (1- or 20-h recovery groups) before their nAChR
channel function was measured. Data are the mean Ϯ S.E.M. of three to
five independent experiments. Nicotine-stimulated 86Rbϩ efflux in both
recovery groups (1- and 20 h) are significantly higher than in their
corresponding no recovery groups (analysis of variance, Dunnett’s mul-
tiple comparison test; ء, P Ͻ 0.05).
Fig. 6. Increase in ␣4␤2 nAChR binding sites induced by Sazetidine-A
and nicotine. Cells expressing human ␣4␤2 nAChRs were grown in the
presence of 1 ␮M sazetidine-A or 10 ␮M nicotine for 5 days before
membrane homogenates were prepared and nAChR binding sites were
measured with 2.4 nM [3H]EB, a concentration that saturates these
receptors and thus reflects the binding site density. Values are the
mean Ϯ S.E.M. of three independent experiments. Binding values in both
treated groups are significantly higher than in the control group (analysis
of variance, Dunnett’s multiple comparison test; ء, P Ͻ 0.01).

A Silent Desensitizer of ␣4␤2 nAChRs
1459
fact that nAChRs can exist in a resting state and two or more suggest that the ␣4␤2 nAChR is the major brain nAChR
desensitized states, which represent distinct but intercon- subtype that mediates nicotine addiction (Flores et al., 1992;
vertible conformations of the receptor (Katz and Thesleff, Picciotto et al., 1998; Marubio et al., 2003; Tapper et al.,
1957; Karlin, 1967; Changeux and Edelstein, 1998). The ag- 2004; Maskos et al., 2005). In addition, this receptor may also
onist-induced desensitization of nAChRs has been studied mediate some of the cognitive enhancing effects of nicotine
extensively (Grady et al., 1994; Marks et al., 1994, 1996; (Picciotto et al., 1995; Maskos et al., 2005).
Fenster et al., 1997). We propose that sazetidine-A has very
The actions of nicotine to both stimulate and block re-
low affinity for the resting state of the ␣4␤2 nAChRs, but sponses of autonomic ganglia were demonstrated more than
very high affinity for one or more of the desensitized states of 100 years ago (Langley and Dickinson, 1889); and the concept
the receptor. Thus, when added to cells with nAChRs in the of desensitization of nAChRs in muscle was proposed almost
resting state, sazetidine-A, because of its low affinity, would 50 years ago (Katz and Thesleff, 1957). Thus, considering
act neither as an agonist to open the channel nor as an nicotine’s dual actions to activate and then desensitize
antagonist to block nicotine’s effects. But preincubation nAChRs, an important question is how each of these two
would allow sazetidine-A to bind to receptors that spontane- essentially diametrically opposed actions contributes to the
ously convert to a desensitized state, and because of its high pharmacological effects of nicotine in the CNS. Most investi-
affinity for those receptors, sazetidine-A would maintain gations of nicotine’s pharmacological actions have focused on
them in that high-affinity, desensitized state. Over the 10- its activity as an agonist and the stimulatory effects it pro-
min preincubation time period used here, as more receptors duces; consequently, there is a rich literature on the agonist
are trapped in the desensitized state, channel function would actions of nicotine in both ganglia and the CNS. However, in
be progressively inhibited. This action would thus allow saze- almost all studies of nicotine and nAChR function, the de-
tidine-A to bind to these receptors in their desensitized state sensitizing actions of nicotine are recognized (Quick and
without first activating them. Sazetidine-A is therefore dif- Lester, 2002; Giniatullin et al., 2005). In rats, for example, a
ferent from nicotine and other nicotinic agonists, which can single injection of nicotine initially stimulates prolactin re-
desensitize nAChRs but only after first activating them to lease, but it then blocks subsequent nicotine-stimulated pro-
some measurable extent (Hulihan-Giblin et al., 1990; Marks lactin release for several hours or longer (Sharp and Beyer,
et al., 1994; Fenster et al., 1997). We propose that sazeti- 1986; Hulihan-Giblin et al., 1990). To explain this observa-
dine-A and other nicotinic ligands with similar properties be tion, nicotine was proposed to act as a “time-averaged antag-
classified as “silent desensitizers” of nAChRs.
onist” of nAChRs (Hulihan-Giblin et al., 1990), meaning that
Compared with nicotine, sazetidine-A has at least 10 times after a brief initial stimulation of brain nAChRs, it causes a
higher binding affinity for ␣4␤2 nAChRs and is ϳ500 times long-lasting desensitization of the receptors that prevents
more selective for the ␣4␤2 subtype over the ␣3␤4 subtype their function for an extended period of several hours or
(Table 1). Sazetidine-A doesn’t activate nAChRs, but it is ϳ6 longer. Our present results suggest that the primary func-
times more potent than nicotine in inhibiting ␣4␤2 nAChR tional effect of sazetidine-A is to desensitize ␣4␤2 nAChRs
function after a 10-min pretreatment (Table 2), presumably without any detectable activation of the receptor population.
by the desensitizing action described above. The desensitiz-
nAChRs are allosteric proteins, and their behavior at the
ing effects of both sazetidine-A and nicotine are reversible. molecular level is complex (Monod et al., 1965; Karlin, 1967;
Thus, after removing sazetidine-A, receptor function recov- Changeux and Edelstein, 1998; Karlin, 2002). The action of
ered ϳ35% in 1 h and nearly 100% in 20 h, whereas in the sazetidine-A might be explained by an allosteric model of
case of nicotine, function recovered fully in 1 h (Fig. 5). ligand-gated channel function of nAChRs. In fact, many of
Long-term exposure to nicotine increases ␣4␤2 nAChRs the important pharmacological effects of nicotine on the CNS
(Flores et al., 1992; Peng et al., 1994; Gopalakrishnan et al., might be due to its ability to desensitize the ␣4␤2 nAChR.
1997). Likewise, incubation of cells with sazetidine-A for 5 Thus, as long as its concentration is high enough to occupy
days significantly increased ␣4␤2 nAChR density (Fig. 6). the desensitized receptors, it acts essentially as an antago-
Although the increase was less than that seen after incuba- nist. After a 10-min pretreatment, nicotine potently inhibited
tion with nicotine, the difference could have been due to the ␣4␤2 receptor function with an IC50 value 180 nM (Table 2).
lower concentration of sazetidine-A used. The concentrations Similar inhibitory effects of nicotine and other agonists were
used here for both sazetidine-A and nicotine were ϳ1200 to also observed with a 3-min pretreatment of both human and
1500 times their affinity (Ki) at the agonist binding site, but rat ␣4␤2 nAChRs (Fitch et al., 2003). Moreover, a recent
other factors related to the mechanisms underlying up-reg- study found that after a 6-h pretreatment, nicotine blocked
ulation, such as penetration into the cell, may be involved. ␣4␤2 receptor function with an IC50 of 6 nM (Kuryatov et al.,
Therefore, of the three actions of nicotine at ␣4␤2 nAChRs— 2005). The nicotine concentration in arterial blood typically
activation of the receptor channel, desensitization of the re- exceeds 200 nM during cigarette smoking and is maintained
ceptor and up-regulation of the receptor density—sazeti- above 100 nM for more than 10 min after smoking (Henning-
dine-A displays only two: desensitization and up-regulation. field et al., 1993; Gourlay and Benowitz, 1997). The concen-
This property may make sazetidine-A a very useful research tration of nicotine in brain is thought to be even higher
tool with which to study the relationships between receptor (Ghosheh et al., 2001). A recent PET imaging study in human
activation, desensitization, and up-regulation.
smokers found that after subjects smoked one cigarette, more
Nicotine, the classic agonist that defines nAChRs, is prob- than 80% of their brain ␣4␤2 nAChRs appeared to be occu-
ably the most widely used addictive drug. Moreover, with pied by nicotine for several hours (Brody et al., 2006).
more than a billion cigarette smokers worldwide, smoking is
Taken together, these findings suggest that nicotine acts
estimated to be responsible for ϳ5 million premature deaths primarily as an antagonist in vivo as long as its concentration is
each year (Ezzati and Lopez, 2003). Several lines of evidence high enough to maintain receptor desensitization. Further-

1460
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Acknowledgments
We thank Dr. Ronald J. Lukas of Barrow Neurological Institute
(Phoenix, AZ) for generously providing cell line SH-EP1-pcDNA-h␣4␤2.
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Address correspondence to: Kenneth J. Kellar, Department of Pharmacol-
ogy, Georgetown University School of Medicine, Washington, DC 20057. E-
mail: kellark@georgetown.edu