Biochimica et Biophysica Acta 1848 (2015) 731–741
Contents lists available at ScienceDirect
Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbamem
Pharmacological and molecular studies on the interaction of varenicline
with different nicotinic acetylcholine receptor subtypes. Potential
mechanism underlying partial agonism at human α4β2 and
α3β4 subtypes
Hugo R. Arias a,⁎, Dominik Feuerbach b, Katarzyna Targowska-Duda c, Agnieszka A. Kaczor d,
Antti Poso e, Krzysztof Jozwiak c
a
Department of Medical Education, California Northstate University College of Medicine, Elk Grove, CA, USA
Neuroscience Research, Novartis Institutes for Biomedical Research, Basel, Switzerland
Department of Chemistry, Laboratory of Medicinal Chemistry and Neuroengineering, Medical University of Lublin, Lublin, Poland
d
Department of Synthesis and Chemical Technology of Pharmaceutical Substances with Computer Modeling Lab, Medical University of Lublin, Lublin, Poland
e
School of Pharmacy, University of Eastern Finland, Kuopio, Finland
b
c
a r t i c l e
i n f o
Article history:
Received 12 August 2014
Received in revised form 29 October 2014
Accepted 6 November 2014
Available online 2 December 2014
Keywords:
Varenicline
Nicotinic acetylcholine receptors
Partial agonist
Full agonist
Molecular modeling
a b s t r a c t
To determine the structural components underlying differences in affinity, potency, and selectivity of varenicline
for several human (h) nicotinic acetylcholine receptors (nAChRs), functional and structural experiments were
performed. The Ca2+ influx results established that: (a) varenicline activates (μM range) nAChR subtypes with
the following rank sequence: hα7 N hα4β4 N hα4β2 N hα3β4 >>> hα1β1γδ; (b) varenicline binds to nAChR
subtypes with the following affinity order (nM range): hα4β2 ~ hα4β4 N hα3β4 N hα7 >>> Torpedo α1β1γδ.
The molecular docking results indicating that more hydrogen bond interactions are apparent for α4containing nAChRs in comparison to other nAChRs may explain the observed higher affinity; and that
(c) varenicline is a full agonist at hα7 (101%) and hα4β4 (93%), and a partial agonist at hα4β2 (20%) and
hα3β4 (45%), relative to (±)-epibatidine. The allosteric sites found at the extracellular domain (EXD) of
hα3β4 and hα4β2 nAChRs could explain the partial agonistic activity of varenicline on these nAChR subtypes.
Molecular dynamics simulations show that the interaction of varenicline to each allosteric site decreases the
capping of Loop C at the hα4β2 nAChR, suggesting that these allosteric interactions limit the initial step in the
gating process. In conclusion, we propose that in addition to hα4β2 nAChRs, hα4β4 nAChRs can be considered
as potential targets for the clinical activity of varenicline, and that the allosteric interactions at the hα3β4- and
hα4β2-EXDs are alternative mechanisms underlying partial agonism at these nAChRs.
© 2014 Elsevier B.V. All rights reserved.
1. Introduction
Abbreviations: nAChR, nicotinic acetylcholine receptor; [3H]MLA, [3H]methyllycaconitine;
[ H]TCP, piperidyl-3, 4-3H(N)]-(N-(1-(2 thienyl)cyclohexyl)-3, 4-piperidine; α-BTx, αbungarotoxin; Varenicline, 7,8,9,10-tetrahydro-6,10-methano-6H-azepino[4,5-g]quinoxaline;
RT, room temperature; BS, binding saline; A, allosteric; EXD, extracellular domain, ORT,
orthosteric; A, allosteric; TMD, transmembrane domain, Ki, inhibition constant; Kd, dissociation constant; IC50, ligand concentration that produces 50% inhibition (of binding or of
agonist activation); EC50, agonist concentration that produces 50% nAChR activation; nH,
Hill coefficient; MD, molecular dynamics; NVT, constant number of particles, volume,
and temperature; NPT, constant number of particles, pressure, and temperature; RMSD,
root mean square deviation; DMEM, Dulbecco's Modified Eagle Medium; FBS, fetal bovine
serum; FLIPR, fluorescent imaging plate reader
⁎ Corresponding author at: Department of Medical Education, California Northstate
University College of Medicine, 9700 W. Taron Dr., Elk Grove, CA 95757, USA. Tel.: +1
916 686 7304; fax: +1 916 686 7310.
E-mail address: hugo.arias@cnsu.edu (H.R. Arias).
3
http://dx.doi.org/10.1016/j.bbamem.2014.11.003
0005-2736/© 2014 Elsevier B.V. All rights reserved.
The addictive properties of nicotine (the active alkaloid involved in
smoking addiction) as well as the activity of varenicline (Chantix®,
Champix®) for smoking cessation therapy [1] are primarily mediated
by their interactions with nicotinic acetylcholine receptors (nAChRs).
The interaction of nicotine with α4β2 nAChRs in the mesocorticolimbic
system, the so-called “brain reward system”, increases the synaptic
levels of dopamine, which in turn produces the pleasurable effects
mediated by nicotine [2–4]. There is a large amount of experimental
evidence supporting an important role of α4β2 nAChRs in the mechanism of nicotine addiction. For example, animal studies show that agonists specific for α4β2 nAChRs produce similar discriminative stimulus
as nicotine [5], and knockout animal results indicates that the β2 subunit is necessary for the reinforcing [6] and discriminative [7] properties
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H.R. Arias et al. / Biochimica et Biophysica Acta 1848 (2015) 731–741
of nicotine and for nicotine-induced dopamine release [8]. Nevertheless,
other nAChR subtypes (i.e., α7, α3β4, and α6β2-containing nAChRs)
are also involved in the mechanism of nicotine addiction [2,3].
Pharmacologically, varenicline behaves as a partial agonist of α4β2
nAChRs and a full agonist of α7 nAChRs [9–12]. Through its intrinsic
partial activation of α4β2 nAChRs, varenicline elicits a moderate and
sustained increase of dopamine levels in the brain reward system,
which would elevate low dopamine levels observed during smoking
cessation attempts [4,11–14]. In addition, varenicline competitively inhibits nicotine binding to α4β2 nAChRs, preventing nicotine-induced
dopaminergic activation. This dual effect ultimately decreases craving,
withdrawal symptoms, smoking satisfaction and reward. Studies using
β2-subunit knockout animals and animals where the β2 subunit has
been re-expressed indicate that β2-containing nAChRs are involved in
the dopaminergic effects mediated by varenicline [15]. Although there
is a good idea of how varenicline acts clinically, we still do not have a
complete understanding of the structural and functional aspects underlying its receptor selectivity, specifically regarding the α4β4 nAChR.
α4 and β4 subunits, potentially forming α4β4-containing nAChRs, are
also expressed in several brain regions implicated in drug addiction, including basal ganglia, cerebellum, midbrain, ventral tegmental area,
hippocampus, and cortex [16,17]. To have a more comprehensive idea
of the interaction of varenicline with different nAChR subtypes, we decided to determine which structural components are important for the
different binding affinities, agonistic and antagonistic potencies, and receptor selectivity of varenicline for several nAChR subtypes including,
the human (h) α4β2, hα4β4, hα3β4, hα7, Torpedo and hα1β1γδ
nAChRs. In this study we applied structural and functional approaches
including radioligand binding assays, Ca2+ influx-induced fluorescence
detections, as well as homology modeling, molecular docking, and molecular dynamics studies.
2. Materials and methods
2.1. Materials
[3H]Epibatidine (45.1 Ci/mmol), [3H]cytisine (34.1 Ci/mmol),
[piperidyl-3,4-3H(N)]-(N-(1-(2 thienyl)cyclohexyl)-3,4-piperidine)
([3H]TCP; 45.0 Ci/mmol), and [3H]imipramine (47.5 Ci/mmol) were obtained from PerkinElmer Life Sciences Products, Inc. (Boston, MA, USA).
[3H]Methyllycaconitine (100 Ci/mmol) was purchased from American
Radiolabeled Chemicals Inc. (Saint Louis, MO, USA). The radioligands
were stored at − 20 °C. Methyllycaconitine citrate, carbamylcholine
dihydrochloride, imipramine hydrochloride, and polyethylenimine
were purchased from Sigma Chemical Co. (St. Louis, MO, USA). (±)Epibatidine hydrochloride was obtained from Tocris Bioscience
(Ellisville, Missouri, USA). Fetal bovine serum (FBS) and trypsin/EDTA
were purchased form Gibco BRL (Paisley, UK). Ham's F-12 Nutrient Mixture was obtained from Invitrogen (Paisley, UK). Varenicline hydrochloride and phencyclidine hydrochloride (PCP) were obtained through the
National Institute on Drug Abuse (NIDA) (NIH, Baltimore, USA). Salts
were of analytical grade.
2.2. Ca2+ influx measurements in cells containing different nAChR subtype
Ca2+ influx measurements were performed in GH3-hα7, HEK293hα4β2, HEK293-hα3β4, and TE671-hα1β1γδ cells incubated at 37 °C
as previously described [17–19]. In the particular case of CHO-hα4β4
cells, a density of 5 × 104 per well was used. Under these conditions,
the majority of expressed nAChRs has the (αx)3(βx)2 stoichiometry
(see [18] and references therein). To determine the agonistic activity,
varenicline or (±)-epibatidine was added to the cell plate using the
96-tip pipettor simultaneously to fluorescence recordings for a total
length of 3 min. To determine the antagonistic activity, cells were
pretreated (5 min) with different concentrations of varenicline before
testing the activity of (±)-epibatidine (0.1 μM for neuronal nAChRs,
and 1 μM for hα1β1γδ nAChRs).
2.3. Radioligand competition binding experiments
To determine receptor selectivity, the effect of varenicline on [3H]
MLA (4.1 nM) binding to hα7 nAChRs, on [3H]epibatidine (4.6 nM)
binding to hα3β4 nAChRs, and on [3H]cytisine (9.1 nM) binding to
hα4β2, hα4β4, and Torpedo nAChRs, respectively, was studied as previously described [19–22]. To determine whether varenicline interacts
with the Torpedo and hα4β2 nAChR ion channels, additional studies
were conducted using [3H]TCP (20 nM) [21] and [3H]imipramine
(13 nM) [23]. The effect of varenicline on [3H]cytisine, in the absence
(nAChRs are in the resting but activatable state) and presence of
200 μM proadifen [24], and [3H]TCP binding, in the presence of 1 mM
CCh (nAChRs are mainly in the desensitized state), was also determined
as previously described [21,23]. Nonspecific binding was determined
in the presence of 10 μM MLA ([3H]MLA experiments), 1 mM CCh
([3H]cytisine experiments), 0.2 μM (±)-epibatidine ([3H]epibatidine
experiments), 100 μM PCP ([3H]TCP experiments), or 100 μM imipramine ([3H]impramine experiments).
After incubation (2 h), nAChR-bound radioligand was separated
from free radioligand by a filtration assay [19–23]. The concentration–
response data were curve-fitted by nonlinear least squares analysis
using the Prism software (GraphPad Software, San Diego, CA). The observed IC50 values were transformed into inhibition constant (Ki) values
using the Cheng–Prusoff relationship [25]:
Ki ¼ IC50
=
i
n
hh i
o
3
ligand
1þ
H ligand Kd
=
ð1Þ
where [[3H]ligand] is the initial concentration of [3H]MLA, [3H]cytisine,
is the dissociation constant for [3H]MLA
or [3H]epibatidine, and Kligand
d
(1.86 nM for the hα7 nAChR [26]), [3H]cytisine (0.1 nM for the hα4β4
nAChR [27], 0.3 nM for the hα4β2 nAChR [28] and 0.45 μM for
the desensitized Torpedo nAChR [22]), and [3H]epibatidine (89 pM for
the hα3β4 AChR [29]). The calculated Ki values were summarized in
Table 2.
2.4. Homology models of the hα3β4, hα4β4, hα4β2, and hα7 nAChRs
The crystal structure of the acetylcholine binding protein (AChBP)
(PDB 4AFT) [30] was used as a template for the extracellular domain
of the human (h)α3β4, hα4β4, hα4β2, and hα7 nAChRs, whereas the
Torpedo nAChR model (PDB 2BG9) [31] was used as a template for
the transmembrane domains (TMD). Water molecules were added to
the model according to the AChBP-cytisine structure (PDB 4AFO) [30].
The amino acid sequence of each nAChR subunit (i.e., hα7, hα3, hα4,
hβ2, and hβ4) was first aligned with corresponding sequences of the
AChBP and Torpedo nAChR subunits by using the ClustalW2 server
(www.ebi.ac.uk/Tools/msa/clustalw2) [32]. A hundred homology
models for each nAChR subtype were generated using Modeller v.9.9
[33], and subsequently assessed by Modeller objective function and
Discrete Optimized Protein Energy profiles [34]. The best model of
each nAChR subtype was subjected to quality assessments using the
Molecular Environment module for Ramachandran plots (http://www.
chemcomp.com/software.htm) and the web-based tools of Annolea
[35], Verify3D [36], and ProCheck [37].
2.5. Molecular docking
Docking simulations were performed using the same protocol as
reported previously [19]. In addition, water molecules were incorporated within the binding pockets. The crystal structure of varenicline,
transferred from its crystal model with AChBP, was used for the subsequent step of molecular docking. Molegro Virtual Docker (MVD v 5.0.0,
H.R. Arias et al. / Biochimica et Biophysica Acta 1848 (2015) 731–741
733
Molegro ApS Aarhus, Denmark) was used for docking simulations of
flexible ligands (i.e., varenicline in the neutral and protonated states)
into the rigid nAChR target. The docking space was extended to ensure
covering two subunits (i.e., α3/β4, α4/β4, α4/β2, α7/α7, α1/δ, and
α1/γ) containing the orthosteric (ORT) site, as well as the extracellular
domain (EXD) from each nAChR model for additional allosteric (A) sites.
The actual docking simulations were performed using the settings
described previously [19,23,38]. The lower energy conformations were
selected from each cluster of superposed poses.
To determine how the binding of varenicline to each allosteric site
found at the hα4β2 nAChR modifies the interaction of the ligand at
the ORT site, only the EXD was studied. The MD procedure was similar
to that described before, although 25-ns MD in NPT ensemble with
protein backbone restrains and additional 25-ns MD without restraints
was used instead. Four models were simulated, when varenicline is
bound to: (1) two ORT sites only (control), (2) two ORT and A1 allosteric sites, (3) two ORT and A2 allosteric sites, and to (4) two ORT and A3
allosteric sites.
2.6. Molecular dynamics simulations
3. Results
To investigate the stability of varenicline at the suggested docking
sites, molecular dynamics (MD) simulations were performed by using
Desmond v. 3.0.3.1 [39] and OPLS-2005 force field. Each nAChRvarenicline model was inserted into 1-palmitoyl-2-oleoyl phosphatidylcholine membranes, solvated with water as described previously [40]
and ions were added to neutralize the protein charges as described elsewhere [41]. Each nAChR model was first minimized and then subjected
to 1-ns MD in NVT (constant number of particles, volume, and temperature) ensemble, followed by 15-ns MD in NPT (constant number of
particles, pressure, and temperature) ensemble. Although fixing constrains for backbone atoms were assigned, all side-chain atoms were
left free to move during the simulations. The total potential energy
of each docked model was calculated using the OPLS-2005 force field
according to Bowers et al. [39].
3.1. Pharmacologic activity of varenicline assessed by Ca2+ influx in cells
expressing different nAChR subtypes
The agonistic potency of varenicline was first determined by
assessing the fluorescence change on each nAChR-expressing cell line
after its direct stimulation (Fig. 1). The observed receptor selectivity
follows the order (EC50s in μM): hα7 (0.18 ± 0.02) N hα4β4 (0.37 ±
0.08) N hα4β2 (1.30 ± 0.18) N hα3β4 (6.4 ± 1.2) ≫ N hα1β1γδ
(N100) (Table 1). Based on the observed efficacy (Emax), varenicline is
a full agonist of the hα7 (101 ± 8%) and hα4β4 (93 ± 7%) nAChRs,
and a partial agonist of the hα4β2 (20 ± 6%) and hα3β4 (45 ± 10%)
nAChRs. In general, our results support previous data [10–12,42]. However, this is the first report showing that varenicline behaves as a full
agonist of hα4β4 nAChRs. The competitive properties of varenicline
Fig. 1. Functional activity of varenicline on (A) HEK293-hα4β2, (B) CHO-hα4β4, (C) HEK293-hα3β4, (D) GH3-hα7, and (E) TE671-hα1β1γδ cells using Ca2+ influx measurements.
Increased concentrations of (±)-epibatidine (■) or varenicline (●) enhance intracellular calcium. The competitive effect of varenicline was investigated by pretreating (5 min) the
cells with different concentrations of varenicline followed by activation of the receptor with (±)-epibatidine (□). The plots for the agonistic action of varenicline are the combination
of 6 (A and D), 4 (B), and 3 (C and E) experiments, respectively. The plots for the antagonistic action of varenicline are the combination of 4 (A), 6 (B), 5 (C), 8 (D), and 3
(E) experiments, respectively. The error bars represent the standard deviation (S.D.). Ligand response was normalized to the maximal (±)-epibatidine response, which was set as
100%. The calculated Emax, EC50, IC50, and nH values are summarized in Table 1.
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H.R. Arias et al. / Biochimica et Biophysica Acta 1848 (2015) 731–741
Table 1
Stimulation and inhibition elicited by varenicline at different nAChR subtypes assessed by
Ca2+ influx assays.
nAChR
subtype
EC50
(μM)
Emax
nH a
hα4β2
hα4β4
hα3β4
hα7
hα1β1γδ
1.30 ± 0.18
0.37 ± 0.08
6.4 ± 1.2
0.18 ± 0.02
N100
20 ± 6
93 ± 7
45 ± 10
101 ± 8
–
1.51
1.15
1.80
4.04
–
±
±
±
±
0.21
0.22
0.32
0.97
IC50
(nM)
nHa
2.8 ± 1.2
26.7 ± 2.4
2336 ± 762
69.0 ± 8.1
N100,000
1.87
0.93
1.50
3.85
–
±
±
±
±
0.46
0.06
0.21
0.59
These values were obtained from Fig. 1A–E, respectively.
a
Hill coefficient.
were also investigated by pre-incubating the nAChR with varenicline
before the (±)-epibatidine-induced nAChR activation (Fig. 1). In this regard, the inhibitory (i.e., desensitizing) potency follows the order (IC50s
in nM): hα4β2 (2.8 ± 1.2) N hα4β4 (26.7 ± 2.4) N hα7 (69.0 ± 8.1) N
hα3β4 (2336 ± 762) ≫ N hα1β1γδ (N 100,000).
The observed nH values are higher than unity, except that for hα4β4
nAChRs (Table 1). This suggests that varenicline activates the nAChRs
and antagonizes the agonist-activated nAChRs in a cooperative manner,
supporting the existence of more than one binding site on each nAChR,
except on hα4β4 nAChRs. This observation is more apparent in the hα7
nAChR, coinciding with the experimental results showing that at least
three agonist sites need to be occupied to activate this ion channel
[43]. nH values higher than unity were also found on the hα4β2
nAChR by voltage-clamp measurements [12].
containing nAChRs. Previous studies support our binding affinity results
[9,11,12,44]. In a simplistic way, the calculated nH values suggest that
varenicline inhibits radioligand binding in a non-cooperative manner
(Table 2). In addition, varenicline enhances [3H]cytisine binding to
Torpedo nAChRs in the resting but activatable state (i.e., in the absence
of proadifen) (Fig. 2D), whereas in the desensitized state (i.e., in the
presence of proadifen) it binds to the nAChR agonist sites with very
low affinity (Table 2).
To further determine whether varenicline binds to the nAChR channel or not, additional [3H]TCP competition experiments were performed
(Fig. 3A). The results indicate that varenicline enhances [3H]TCP binding
when the nAChR is in the resting but activatable state (in the absence of
CCh), with the same apparent EC50 value as that for [3H]cytisine binding
enhancement (Table 2). This result suggests that varenicline does not
bind to the TCP site when the Torpedo nAChR is in the resting (i.e., in
the absence of CCh) or desensitized (i.e., in the presence of CCh) state
(Fig. 3A). A simple explanation for the result in the resting state is that
when varenicline binds to its orthosteric sites at the Torpedo nAChR,
the receptor becomes desensitized, and thus [3H]TCP binding to the
ion channel is enhanced. The [3H]TCP results were corroborated by additional [3H]imipramine competition experiments in the hα4β2 nAChR
(Fig. 3B), where an apparent IC50 value of ~12 mM (nH b 0.5) was estimated from these experiments (Table 2). These data support the idea
that varenicline inhibits [3H]imipramine binding with extremely low
affinity. However, we cannot rule out the possibility that varenicline
binds to a luminal site located apart from the TCP/imipramine locus.
3.3. Molecular docking of varenicline with different nAChR subtypes
3.2. Radioligand binding experiments
The binding affinity of varenicline for each nAChR subtype was determined by radioligand binding (Fig. 2A–C). The calculated Ki values
demonstrate that varenicline binds to the hα4β4 (121 ± 4 pM) and
hα4β2 (90 ± 7 pM) nAChRs with practically the same affinity
(Table 2). Moreover, varenicline binds to the hα7 (100 ± 7 nM),
hα3β4 (13 ± 1 nM), and desensitized Torpedo (116 ± 8 μM) nAChRs
with ~ 100–1,000,000-fold lower affinity compared to that for α4-
Varenicline, in the neutral and protonated state, was docked to the
EXD from each nAChR subtype (i.e., hα3β4, hα4β4, hα4β2, and hα7).
Since varenicline is highly protonated (~ 100%) at physiological pH
[pKa values of 9.8 ± 0.2 calculated by the ACD/ADME Suite software
(Advanced Chemistry Development, Inc., Toronto, Canada)] the different modes of binding are shown only for the protonated state. The
docking results indicate that varenicline interacts with ORT and allosteric (A) binding sites (Table 3).
Fig. 2. Varenicline-induced inhibition of (A) [3H]cytisine binding to hα4β4 (■) and hα4β2 (□) nAChRs, (B) [3H]MLA binding to hα7 nAChRs, (C) [3H]epibatidine binding to
hα3β4 nAChRs, and of (D) [3H]cytisine binding to Torpedo nAChRs in the resting but activatable (●) and desensitized (○) states, respectively. nAChR membranes (1–2 mg/mL) were
pre-incubated (30 min) with 9.1 nM [3H]cytisine, 4.6 nM [3H]epibatidine, or 4 nM [3H]MLA, respectively, and then equilibrated (2 h) with increasing concentrations of varenicline.
To maintain the Torpedo nAChR in the desensitized state, Torpedo nAChR membranes were pre-incubated with 200 proadifen (○). Nonspecific binding was determined at 1 mM CCh
(A and D), 10 μM MLA (B), and 0.2 μM (±)-epibatidine (C), respectively. Each plot is the combination of 2–3 separated experiments each one performed in triplicate, where the error
bars correspond to the S.D. The observed IC50 values were used to calculate the Ki values according to Eq. (1). The apparent EC50, Ki, and nH values were summarized in Table 2.
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H.R. Arias et al. / Biochimica et Biophysica Acta 1848 (2015) 731–741
Table 2
Binding affinity of varenicline for different binding sites at various nAChR subtypes.
nAChR subtype
Radioligand
Ki a
nH b
hα4β2
[3H]Cytisine
[3H]Imipramine f
[3H]Cytisine
[3H]Epibatidine
[3H]MLA
[3H]Cytisine
[3H]TCP
90 ± 7 pM
~12 mM
121 ± 4 pM
13 ± 1 nM
77 ± 6 nM
116 ± 8 μM d
No binding e
1.00
0.46
1.11
1.26
1.15
1.21
–
hα4β4
hα3β4
hα7
Torpedo
±
±
±
±
±
±
0.07
0.15
0.03
0.10
0.10
0.14
Apparent EC50c
nHb
–
–
–
–
–
1.1 ± 0.2 μM
1.4 ± 0.2 μM
–
–
–
–
–
1.36 ± 0.33
1.10 ± 0.12
a
Ki values were calculated from Fig. 2A ([3H]cytisine experiments), Fig. 2B ([3H]MLA experiments), Fig. 2C ([3H]epibatidine experiments), and Fig. 3A ([3H]TCP experiments),
respectively, according to Eq. (1).
b
Hill coefficients.
c
These values were obtained in the absence of any ligand (receptors are in the resting but activatable state).
d
This value was obtained in the presence of proadifen (receptors are mainly in the desensitized state).
e
This result was obtained in the presence of CCh (receptors are mainly in the desensitized state).
f
These apparent IC50 and apparent nH values were obtained from Fig. 3B.
The results obtained for hα4β2 nAChR suggest that varenicline
interacts with the ORT sites (Fig. 4B) and three A binding sites
(i.e., A1–3). A1 is located within the α4 subunit (Fig. 4C), whereas A2
and A3 are located at the inner (Fig. 4D) and outer surface, respectively,
formed between the α4- and β2-EXDs. Although the interaction of
varenicline with the A3 site was considered stable (see Fig. S1D;
Supporting information), this interaction becomes unstable when
varenicline also interacts with the ORT sites (see Fig. S2D; Supporting
information), and consequently, this site is not included in Fig. 4 and
Table 3.
At the ORT site, the aromatic portion of varenicline is stabilized by
hydrophobic interactions with the aromatic cage formed by residues
from the principal component (i.e., α4-Tyr98, α4-Tyr-202, α4-Tyr195,
and α4-Trp154) (Fig. 4B; Table 3). Moreover, varenicline interacts
with Loop C (i.e., Cys197–Cys198), two polar residues (i.e., α4-Ser153
and α4-Thr155) and several residues from the complementary component (i.e., β2-Leu121 and β2-Asn109). Two hydrogen bonds are additionally formed, one between the piperidine nitrogen of the ligand
Fig. 3. Modulation of (A) [3H]TCP binding to Torpedo nAChRs and of (B) [3H]imipramine
binding to hα4β2 nAChRs by varenicline. AChR membranes were pre-incubated
(20 min) with 20 nM [3H]TCP or 13 nM [3H]imipramine, in the presence of 1 mM CCh
(Torpedo nAChRs are mainly in the desensitized state) (■) or in the absence of any ligand
(nAChRs are in the resting but activatable state) (○), and then equilibrated with increasing concentrations of varenicline. Nonspecific binding was determined at 1 mM
CCh (A) or 100 μM imipramine (B). Each plot is the combination of two separated experiments each one performed in triplicate, where the error bars correspond to the S.D. The
observed IC50 values were used to calculate the Ki values according to Eq. (1). The apparent
EC50, Ki, and nH values were summarized in Table 2.
and the hydroxyl group of α4-Tyr98, and another between the same
nitrogen and the carbonyl from the β2-Trp154 backbone. In A1,
varenicline is stabilized within the α4 subunit through van der Waals
interactions with Leu40, Ile42, Leu45, Met55, Val59, Tyr129, Met149,
Phe151, Tyr207, and Phe209 (Fig. 4C; Table 3). In addition, varenicline
forms one hydrogen bond between its pyrazine nitrogen and the
carbonyl from the Cys147 backbone. In A2, varenicline is stabilized
by van der Waals contacts with α4-Ile95, α4-Val96, α4-Leu97, α4Pher105, α4-Gly103, β2-Val104, β2-Phe106, and β2-Tyr107 (Fig. 4D;
Table 3). The aromatic portion of varenicline may act as hydrogen
bond acceptor, whereas β2-Thr155 (ORT site), Thr57 (A1 site), and
Tyr107 and a water molecule (A2 site), may serve as hydrogen bond
donors. Furthermore, an electrostatic interaction is formed between
the positively charged pyrazine nitrogen of varenicline and the negatively charged amino moiety of α4-Asp104 (A2 site). Three additional
water-mediated hydrogen bonds are formed with the carbonyl from
the α4-Gly103 and α4-Asp104 backbones as well as with the hydroxyl
group of β2-Ser105 (A2 site) (Fig. 4D). Another water molecule is
present in A2, forming an internal network of hydrogen bonds that
stabilizes the ligand in this binding pocket.
In the hα4β4 nAChR, varenicline interacts only with the ORT site
(Fig. 5A; Table 3). The ligand is stabilized by hydrophobic interactions
with aromatic residues from the principal component as well as nonpolar residues from the complementary component as suggested for
the hα4β2 nAChR, as well as with β4-Ile113. Moreover, three hydrogen bonds are formed, two between the piperidine nitrogen of the ligand and the hydroxyl group of α4-Tyr98 or the carbonyl from the
α4-Trp154 backbone, and the third between the pyrazine nitrogen of
the molecule and the indole nitrogen of α4-Trp154. In addition, the
aromatic portion of varenicline acts as a hydrogen bond acceptor,
whereas β2-Tyr202 serves as a hydrogen bond donor.
Regarding the hα3β4 nAChR, varenicline interacts with the ORT
and A2 sites (Table 3). In the ORT site, varenicline interacts with practically the same residues proposed for the hα4β2 and hα4β4 nAChRs
(Table 3). In addition, two hydrogen bonds are formed with α3-Tyr93
and α3-Trp149, respectively (data not shown). In A2, varenicline interacts by van der Waals contacts with nonpolar residues as suggested for
hα4β2 nAChR (Fig. 4B; Table 3). In addition, its pyrazine nitrogen forms
a hydrogen bond with the carbonyl from the α3-Phe100 backbone
(data not shown).
Regarding the hα7 nAChR, varenicline interacts with the ORT site
(Fig. 5B) as well as with an additional allosteric site (i.e., A4), located
at the complementary component (Fig. 5C). In the ORT site, varenicline
is stabilized by hydrophobic interactions with aromatic residues
(Fig. 5B; Table 3) as suggested for the hα4β2 nAChR. Moreover, the ligand interacts with residues from Loop C (i.e., Cys190) and the complementary component (i.e., Asn107, Gln117, Leu119, and Ser150), and a
hydrogen bond is formed between the piperidine nitrogen of the ligand
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H.R. Arias et al. / Biochimica et Biophysica Acta 1848 (2015) 731–741
Table 3
Residues involved in the binding of protonated varenicline at the orthosteric and allosteric
(A) binding sites from the hα7, hα4β4, hα4β2, and hα3β4 nAChR models.
nAChR subtype
Orthosteric site
A1 site
A2 site
hα4β2
α4-C197
α4-C198
α4-Y195
α4-Y202
α4-Y98
α4-W154
α4-S153
α4-T155
β2-N109
β2-L121
α4-S131
α4-Y207
α4-A208
α4-C147
α4-T148
α4-F209
α4-M149
α4-L40
α4-I42
α4-V59
α4-F151
α4-T57
α4-Y129
α4-M55
α4-I95
α4-V96
α4-L97
α4-G103
α4-D104
α4-F105
β2-V104
β2-S105
β2-F106
β2-Y107
hα4β4
α4-C197
α4-C198
α4-Y195
α4-Y202
α4-Y98
α4-W154
α4-T155
β4-N111
β4-I113
β4-L123
hα7
α7-C190 (P)
α7-Y188 (P)
α7-Y195 (P)
α7-Y93 (P)
α7-W149 (P)
α7-W55 (C)
α7-N107 (C)
α7-Q117 (C)
α7-L119 (C)
α7-S150 (C)
hα3β4
α3-Y93
α3-Y190
α3-Y197
α3-C192
α3-C193
α3-W149
β4-L112
β4-I113
β4-L123
β4-S150
β4-N111
A4 site
α7-F33
α7-I54
α7-L56
α7-M58
α7-K87
α7-P88
α7-I90
α7-F100
α7-P120
α7-P121
α7-G122
α7-F146
α3-V91
α3-L92
α3-F124
α3-A96
α3-F100
β4-Y109
β4-V106
β4-V108
P, principal component; C, complementary component.
and the hydroxyl group of Trp149. In A4, varenicline is stabilized by
a hydrogen bond formed between its piperidine nitrogen and the
carbonyl from the Pro120 backbone (Fig. 5C). The ligand also interacts
with nonpolar as well as ionized residues (Table 3), whereas its aromatic portion acts as a hydrogen bond acceptor interacting with the
hydrogen bond donor Phe100 (Fig. 5C).
3.4. Molecular dynamics simulations
The MD simulations of varenicline docked to each binding site from
each studied nAChRs subtype indicated that the complexes are stable
during the 15-ns simulations (Fig. S1A–E; Supporting information).
The calculated root mean square deviation (RMSD) values for each
suggested site (Table 3) are below 0.5 Å, supporting the view that the
observed interactions (Figs. 4 and 5) are stable. Furthermore, the potential energy value for each stable interaction was calculated from the MD
simulations (Figs. S1F and S2E; Supporting information). The results
indicating that the potential energy values are constant support the
quality of the performed MD simulations and that they can be used to
measure the distance between the amino acids involved in the capping
of Loop C.
Additional MD simulations, performed when varenicline is bound to
each allosteric site while the ORT sites are also occupied, indicate that the
interaction with A1 (Fig. S2B) or A2 (Fig. S2C) is stable, whereas the interaction with A3 is unstable (Fig. S2D) (see Supporting information).
To determine how the binding of varenicline to A1 or A2 modifies the
capping of Loop C, the average distance between the hydroxyl group
of α4-Tyr195 (Loop C) and the amino group of α4-Lys150 (β7 strand)
as well as the average distance between the amino group of α4Lys150 and the carboxyl group of α4-Asp204 (β10 strand) were first
calculated when varenicline interacts with only the ORT sites and
then contrasted to that when the A1 or A2 site is also occupied. Under
these conditions, our results indicate that the distance between α4Tyr195 and α4-Lys150 is increased, whereas the distance between
α4-Lys150 and α4-Asp204 is decreased (Table 4). When varenicline interacts with the ORT and any of the allosteric sites, the average distance
between the carboxyl group of α4-Glu200 (Loop C) and the amino
group of β2-Lys79 (β1–β2 loop) was also increased and subsequently
the corresponding salt bridge is broken, whereas the average distance
required for the stable hydrogen bond between the backbone N–H of
α4-Lys158 and the backbone carbonyl of α4-Ile201 was not affected
(Fig. 6A; Table 4). In conclusion, the interaction of varenicline to A1 or
A2 modifies the capping of Loop C, compared to that in which none of
the allosteric sites is occupied (Fig. 6).
4. Discussion
As part of our attempt to characterize the interaction of varenicline
with several nAChRs as well as to define why this ligand behaves as a
full or partial agonist depending on the studied nAChR subtype, functional and structural approaches were used.
Our Ca2+ influx results indicate that varenicline activates nAChRs
with the following rank sequence (EC50s in μM): hα7 (0.18 ± 0.02) N
hα4β4 (0.37 ± 0.08) N hα4β2 (1.30 ± 0.18) N hα3β4 (6.4 ± 1.2) ≫ N
hα1β1γδ (N 100). Based on the observed efficacy (Emax), varenicline behaves as a full agonist of hα7 and hα4β4 nAChRs, and as a partial agonist of hα4β2 and hα3β4 nAChRs. This is the first report showing that
varenicline behaves as a full agonist of hα4β4 nAChRs. Different intrinsic properties and functional roles have been described for the α4β4
and α4β2 nAChRs. For instance, α4β4 nAChRs showed higher agonist
potencies and slower desensitization [45,46] as well as lower propensity to nicotine-induced upregulation [47] compared to that for α4β2
nAChRs. Subsequently, hα4β4 nAChRs might still maintain cholinergic
function after chronic exposure of nicotine concentrations found during
smoking (i.e., ~ 0.3 μM). In addition, knockout mice studies demonstrated that β2-containing nAChRs are implicated in the reinforcing
properties of nicotine [6] whereas β4-containing nAChRs play important
roles in nicotine withdrawal symptoms [48]. Finally, individuals with
the β4 subunit mutations (e.g., Arg136Trp and Met467Val) have higher
sensitivity to ACh [49], and subsequently, they could be more sensitive
to the addictive activity of nicotine. This evidence points out important
differences between α4β2 and α4β4 nAChRs, and suggests that α4β4
AChRs could also be involved in the clinical activity of varenicline.
Combining our and previous [12] radioligand binding results,
the following receptor selectivity for varenicline is obtained: hα4β2
(90 ± 7 pM) N hα4β4 (121 ± 4 pM) N hα3β4 (13 ± 1 nM) N hα7
(77 ± 6 nM) N hα4α6β4 AChR (110 ± 13 nM; [12]) ≫ N Tα1β1γδ
(116 ± 8 μM). Based on previous studies, the subunit α6 seems to decrease the varenicline binding affinity [12,44]. Based on the molecular
docking and MD results, we propose that the higher affinity of
varenicline for the hα4β4 and hα4β2 nAChRs compared to that for
the hα7, hα3β4, and Torpedo nAChRs may be ascribed to the observed
higher number of hydrogen bonds at α4-containing AChRs compared
to other nAChR subtypes.
H.R. Arias et al. / Biochimica et Biophysica Acta 1848 (2015) 731–741
737
Fig. 4. Docking of protonated varenicline to the extracellular domain of the hα4β2 nAChR model. (A) Side view of the orthosteric (ORT) and allosteric (A) sites for varenicline (yellow) at
the hα4β2 nAChR. The A1 site is located within the α4 subunit (orange), whereas the A2 site (green) is located at the inner surface formed between the α4- and β2-EXDs. (B) 3D and
2D (scheme) views of varenicline (yellow) interacting with the ORT site. The aromatic portion of the ligand is stabilized by hydrophobic interactions with the aromatic cage formed by
residues from the principal component (i.e., α4-Tyr98, α4-Tyr-202, α4-Tyr195, and α4-Trp-154). Varenicline also interacts with Loop C (i.e., Cys197–Cys198), two polar residues
(i.e., α4-Ser153 and α4-Thr155) and several residues from the complementary component (i.e., β2-Leu121 and β2-Asn109). Moreover, two hydrogen bonds are formed, one between
the piperidine nitrogen of the ligand and the hydroxyl group of α4-Tyr98, and another with the carbonyl from the α4-Trp154 backbone. (C) 3D and 2D (scheme) views of varenicline
(orange) interacting with the A1 site, located within the α4 subunit. The molecule is stabilized by van der Waals interactions with Leu40, Ile42, Met55, Val59, Tyr129, Met149, Phe151,
Tyr207, and Phe209. In addition, varenicline forms a hydrogen bond between its pyrazine nitrogen and the carbonyl from the Cys147 backbone. (D) 3D and 2D (scheme) views of
varenicline (green) interacting with the A2 site, located at the inner surface of the α4-/β2-EXD interface. The ligand is stabilized by van der Waals contacts with α4-Ile95, α4-Val96,
α4-Leu97, α4-Gly103, β2-Val104, α4-Pher105, β2-Phe106, and β2-Tyr107. In addition, an electrostatic interaction is formed between its positively charged pyrazine nitrogen and the
negatively charged amino moiety of α4-Asp104. Three additional water-mediated hydrogen bonds are formed, two with the carbonyls from the α4-Gly103 and α4-Asp104 backbone,
and another with the hydroxyl group of β2-Ser105. Another water molecule in A2 forms an internal network of hydrogen bonds that stabilizes the ligand. For clarity, one α4 and two
β2 subunits are hidden, whereas the other α4 and β2 subunits are shown in magenta and gray, respectively. The ligand is rendered in ball (A) or stick (B–E) mode. The residues involved
in ligand binding are presented in 2D or shown explicitly in stick mode and are colored in red or gray, depending on the subunit. The nonpolar hydrogen atoms are hidden. When interactions occur within one receptor subunit, chain labels are not shown.
738
H.R. Arias et al. / Biochimica et Biophysica Acta 1848 (2015) 731–741
Fig. 5. Docking results of protonated varenicline to the extracellular domain of the hα4β4 and hα7 nAChR models. (A) 3D and 2D (scheme) views of varenicline (yellow) interacting with
the ORT site at the hα4β4 nAChR. The ligand is stabilized by hydrophobic interactions with the aromatic cage formed by the same residues from the principal component as suggested for
the hα4β2 nAChR (see Fig. 4). Three hydrogen bonds are formed, two between the piperidine nitrogen of the ligand and either the hydroxyl group of α4-Tyr98 or the carbonyl from the
α4-Trp154 backbone, and another between the pyrazine nitrogen of the molecule and the indole nitrogen of α4-Trp154. Moreover, varenicline interacts with the same residues from Loop
C as well as nonpolar residues (i.e., β4-N111, β4-Ile113, and β4-Leu123) that were suggested for the hα4β2 nAChR (see Fig. 4). (B) Detailed view and scheme interactions of varenicline
(yellow) binding to the ORT site at the hα7 nAChR. In this site, the ligand is stabilized by hydrophobic interactions with aromatic residues from the principal (i.e., Tyr93, Tyr188, Tyr195,
and Trp149) and complementary (i.e., Trp55) components. In addition, the ligand interacts with residues from Loop C (i.e., Cys190), and from the complementary component (i.e., Asn107,
Gln117, Leu119, and Ser150), and a hydrogen bond is formed between the piperidine nitrogen of the ligand and the hydroxyl group of Trp149. (C) Detailed view and scheme interactions of
varenicline (purple) at the A4 site, located within the α7 subunit. In this site, varenicline is stabilized by a hydrogen bond formed between its piperidine nitrogen and the carbonyl from the
Pro120 backbone. In addition, the molecule interacts with nonpolar (i.e., Phe33, Ile54, Leu56, Met58, Pro88, Ile90, Phe100, Pro120, Pro121, Gly122, and Phe146) as well as ionized
(i.e., Lys87) residues, whereas its aromatic portion acts as a hydrogen bond acceptor where Phe100 serves the hydrogen bond donor. For clarity, the principal and complementary components at the hα4β4 and hα7 nAChR-EXDs are shown in magenta and gray, respectively. The ligand is rendered in stick mode. The residues involved in ligand binding are presented in 2D
or shown explicitly in stick mode, and colored in red or gray, depending on the subunit. The nonpolar hydrogen atoms are hidden.
The Ca2+ influx results also indicate that varenicline is more potent
as an antagonist (nM concentration range) than as an agonist (μM concentration range). However, differences among nAChR subtypes are
observed: the EC50/IC50 ratio for the hα4β2 is the highest (464) compared to that for the hα4β4 (14), hα7 (2.6), and hα3β4 (2.7) nAChRs.
Further experimental evidence is needed to understand these unexpected findings. The Ca2+ results are in agreement with the experimental evidence indicating that varenicline (1 mg twice-daily) activates only
Table 4
Average distance between residues involved in varenicline interaction to the hα4β2
nAChR when the agonist binds only to the orthosteric sites compared to that when it binds
to both the orthosteric site and the respective A1 or A2 allosteric site.
Interactive residues
α4-Tyr195/α4-Lys150
α4-Lys150/α4-Asp204
α4-Glu200/β2-Lys79
α4-Lys158/β2-Ile201
Interaction
type
Average distance [Å]
Hydrogen bond
Salt bridge
Salt bridge
Hydrogen bond
6.00
8.03
4.08
1.97
Orthosteric Orthosteric Orthosteric
site (only) and A1 sites and A2 sites
8.83
6.09
6.37
2.07
7.80
7.04
9.47
2.10
minimal fractions of α3β4 and α4β2 (b 2%), and α7 (b0.05%) AChRs,
whereas it inhibits these receptors in a selective manner: α4β2 (42%–
56%) N α7 (16%) N α3β4 (11%) [50]. The 10-fold higher antagonistic potency of varenicline for the hα4β2 compared to that for the hα4β4
nAChR can be explained by its higher desensitizing activity at the
hα4β2 nAChR [45]. Moreover, the functional effects of therapeutic
varenicline concentrations at α7 nAChRs are not related to α7 nAChR activation but to desensitization of moderate fractions of this subtype [50].
Our radioligand results indicate that varenicline enhances [3H]cytisine
and [3H]TCP binding to Torpedo nAChRs in the resting but activatable
state, consistent with the idea that varenicline induces nAChR desensitization, but ruling out the possibility of AChR ion channel blocking by
binding to the TCP/imipramine site located in the middle of the ion
channel [52]. Our molecular docking and MD simulation results support
the possibility that varenicline modulates α4β2 and α3β4 nAChRs by
binding to allosteric sites similar to that reported for the partial agonist
(−)-cytisine [51]. Since (−)-cytisine also binds to a homologous A1 site
at Torpedo nAChRs [51], we suggest that these extracellular allosteric
binding sites are part of a novel mechanism underlying partial agonism
at selective AChRs.
H.R. Arias et al. / Biochimica et Biophysica Acta 1848 (2015) 731–741
Fig. 6. Capping of Loop C induced by varenicline binding to the allosteric A1 (A) or A2
(B) site when the orthosteric (ORT) sites at the hα4β2 nAChR are also occupied compared to that when the agonist binds only to the ORT sites. (A) The MD results show
an increased distance between the hydroxyl moiety of α4-Tyr195 (Loop C) and the
amino moiety of α4-Lys150 (β7 strand), and concomitantly a decreased distance
between the amino moiety of α4-Lys150 and the carboxyl group of α4-Asp204
(β10 strand). On the other hand, a stable hydrogen bond interaction between the
carboxyl group of α4-Lys158 (Loop B) and the carbonyl group from the α4-Ile201
(Loop C) backbone is observed, whereas the salt bridge between the carboxyl group
of α4-Glu200 (Loop C) and the amino moiety of β2-Lys79 (β1-β2 loop) is broken
when varenicline interacts with the A1 site (orange) while the ORT sites are also occupied (yellow). The calculated average distances between residues are summarized in
Table 4. (B) The MD results show a wider gap between the Loop C and the surface of
the adjacent β2 subunit, indicating a decreased capping of Loop C when varenicline
binds to A2 (green) while the ORT sites are also occupied (yellow). Similar results
were obtained for A1. For a better visualization, the α4 subunit is rotated 90° compared
to that in (A). Varenicline and the most important residues are presented in stick mode,
whereas the nonpolar hydrogen atoms are hidden. The nAChR subunits are shown in
magenta when varenicline binds only to the ORT sites, whereas they are in gray when
the agonist also interacts with the allosteric site.
When agonists bind to AChBPs and nAChRs, the capping of Loop C
draws a conserved Tyr185 (corresponding to α4-Tyr195 from Loop
C) into register with a conserved Lys139 (corresponding to α4-Lys150
739
from β7 strand), forming a hydrogen bond or possibly a salt bridge,
and subsequently decreasing the distance between these two residues
[53]. In this regard, the calculated distances for nicotine and CCh, two
full agonists, are in the range of 2–3 Å [54]. These changes also weaken
the electrostatic link between Lys139 and Asp194 [corresponding to
α4-Lys150 (β7 strand) and α4-Asp204 (β10 strand), respectively]. Interestingly, the capping of Loop C produced by partial agonists is inbetween the movements elicited by full agonists (i.e., Loop C is in closed
conformation) and that in the absence of ligands (i.e., Apo state), when
Loop C is in the open conformation [55–57]. When varenicline binds to
the ORT sites, the concomitant movement of Loop C (i.e., intermediate
capping) produced a distance of 6.00 Å between α4-Tyr195 and
α4-Lys150. Our results with varenicline using the hα4β2 nAChR
model are in agreement with previous measurements (6.85 Å) at
the AChBP [30]. These results support the notion that this ligand is
a partial agonist of both hα4β2 and hα3β4 nAChRs. As an alternative
explanation, this partial agonism might be attributed to the interaction of varenicline with the allosteric sites observed at these receptor subtypes. In this regard, we tested the hypothesis that when
varenicline binds to either A1 or A2 site at the hα4β2 nAChR it induces conformational changes at Loop C, limiting the course of capping, the first step in the gating process [56,57]. The results show
an increase in the average distance between α4-Tyr195 and α4Lys150 and a concomitant decrease in the average distance between
α4-Asp204 and α4-Lys150 when varenicline is bound to either A1 or
A2 site, compared to that in the absence of allosteric site occupancy.
In this regard, the observed increased distance elicited by varenicline
(7.80–8.83 Å), which is close to that in the Apo state (8.5 Å) [30] suggests a decreased capping of Loop C, impairing the first step in the
process of nAChR gating.
The electrostatic interactions involving negatively charged residues
at the β1–β2 loop are important for intersubunit contacts, whereas
several of its residues are essential for gating [58]. In our study, a stable
salt bridge was observed between α4-Glu200 (Loop C) and β2-Lys79
(β1–β2 loop) when varenicline interacts only with the ORT sites.
The distance between these two residues (4.08 Å) was increased
(6.37–9.47 Å) when varenicline also interacts with each allosteric site.
In this regard, when varenicline binds to any of the allosteric sites, it
weakens this salt bridge, limiting the capping of Loop C at the hα4β2
nAChR. The same mechanism was observed for halothane at the
hα4β2 nAChR, although this general anesthetic interacts with an allosteric locus near the agonist site but differs from the A1 or A2 site [59]. This
suggests that varenicline, in addition to be an agonist, has negative allosteric modulatory properties at hα4β2 and hα3β4 nAChRs. On the other
hand, the hydrogen bonds and salt bridges in the region formed between
Loop C residues and Lys158, a critical residue from Loop B involved in
nicotine affinity [54] was not altered when varenicline interacts with
the allosteric sites, ruling out the possibility that this interaction decreases the affinity for these receptors.
The allosteric sites A1 and A2 have not been found in all studied
nAChR subtypes. At the hα4β2 nAChR, the A1 site is formed only by
α4 subunit residues (i.e., intrasubunit site), whereas no A1 site was
found at the hα4β4 nAChR (see Table 3). The A2 site at the hα4β2
nAChR is formed by several α4 and β2 residues (i.e., intersubunit site),
whereas at the hα3β4 nAChR, this site is formed by several α3 residues
but only one β4 residue (Y104) (the sequence similarity between the
β2 and β4 subunits is ~ 60%). Since the A1 site at the hα4β2 nAChR is
formed only by α4 residues, the allosteric effect of the β4 subunit
from the hα4β4 nAChR might be precluding the formation of the
intrasubunit pocket within the α4 subunit. We tested whether the β4
subunit changes the shape (e.g., by decreasing the volume) and properties (e.g., by preventing the formation of the hydrogen bonds network)
of the varenicline binding pocket as seen in the hα4β2 nAChR, and the
differences are too small to confirm this possibility. An alternative possibility is a difference in the varenicline access route to reach its pocket
between both AChRs. However, to test this hypothesis would require far
740
H.R. Arias et al. / Biochimica et Biophysica Acta 1848 (2015) 731–741
more demeaning MD simulations and other modeling studies that are
beyond the scope of this manuscript.
Based on our work, we propose that hα4β4 nAChRs are also potential targets for the clinical activity of varenicline. From the molecular point of view, the observed higher number of hydrogen bonds at
α4-containing nAChRs may explain the higher affinity of varenicline
for the hα4β4 and hα4β2 nAChRs compared to that for other receptor
subtypes. Although varenicline can activate hα4β2 and hα3β4 nAChRs
by binding to the ORT sites, its interaction with either allosteric site may
limit the first step in the gating process, contributing to the observed
partial agonistic activity at these nAChR subtypes.
Acknowledgement
This work was partially supported by grants from the TEAM research
subsidy from the Foundation for Polish Science (to K.J.), and from the
National Science Center, Poland (SONATA funding, UMO-2013/09/D/
NZ7/04549) [to K.T-D. (PI) and H.R.A. (Co-PI)]. The molecular modeling
experiments were developed using the equipment purchased within
the project “The equipment of innovative laboratories doing research
on new medicines used in the therapy of civilization and neoplastic diseases” within the Operational Program Development of Eastern Poland
2007–2013, Priority Axis I modern Economy, operations I.3 Innovation
promotion (to K.J. and A.K.). The research was partially performed during the postdoctoral Marie Curie fellowship of Dr. Kaczor at University of
Eastern Finland, Kuopio, Finland, and during the scholar visit of Dr.
Targowska-Duda at this University. Calculations were partially performed under a computational grant by the Interdisciplinary Center
for Mathematical and Computational Modeling (ICM), Warsaw,
Poland (grant number G30-18, to A.K.) and under resources and licenses
from CSC, Finland (to A.K. and A.P.). The authors thank to National Institute on Drug Addiction (NIDA, NIH, Bethesda, Maryland, USA) for its gift
of varenicline hydrochloride and phencyclidine hydrochloride.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.bbamem.2014.11.003.
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