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Arias, J Thermodynam Cat 2012, 3:4
DOI: 10.4172/2157-7544.1000116
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Molecular Interactions between Ligands and Nicotinic Acetylcholine
Receptors Revealed by Studies with Acetylcholine Binding Proteins
Hugo R. Arias*
Department of Medical Education, College of Medicine, California Northstate University, CA, USA
Abstract
Nicotinic acetylcholine receptors (AChRs) are the best characterized ion channels representing the Cys-loop
ligand-gated ion channel superfamily. Studies using Torpedo AChRs in the closed and open states and acetylcholine
binding proteins (AChBPs) from different origins have elucidated the most important structural and functional features
of the agonist/competitive antagonist binding sites. The first step in recognizing the neurotransmitter ACh and other
agonists is fundamental in the process of agonist-induced activation, including the opening of the intrinsic cation
channel. The AChBP studies demonstrated that Loop C is an important structural feature that is modified by ligand
binding. These studies defined important pharmacologic features of AChR ligands, including the differences between
full and partial agonists, agonists and competitive antagonists, peptidic and non-peptidic ligands, and between high
affinity and high selectivity. The studies showing the structural mechanisms by which specific ligands can activate,
inhibit, and potentiate different AChR subtypes could be of therapeutic importance.
Keywords: Cys-loop ligand-gated ion channels; Nicotinic
acetylcholine receptors; Acetylcholine binding proteins; Loop C;
Crystallography
Abbreviations: AChR: Nicotinic acetylcholine receptor; AChBP:
Acetylcholine bound protein; 5-HT: 5-Hydroxytryptamine (serotonin);
ACh: Acetylcholine; GABA: γ-aminobutyric acid; DMXBA: 3-(2,4
dimethoxybenzylidene)-anabaseine; α-BTx: α-bungarotoxin; κ-BTx:
κ-bungarotoxin; α-CbTx: α-cobrotoxin; MLA: Methyllycaconitine;
d-TC: d-tubocurarine; DHβE: Dihydro-β-erythroidine; SPX:
13-desmethyl spirolide C; GYM: Gymnodimine A; α-CTx: α-conotoxin;
MTSET+:
2-(trimethylammonium)
ethylmethanethiosulfonate;
MMTS: methylmethanethiosulfonate; Ls: Lymnaeastagnalis; Ac:
Aplysia californica; Bt: Bulinus truncates; Bg: Biomphalaria glabrata;
Ct: Capitella teleta; GLIC: Gloebacter violaceus; ELIC: Erwinia
chrysanthemi
Introduction
A vast amount of evidence indicates that nicotinic acetylcholine
receptors (AChRs) are important for the homeostasis and function
of our body. AChRs are cation channels members of the Cys-loop
ligand gated ion channel superfamily, including type 3 serotonin
(5-hydroxytryptamine; 5-HT) cation channels as well as anion
channels such as type A and C γ-aminobutyric acid (GABA) and
glycine receptors [1-3]. A large number of subunits have been cloned
for all members of the Cys-loop super family from vertebrates and
invertebrates (Ligand-gated ion channel database, http://www.ebi.
ac.uk/compneur-srv/LGICdb/cys-loop.php) [4]. In vertebrates, AChR
subunits are classified in two types, α (i.e., α1-α10) and non-α (i.e., β1β4, γ, δ, and ε), where α subunits contain a disulphide bridge in the
binding site, whereas non-α subunits do not. AChRs can be formed
by the same subunit comprising homomeric receptors [e.g., α7, α8
(expressed only in chicks), and α9] or by different subunits comprising
heteromeric receptors (e.g., α4β2, α3β4) [5,6]. However, not all possible
subunit arrangements are functional. For example, the neuronal AChR
subunits α2-α4, β2, and β4 can co-assemble in pair wise combinations
forming functional AChRs (e.g., α4β2, α3β4). Although the α9 subunit
is actually expressed in the cochlea, the functional receptor in hear
cells is the α9α10 AChR subtype. Although the homomeric α7 AChR
is functional, α7 AChRs containing other subunits (e.g., α7β2) have
J Thermodynam Cat
ISSN: 2157-7544 JTC, an open access journal
been characterized endogenously and probably these are the native
AChRs. Each α5, α6, and β3 subunit does not form functional AChRs
by binary combinations with another subunit, instead they prefer
ternary combinations [e.g., (α4)2α5(β2)2]. AChRs can even be formed
by assembling four different subunits [e.g., α4α6α5(β2)2], but there is
no evidence of a receptor subtype formed by five different subunits. In
addition, two different stoichiometries of the α4β2 AChR (and probably
for the α3β4 AChR as well), (α4)3(β2)2 and (α4)2(β2)3, have been found
in heterologous [7-10] and endogenous [11] cells. The (α4)2(β2)3
stoichiometry is more sensitive to the action of agonists including
agonist induced up-regulation, desensitizes less rapidly, and has lower
Ca2+ permeability compared to that for the (α4)3(β2)2 stoichiometry.
The use of subunit concatemers might be useful in determining the
subunit composition and stoichiometry of native AChRs [5].
Vertebrate AChRs are expressed in neuronal and non-neuronal
tissues. In the peripheral and central nervous systems, presynaptic
AChRs modulate the release of several neurotransmitters, including
ACh, 5-HT, GABA, dopamine, norepinephrine, and glutamate,
whereas postsynaptic AChRs mediate rapid transmission by converting
a chemical signal into membrane depolarization (i.e., electrical signal),
or trigger cytoplasmic cascades. These combined actions modulate
several important functions in our body, including cognition, memory,
pain perception, auditory response, and muscle contraction. In nonneuronal tissues, AChRs are involved in angiogenesis and immune
responses [12,13]. Since AChRs are involved in such important
physiological functions, their improper activities (e.g., decreased
number, mutations, or hypo/hyperactivity) can produce several diseases,
*Corresponding author: Hugo R. Arias, Department of Medical Education, College
of Medicine, California Northstate University, USA, Tel: 916-686-7300; Fax: 916-6867310; E-mail: hugo.arias@cnucom.org
Received October 12, 2012; Accepted October 18, 2012; Published October 22,
2012
Citation: Arias HR (2012) Molecular Interactions between Ligands and Nicotinic
Acetylcholine Receptors Revealed by Studies with Acetylcholine Binding Proteins.
J Thermodynam Cat 3:116. doi:10.4172/2157-7544.1000116
Copyright: © 2012 Arias HR. This is an open-access article distributed under the
terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and
source are credited.
Volume 3 • Issue 4 • 1000116
Citation: Arias HR (2012) Molecular Interactions between Ligands and Nicotinic Acetylcholine Receptors Revealed by Studies with Acetylcholine
Binding Proteins. J Thermodynam Cat 3:116. doi:10.4172/2157-7544.1000116
Page 2 of 7
including Alzheimer’s disease, Parkinson’s disease, schizophrenia,
nocturnal frontal lobeepilepsy, attention deficit hyperactivity disorder,
Tourette’s syndrome, drug and nicotine addictions, depression and
anxiety, myasthenia gravis, myasthenic syndromes, tumor growth, and
decreased immune response [2,3,12-15].
Several AChR subunits have been also cloned in insects and
invertebrates [16]. In invertebrates, AChRs have become key elements
for the development of neuroactive pesticides. For example, several
neonicotinoids (e.g., imidacloprid, clothianidin, and thiacloprid) have
insecticide activities but extremely low mammalian toxicities due to
their high affinity and specificity for insect AChRs [16].
Breakthroughs in the Study of AChRs
Three main breakthroughs in the last 10 years have helped in
elucidating the functionally relevant structural features of AChRs and
their cousins [1,17,18]: (1) the Torpedo AChR structures in the closed
[19] and open [20] states, showing the main features at the extracellular,
transmembrane, and intracellular domains (Figure 1), (2) the crystal
structures of several acetylcholine binding proteins (AChBPs) showing
details of the binding sites for agonists and competitive antagonists
[21], and (3) the recently elucidated prokaryotic cation channels
showing subtle differences between the open and closed conformations,
as well as between activated (i.e., with several primary amines [22]),
blocked (i.e., with several cations and open-channel blockers [23]), and
inhibited (i.e., with several allosteric modulators [24]) states. Table 1
summarizes the different three dimensional structures obtained so far
for Torpedo AChRs and different AChBPs bound to a variety of ligands.
Structurally, AChRs are pentameric proteins with an extracellular
domain that carries the binding sites for agonists (e.g., the
neurotransmitter ACh and nicotine) and competitive antagonists
[e.g., methyllycaconitine (MLA)]. In addition to these orthosteric sites,
several additional sites have been characterized in this domain for
allosteric modulators [25,26]. The transmembrane domain, specifically
the M2 transmembrane segments from each subunit, forms the ion
channel that is essential for cation flux (i.e., Na+ and Ca2+influx and K+
efflux), finally producing membrane depolarization, and in the case of
Ca2+, triggering different intracellular pathways.
M3) separated by short loops; (3) a cytoplasmic loop of variable size
and amino acid sequence; and (4) a fourth transmembrane domain
(M4) with a relatively short and variable extracellular COOH-terminal
sequence (Figure 1). AChRs are therefore built on a modular basis,
with the extracellular domain containing the agonist binding sites,
the transmembrane domain containing the pore, selectivity filter,
and channel gate, and the cytoplasmic domain performing additional
modulatory activities [19,20].
Recent structural studies have provided details of the three
dimensional structure of AChRs and consequently for other members
of this receptor superfamily. In particular, the structural model of
the Torpedo AChR at 4 Å resolution [19] has revealed important
information and has been invaluable in the interpretation of functional
and pharmacological data. Although no structural information is
available for any Cys-loop receptor at the atomic resolution level,
the extracellular domain of the AChR α1 subunit has been resolved
at 1.94 Å [28]. Additional high resolution structural information has
become available from studies of proteins which show close structural
similarity to AChRs, including soluble AChBPs from a variety of
animals from the Mollusca [21,29] and Annelida [30] phyla [1,17],
as well as prokaryotic proton-gated ion channels from the bacteria
Erwinia chrysanthemi (ELIC) and Gloebacter violaceus (GLIC),
respectively [1,18]. The characterization of GLIC showed that it forms a
cation-selective channel that is activated by protons, where currents do
not decay during activation, suggesting no or very slow desensitization
[31]. Recent results using ELIC showed that different primary amines,
including GABA, can also activate this channel [22]. The ELIC X-ray
structure shows 16% sequence identity to αAChR subunits. In general,
the extracellular domain is very similar to its eukaryotic counterpart
and to AChBPs, but lacks the N-terminal α-helix. However, the putative
binding site and several of the aromatic residues found in AChRs are
conserved. The central part of the Cys-loop is also conserved but lacks
the flanking disulfide-bridge.
The ion channel is the maindomain for the interaction with a
very broad group of compounds called noncompetitive antagonists
[14,15,27]. In addition, several binding sites for negative allosteric
modulators have been characterized between the four M1-M4
transmembrane segments [25,26].
Agonist binding at the main domain domain triggers the opening
of the ion channel, an intrinsic process called gating [1]. Determining
how the agonist-induced structural changes, that start in the agonist
binding pocket, are propagated through a distance of ~50 Å to the
gate is central for the understanding of the receptor function. One
of the AChR domains that have recently attracted attention among
researchers is the extracellular–transmembrane interface. This is a very
unique transitional zone where β-sheets from the extracellular domain
merge with α-helices from the transmembrane domain, finally allowing
functional communication between both domains [1,25,26].
Overall Structure of AChRs
AChRs are pentameric proteins where the subunits are arranged
around an axis perpendicular to the membrane, and each subunit
shares a basic scaffold composed of: (1) a large N-terminal extracellular
domain of ~200 amino acids; (2) three transmembrane domains (M1J Thermodynam Cat
ISSN: 2157-7544 JTC, an open access journal
Figure 1: Torpedo AChR model showing the structural differences between
the open (magenta) and closed (blue) states at 6.2 Å resolution (modified
from [20] ). Only subtle differences can be seen between both conformational
states. AChRs are formed by three main domains: The extracellular domain
shows the β-strands forming the β-sandwich structure.The transmembrane
domain is formed by 20 α-helices, four α-helices per subunit. The cytoplasmic
domain is the smallest domain.
Volume 3 • Issue 4 • 1000116
Citation: Arias HR (2012) Molecular Interactions between Ligands and Nicotinic Acetylcholine Receptors Revealed by Studies with Acetylcholine
Binding Proteins. J Thermodynam Cat 3:116. doi:10.4172/2157-7544.1000116
Page 3 of 7
The Extracellular Domain
The solution of the high-resolution structure of the AChBP from
Lymnaea stagnalis (Ls-AChBP) was a giant step forward for our
knowledge of the structure of the extracellular domain of AChRs
[1,17,21]. Since then, several other AChBPs from mollusks and
annelids have been characterized (Table 1). In general, AChBPs lack
the transmembrane region but contain many of the structural features
that give AChRs their unique signature and have therefore become
functional and structural surrogates of the extracellular domain of
the Cys-loop receptor superfamily. The AChR extracellular domain
contains 210 amino acids and shares ∼15-24% sequence identity
to aligned sequences of the amino-terminal, extracellular halves of
Cys-loop receptor subunits. Each AChBP monomer consists of an
N-terminal α-helix, two short 310 helices, and a core of 10 β-strands
that form a β-sandwich structure. The inner β-sheet is formed by the
β1, β2, β3, β5, β6 and β8 strands, and the outer β-sheet by the β4, β7,
β9 and β10 strands. The N- and C- terminals are located at the top and
bottom of the pentamer, respectively. In Cys-loop receptors, the end of
β10 connects to the start of M1. Located at the bottom of the subunit;
the linker between β6 and β7 strands is the signature Cys-loop found in
all members of the superfamily, including bacterial ion channels.
Agonist binding sites are located at the subunit interfaces [13,17,18]. Each binding site isformed by two faces. One face, called the
principal or “positive” face at the α-subunit, is formed by β-strands
connected by three loops harboring key aromatic residues [i.e., Loop
A (β4β5 loop), Loop B (β7β8 loop), and Loop C (β9β10 loop)]. The
complementary or “negative” face at the non-α-subunit contributes
with three β-strands clustered in segments by Loops D-F. Thus, key
residues (corresponding to Torpedo α1-subunit) from the principal face
come from Loop A (Trp86 and Tyr93), Loop B (Trp149 and Gly153)
and Loop C (Tyr190, Cys192, Cys193 and Tyr198). The complementary
face is formed by residues from Loop D (Trp55 and Asp57), Loop
E (Leu109, Arg111, Thr117 and Leu119), and Loop F (Asp174 and
Glu176) (residues from the Torpedo δ- or γ-subunit) [1,21,32].
β-sheet, causing tilting of the Cys-loop (Loop C) away from the fivefold axis [20].
The results using AChBPs with different ligands indicate that Loop
C from the principal face is in an extended (“open”) conformation
in the resting AChR (no agonist), whereas in the presence of full
agonists, Loop C is contracted (“closed”) and caps the entrance to the
binding cavity, trapping the agonist [36,37]. Cysteine substitution and
subsequent oxidation studies on Loop C of muscle AChRs indicate that
Loop C capping is involved in the transition of the closed receptor to an
activated pre-open intermediate state [38].
The interaction of several agonists (full and partial), competitive
antagonists, and allosteric modulators with their binding sites was
studied in minute detail by co-crystallization of various ligands with
AChBPs from different species (Table 1). In addition to structural
differences between ligands, diverse AChBP selectivity for several
ligands was observed [17,39]. The structural basis to distinguish high
affinity vs high selectivity for different ligands [e.g., d-tubocurarine
(d-TC) and strychnine] was also described [40]. Another important
structural difference is the interaction between full and partial agonists.
In general, AChBP-agonist complexes show a fully contracted (closed)
state, whereas AChBP-antagonist complexes show a more extended
(open) state. However, additional experiments comparing the full
agonist nicotine and the partial agonists cytisine and varenicline could
not discriminate any variation in the Loop C closure [41]. In particular,
varenicline interacts with highly conserved aromatic amino acids at the
principal face of the binding site, and with less conserved hydrophobic
residues at the complementary face (e.g., at Loop E) [30]. Interestingly,
dihydro-β-erythroidine (DHβE), a potent competitive antagonist of β2containing AChRs, imposes closure of the Loop C as agonists do, but
also induces a structural change perpendicular to the observed Loop
C movements [42]. To illustrate some important differences between
agonists and competitive antagonists, AChBP structures complexed
with the partial agonists varenicline and lobeline (Figure 2) and the
competitive antagonist DHβE (Figure 3) are shown.
The ancestral Cys-loop receptor was likely homomeric and
contained five identical binding sites, similarly to present day
homomeric receptors, such as α7 and 5-HT3A receptors [32,33].
Evolution led to the appearance of new subunits which lost the ability
to form agonist binding sites, giving rise to heteromeric receptors with
fewer than five binding sites. The prototypic heteromeric receptors,
muscle AChR and GABAARs, contain only two agonist binding sites,
which have to be both occupied to allow appropriate gating. Although
homomeric receptors contain five identical binding sites, it was shown
that occupancy of only three of the five sites is required for optimal
activation [34].
Structural Changes of Loop C when Interacting with
Agonists and Small Competitive Antagonists
Several lines of evidence indicate that ligands at the agonist binding
site are stabilized by π-cation, dipole-cation, hydrogen bonding, and
van der Waals interactions [35,36]. Agonist interaction produces
activation (opening) of the AChR ion channel. The transition from
the resting to the activated state is relatively fast, although the time
regime is different among receptor subtypes. Molecular details about
the activation process were determined by studies on AChBP- and
AChR-bound ligand structures. For example, structural differences
between the closed and open states permitted to determine that ACh
elicits clockwise rotation of the inner β-sheet with respect to the outer
J Thermodynam Cat
ISSN: 2157-7544 JTC, an open access journal
Figure 2: Structure of AChBP from Capitella teleta (Ct-AChBP) complexed
with the partial agonists lobeline (A-C) and varenicline (D) respectively
(modified from [30]). AChBP-lobeline structures showing the N-terminus
pointing away (A) or toward (B) the viewer, and the subunit interface (principal
face in yellow and complementary face in blue) towards the viewer (C). Each
of the five subunits is displayed in a different color. Oxygen, red; nitrogen,
blue; sulfur, green. The glycosyl chain N-linked to N122 is shown in magenta.
The paucimannose chain is composed of N-acetylglucosamine (NAG), α-Dmannose (α-MAN), and β-D-mannose (β-MAN) (D) Ct-AChBP-varenicline
complex. Lobeline and varenicline complexed with Ct-AChBP are shown as
spheres, whereas the insets show the electron density of each molecule.
Volume 3 • Issue 4 • 1000116
Citation: Arias HR (2012) Molecular Interactions between Ligands and Nicotinic Acetylcholine Receptors Revealed by Studies with Acetylcholine
Binding Proteins. J Thermodynam Cat 3:116. doi:10.4172/2157-7544.1000116
Page 4 of 7
Structure
Ligand bound
Conformational state
PDB
Resolution (Å)
Reference
AChR
AChR
None
ACh
ACh
α-BTx
α-BTx
HEPES
HEPES
(-)-Nicotine
CCh
α-CbTx
DHβE
n-Acetyl-d-glucosamine
(+)-Epibatidine
CAPS
None
(+)-Epibatidine
α-Lobeline
MLA
α-CTx ImI
HEPES
α-CTx PnIA(A10L/D14K)
SPX
GYM
α-CTx ImI
α-CTx TxIA(A10L)
Cocaine
Galantamine
Imidacloprid
Thiacloprid
Clothianidin
Imidacloprid
Sulfate
Anabaseine
DMXBA
4OH-DMXBA
Tropisetron
None
Compound 31
Compound 35
Metocurine
d-TC
Fragment 1
Compound 3
Compound 4
Compound 6
MTSET+
MMTS and ACh
d-TC
Strychnine
Mutant I
Mutant II + MLA
Mutant III + MLA
Compound 18 (complex I)
Compound 6 (complex II)
Compound 6 (complex III)
Varenicline
Cytisine
Triazole 18
None
None
None
Varenicline
α-Lobeline
Compound 5
Closed (Resting)
Closed (Desensitized)
Open
Open
Intermediate
Intermediate
Closed
Closed
Open
Closed
Apo
Closed
Intermediate
Apo
Closed
Closed; g-to-t
Intermediate
Open
Intermediate
Open
Intermediate
Intermediate
Open
Open
Unchanged
Unchanged
Closed
Closed
Closed
Closed
Coordinated with Lys residues
Closed
Intermediate
Intermediate
Intermediate
Apo; g
Intermediate
Intermediate
Open; g
g-to-t
g-to-t
endo
Open
Closed
Open
Closed
Apo
Open
Open
Open
Closed
Closed
Pentagonal dodecahedron
2BG9
4AQ5
4AQ9
2QC1
1HCB
1I9B
1UX2
1UW6
1UV6
1YI5
4ALX
3SQ9
3SQ6
2BJ0
2BYN
2BYQ
2BYS
2BYR
2BYP
2BR7
2BR8
2WZY
2X00
2C9T
2UZ6
2PGZ
2PH9
3C79
3C84
2ZJV
2ZJU
3GUA
2WNL
2WNJ
2WN9
2WNC
2Y7Y
2W8F
2W8G
3PEO
3PMZ
2Y54
2Y56
2Y57
2Y58
2XZ6
2XZ5
2XYT
2XYS
3T4M
3SH1
3SIO
2XNT
2XNU
2XNV
4AFT
4AFO
4DBM
4AOD
4AOE
4AFG
4AFH
4B5D
4.0
6.2
6.2
1.94
1.8
2.70
2.2
2.2
2.5
4.20
2.51
3.10
2.80
2.0
2.02
3.40
2.05
2.45
2.07
3.0
2.4
2.51
2.40
2.2
2.4
1.76
2.88
2.48
1.94
2.70
2.58
3.10
2.70
1.80
1.75
2.20
1.89
2.7
2.8
2.10
2.44
3.65
3.59
3.30
3.25
3.14
2.80
2.05
1.91
3.00
2.90
2.32
3.21
2.55
2.44
3.20
2.88
2.30
~6
~6
Low resolution
2.0
1.9
2.3
[19]
[20]
α1
α1-13-residue peptide
Ls-AChBP
Ls-AChBP
Ls-AChBP
Ls-AChBP
hα7/Ls-AChBP chimera
Bt-AChBP
Ac-AChBP
Ac-AChBP
Ac-AChBP
Ac-AChBP
Ac-AChBP
Ac-AChBP
Ac-AChBP
Ac-AChBP
Ac-AChBP
Ac-AChBP
Ac-AChBP
Ac-AChBP
Ac-AChBP
Ac-AChBP
(Y53C mutant)
Ac-AChBP
hα7/Ac-AChBP chimera
Ac-AChBP
Ac-AChBP
Ac-AChBP
Bg-AChBP1
Bg-AChBP2
Ct-AChBP
Ct-AChBP
Ct-AChBP
Apo
Intermediate; g-to-t
Closed; g-to-t
Intermediate
[28]
[52]
[21]
[53]
[54]
[42]
[62]
[55]
[46]
[56]
[49]
[57]
[50]
[43]
[58]
[16]
[59]
[45]
[44]
[60]
[39]
[51]
[40]
[61]
[63]
[41]
[64]
[29]
[65]
[30]
[66]
AChRs are obtained from Torpedo marmorata electric fish.
AChBPs are obtained from Lymnaea stagnalis (Ls), Aplysia californica (Ac), Bulinus truncates (Bt), Biomphalaria glabrata (Bg), and Capitella teleta (Ct), respectively.
The close (i.e., contracted) and opened (i.e., extended) states of Loop C in the AChBP-ligand complexes indicate the active/desensitized (i.e., agonists) and resting (i.e.,
antagonists) states, respectively. Several ligands, including partial agonists, adopt an intermediate configuration between that for full agonists and competitive antagonists,
whereas allosteric modulators (e.g., cocaine and galantamine) do not produce any apparent change on Loop C.
The g-to-t (also called Tyr-flip) conformational state corresponds to the opening of the lobeline pocket. The g state is the closed lobeline pocket, whereas in the endo
configuration the ligand is unable to induce the opening of the lobeline pocket.
Table 1: Conformation states of AChRs and AChBPs in unbound and bound conditions.
J Thermodynam Cat
ISSN: 2157-7544 JTC, an open access journal
Volume 3 • Issue 4 • 1000116
Citation: Arias HR (2012) Molecular Interactions between Ligands and Nicotinic Acetylcholine Receptors Revealed by Studies with Acetylcholine
Binding Proteins. J Thermodynam Cat 3:116. doi:10.4172/2157-7544.1000116
Page 5 of 7
to induce the opening of the lobeline pocket. These results suggest
that the different occupation and orientation between full and partial
agonists may produce distinct conformations of Loop C. Interestingly,
α-lobeline binds to Ct- and Ac-AChBPs at different configurations
[30,46]. In Ct-AChBP, the α-lobeline piperidine ring adopts a chair
conformation and its hydroxyl group is in the S-configuration, whereas
in the Ac-AChBP the ring adopts a half-chair conformation and its
hydroxyl group is in the R-configuration.
The AChBP structures in the absence of ligands or buffer molecules
(i.e., Apo) show that the Loop C is more flexible than that in the
presence of ligands, and that there are ordered water molecules filling
the pocket. This structure is reminiscent to the Torpedo AChR model
in the resting state [19]. Instead, buffer molecules (e.g., HEPES, CAPS)
in the binding pocket move Loop C in a more open configuration,
suggesting an intermediate state [46].
Structural Changes of Different Loops when Interacting
with Large Competitive Antagonists
Figure 3: Structure of AChBP from Lymnaea stagnalis (Ls-AChBP) complexed
with the competitive antagonist dihydro-β-erythroidine (DHβE) (modified
from [42] ). (a) Structure of DHβE. (b) Complex viewed along the five-fold
symmetry axis. The five subunits are shown in different colors and DHβE in
red spheres. (c) Interfacial binding pocket formed by the highly conserved
aromatic residues Tyr89, Trp143, Tyr185, and Tyr192 from the principal side of
the interface (yellow) and Trp53 from the complementary side (limon). DHβE
is shown in red, and hydrogen bonds between DHβE and its surroundings are
shown as stippled lines.
Some other ligands produce an intermediate state, whereas the
allosteric modulators cocaine and galantamine do not produce any
apparent conformational change [43]. For example, the dibenzosuberylatropine analogs, compounds 31 and 35, which pharmacologically act
as mixed competitive/noncompetitive antagonists on AChRs [44],
induce an intermediate open configuration of Loop C, suggesting an
intermediate resting/activated state. Interestingly, partial agonists
of the α7 AChR such as DMXBA [3-(2,4-dimethoxybenzylidene)anabaseine], its hydroxyl metabolite 4OH-DMXBA, and tropisetron,
display multiple orientations within the five binding sites and
adopt an intermediate configuration between that for full agonists
and competitive antagonists [45]. Nevertheless, the partial agonist
α-lobeline induces a strong Loop C closure [30,46]. More specifically,
the interaction of α-lobeline with the AChBP site opens a subpocket to
accommodate the α-hydroxyphenetyl moiety, inducing the g-to-t (also
called Tyr-flip) conformational state [39], where the g state is the closed
lobeline pocket, whereas in the endo configuration the ligand is unable
J Thermodynam Cat
ISSN: 2157-7544 JTC, an open access journal
Competitive antagonists overlap the agonist sites and inhibit
their pharmacological action. In addition to this competitive (steric)
mechanism, there is information that some antagonists [e.g., αand κ-bungarotoxin (α- and κ-BTx), α-cobrotoxin (α-CbTx), and
methyllycaconitine (MLA)] maintain the AChR in the resting state
[36,47,48], probably inhibiting the gating process. At the molecular
level, MLA, the potent competitive antagonist of the α7 AChR,
induces an intermediate open configuration of Loop C [46]. Although
the Loop C conformation distinguishes agonists from competitive
antagonists, additional results indicate that Loop F is another structural
component responsible for ligand selectivity; especially for antagonists
[45]. Although the toxins 13-desmethyl spirolide C (SPX) obtained
from Alexandrium ostenfeldii and gymnodimine A (GYM) obtained
from Karenia selliformis are among the most potent non-peptidic
antagonists (i.e., binding affinities in the subnanomolar concentration
range), they are nonselective antagonists, probably because they
interact in less proportion with Loop F in the complementary face [49].
Crystallographic information supports the view that in general peptidic
antagonists (i.e., snail and snake neurotoxins) maintain the AChBP
in the resting state, whereas other nonpeptidic antagonists induce an
intermediate state (Table 1). Different snail α-conotoxins (α-CTxs)
also differ in their intrinsic orientations [50], probably resembling their
receptor selectivities.
The Ac-AChBP-Y53C mutant binds to Cys-modifying agents by
different manners [51]. In this regard, MTSET+ [2-(trimethylammonium)
ethylmethanethiosulfonate] produces a less constricted Loop C, similar
to peptidic antagonists, whereas the conformation of Loop C in the
presence of MMTS (methylmethanethiosulfonate) and ACh resembles
that for agonists (Table 1). Since the location of the conserved amino
acid Y53 is at Loop D, these results also emphasize the concept of
concerted interactions between Loops D and C. Another important
difference between agonists and antagonists is that cation–π interactions
are formed between the conserved Trp residue in Loop B (i.e., W143 in
Ls-AChBP) and agonists such as (-)-nicotine [53], whereas no cation–π
interactions are formed with antagonists such as α-CTxs [46,50,56,57]
and neonicotinoids [16,58]. Taking into account all studied ligand
interactions, a good correlation (with some few exceptions) between
Loop C closure and the type of ligand (i.e., full and partial agonists,
competitive antagonists, and modulators) was found [1]).
In addition to details on ligand interactions, AChBP studies have
also helped in finding an ion selectivity filter in the extracellular domain
Volume 3 • Issue 4 • 1000116
Citation: Arias HR (2012) Molecular Interactions between Ligands and Nicotinic Acetylcholine Receptors Revealed by Studies with Acetylcholine
Binding Proteins. J Thermodynam Cat 3:116. doi:10.4172/2157-7544.1000116
Page 6 of 7
of Cys-loop ligand-gated ion channels [59]. This selectivity filter is
negatively charged in cation ion channels and positively charged in
anion ion channels.
Concluding Remarks
AChRs mediate rapid transmission throughout the nervous
system and also present functional roles in non-neuronal tissues.
Structural and functional studies permitted to highlight the
importance of the extracellular domain for the binding of agonists,
competitive antagonists, and allosteric modulators. It is remarkable
the advancement in our knowledge on ligand recognition and binding
through the study of AChBPs from mollusks and annelids bound to
a variety of ligands at atomic resolution. The current knowledge of
the structural components of the AChR binding sites is paramount in
differentiating agonists (full vs. partial) from competitive antagonists
(small and large) and modulators. The crystallographic results in
combination with mutagenesis, biochemical, electrophysiological,
and animal behavior studies will help in the design of more selective
agonists and competitive antagonists that can be used for the treatment
of AChR-related diseases. For example, compound 5 (3-[(2(S)azetidinyl)methoxy]-5-[(1S,2R)-2-(2-hydroxyethyl)cyclopropyl]
pyridine) is a highly selective partial agonist of the α4β2 AChR with
antidepressant properties that has been recently co-crystallized with an
AChBP [60-66].
Acknowledgement
I would like to thank Katarzyna Targowska-Duda (Medical University of Lublin,
Poland) for superimposing the open and closed Torpedo AChR models.
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