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1521-0111/90/3/288–299$25.00 http://dx.doi.org/10.1124/mol.116.104240
MOLECULAR PHARMACOLOGY Mol Pharmacol 90:288–299, September 2016
Copyright ª2016 by The American Society for Pharmacology and Experimental Therapeutics
MINIREVIEW—A LATIN AMERICAN PERSPECTIVE ON ION CHANNELS
Understanding the Bases of Function and Modulation of a7
Nicotinic Receptors: Implications for Drug Discovery
Jeremías Corradi and Cecilia Bouzat
Instituto de Investigaciones Bioquímicas de Bahía Blanca, Universidad Nacional del Sur, CONICET/UNS, Bahía Blanca,
Argentina
Received March 7, 2016; accepted May 5, 2016
ABSTRACT
The nicotinic acetylcholine receptor (nAChR) belongs to a
superfamily of pentameric ligand-gated ion channels involved
in many physiologic and pathologic processes. Among nAChRs,
receptors comprising the a7 subunit are unique because of their
high Ca
21
permeability and fast desensitization. nAChR agonists
elicit a transient ion flux response that is further sustained by the
release of calcium from intracellular sources. Owing to the dual
ionotropic/metabotropic nature of a7 receptors, signaling path-
ways are activated. The a7 subunit is highly expressed in the
nervous system, mostly in regions implicated in cognition and
memory and has therefore attracted attention as a novel drug
target. Additionally, its dysfunction is associated with several
neuropsychiatric and neurologic disorders, such as schizophre-
nia and Alzheimer’s disease. a7 is also expressed in non-neuronal
cells, particularly immune cells, where it plays a role in immunity,
inflammation, and neuroprotection. Thus, a7 potentiation has
emerged as a therapeutic strategy for several neurologic and
inflammatory disorders. With unique activation properties, the
receptor is a sensitive drug target carrying different potential
binding sites for chemical modulators, particularly agonists and
positive allosteric modulators. Although macroscopic and single-
channel recordings have provided significant information about
the underlying molecular mechanisms and binding sites of mod-
ulatory compounds, we know just the tip of the iceberg. Further
concerted efforts are necessary to effectively exploit a7 as a drug
target for each pathologic situation. In this article, we focus mainly
on the molecular basis of activation and drug modulation of a7,
key pillars for rational drug design.
Introduction
Nicotine has been a key molecule for the advancement of
pharmacology since the beginning of the 20th century, when
Langley (1905), through fundamental experiments, concluded
that muscle contraction was mediated by a “receptive sub-
stance”present on the muscle. The muscle nicotinic acetylcho-
line receptor (nAChR) was thus a pillar in the discovery of
neurotransmitter receptors (Langley, 1905). Still, it was not until
1970 that the first neurotransmitter receptor, nAChR, was
identified (Changeux et al., 1970; Miledi and Potter, 1971). With
the later advent of the molecular biology revolution in the 1980s,
the nAChR family was first identified and an extended family of
homologous pentameric receptors was revealed (Patrick et al.,
1983; Le Novère and Changeux, 1995). This class of receptors
was first known as Cys-loop receptors because all family sub-
units contain a conserved pair of disulfide-bonded cysteines
separated by 13 residues. The discovery of orthologs in pro-
karyotes (Tasneem et al., 2005), which lack the double cyste-
ines, has extended the Cys-loop family to the superfamily of
pentameric ligand-gated ion channels (pLGIC).
In vertebrates, the pLGIC superfamily includes cationic chan-
nels, nAChRs and serotonin 5-HT
3
receptors, and anionic chan-
nels activated by GABA or glycine (Le Novère and Changeux,
2001; Lester et al., 2004; Sine and Engel, 2006; Bartos et al., 2009).
Their vital role in converting chemical recognition into an
electrical impulse makes these receptors prime loci for learn-
ing, memory, and disease processes, as well as targets for clini-
cally relevant drugs.
The nAChR is widely distributed throughout the animal
kingdom, from nematodes to humans (Le Novère and Changeux,
1995). nAChRs are expressed in many regions of the central
This work was supported by grants from Universidad Nacional del Sur
(UNS), Consejo Nacional de Investigaciones Científicas y Técnicas (CONI-
CET), FONCYT, and the Bill and Melinda Gates Foundation to C.B.
dx.doi.org/10.1124/mol.116.104240.
ABBREVIATIONS: ACh, acetylcholine; a-BTX, a-bungarotoxin; ECD, extracellular domain; JAK, Janus kinase; LY-2087101, (2-amino-5-keto)
thiazole), [2-[(4-fluorophenyl)amino]-4-methyl-5-thiazolyl]-3-thienyl-methanone; nAChR, nicotinic acetylcholine receptor; NAM, negative allosteric
modulators; NS-1738, 1-(5-chloro-2-hydroxyphenyl)-3-(2-chloro-5-trifluoromethylphenyl)urea; PAM, positive allosteric modulator; pLGIC, pentameric
ligand-gated ion channels; PNU-120596, 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)urea; SAM, silent allosteric modulator;
STAT, signal transducer and activator of transcription; TMD, transmembranedomain;TQS,4-(naphthalen-1-yl)-3a,4,5,9b-tetrahydro-3H-
cyclopenta[c]quinoline-8-sulfonamide.
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and peripheral nervous system, in addition to non-neuronal
tissues. The muscle nAChR plays a major role in neuromus-
cular transmission and is the target of muscle relaxants (Sine,
2012), whereas nAChRs in the brain represent a broad hetero-
geneous family of ubiquitously expressed receptors. nAChR
responses to endogenous acetylcholine (Ach) and choline and
exogenous nicotine are involved in a number of physiologic
processes and pharmacological effects (Dani and Bertrand,
2007; Albuquerque et al., 2009; Hurst et al., 2013).
The homopentameric a7, one of the most abundant nAChRs
in the nervous system, is also expressed in many non-neuronal
cells. Its unique activation properties, high calcium perme-
ability, ionotropic/metabotropic dual action, ubiquitous distri-
bution, and involvement in a range of neurologic, psychiatric,
and inflammatory disorders have made a7animportant
emerging drug target; the participation of a7 in pathologic
conditions and the therapeutic potential of a7 ligands has been
well documented (see for example Taly and Changeux, 2008;
Wallace and Porter, 2011; Lendvai et al., 2013; Wallace and
Bertrand, 2013; Uteshev, 2014; Dineley et al., 2015). In this
article, wefocus on the unique properties of activation and drug
modulation of a7 and its relationship withdisease and therapy.
nAChR Structure and Function
nAChR subunits are classified as two types, aand non-a,
with the a-type containing a disulfide bridge in the agonist
binding site. Five muscular (a1, b,g,«, and d) and eleven
neuronal (a2–a7, a9, a10, and b2–b4) nAChR subunits have
been identified in the mammalian genome (ligand-gated
ion channel database, http://www.ebi.ac.uk/compneur-srv/
LGICdb/cys-loop.php).
nAChRs are assembled from five identical (a7ora9) or
different subunits (at least two a-type subunits), and can form a
variety of different heteromeric receptors with a broad spec-
trum of pharmacological and kinetic properties (Fig. 1). The
resolution of the three-dimensional structures of pLGICs has
been the subject of intense efforts over the last decade (Brejc
et al., 2001; Dellisanti et al., 2007; Hilf and Dutzler, 2008, 2009;
Bocquet et al., 2009; Hibbs and Gouaux, 2011; Corringer et al.,
2012; Hassaine et al., 2014; Miller and Aricescu, 2014; Sauguet
et al., 2014; Cecchini and Changeux, 2015). However, no high-
resolution structure of the full length a7hasbeenreportedto
date; an extracellular domain of a7/AChBP chimera (Li et al.,
2011; Nemecz and Taylor, 2011) and a nuclear magnetic
resonance (NMR) structure of a7 transmembrane domain have
been described (Bondarenko et al., 2014).
All pLGICs share a conserved organization with five
subunits symmetrically arranged around a central ion pore
(Fig. 2). Functional domains include the extracellular domain
(ECD), which carries the agonist binding sites at subunit
interfaces; the transmembrane domain (TMD), which con-
tains the ion pore and the gate; and the intracellular domain
(ICD), which contains determinants of channel conductance
and sites for regulation and intracellular signaling (Paulo
et al., 2009; Jones et al., 2010; King et al., 2015) (Fig. 2). The
interface between the ECD and TMD, also referred to as the
coupling region, is important for coupling agonist binding to
channel opening (Bouzat et al., 2004; Lee and Sine, 2005;
Castillo et al., 2006; Bartos et al., 2009), as well as for
determining open channel lifetime and rate of desensitization
(Bouzat et al., 2008; Yan et al., 2015) (Fig. 2).
The possible structural events that translate neurotrans-
mitter binding at the ECD into opening of the transmembrane
ion channel 60 Å away is an issue of intense research that has
been discussed in recent reviews (Corringer et al., 2012;
Althoff et al., 2014; Sauguet et al., 2014; Cecchini and Changeux,
2015). On the basis of the Monod-Wyman-Changeux model
(Monod et al., 1965), the functional response of a pLGIC can be
interpreted as a selection from a few global and discrete
conformations elicited by the binding of agonist: closed, open,
and desensitized, the latter showinghighagonistaffinityatthe
same time being impermeable to ions (Zhang et al., 2013) (Fig. 3).
Intermediate states between closed and open or open and
desensitized states have been proposed for nAChRs and several
pLGICs (Lape et al., 2008; Corradi et al., 2009; Mukhtasimova
et al., 2009; Cecchini and Changeux, 2015). Therefore, the
number of states in the main conformations and the rate of
transition between states determine receptor kinetics, and this
tunes each receptor to its physiologic role. In turn, drugs, by
binding to different states, can differently modulate receptor
function.
a7 in the Nervous System in Healthy and Disease
States
a7 is one of the most abundant nAChRs in the central
nervous system. It is particularly expressed in regions impli-
cated in cognitive function and memory, such as hippocampus,
cortex, and several subcortical limbic regions (see Lendvai
Fig. 1. Models of pentameric arrangements of homomeric and some
heteromeric nAChRs. a7 and a9 are the only a-type subunits capable of
forming functional homomeric receptors, which contain five identical
binding sites. In a7, occupancy of only one site is required for activation.
Examples of possible combinations of aand non-asubunits in heteromeric
arrangements are shown. An a-type subunit is required for the principal
face of the binding site. The muscle nAChR contains two functional binding
sites at a/dand a/«(g) interfaces. Some subunits can assemble with
different stoichiometries, such as a4andb2. In addition to the arrange-
ment shown, (a4)
3
(b2)
2
receptors are also functional.
a7 Modulation 289
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et al., 2013). It is also expressed on non-neuronal cells,
including astrocytes, microglia, oligodendrocyte precursor
cells, and endothelial cells, where it plays a role in immunity,
inflammation, and neuroprotection (Shytle et al., 2004; Shen
and Yakel, 2012; Dineley et al., 2015).
In neurons, a7 receptors localize presynaptically on GABAer-
gic and glutamatergic terminals in the hippocampus and other
regions to facilitate release of neurotransmitters. Postsynapti-
cally, a7 receptors mediate fast synaptic transmission, and in
perisynaptic locations they modulate other inputs to neurons and
activate a variety of signaling pathways through volume trans-
mission (Gotti and Clementi, 2004; Jones and Wonnacott, 2004;
Dani and Bertrand, 2007; Dickinson et al., 2008; Albuquerque
et al., 2009; Sinkus et al., 2015) (Fig. 4).
a7 contributes to cognitive functioning, sensory processing
information, attention, working memory, and reward path-
ways, and a large body of evidence shows that enhancing a7
activity improves attention, cognitive performance, and neu-
ronal resistance to injury (reviewed in Uteshev, 2014).
Significant reduction of a7 in the brain, particularly in the
hippocampus, has been reported in Alzheimer disease (Guan
et al., 2000; Kadir et al., 2006) and schizophrenic patients
(Schaaf, 2014; Dineley et al., 2015). The a7 gene, CHRNA7 on
chromosome 15, is genetically linked to multiple disorders
with cognitive deficits, including schizophrenia, intellectual
disability, bipolar disorder, autism spectrum disorders, attention
deficit hyperactivity disorder, epilepsy, Alzheimer disease, and
sensory processing deficit (Sinkus et al., 2009, 2015; Schaaf,
2014; Dineley et al., 2015; Deutsch et al., 2016). A partial dupli-
cation of CHRNA7 resulting in the chimeric gene CHRFAM7A,
which is present only in humans, has been associated with
schizophrenia (Sinkus et al., 2009; Neri et al., 2012). Its gene
product, dupa7, lacks part of the binding site and may act as a
negative modulator of a7 (Wang et al., 2014).
Despite its homomeric character, a7 can assemble with
other subunits to form heteromeric receptors. In particular,
a7b2 heteromeric receptors have been detected in several
Fig. 2. Structural model of pLGICs. The ECD is folded into a
highly conserved immunoglobulin-like b-sandwich. The agonist
binding site is found in a cavity at an interface between two
adjacent subunits (Sine, 2012). The principal or positive face is
provided by an a-type subunit and includes three loops that
span bstrands (named as Loops A–C) that harbor predomi-
nantly aromatic residues essential for binding and gating. The
adjacent subunit, which forms the complementary or minus
face, contributes with residues clustered in segments called
Loops D–F (Brejc et al., 2001; Sine, 2012). ACh docked into the
binding site is shown (a7/AChBP chimera, pdb code 3SQ6). Key
aromatic residues from the principal face are Tyr188, Trp149,
and Tyr93, and from the minus face, Trp55. The transmem-
brane domain (TMD) is composed of four transmembrane-
spanning helices (TM1–TM4). The TM2 forms the walls of the
ion pore, which contains the gate (ring of leucines at 99position)
and determinants for selectivity. The outer ring of fifteen
a-helices (TM1, TM3, and TM4) shields the inner TM2 ring
from the lipids (reviewed in Althoff et al., 2014). The interface
between the ECD and TMD, also named as coupling region,
includes the conserved Cys-loop (b6b7 loop), b1b2andb8b9
loops from the ECD, and the M2–M3 linker from the TMD. The
long intracellular region (ICD) between TM3 and TM4 is highly
variable and particularly short in prokaryotic pLGICs. It is
thought to be associated with cytoskeletal proteins and in-
volved in channel modulation in all pLGICs (Kabbani et al.,
2013). As shown in the figure, in a7, this region contains
determinants of channel conductance (Andersen et al., 2011,
2013) and mediates several intracellular signals (Paulo et al.,
2009; Jones et al., 2010).
Fig. 3. Minimal model of nAChR activation. pLGICs have three main
classes of conformational states: Closed (C), open (O), and desensitized (D).
Intermediate states have also been detected.
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brain areas (Liu et al., 2009, 2012; Moretti et al., 2014;
Thomsen et al., 2015; Zoli et al., 2015). Interestingly, a7b2
are highly sensitive to blockade by Ab1–42, suggesting that
they may play a unique role in the neuropathology of Alzheimer
disease (Liu et al., 2009). Additionally, these heteromeric
receptors exhibit high sensitivity to volatile anesthetics, and
therefore could be targets for anesthetic action (Mowrey et al.,
2013). Thus, a7b2 nAChR may represent a novel molecular
target requiring selective a7b2 ligands.
Extraneuronal a7 and Its Pleiotropic Roles
a7 is present in various non-neuronal tissues, such as glia
(Sharma and Vijayaraghavan, 2001), blood cells (Kawashima
and Fujii, 2004; De Rosa et al., 2005; Báez-Pagán et al., 2015),
keratinocytes (Maus et al., 1998), epithelial cells and fibro-
blasts (Zia et al., 1997), endothelial cells (Macklin et al., 1998),
cells of the digestive system and lung cells (reviewed in
Wessler and Kirkpatrick, 2008), spermatogonia, spermato-
cytes, and seminiferous tubular and Sertoli cells (Schirmer
et al., 2011). The functional role of a7 in these cells is being
intensively investigated and has been associated with differ-
entiation, migration, adhesion, cell contact, apoptosis, and
angiogenesis processes (Ni et al., 2010; Egea et al., 2015;
Zdanowski et al., 2015).
In particular, a7, present in all types of immune cells,
including lymphocytes (T and B cells), dendritic cells and
macrophages (reviewed in Egea et al., 2015), has attracted
considerable attention as an important drug target for in-
flammation. a7 is an important player in the “cholinergic anti-
inflammatory pathway,”which is a linkbetween vagal efferent
fibers and innate immune system (Martelli et al., 2014). a7
modulates intracellular signal pathways [Janus kinase 2
(JAK2)/signal transducer and activator of transcription
(STAT3) and PI3K/Akt] in immune cells, which results in
potent anti-inflammatory effects through cytokine production
inhibition and overexpression of heme oxygenase 1 (Báez-
Pagán et al., 2015; Egea etal., 2015) (Fig. 5). A brain cholinergic
pathway also exists that regulates microglial activation
through a7 (Shytle et al., 2004). This pathway is crucial for
neuroprotection (Park et al., 2007) and is probably important
in Parkinson disease (Stuckenholz et al., 2013), oxygen and
glucose deprivation (Parada et al., 2013), and global ischemia
(Guan et al., 2015).
Therefore, a7 nAChR is emerging as an important drug
target for the modulation of inflammation in different path-
ologic contexts, including sepsis, ischemia/reperfusion, rheu-
matoid arthritis, and pancreatitis.
a7 Pharmacology and Ion Selectivity
Hallmark features of a7 receptors include high Ca
21
permeability, relatively low sensitivity to ACh, full activation
by choline, high-affinity for a-bungarotoxin (a-BTX), relative-
ly low affinity for nicotine, and fast desensitization that occurs
on the submillisecond time scale.
Dose-response curves show EC
50
values of ∼100–200 mM for
ACh [Hill coefficient (nH) ∼1] (Andersen et al., 2013), 0.4–
1.6 mM for choline, and 18–91 mM for nicotine (Wonnacott and
Barik, 2007). Several a7-specific agonists have been synthe-
tized, including PNU-282987 (EC
50
128 nM), AR-R17779
(EC
50
∼10–20 mM), compound A (EC
50
∼14 nM to 0.95 mM),
and partial agonists GTS-21 (EC
50
∼6–26 mM), and SSR180711
(EC
50
∼1–4mM). Selective competitive antagonists are a-BTX
(IC
50
∼1–100 nM), which has been widely used to detect a7
in tissues, and methyllycaconitine (MLA, IC
50
10–200 nM)
(Wonnacott and Barik, 2007).
a7 allows flux of Na
1
and K
1
and is highly permeable to
Ca
21
. The PCa
21
/PNa
1
ratio is ∼10–20, which is greater than
that of other nAChRs and similar to N-methyl-D-aspartate
receptors (Séguéla et al., 1993; Albuquerque et al., 1997). The
high Ca
21
permeability underlies most of a7 functions: facil-
itation of neurotransmitter release, depolarization of post-
synaptic cells, and initiation of many cellular processes through
its action as a second messenger (Gotti and Clementi, 2004).
The transient increase in intracellular Ca
21
is converted into a
sustained, wide-ranging phenomenon by calcium release from
intracellular stores through a calcium-induced calcium release
mechanism, a process involving IP
3
and ryanodine receptors
(Tsuneki et al., 2000; Dajas-Bailador et al., 2002; Guerra-
Álvarez et al., 2015) (Fig. 5).
The concept of a7 as a dual metabotropic/ionotropic receptor
is attracting increasing attention (Kabbani et al., 2013). a7
binds both Gaand Gbg proteins through the M3–M4 loop and
enables a downstream calcium signaling response that can
persist beyond the expected time course of channel activation
(Fig. 5) (Kabbani et al., 2013; King et al., 2015). Moreover,
in lymphocyte T cells, mobilization of Ca
21
through the a7
channel is not necessarily required for the nicotine-induced
release of Ca
21
from the internal stores (Razani-Boroujerdi
et al., 2007). Channel-independent signal transduction has
been related to the role of a7 in inflammation (de Jonge and
Ulloa, 2007; Thomsen and Mikkelsen, 2012) and in neurite
growth (Nordman and Kabbani, 2012; Kabbani et al., 2013).
a7 is not only permeable to Ca
21
but is also modulated by
the ion; Ca
21
has been shown to regulate agonist efficacy and
cooperativity (Bonfante-Cabarcas et al., 1996; Albuquerque
et al., 1997). As in other pLGICs (Zimmermann et al., 2012),
the divalent modulatory sites may be located at the ECD.
a7 Channel Kinetics
Heterologous expression of a7 in oocytes and mammalian
cells combined with electrophysiological experiments has
provided information about the receptor’s unique activation
properties. The surface expression of recombinant a7 requires
Fig. 4. Neurotransmission mediated by a7 in the mammalian brain. a7
receptors can be postsynaptic, presynaptic (with a role in regulation of
neurotransmitter release), or perisynaptic when they are involved in
volume transmission.
a7 Modulation 291
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coexpression of the chaperone RIC-3 (Williams et al., 2005), a
transmembrane protein that is required for efficient receptor
folding, assembly, and functional expression (Castillo et al.,
2005; Millar, 2008). Recently, another chaperone, NACHO,
has been identified. NACHO is a transmembrane protein of
neuronal endoplasmic reticulum that mediates assembly of
a7 by promoting protein folding, maturation through the Golgi
complex, and expression at the cell surface (Gu et al., 2016).
Typical a7 receptor-mediated currents decay rapidly in the
presence of agonist as a result of desensitization (Fig. 6A).
Given the fast kinetics, the temporal resolution of agonist
exchange and most recording systems limit the accurate
estimation of the true desensitization rate (Zhou et al., 1998;
Lovinger et al., 2002; Bouzat et al., 2008), which may partially
account for the great variability of desensitization rates found
in the literature. Outside-out patches rapidly perfused with
ACh, which allows for a more accurate determination of the
desensitization rate, show current decay time constants of
∼0.4 milliseconds (Bouzat et al., 2008). Owing to their fast
decay, a7 peak responses occur in advance of complete solu-
tion exchange. Therefore, more accurate EC
50
values are
obtained if the net charge, which represents the time in-
tegration of all channel activation, rather than the peak
current, is used for the analysis of the concentration-effect
relationship (Papke and Porter Papke, 2002) (Fig. 6A).
At the single-channel level, channel activity appears as
isolated brief pulses (0.1–0.3 milliseconds) flanked by long
closed periods and, less often, as two or three brief pulses in
quick succession (bursts) (Fig. 6B). Thus, a7 has a very low
open probability. Single-channel openings exhibit a broad
distribution of current amplitudes, probably owing to limited
time resolution of the brief openings, with a maximum of ∼10 pA
at –70 mV (Mike et al., 2000; Bouzat et al., 2008; Andersen et al.,
2013; daCosta and Sine, 2013; Yan et al., 2015).
Although there is no general consensus for an a7 kinetic
model, an interesting aspect is that the temporal pattern of
single ACh-activated currents is similar at 10 mMor1mM
ACh (Bouzat et al., 2008) (Fig. 6B). This lack of concentration-
dependence combined with the fact that most receptor acti-
vation episodes consist of a single opening with a duration
similar to the desensitization time constant suggests that
desensitization is the predominant pathway for channel
closing, a unique feature among nAChRs. Control of open-
channel lifetime through desensitization has potential conse-
quences for inter-response latency at a synapse where the
neurotransmitter pulse is transient. Recovery from desensiti-
zation depends on agonist concentration and exposure dura-
tion, since different desensitized states may exist. Desensitized
a7 receptors expressed in human embryonic kidney cells
recover with a time constant of ∼1 second (Bouzat et al.,
2008), whereas 15–30 seconds are required for full recovery in
the hippocampus (Frazier et al., 1998). Thus, after a7 brief
response, a latency of several seconds is required to generate
another response of full amplitude. The fast desensitization
and brief open duration may avoid cell toxicity caused by
increased intracellular Ca
21
owing to a7 overstimulation.
Fig. 5. Dual ionotropic/metabotropic nature of a7: Intracellular pathway signaling mediated by a7 activation. a7 allows transient flux of Na
+
,K
+
, and
Ca
2+
. The transient increase of calcium may also lead to a sustained calcium response by a calcium-induced calcium release (CICR) mechanism through
IP
3
receptors. As a metabotropic receptor, a7 mediates intracellular signals by binding to G aand Gbg proteins and several other partners (Kabbani et al.,
2013; King et al., 2015). In immune cells, a7 has been shown to be involved in several intracellular pathways; although not yet fully deciphered
mechanistically, these pathways lead to potent anti-inflammatory effects. For example, a7 has been shown to activate JAK2/STAT3 in some immune
cells, which leads to blockade of nuclear factor (NF)-kB nuclear translocation and inhibition of NF-kB, resulting in inhibition of proinflammatory cytokine
production. Also, a7 has been shown to activate the PI3K/Akt pathway that promotes Nrf-2 translocation to the nucleus and overexpression of heme
oxygenase 1 (HO-1), resulting in potent anti-inflammatory effects (see de Jonge and Ulloa, 2007; Báez-Pagán et al., 2015; Egea et al., 2015). DAG,
diacylglycerol; GPCR, G protein-coupled receptor; PKC, protein kinase C; ROS, reactive oxygen species.
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Another intriguing aspect of a7 activation is the relation-
ship between ACh occupancy and activation. We set out to
determine how many of the five identical agonist binding sites
are required to activate a7 by developing a strategy that
utilizes coexpression of an inactivated binding-site subunit
and a reporter amplitude subunit. This allows for the de-
termination of the number of ACh-occupied sites from the
amplitude of each individual single-channel opening (Rayes
et al., 2009; Andersen et al., 2011, 2013). The results revealed
that ACh occupancy of only one of five a7 binding sites is
necessary for activation, and that open-channel lifetime of
a single-occupied receptor is indistinguishable from that of
receptors containing five intact binding sites (Andersen et al.,
2013). Also, occupancy of a single site by the antagonist
methylcaconitine (Palma et al., 1996) or a-BTX (daCosta
et al., 2015) will inhibit a7 function.
Whole-cell experiments have revealed the physiologic con-
sequence of having more sites than those required for
activation; saturation of receptors in cells expressing a wild-
type and a binding-site mutant subunit (a7Y188T) is achieved
at higher ACh concentrations when the proportion of recep-
tors with fewer functional binding sites increases (Andersen
et al., 2013; Williams et al., 2011a). This suggests that a7is
a highly sensitive sensor of ACh and is therefore adapted to
function with submaximal occupancy of its sites, a property
appropriate for volume transmission.
The unique activation properties of a7 also suggest that any
slight change in the energy barriers between active, closed,
and/or desensitized states may have a deep impact on receptor
function, which makes these receptors very sensitive drug
targets.
a7 Modulation as a Therapeutic Strategy
In addition to agonists and antagonists that bind to the
orthosteric site, a large number of compounds modulate a7
function by binding to allosteric sites. These compounds may
act as: 1) positive allosteric modulators (PAMs) that potenti-
ate currents only in the presence of the agonist; 2) allosteric
agonists that activate receptors from nonorthosteric sites; 3)
negative allosteric modulators (NAMs), which either act as
open-channel blockers by binding to the pore or inhibit
activation allosterically; and 4) silent allosteric modulators
(SAMs) that have no effect on orthosteric agonist responses
but block allosteric modulation (Fig. 7).
Since stimulation of a7 improves attention, cognitive per-
formance, and neuronal resistance to injury in addition to
eliciting robust analgesic and anti-inflammatory effects, a7
potentiation has emerged as a potential therapeutic strategy,
and the search for novel potentiators is an active research
field. The potential therapeutic use of several a7 partial
agonists and PAMs on animals and humans has been docu-
mented in several recent reviews (e.g., Wallace and Porter,
2011; Thomsen and Mikkelsen, 2012; Lendvai et al., 2013;
Dineley et al., 2015) (Table 1). Still, no drug has reached phase
III clinical stage.
Compared with agonists, PAMs are promising therapeutic
tools because they: 1) better maintain the temporal spatial
characteristics of endogenous activation; 2) show higher
selectivity, since the orthosteric site is more conserved among
nAChRs than allosteric sites (Yang et al., 2012); 3) allow
greater diversity in structure and final effects; 4) reduce
tolerance attributable to a7 desensitization; and 5) act as
neuronal protectors (Kalappa et al., 2013; Sun et al., 2013;
Uteshev, 2014). In particular, because neuronal damage
elevates choline levels near the site of injury, the presence of
a PAM may not require coapplication of an agonist for the
mediation of a local neuroprotective effect (Uteshev, 2014).
Therefore, it is increasingly accepted that targeting allosteric
sites can provide novel medications with greater structural
diversity and specificity.
PAMs have been classified on the basis of their macroscopic
effects as type I or type II (Fig. 8). Type I PAMs mainly
enhance agonist-induced peak currents without significantly
affecting current decay and do not reactivate desensitized
receptors, whereas type II PAMs delay desensitization and
reactivate desensitized receptors (Bertrand and Gopalakrishnan,
2007; Arias and Bouzat, 2010; Williams et al., 2011b). The
ratio of the changes in net charge/peak current induced by
type I PAMs is close to one, whereas it is higher than one in
the presence of type II PAMs (Andersen et al., 2016; Williams
et al., 2011c).
Single-channel recordings provide an invaluable tool for un-
derstanding the foundations of these macroscopic effects. In
the presence of either type I or type II PAMs, ACh-activated a7
channels show prolonged open durations and appear in longer
activation episodes, revealing that both PAM types affect
activation kinetics (Andersen et al., 2016) (Fig. 8). The most
efficacious PAM to date is PNU-120596, a type II PAM (Hurst
et al., 2005). This compound elicits significantly prolonged
openingsthatappeargroupedinbursts,whichinturncoalesce
into long activation periods of several seconds, referred to as
clusters (daCosta et al., 2011, 2015; Williams et al., 2011b;
Pałczy
nska et al., 2012; Andersen et al., 2016). However, the
Fig. 6. a7 single-channel and macroscopic currents. (A) Typical whole-cell
currents elicited by different ACh concentrations (from 10 to 1000 mM)
from cells transfected with human a7. At left, a schematic representation
of a macroscopic current with the measured parameters. Both the maximal
current (peak) and the net charge (net) can be used to construct dose-
response curves as shown in the figure. (B) Single-channel activity from
cell-attached patches of cells expressing human a7 appears as very brief
(∼0.1–0.3 milliseconds) and isolated openings or less often as short bursts.
Channel openings are shown as upward deflections. Membrane potential,
–70 mV; filter, 9 kHz. At right, a typical open duration histogram (Bouzat
et al., 2008).
a7 Modulation 293
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M254
F456
I281
V288
S223
C460
A226 TM1
TM2 TM3 TM4
Extracellular pockets
Intrasubunit cavity
Pore
PAMs
NAMs
SAMs
Allosteric agonists
NAMs
PAMs
MODULATORY SITES
PNU-120596
Open-channel blockers
Fig. 7. Allosteric modulatory sites in a7. Structural model of the a7 receptor viewed from the side representing several potential sites for allosteric
modulators. At the TMD, the intrasubunit transmembrane cavity is a site for a great variety of compounds that may elicit different pharmacological
effects (potentiation (PAMs), inhibition (NAMs), no effect (SAMs), or activation (allosteric agonists)). PNU-120596 docked into this cavity is shown. Open-
channel blockers inhibit response by transiently blocking the flux of ions through the pore. At the ECD, different potential sites for inhibitors and
potentiators have been proposed (Ludwig et al., 2010; Spurny et al., 2015).
TABLE 1
a7 modulators with potential clinical applications.
Drug Potential Therapeutic Effects and Uses References
Agonists GTS-21/DMXB-A Cognitive disorders, schizophrenia, Alzheimer
disease, attention deficit hyperactivity disorder Olincy et al. (2006)
Pain van Westerloo et al. (2006)
Inflammatory processes Vukelic et al. (2013)
Terry et al. (2015)
AR-R17779 Cognitive disorders Levin et al. (1999)
Inflammatory processes Hashimoto et al. (2014)
Atherosclerotic vascular diseases Van Kampen et al. (2004)
PNU-282987 Cognitive disorders Hajós et al. (2005)
Inflammatory processes Terry et al. (2015)
SSR180711 Cognitive disorders Pichat et al. (2007)
Terry et al. (2015)
ABBF Cognitive disorders Boess et al. (2007)
Terry et al. (2015)
EVP-6124 Cognitive disorders Prickaerts et al. (2012)
Terry et al. (2015)
TC-5619 Cognitive disorders Hauser et al. (2009)
Terry et al. (2015)
RG3487 Cognitive and sensorimotor gating disorders Wallace et al. (2011)
Terry et al. (2015)
PHA-568487 Cognitive disorders Karamihalev et al. (2014)
Terry et al. (2015)
CP-810123 Cognitive disorders O’Donnell et al. (2010)
Terry et al. (2015)
AZD0328 Cognitive disorders Sydserff et al. (2009)
Terry et al. (2015)
Tropisetron Cognitive disorders Shiina et al. (2010)
Hashimoto, (2015)
Terry et al. (2015)
ABT-107 Cognitive disorders Bitner et al. (2010)
Neuroprotection Quik et al. (2015)
Terry et al. (2015)
JN403 Cognitive disorders Feuerbach et al. (2007, (2009)
Anticonvulsive
Pain
Type I PAMs Genistein Neuroprotection Menze et al. (2015, 2016)
Memory disorders Terry et al. (2015)
NS-1738 Cognitive disorders Timmermann et al. (2007)
Terry et al. (2015)
AVL-3288 Cognitive disorders Ng et al. (2007)
Terry et al. (2015)
Galantamine Cognitive disorders Nikiforuk et al. (2015)
Alzheimer disease Terry et al. (2015)
Type II PAMs PNU-120596 Cognitive disorders Nikiforuk et al. (2015)
Inflammatory processes Callahan et al. (2013)
Terry et al. (2015)
PAM-2 Cognitive disorders Targowska-Duda et al. (2016)
Pain Bagdas et al. (2015)
Inflammatory processes
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single-channel profile in the presence of a weaker type II PAM,
PAM-2 (3-furan-2-yl-N-p-tolyl-acrylamide) (Arias et al., 2011),
more closely resembles that of type I 5-HI or NS-1738 PAMs
than that of type II PNU-120596 (Fig. 8). Thus, when analyzed
at the molecular level, potentiation is more complex than
initially believed. Moreover, PAMs showing macroscopic in-
termediate type I/II properties were proposed (Dunlop et al.,
2009; Malysz et al., 2009; Dinklo et al., 2011; Sahdeo et al.,
2014; Chatzidaki and Millar, 2015). Therefore, the classifica-
tion of type I and type II appears to be an oversimplification
resulting mainly from macroscopic observations, and a more
thorough classification may be required.
It is a generally accepted statement that type II PAMs may
increase the energetic barrier for desensitization, which allows
successive opening/closing events (daCosta et al., 2011) and/or
reversal of some forms of agonist-induced desensitization
(Williams et al., 2011b). Also, it has been proposed that PNU-
120596 binds predominantly to a fast desensitized state and
induces a set of conformations in which the opening of the pore
is energetically more favorable (Szabo et al., 2014). On the other
hand, only the decrease inthe energetic barrier for opening has
been proposed as the underlying mechanism for enhancement
of peak currents by type I PAMs (Williams et al., 2011b; Hurst
et al., 2013). Such a decrease might explain the appearance of
bursts of openings owing to rapid reopening of the closed
channel. However, it would only explain the increase in open-
channel duration if reopening were so fast that the associated
brief closings could not be detected, thus making openings ap-
pear longer. Alternatively, the increase in open duration could
be the result of either slight changes in desensitization that are
not detectable from whole-cell macroscopic currents or to the
induction of different open states. Thus, there seems to be more
than one mechanism by which PAMs prolong open-channel
lifetime and activation episodes.
a7 PAM potentiation is particularly dependent on temper-
ature. For PNU-120596, such dependence is revealed by
decreased potentiated macroscopic currents (Dunlop et al.,
2009; Williams et al., 2012) and the absence of long clusters
(Andersen et al., 2016) at physiologic temperature compared
with room temperature. Thus, for better extrapolation to the
in vivo situation, in vitro studies should be carried out at
physiologic temperatures.
a7 Allosteric Binding Sites
Computational studies (Dey and Chen, 2011), electrophysio-
logical studies from mutant or chimeric receptors (Bertrand
et al., 2008; Young et al., 2008; Collins et al., 2011; daCosta and
Sine, 2013), crystallographic studies, (Spurny et al., 2015), and
NMR studies (Bondarenko et al., 2014) have suggested the
existence of several allosteric binding sites, some of which are
common to other pLGICs (Sauguet et al., 2014).
The Intrasubunit Transmembrane Cavity. In silico
and electrophysiological studies show that several PAMs, in-
cluding LY-2087101, PNU-120596, and TQS bind to an intra-
subunit transmembrane cavity (Young et al., 2008) (Fig. 7). An
a7 receptor with mutations at five residues lining this cavity
was not potentiated by PNU-120596, PAM-2, or type I PAM
NS-1738, indicating common structural determinants for
their potentiation (daCosta et al., 2011; Andersen et al., 2016).
The macrocyclic lactone ivermectin (a type I PAM) also appears
to bind in close proximity to this intrasubunit site (Collins and
Millar, 2010), although it binds at an intersubunit transmem-
brane site in the glutamate-activated Cys-loop receptor (Hibbs
and Gouaux, 2011).
This cavity is also the site for allosteric agonists that me-
diate a7 activation in the absence of an orthosteric agonist
(Gill et al., 2011, 2012, 2013; Pałczy
nska et al., 2012). Single-
channel activity of a7 in the presence of allosteric agonists
resembles that of the receptor in the presence of ACh and a
PAM. Both are characterized by long bursts instead of isolated
brief ACh-elicited openings, indicating activation with signif-
icantly reduced desensitization (Pałczy
nska et al., 2012).
Moreover, a7-selective allosteric modulators showing subtle
structural changes and displaying distinct pharmacological
effects (typical of type I PAMs, type II PAMs, NAMs, SAMs,
and allosteric agonists) may bind to this common site (Gill
et al., 2013; Gill-Thind et al., 2015). Thus, the intrasubunit trans-
membrane cavity appears to be a promiscuous binding site
with a strategic location for allosteric modulation. Further-
more, this cavity may be a common modulatory site within the
pLGIC superfamily from which a large variety of different
compounds that bind to and mediate a great spectrum of
allosteric effects (Corradi et al., 2011; Nury et al., 2011;
Jayakar et al., 2013; Sauguet et al., 2014).
Fig. 8. Macroscopic and single-channel current profiles of human a7in
the presence of typical type I and type II PAMs. Macroscopic current
profiles have been used to classify PAMs into type II (i.e., PNU-120596 and
PAM-2), which increase the decay time constant, or type I, which only
increase the peak current (5-HI and NS-1738). Left: Macroscopic currents
elicited by ACh in the absence (black) and presence of the specified PAM
(gray traces). Right: Traces of 50–100 mM ACh-activated single a7
channels in the absence or in the presence of 1 mM PNU-120596, 5 mM
PAM-2, 2 mM 5-HI, 10 mM NS-1738. Membrane potential: –70 mV.
Channel openings are shown as upward deflections. All PAMs enhance
open channel lifetime and elicit activation episodes formed by successive
opening events.
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Extracellular Allosteric Binding Sites. The putative
binding site of galantamine, an a7 potentiator, is located at
the outer surface of the ECD in the vicinity of the ACh site
(Hansen and Taylor, 2007; Ludwig et al., 2010). Three
different allosteric sites in the ECD of the a7/AChBP chimera
were also identified by X-ray crystallography (Spurny et al.,
2015; Fig. 7). Although all the allosteric binders behaved on
human a7 as negative allosteric modulators, it was proposed
that their chemical modification could lead to a change in
functional activity.
Binding to potential ECD sites has been proposed for the
type I PAMs 5-HI and NS-1738, although other reports
support a TMD location (Placzek et al., 2004; Bertrand et al.,
2008; Hu and Lovinger, 2008; Gronlien et al., 2010; Collins
et al., 2011; Andersen et al., 2016). A virtual screening re-
vealed that some PAMs that bind to the TMD, such as PNU-
120596 and TQS, also dock into potential allosteric sites at
the ECD (Dey and Chen, 2011). Therefore, for any given PAM,
multiple binding sites or domains may be involved in the
conformational changes associated with potentiation, which
could account for these controversial results. Also, until the
location of an allosteric binding site is unequivocally defined,
it is advisable to refer to structural determinants of potentia-
tion instead of a binding site.
Despite the large body of experimental evidence supporting
a7 potentiation as a promising therapeutic strategy, there are
still many unsolved challenges: 1) Potentiation by exogenous
agonists may inhibit a7 response owing to desensitization.
Thus, PAMs might have therapeutic benefits in situations
where stronger agonist responses are desirable. 2) Given the
presence of other nAChRs and homologous receptors, high
PAM selectivity is required. However, PAMs targeting multi-
ple receptors might show better efficacy (Iturriaga-Vásquez
et al., 2015; Möller-Acuña et al., 2015). 3) Excessive receptor
activation, particularly with efficacious nondesensitizing
PAMs, might lead to cytotoxicity, which is an issue of concern
and controversy (Ng et al., 2007; Liu et al., 2009; Williams
et al., 2012; Guerra-Álvarez et al., 2015; Uteshev, 2016). 4)
The ubiquitous distribution of a7 and its interplay with
different signal pathways could make the cell response to a
given PAM variable among cell types or conditions. Given the
broad spectrum of effects and molecular mechanisms of PAMs,
it is probable that each patient or pathologic situation could
require a unique PAM.
Concluding Remarks
a7 has emerged as an important drug target for improving
cognition and memory in several neuropsychiatric disorders,
and as a target for inflammatory processes. a7 is unique owing
to its high calcium permeability and fast desensitization, and
it behaves as a ACh-sensitive sensor harboring a built-in
filtering mechanism against excessive stimulation. Transient
calcium responses are further sustained by the release of
calcium from intracellular sources, and several signaling
pathways are also activated because a7 has a dual ionotropic/
metabotropic nature. Its ubiquitous location and pleiotropic
effects make a7 an interesting but complex drug target. A bet-
ter understanding of the molecular basis underlying allosteric
modulation and its wide spectrum of effects, as well as the
availability of high resolution structures of a7, will help in the
rational design of therapeutics for the receptor.
Acknowledgments
ASPET thanks Dr. Katie Strong for copyediting of this article.
Authorship Contributions
Wrote or contributed to the writing of the manuscript: Bouzat,
Corradi.
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