Rhesus monkey alpha7 nicotinic acetylcholine receptors: comparisons to human alpha7 receptors expressed in Xenopus oocytes.
ABSTRACT An alpha7 nicotinic acetylcholine receptor sequence was cloned from Rhesus monkey (Macaca mulatta). This clone differs from the mature human alpha7 nicotinic acetylcholine receptor in only four amino acids, two of which are in the extracellular domain. The monkey alpha7 nicotinic receptor was characterized in regard to its functional responses to acetylcholine, choline, cytisine, and the experimental alpha7-selective agonists 4OH-GTS-21, TC-1698, and AR-R17779. For all of these agonists, the EC(50) for activation of monkey receptors was uniformly higher than for human receptors. In contrast, the potencies of mecamylamine and MLA for inhibiting monkey and human alpha7 were comparable. Acetylcholine and 4OH-GTS-21 were used to probe the significance of the single point differences in the extracellular domain. Mutants with the two different amino acids in the extracellular domain of the monkey receptor changed to the corresponding sequence of the human receptor had responses to these agonists that were not significantly different in EC(50) from wild-type human alpha7 nicotinic receptors. Monkey alpha7 nicotinic receptors have a serine at residue 171, while the human receptors have an asparagine at this site. Monkey S171N mutants were more like human alpha7 nicotinic receptors, while mutations at the other site (K186R) had relatively little effect. These experiments point toward the basic utility of the monkey receptor as a model for the human alpha7 nicotinic receptor, albeit with the caveat that these receptors will vary in their agonist concentration dependency. They also point to the potential importance of a newly identified sequence element for modeling the specific amino acids involved with receptor activation.
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ABSTRACT: Voltage-clamp techniques are typically used to study the plasma membrane proteins, such as ion channels and transporters that control bioelectrical signals. Many of these proteins have been cloned and can now be studied as potential targets for drug development. The two approaches most commonly used for heterologous expression of cloned ion channels and transporters involve either transfection of the genes into small cells grown in tissue culture or the injection of the genetic material into larger cells. The standard large cells used for the expression of cloned cDNA or synthetic RNA are the egg progenitor cells (oocytes) of the African frog, Xenopus laevis. Until recently, cellular electrophysiology was performed manually by a single operator, one cell at a time. However, methods of high throughput electrophysiology have been developed which are automated and permit data acquisition and analysis from multiple cells in parallel. These methods are breaking a bottleneck in drug discovery, useful in some cases for primary screening as well as for thorough characterization of new drugs. Increasing throughput of high-quality functional data greatly augments the efficiency of academic research and pharmaceutical drug development. Some examples of studies that benefit most from high throughput electrophysiology include pharmaceutical screening of targeted compound libraries, secondary screening of identified compounds for subtype selectivity, screening mutants of ligand-gated channels for changes in receptor function, scanning mutagenesis of protein segments, and mutant-cycle analysis. We describe here the main features and potential applications of OpusXpress, an efficient commercially available system for automated recording from Xenopus oocytes. We show some types of data that have been gathered by this system and review realized and potential applications.Combinatorial chemistry & high throughput screening 12/2009; 12(1):38-50. · 2.46 Impact Factor
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ABSTRACT: Nicotinic acetylcholine receptors (nAChRs) that contain α6 and β4 subunits have been demonstrated functionally in human adrenal chromaffin cells, rat dorsal root ganglion neurons, and on noradrenergic terminals in the hippocampus of adolescent mice. In human adrenal chromaffin cells, α6β4* nAChRs (the asterisk denotes the possible presence of additional subunits) are the predominant subtype whereas in rodents, the predominant nAChR is the α3β4* subtype. Here we present molecular and pharmacological evidence that chromaffin cells from monkey (Macaca mulatta) also express α6β4* receptors. PCR was used to show the presence of transcripts for α6 and β4 subunits and pharmacological characterization was performed using patch-clamp electrophysiology in combination with α-conotoxins that target the α6β4* subtype. Acetylcholine-evoked currents were sensitive to inhibition by BuIA[T5A,P6O] and MII[H9A,L15A]; α-conotoxins that inhibit α6-containing nAChRs. Two additional agonists were used to probe for the expression of α7 and β2-containing nAChRs. Cells with currents evoked by acetylcholine were relatively unresponsive to the α7-selctive agonist choline but responded to the agonist 5-I-A-85380. These studies provide further insights into the properties of natively expressed α6β4* nAChRs.PLoS ONE 01/2014; 9(4):e94142. · 3.53 Impact Factor
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ABSTRACT: OBJECTIVE Deficient cerebral inhibition is a pathophysiological brain deficit related to poor sensory gating and attention in schizophrenia and other disorders. Cerebral inhibition develops perinatally, influenced by genetic and in utero factors. Amniotic choline activates fetal α7-nicotinic acetylcholine receptors and facilitates development of cerebral inhibition. Increasing this activation may protect infants from future illness by promoting normal brain development. The authors investigated the effects of perinatal choline supplementation on the development of cerebral inhibition in human infants. METHOD A randomized placebo-controlled clinical trial of dietary phosphatidylcholine supplementation was conducted with 100 healthy pregnant women, starting in the second trimester. Supplementation to twice normal dietary levels for mother or newborn continued through the third postnatal month. All women received dietary advice regardless of treatment. Infants' electrophysiological recordings of inhibition of the P50 component of the cerebral evoked response to paired sounds were analyzed. The criterion for inhibition was suppression of the amplitude of the second P50 response by at least half, compared with the first response. RESULTS No adverse effects of choline were observed in maternal health and delivery, birth, or infant development. At the fifth postnatal week, the P50 response was suppressed in more choline-treated infants (76%) compared with placebo-treated infants (43%) (effect size=0.7). There was no difference at the 13th week. A CHRNA7 genotype associated with schizophrenia was correlated with diminished P50 inhibition in the placebo-treated infants, but not in the choline-treated infants. CONCLUSIONS Neonatal developmental delay in inhibition is associated with attentional problems as the child matures. Perinatal choline activates timely development of cerebral inhibition, even in the presence of gene mutations that otherwise delay it.American Journal of Psychiatry 01/2013; · 14.72 Impact Factor
Rhesus monkey α7 nicotinic acetylcholine receptors: Comparisons to human
α7 receptors expressed in Xenopus oocytes
Roger L. Papkea,⁎, Thomas J. McCormacka, Brian A. Jacka, Daguang Wangb,
Bozena Bugaj-Gawedab, Hillary C. Schiffa, Joshua D. Buhra, Amanda J. Wabera, Clare Stokesa
aDepartment of Pharmacology and Therapeutics, 100267 JHMHSC, 1600 SW Archer Rd., University of Florida, College of Medicine, Gainesville, FL 32610, USA
bMemory Pharmaceuticals Corp., 100 Philips Parkway, Montvale, NJ 07645, USA
Received 12 May 2005; received in revised form 22 August 2005; accepted 29 August 2005
An α7 nicotinic acetylcholine receptor sequence was cloned from Rhesus monkey (Macaca mulatta). This clone differs from the mature
human α7 nicotinic acetylcholine receptor in only four amino acids, two of which are in the extracellular domain. The monkey α7 nicotinic
receptor was characterized in regard to its functional responses to acetylcholine, choline, cytisine, and the experimental α7-selective agonists
4OH-GTS-21, TC-1698, and AR-R17779. For all of these agonists, the EC50for activation of monkey receptors was uniformly higher than for
human receptors. In contrast, the potencies of mecamylamine and MLA for inhibiting monkey and human α7 were comparable. Acetylcholine and
4OH-GTS-21 were used to probe the significance of the single point differences in the extracellular domain. Mutants with the two different amino
acids in the extracellular domain of the monkey receptor changed to the corresponding sequence of the human receptor had responses to these
agonists that were not significantly different in EC50from wild-type human α7 nicotinic receptors. Monkey α7 nicotinic receptors have a serine at
residue 171, while the human receptors have an asparagine at this site. Monkey S171N mutants were more like human α7 nicotinic receptors,
while mutations at the other site (K186R) had relatively little effect. These experiments point toward the basic utility of the monkey receptor as a
model for the human α7 nicotinic receptor, albeit with the caveat that these receptors will vary in their agonist concentration dependency. They
also point to the potential importance of a newly identified sequence element for modeling the specific amino acids involved with receptor
© 2005 Elsevier B.V. All rights reserved.
Keywords: Voltage-clamp; Alzheimer's disease; Acetylcholine binding protein; α7 nicotinic receptor
The α-bungarotoxin sensitive α7-type nicotinic acetyl-
choline receptor is expressed throughout the brain and also in
the peripheral nervous system and some peripheral tissues
(Sharma and Vijayaraghavan, 2002). In the brain, α7nicotinic
acetylcholine receptor are located in high concentrations in the
hippocampus, neocortex, and hypothalamus as seen by binding
sites (Clarke et al., 1985). The use of nicotinic agonists has
improved delay matching in primates (Terry et al., 2002), eye
blink memory in rabbits, and spatial-memory related behavior
in rats, as well as social memory relationships in rats (Arendash
et al., 1995a,b; Meyer et al., 1994; Van Kampen et al., 2004).
Some mutations and/or splice variants of the α7 gene have been
linked to a decrease in hippocampal auditory gating, which is
a symptom of some schizophrenics and approximately 50% of
their family members (Freedman et al., 1994, 2000). This may
be due to the roles played by α7nicotinic acetylcholine receptor
in the activation of GABAergic inhibitory interneurons in
hippocampus (Adler et al., 1998; Frazier et al., 2003). The α7
nicotinic receptor may also be involved with the etiology and/or
possible treatment of other conditions such as Alzheimer's
Disease and Down's Syndrome. Nicotinic receptor agonists
have been shown to improve memory and are neuroprotective.
Moreover, α7 has been found to coprecipitate with the Aβ1-42
within the histopathological amyloid beta plaques (Wang et al.,
2000) and the functional interactions between Aβ1-42and α7
European Journal of Pharmacology 524 (2005) 11–18
⁎Corresponding author. Tel.: +1 352 392 4712; fax: +1 352 392 9696.
E-mail address: firstname.lastname@example.org (R.L. Papke).
0014-2999/$ - see front matter © 2005 Elsevier B.V. All rights reserved.
nicotinic acetylcholine receptor (Liu et al., 2001) further support
α7 nicotinic acetylcholine receptor as a therapeutic target for
The adverse side effects of nicotine or other non-selective
cholinergic agonists have promoted the development of more
selective α7agonists for therapeutics. GTS-21 (2,4-dimethoxy-
benzylidene anabaseine or DMBX), one such selective agonist
was tested in phase 1 clinical trials and was found to have no
adverse side effects and to increase cognitive functioning in
healthy subjects (Kitagawa et al., 2003). With the identification
of α7 nicotinic receptors as potential therapeutic targets has also
come the need to develop animal models for the testing of novel
therapeutic agents. While rodent models are most commonly
used, there are numerous pharmacological differences between
rat and human α7 nicotinic receptors (Papke and Papke, 2002).
Monkey models have the intrinsic advantage of being amenable
to more complex behavioral testing than rodents and therefore
may have special usefulness for evaluating potential drugs for
human therapeutics. We report the cloning and functional
characterization of a Rhesus monkey (Macaca mulatta) α7
nicotinic acetylcholine receptor (mkα7) in regard to its
responses to a series of nicotinic agonists including acetyl-
choline (acetylcholine), choline, and cytisine, as well as the α7-
selective agonists 4OH-GTS-21 (4-hydroxy 2-methoxybenzy-
lidene anabaseine), TC-1698 (2-(3-pyridyl)-1-azabicyclo[3.2.2]
nonane) and AR-R17779 ((−)-spiro[1-azabicyclo[2.2.2]octane-
3′,5′-oxazolidin-2′-one) (Marrero et al., 2003; Meyer et al.,
1998; Papke et al., 2004). These agonists had lower EC50s for
activating human α7 (hα7) nicotinic receptors than for
monkey α7(mkα7) nicotinic receptors. Only two amino acids
differ between the mkα7 and hα7 sequence in the extracellular
domain. Mutations were made of mkα7 sequence to the amino
acids present in hα7, and the resulting mutants were tested to
determine if changing either or both of the amino acids shifted
the concentration–response relationships towards that of hα7.
2.1. Rapid amplification of cDNA ends (RACE) for the 5′- and,
3′-ends of rhesus monkey α7
To identify the 5′- and 3′-ends of rhesus monkey α7,
GGTGCAG), mkα7-5′N (CGCACCTTATCCTCTCCCGGC-
CTCTTCATG), mkα7-3′R (CATGAAGAGGCCGGGAGA-
GGATAAGGTGCG) and mkα7-3′N (CTGCACCTGGCCA-
GCGTGGAGATGAG), were designed based on Genbank
sequence AJ245976 and a polymerase chain reaction (PCR)
was used with a Gene Racer cDNA library generated using
rhesus monkey brain mRNA (Biochain). The cDNA frag-
ments from the nested PCR were cloned and sequenced. The
3′ fragment contained a stop codon (TAA) and a polyA+
signal (AATAAA) indicating the end of the transcript. The 5′
fragment extended further upstream of AJ245976, however,
failed to reach the starting Met. Additional primers were
designed based on new sequence information and used for the
second round of 5′ RACE. After the third 5′ RACE, the
resulting cDNA fragment contained a Met and an in-frame
stop codon, suggesting identification of the starting Met.
Additional 5′ RACE primers: mkα7-5′R1 (GACCAGCCTC-
CATAAGACCAGGATCCAAACTTCAG), mkα7-5′N1 (CG-
5′R2 (GGTTCTTCTCATCCGCGTCCATGATCTGCAG), and
2.2. Full-length cloning of rhesus monkey α7
A cDNA contig of rhesus monkey α7was built by
combining the sequences of the 5′ and 3′ RACE fragments.
Two primers spanning the coding sequence of the monkey α7,
a 5′ primer CTCAACATGCGCTGCTCGCAGGGAGG and
a 3′ primer CCAAGCCAGAGGCCTTGCCCATCTGTGAG,
were designed based on the contig and were used to PCR
a monkey brain cDNA library. The resulting PCR product
was cloned into pcDNA3.1 TOPO vector (Invitrogen) and
confirmed by sequencing.
The full length clone we isolated has a predicted amino acid
sequence that is 100% identical to that of the unpublished
Genbank M. mulatta sequence AF486623, although differing
by a total of 5 nucleotides in the open reading frame.
2.3. Sequence comparisons and selection of mutations
Two differences were noted within the extracellular region
of the rhesus monkey α7 and human α7. Numbering the
amino acids as for human α7 (vicinal cysteines at positions
190 and 191), these differing residues were located at positions
171 and 186. Two single point mutants were made in mkα7
(mkα7S171N and mkα7K186R) as well as the double mutant
(mkα7S171N,K186R). Mutants were constructed using the
QuikChange site-directed mutagenesis kit (Stratagene, La
Jolla, CA, USA) according to the manufacturer's instructions,
and were confirmed by automated fluorescent sequencing
(University of Florida ICBR core facility, Gainesville, FL,
2.4. Preparation of RNA
The hα7 clone was obtained from Dr. Jon Lindstrom
(University of Pennsylvania), and the M. mulatta (rhesus) α7
cloned as described above. After linearization and purification
of cDNA templates, RNA was prepared using the appropriate
mMessage mMachine kit from Ambion, Inc. (Austin, TX,
USA), according to the manufacturer's instructions.
2.5. Expression in Xenopus oocytes
The preparation of Xenopus laevis oocytes for RNA
expression was conducted as previously described (Papke and
Papke, 2002). In brief, mature (N9 cm) female X. laevis African
frogs (Nasco, Ft. Atkinson, WI) were used as a source of
oocytes. Prior to surgery, the frogs were anesthetized by placing
the animal in a 1.5g/l solution of MS222 (3-aminobenzoic acid
12 R.L. Papke et al. / European Journal of Pharmacology 524 (2005) 11–18
ethyl ester) for 30 min. Oocytes were removed from an incision
made in the abdomen.
In order to remove the follicular cell layer, harvested oocytes
were treated with 1.25 mg/ml Type 1 collagenase (Worthington
Biochemical Corporation, Freehold, NJ) for 2 h at room
temperature in calcium-free Barth's solution (88 mM NaCl,
1 mM KCl, 0.33 mM MgS04, 2.4 mM NaHCO3, 10 mM
HEPES (pH 7.6), 50 mg/l gentamicin sulfate). Subsequently,
stage 5 oocytes were isolated and injected with 50 nl (5–20 ng)
each of the appropriate subunit cRNAs. Recordings were made
5 to 15 days after injection.
The source of the 4OH-GTS-21 was Taiho Pharmaceuticals
(Tokyo, Japan) and TC-1698 was provided by Targacept
(Winston-Salem, NC). All other chemicals were obtained from
Sigma Chemical Co. (St. Louis, MO) with the exception of AR-
R17779 which was synthesized and supplied by Memory
Experiments were conducted using OpusXpress 6000A
(Axon Instruments, Union City, CA, USA). OpusXpress is an
integrated system that provides automated impalement and
voltage clamp of up to eight oocytes in parallel. Both the
voltage and current electrodes were filled with 3 M KCl.
Cells were voltage-clamped at a holding potential of −60
mV. Data were collected at 50 Hz and filtered at 20 Hz. Cells
were bath-perfused with Ringer's solution, and agonist
solutions were delivered from a 96-well plate via disposable
tips, which eliminated any possibility of cross-contamination.
Flow rates were set at 2 ml/min. Drug applications alternated
between acetylcholine controls and experimental agonists.
Applications were 12 s in duration followed by 181-s washout
2.8. Experimental protocols and data analysis
Responses were calculated as net charge (Papke and
Papke, 2002). Except where noted, each oocyte received two
initial control applications of 300 μM acetylcholine, then an
experimental drug application, and then a follow-up control
application of 300 μM acetylcholine, a concentration which
is sufficient to evoke a maximal net charge response (Papke
and Papke, 2002). Responses to experimental drug appli-
cations were calculated relative to the preceding acetylcholine
control responses in order to normalize the data, com-
pensating for the varying levels of channel expression among
the oocytes. Means and standard errors (S.E.M.) were
calculated from the normalized responses of at least four
oocytes for each experimental concentration. For concen-
tration–response relations, data derived from net charge
analyses were plotted using Kaleidagraph 3.0.2 (Abelbeck
Software, Reading, PA), and curves were generated from the
where Imax denotes the maximal response for a particular
agonist/subunit combination, and n represents the Hill
coefficient. Imax, n, and the EC50were all unconstrained for
the fitting procedures, except in the case of the acetylcholine
response curves. Since acetylcholine is our reference full
Fig. 1. Protein sequence comparison between human and monkey α7.The sequence alignment was created with Lasergene 6 software using ClustalV. The identical
amino acids were boxed. The predicted signal peptide cleavage site was indicated by an arrow head. The predicted transmembrane helices were shaded and labeled as
M1, M2, M3 and M4.
13 R.L. Papke et al. / European Journal of Pharmacology 524 (2005) 11–18
agonist, for the acetylcholine concentration response curves
the data were normalized to the observed acetylcholine
maximum and the Imaxof the curve fits were constrained to
equal one. For the evaluation of antagonists the control
acetylcholine concentration used were the EC50 values of 30
μM and 70 μM for human and monkey α7, respectively.
Significant differences between responses of the wild-type
human and monkey receptors were determined by t-tests on
the normalized data. Responses of the mutant receptors were
compared in separate t-tests to the responses of each of the
2.9. Molecular modeling
We created a structural model for the monkey and human α7
based on the structure of acetylcholine binding protein (PDB
entry 1I9B). The agonist (nicotine) was drawn in 3-dimensional
space using a text-editor and RASMOL version 2.6, adapted
from the PDB structure of nicotine. The superimposed
backbones of the rat and human receptor were staggered by
a small distance (roughly 0.2 Å) such that regions of both
backbones could be seen. The sidechains of residues within
loops C and F were oriented according to a local energy
minimization protocol on SwissPDB. The resultingfigures were
imported as a BMP files into Canvas 5.0 (Deneba Software,
3.1. The sequence of monkey α7 acetylcholine receptor
The cloned monkey α7 subunit is 502 amino acids long, with
a calculated molecular weight of 56.4 kDa. There are five amino
acid changes when compared to human α7 (Fig. 1). Two of the
changes are localized in the extracellular N-terminal domain
that involves in ligand binding; two more changes in the second
intracellular loop between transmembrane domain M3 and M4;
one more in the predicted signal peptide which is eliminated in
3.2. Electrophysiological responses of human and monkey α7
As shown in Fig. 2, both the human and monkey α7 nicotinic
receptors responded well to the relatively high acetylcholine
control concentrations. However while 30 μM acetylcholine
and 300 μM Choline evoked responses from human receptors
Fig. 2. (A) Representative responses of human (top) and monkey (bottom) α7
receptors expressed in Xenopus oocytes to applications of acetylcholine and
choline. Both receptors gave similar robust responses to the saturating 300 μM
acetylcholine control applications, however the relative responses of the human
receptors to 30 μM acetylcholine (top) and 300 μM choline (bottom) were much
greater than the responses of the monkey receptors. (B) Concentration response
relationships of human and monkey α7 to the endogenous ligands acetylcholine
and choline. For both acetylcholine (A) and choline (B), mkα7 is significantly
shifted to the right from hα7. Data were normalized to the net charge of control
300μM acetylcholine responses obtained 5 min before the experimental agonist-
evoked responses. Each point represents the average±S.E.M. of the normalized
responses of at least 4 oocytes. Since acetylcholine is our reference full agonist
the acetylcholine maximum responses were defined as 1 and the Imaxvalues for
the curve constrained to equal 1. Concentrations where the responses of the
monkey and human α7 receptors to the same concentration of agonist differed
significantly (pb.01) are indicated (*). (C) Kinetic parameters of human and
monkey α7 acetylcholine-evoked responses. As shown on the left, the 10–90%
rise time of acetylcholine evoked were several seconds long at relatively low
acetylcholine concentrations for both human and monkey α7 responses and
decreased to values less that a second at higher concentrations. Consistent with
the reduced potency of acetylcholine for monkey rise times of the human
responses to 30 μM acetylcholine were significantly faster than those of the
monkey receptors (pb0.05) to the same acetylcholine concentration. However,
the rise times of the monkey responses to 100 μM acetylcholine were not
significantly different from the rise times of human responses to 30 μM
acetylcholine. Similar results were apparent in the 90–50% fall times (right).
Decay times of the human responses to 100 μM acetylcholine were significantly
briefer than those of the monkey receptors (pb0.01) to the same acetylcholine
concentration, while the decay times of the monkey responses to 300 μM
acetylcholine were not significantly different from the decay times of human
receptor responses to 100 μM acetylcholine.
14R.L. Papke et al. / European Journal of Pharmacology 524 (2005) 11–18
that had approximately 50% the net charge of the 300 μM
acetylcholine controls, the relative responses of monkey
receptors were much lower. Fig. 2B shows the net charge
concentration response relationships of wild-type human and
monkey α7 to the endogenous agonists acetylcholine and
choline. The EC50values for these agonists were lower for hα7
than for mkα7 (Fig. 2B) and there were also differences in Hill
slope (Table 1). We evaluated a series of experimental agonists;
cytisine, 4OH-GTS-21, TC-1698, and AR-R17779, and in each
case, the EC50values were significantly higher for monkey α7
than for human α7(Fig. 3 and Table 1). Specifically, for mkα7
the EC50 values of the agonists tested were on the average
232±13% (S.E.M.) of those for hα7.
The kinetics of macroscopic responses mediated α7
nicotinic acetylcholine receptor are strongly affected by the
agonist concentration applied (Papke et al., 2000; Papke and
Papke, 2002; Papke and Thinschmidt, 1998; Uteshev et al.,
2002). Responses to high effective concentrations of agonist
are very brief, showing both rapid rise and rapid decay with the
most synchronous channel activation occurring during the
rising phase of solution exchange. As shown in Fig. 2C, the
rise time and decay times of the responses of monkey α7
nicotinic receptorsshow similar
concentration of agonist applied, with an appropriate shift
reflecting the reduced potency of acetylcholine for activating
monkey α7 nicotinic receptors compared to human α7nicotinic
Two antagonists, mecamylamine and MLA (methyllyca-
potency for inhibiting monkey and human α7 (Fig. 4). The
Mecamylamine IC50values were 3.3±0.6 μM and 3.0±0.5
μM for monkey and human receptors, respectively and the
MLA IC50values were 13.9±1.2 nM and 13. 3±6.5 nM for
monkey and human receptors, respectively.
3.3. Human and monkey sequence comparison
The sequences of the human and monkey α7 nicotinic
receptor N-terminal extracellular domains are shown in Fig. 5.
The proposed arrangement of helical and beta strands (Brejc et
al., 2001) is indicated, as well as the putative agonist binding
subdomains, including the proposed loops A–F (Corringer et
al., 2000). Only two amino acids differ throughout this entire
region of the mature protein. One of the point differences (186)
is located in the putative C-loop of the positive face of the
agonist binding site. The other point difference is near the F-
loop of the complementary or negative face of the agonist
binding site. This is the portion of the agonist binding site which
is associated with the non-alpha subunits in heteromeric
In addition to the two amino acids which differ in the
extracellular domain, there are also two amino acids differences
Fig. 3. Concentration response relationships of human and monkey α7 to
experimental agonists. For the agonists tested: A) cytisine, B) 4OH-GTS-21, C)
TC-1698, and D) AR-R17779, there is a significant potency shift to the right
from hα7 to mkα7. Data were normalized to the net charge of control 300μM
acetylcholine responses obtained 5 min before the experimental agonist-evoked
responses. Each point represents the average±S.E.M. of the normalized
responses of at least 4 oocytes. Concentrations where the responses of the
monkey and human α7 receptors to the same concentration of agonist differed
significantly (pb.01) are indicated (*).
Curve fit valuesafor wild-type human and monkey α7 nicotinic receptors
AgonistHuman receptor Monkey receptor
aError estimates represent the 95% confidence values from the curve fits.
bSince acetylcholine is defined as the reference full agonist, the maximal responses obtained with acetylcholine were defined as 1, and the curve fits for
acetylcholine were constrained to have Imaxequal to 1. For all other agonists, Imaxis relative to the saturating 300 μM acetylcholine control responses. Note that
n represents the Hill coefficients of the fits.
15 R.L. Papke et al. / European Journal of Pharmacology 524 (2005) 11–18
in the intracellular domain (not shown), but no differences in the
putative transmembrane domains.
To test the hypothesis that the sequence differences in the
extracellular domain would be most important for the agonist
activation differences seen, mutations were made (see methods)
in the monkey sequence to switch these residues to those of the
human sequence, either singly (mkα7S171N and mkα7K186R)
or in tandem (mkα7S171N,K186R).
As shown in Fig. 6A the mkα7S171N,K186R mutant was
not significantly different from hα7 in response to acetylcholine
application, supporting the hypothesis that the difference in
acetylcholine EC50could be attributed to the two single amino
acid differences between human and monkey sequences (see
also Table 2). Analysis of the single point mutants indicated that
the residue at 171 was potentially most important for the effects
observed (Fig. 6B), while the mutation at position 186 had no
apparent effect on the EC50 (Fig. 6C). We also evaluated
responses of the monkey mutants to the α7-selective partial
agonist 4OH-GTS-21. As shown in Fig. 6D, the double mutant
mkα7S171N,K186R was not significantly different from hα7in
was essentially like the wild-type monkey receptor in its
responses to 4OH-GTS-21 (Fig. 6F), the single point mutant
mkα7S171N was apparently more responsive to this partial
agonist than even the wild-type human α7 (Fig. 6E). It is
interesting to note that while the S171N mutation was effective
at shifting the concentration response curves to the left (Figs. 6B
and E), the curves were not identical to the wild-type human α7.
Human and monkey α7 differ significantly in their EC50
values for the endogenous agonists acetylcholine and choline,
as well as for the experimental agonists, cytisine, 4OH-GTS-21,
TC-1698, and AR-R17779. This basic observation should be
considered if the rhesus monkey is used as a model system to
test α7 agonists for human therapeutics. Specifically, our results
suggest that any nicotinic agonist targeting α7 may be less
potent in the monkey than it would be in humans, although
antagonist activities would be similar.
The observation that the double mutant mkα7S171N,K186R
does not differ significantly in EC50for the agonists tested
supports the hypothesis that the two amino acid differences in
the extracellular domain account for much of the functional
higher Hill coefficients for most agonists than the human
receptor. While the mutations do reduce the EC50's they
appeared to have less effect on the Hill coefficients. The reason
Fig. 4. Concentration response relationships of human and monkey α7 to the
antagonists mecamylamine (A) and MLA (B). For these experiments the control
acetylcholine concentration used were the EC50 values of 30 μM and 70 μM for
human and monkey α7, respectively. Data were normalized to the net charge of
the respective control acetylcholine responses obtained 5 min before the co-
application of acetylcholine and antagonist at the indicated concentration. Each
point represents the average±S.E.M. of the normalized responses of at least
4 oocytes. As indicated (*), monkey receptors responses to 70 μM acetylcholine
showed significantly less inhibition (pb.05) by 3 μM MLA than did the
responses of human α7 receptors to 30 μM acetylcholine.
Fig. 5. Sequence alignment of human and monkey α7 sequence in the extracellular domain. The proposed arrangement of helical and beta sheet structures are
indicated, as well as the putative agonist binding subdomains. Sites of sequence difference between human and monkey α7 are highlighted.
16 R.L. Papke et al. / European Journal of Pharmacology 524 (2005) 11–18
for this is unclear; however, in addition to the two extracellular
sites we investigated, there are two amino acids in the
intracellular portion of the α7 subunit which differ between
human and monkey. Specifically, human has a valine at position
373, where monkey has methionine, and human has valine also
at position 376, where monkey has alanine. These residues are
all considered to be hydrophobic and so the differences between
monkey and human are relatively conservative.
The data obtained with the S171N mutant indicates that this
site, which is somewhat outside of the canonical binding site as
defined by the hypothetical 6-loop structure (see Fig. 5), is
nonetheless important for agonist activation and/or binding. The
S171N is a semiconservative difference. However, the 171
residue is almost invariant among species in the α7 nicotinic
receptor, with an asparagine present in the α7 sequence of rat,
bovine, mouse, human, chimpanzee and zebra fish, making the
serine found in the rhesus monkey sequence rather unusual (see
for α7 subunit sequence accession numbers). Interestingly,
although the 171N residue is usually found in α7 subunits,
a serine at this site is about equally common in other subunits.
Residue 171 is located near the putative F-loop of the
complementary face of agonist binding domain. In a structural
model of the receptor this residue appears near one of two
Fig. 6. The effect of mutations on the responses of mkα7 to acetylcholine and
4OH-GTS-21. The responses of the double mutant (mkα7S171N,K186R) were
not significantly different from those of wild-type hα7 to either acetylcholine
(A) or 4OH-GTS-21 (D). The effects of the single point mutations are shown in
the lower panels. See Table 2 for curve fit values. Data were normalized to the
net charge of control 300μM acetylcholine responses obtained 5 min before the
experimental responses. Each point represents the average±S.E.M. of the
were compared to responses of each wild-type receptor. Concentrations at which
there were significant (pb.05) differences between mutant and human (*) and
between mutant and monkey (#) are indicated.
Curve fit valuesafor mutant monkey α7 nicotinic receptors
36±5.1 2.1±0.3 1b
74±9.1 3.0±1.0 1
Imax EC50μM nImax
4.7±0.2 1.9±0.1 0.38±0.01
10.0±0.2 4.2±1.7 0.24±0.01
25.5±2.3 1.9±0.3 1
22.5±2.0 2.8±0.6 1
85.6±2.9 2.3±0.2 1
3.5±2.7 7.1±3.7 0.35±0.01
3.5±0.4 3.8±2.2 0.44±0.1
10.5±2.8 1.5±0.5 0.20±0.02
aError estimates represent the 95% confidence values from the curve fits.
bSince acetylcholine is defined as the reference full agonist, the maximal
responses obtained with acetylcholine were defined as 1 and the curve fits for
acetylcholine were constrained to have Imaxequal to 1.
Fig. 7. Structural alignment of human and rhesus α7 nicotinic receptors showing
the interface between two contiguous subunits, as they would be arranged in the
pentamer. The human receptor backbone is black (positive face) and grey
(negative face) while the Rhesus receptor subunits are yellow. An agonist
(nicotine) is docked in the putative binding site and shown in spacefill format.
The amino acids which differ between human and rhesus α7 are represented as
“sticks” on the protein backbone models. The amino acids of the human
sequence are colored brown and Rhesus amino acids are colored orange.
Backbone structures are staggered by a small distance (0.2 A), so that both
protein backbones are visible. The 171 residue appears as part of the negative
(complementary) face, below the docked agonist. The 186 residue is in the C-
loop of the positive (primary) face and is shown above the nicotine molecule.
The human α7 structure was created using Jigsaw3d, and the rhesus α7 was
alignedtothe humanusingtheSwissModelsoftware package.(Forinterpretation
of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
17 R.L. Papke et al. / European Journal of Pharmacology 524 (2005) 11–18
possible entrance to the agonist binding site (Fig. 7) (Stokes et
The K/R substitution at residue 186 is a conservative
difference, and although this site is located in the C loop of the
agonist binding domain, it had relatively little effect on the
activation by acetylcholine or 4OH-GTS-21. This particular
residue is rather variable among the different nicotinic
acetylcholine receptor subunits, but is most often either arginine
It is lysine in the rat, bovine, and mouse α7 sequences, as well as
in the rhesus, but arginine in human, chimpanzee, and zebra fish
(Genbank: AY247962), although in chicken α7 it is serine.
The observations discussed above suggest that there would
have been a common ancestral sequence in early primates with
asparagine at residue 171 and lysine at 186. This is consistent
with the ancestral sequence as predicted by the PAML software
package (Yang, 1997). In this scenario, the two amino acid
differences between rhesus and humans in the α7 extracellular
domain would have arisen separately as those evolutionary lines
diverged. Additionally, the sequence identity present between the
R mutation at 186 occurred prior to the relatively recent human–
chimp divergence but after the divergence of the rhesus line.
In conclusion, our results bring to light an important
consideration for the use of the rhesus monkey as a model for
nicotinic receptor-based therapies related to agonist potency.
Additionally, further investigation of the amino acids around
171 in the α7 sequence may delineate the importance of this part
of the receptor for agonist activation and/or binding, improving
our models for this process. This may ultimately be useful for
thedevelopment of improved α7agonistsastherapeutics,and to
the more complete understanding of the mechanisms of α7-
related diseases such as Alzheimer's Disease and Schizophrenia.
This work was supported by NIH grant GM57481-01A2 and
Memory Pharmaceuticals. We thank Taiho Pharmaceuticals for
providing 4-OH-GTS-21 and Targacept for providing TC-1698.
We particularly thank Dr. Cathy Smith-Maxwell, and are very
grateful to Axon Instruments/Molecular Devices for the use of
an OpusXpress 6000A and pClamp9.1.
Adler, L.E., Olincy, A., Waldo, M., Harris, J.G., Griffith, J., Stevens, K., Flach,
K., Nagamoto, H., Bickford, P., Leonard, S., Freedman, R., 1998.
Schizophrenia, sensory gating, and nicotinic receptors. Schizophr. Bull.
Arendash, G.W., Sanberg, P.R., Sengstock, G.J., 1995a. Nicotine enhances the
learning and memory of aged rats. Pharmacol. Biochem. Behav. 52,
Arendash, G.W., Sengstock, G.J., Sanberg, P.R., Kem, W.R., 1995b. Improved
learning and memory in aged rats with chronic administration of the
nicotinic receptor agonist GTS-21. Brain Res. 674, 252–259.
Brejc, K., van Dijk, W.J., Klaassen, R.V., Schuurmans, M., van Der Oost, J.,
Smit, A.B., Sixma, T.K., 2001. Crystal structure of an ACh-binding protein
reveals the ligand-binding domain of nicotinic receptors. Nature 411,
Clarke, P.B.S., Schwartz, R.D., Paul, S.M., Pert, C.B., Pert, A., 1985. Nicotinic
binding in rat brain: autoradiographic comparison of [3H] acetylcholine [3H]
nicotine and [125I]-alpha-bungarotoxin. J. Neurosci. 5, 1307–1315.
Corringer, P.J., Le Novere, N., Changeux, J.P., 2000. Nicotinic receptors at the
amino acid level. Annu. Rev. Pharmacol. Toxicol. 40, 431–458.
Frazier, C.J., Strowbridge, B.W., Papke, R.L., 2003. Nicotinic acetylcholine
receptors on local circuit neurons in the dentate gyrus: a potential role in the
regulation of granule cell excitability. J. Neurophysiol. 89, 3018–3028.
Freedman, R., Adler, L., Bickford, P., Byerley, W., Coon, H., Cullum, C.,
Griffith, J., Harris, J., Leonard, S., Miller, C., 1994. Schizophrenia and
nicotinic receptors. Harv. Rev. Psychiatr. 2, 179–192.
Freedman, R., Adams, C.E., Leonard, S., 2000. The alpha7-nicotinic acetylcho-
line receptor and the pathology of hippocampal interneurons in schizo-
phrenia. J. Chem. Neuroanat. 20, 299–306.
Kitagawa, H., Takenouchi, T., Azuma, R., Wesnes, K.A., Kramer, W.G., Clody,
D.E., Burnett, A.L., 2003. Safety, pharmacokinetics, and effects on
cognitive function of multiple doses of GTS-21 in healthy, male volunteers.
Neuropsychopharmacology 28, 542–551.
Le Novere, N., Changeux, J.P., 1995. Molecular evolution of the nicotinic
acetylcholine receptor: an example of multigene family in excitable cells. J.
Mol. Evol. 40, 155–172.
Liu,Q.,Kawai, H.,Berg,D.K.,2001.Beta-Amyloidpeptide blocksthe response
of alpha 7-containing nicotinic receptors on hippocampal neurons. Proc.
Natl. Acad. Sci. U. S. A. 98, 4734–4739.
Marrero, M.B., Papke, R.L., Bhatti, B.S., Shaw, S., Bencherif, M., 2003. The
neuroprotective effect of TC-1698, a novel alpha7-selective ligand, is
prevented through angiotensin II activation of a tyrosine phosphatase. J.
Pharmacol. Exp. Ther. 309, 16–27.
Meyer, E., deFiebre, C.M., Hunter, B., Simpkins, C.E., Frauworth, N., deFiebre,
N.C., 1994. Effects of anabaseine-related analogs on rat brain nicotinic
receptor binding and on avoidance behaviors. Drug Dev. Res. 31, 127–134.
Meyer, E., Kuryatov, A., Gerzanich, V., Lindstrom, J., Papke, R.L., 1998.
Analysis of 40H-GTS-21 selectivity and activity at human and rat α7
nicotinic receptors. J. Pharmacol. Exp. Ther. 287, 918–925.
Papke, R.L., Papke, J.K.P., 2002. Comparative pharmacology of rat and human
alpha7 nAChR conducted with net charge analysis. Br. J. Pharmacol. 137,
Papke, R.L., Thinschmidt, J.S., 1998. The Correction of alpha7 nicotinic
acetylcholine receptor concentration–response relationships in Xenopus
oocytes. Neurosci. Lett. 256, 163–166.
Papke, R.L., Meyer, E., Nutter, T., Uteshev, V.V., 2000. Alpha7-selective
agonists and modes of alpha7 receptor activation. Eur. J. Pharmacol. 393,
Papke, R.L., Papke, J.K.P., Rose, G.M., 2004. Activity of alpha7-selective
agonists at nicotinic and serotonin receptors expressed in Xenopus oocytes.
Bioorg. Med. Chem. Lett. 14, 1849–1853.
Sharma, G., Vijayaraghavan, S., 2002. Nicotinic receptor signaling in
nonexcitable cells. J. Neurobiol. 53, 524–534.
Stokes, C., Papke, J.K.P., McCormack, T., Kem, W.R., Horenstein, N.A., Papke,
R.L., 2004. The structural basis for drug selectivity between human and rat
nicotinic alpha7 receptors. Mol. Pharm. 66, 14–24.
Terry Jr., A.V., Risbrough, V.B., Buccafusco, J.J., Menzaghi, F., 2002. Effects of
(+/−)-4-[[2-(1-methyl-2-pyrrolidinyl)ethyl]thio]phenol hydrochloride (SIB-
1553A), a selective ligand for nicotinic acetylcholine receptors, in tests of
visual attention and distractibility in rats and monkeys. J. Pharmacol. Exp.
Ther. 301, 284–292.
Uteshev, V.V., Meyer, E.M., Papke, R.L., 2002. Activation and inhibition of
native neuronal alpha-bungarotoxin-sensitive nicotinic ACh receptors. Brain
Res. 948, 33–46.
Van Kampen, M., Selbach, K., Schneider, R., Schiegel, E., Boess, F., Schreiber,
R., 2004. AR-R 17779 improves social recognition in rats by activation of
nicotinic alpha7 receptors. Psychopharmacology (Berl.) 172, 375–383.
Wang, H.Y., Lee, D.H., D'Andrea, M.R., Peterson, P.A., Shank, R.P., Reitz, A.
B., 2000. beta-Amyloid(1-42) binds to alpha7 nicotinic acetylcholine
receptor with high affinity. Implications for Alzheimer's disease pathology.
J. Biol. Chem. 275, 5626–5632.
Yang, Z., 1997. PAML: a program package for phylogenetic analysis by
maximum likelihood. Comput. Appl. Biosci. 13, 555–556.
18 R.L. Papke et al. / European Journal of Pharmacology 524 (2005) 11–18