Activation of the Torpedo nicotinic acetylcholine receptor
The contribution of residues aArg55 and cGlu93
Ankur Kapur, Martin Davies, William F. Dryden and Susan M.J. Dunn
Department of Pharmacology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Canada
The muscle-type nicotinic acetylcholine receptor
(nAChR) is the prototype of the Cys-loop ligand-gated
ion channel (LGIC) super-family that includes the
neuronal nicotinic, c-aminobutyric acid (GABA) type
A, 5-hydrotryptamine type 3 (5-HT
3
) and glycine
receptors. This is largely a consequence of the abun-
dance of this receptor in Torpedo electric organ, which
facilitated its early purification and characterization.
The Torpedo nAChR is a pentameric transmembrane
protein complex in which four structurally related
subunits (a, b, c, d) in a stoichiometry of 2 : 1 : 1 : 1
assemble to form a central cation-selective ion channel
[1,2]. The a and b subunits of the Torpedo receptor
referred to in this report correspond to the a1 and b1
subunits in the nomenclature recommended by the
International Union of Pharmacology [3]. Radioligand
binding studies have demonstrated that, under equilib-
rium conditions, the nAChR carries two high affinity
Keywords
acetylcholine; loop D; mutagenesis; nicotinic
receptor; oocytes
Correspondence
S.M.J. Dunn, Department of Pharmacology,
University of Alberta, Edmonton, Alberta,
T6G 2H7 Canada

mutations, aR55E-cE93R or aR55F-cE93R, the potency for acetylcholine
activation was partially restored to that of the wild-type. The results sug-
gest that, although individually these residues influence receptor activation,
direct interactions between them are unlikely to play a major role in the
stabilization of different conformational states of the receptor.
Abbreviations
5-HT
3A
receptor, serotonin type 3 A receptor; a-BgTx, alpha-bungarotoxin; ACh, acetylcholine; AChBP, acetylcholine binding protein;
dTC, d-tubocurarine; GABA, c-aminobutyric acid; LGIC, ligand-gated ion channel; nAChR, nicotinic acetylcholine receptor; PTMA,
phenyltrimethylammonium; WT, wild-type.
960 FEBS Journal 273 (2006) 960–970 ª 2006 The Authors Journal compilation ª 2006 FEBS
binding sites for agonists and competitive antagonists
[4,5]. It is now generally agreed that these sites lie at
the interfaces between the a–c and the a–d subunits
[6]. Labeling and mutational studies have identified
several key amino acids lying in discrete noncontigu-
ous ‘loops’ of the a-subunits (designated as loops
A–C, the ‘primary component’), together with amino
acids in the neighboring c and d subunits (lying in
loops D–F, the ‘secondary component’) that partici-
pate in forming these binding pockets [7–9].
Although none of the ligand-gated ion channel fam-
ily to which the nAChR belongs has been crystallized,
the published structure of a related protein [10], the
acetylcholine binding protein (AChBP), lends credence
to current ideas of high affinity binding site location.
The AChBP, which is secreted by the glial cells of the
snail, Lymnaea stagnalis , is a truncated homologue
of the extracellular amino terminal domains of the

of the a-subunit in ligand binding has long been recog-
nized [11-15], but the involvement of non a-subunit
residues has become clear only more recently. The first
direct evidence for the contribution of the c and d sub-
units to ligand recognition came from photoaffinity
labeling studies using [
3
H]nicotine and [
3
H]d-tubocura-
rine (dTC), which identified residues cW55 and the
homologous dW57 (lying in what is now referred to as
the loop D domain) as specific sites of ligand incorpor-
ation [5,15-17]. In the present study, we have investi-
gated the effects of mutations of the equivalent residue
(aR55) lying in loop D of the a-subunit, i.e. at the
opposite side of the subunit from residues (in loops A–
C) that have previously been implicated in agonist
binding (Fig. 1). Within the LGIC family, this residue
in the peripheral nAChR is unique; whereas almost all
subunits in the family have an aromatic residue at this
position, a positively charged arginine residue is con-
served in all peripheral a-subunits (see Fig. 1). Previ-
ous comparative modeling studies have revealed that
E93 of the c-subunit (lying in putative binding loop A)
may lie in close proximity to aR55, leading to the pro-
posal that an ionic interaction between these two resi-
dues may stabilize receptor conformation [18,19]. This
53
A

A
C
h
A
B
C
D
E
F
A
B
C
D
D
D
E
F
-
+
+
-
+-
α
α
β
δ
γ
Fig. 1. Loop D of the LGIC family. (A) Amino acid sequence align-
ments of residues lying in loop D of the a1, c and d subunits from
Torpedo californica (T. Ca) nAChR, human (H) a1 nAChR subunit,

using two-electrode voltage clamp techniques. Figure 2
shows the concentration-effect curves for ACh-medi-
ated responses. The WT nAChR receptor has an EC
50
value for ACh-induced activation of $24 lm with an
estimated Hill coefficient of 1.6. The substitution of
aArg55 with glutamic acid (aR55E) or lysine (aR55K)
resulted in a statistically insignificant shift in the EC
50
values for ACh activation to 29 and 47 lm, respect-
ively, and had no significant effect on the cooperativity
of receptor activation. In contrast, the aR55F and
aR55W mutations caused a five- to six-fold shift in the
EC
50
for ACh activation to 112 and 151 lm, respect-
ively. In addition, the Hill coefficients for Ach-induced
activation for these mutant receptors were significantly
reduced in comparison with the WT nAChR (Table 1).
The effects of phenyltrimethylammonium (PTMA)
on activation of WT and mutant receptors were also
investigated. PTMA is a poor partial agonist of the
WT nAChR and it elicits a maximum current of only
1.5 ± 0.1% of the ACh response (data not shown).
The WT receptor was activated by PTMA with an
EC
50
of 57 lm and a Hill coefficient of 2.1 ± 0.2
(Fig. 3A, Table 2). In contrast, PTMA failed to acti-
vate the aR55F and aR55W mutant receptors, even at

represent the mean ±
SEM. Values for log EC
50
and Hill coefficient
(n
H
) were determined from concentration-effect curves using GRAPH-
PAD PRISM
software. Log EC
50
and Hill coefficients from individual
curves were averaged to generate final mean estimates. The val-
ues in parentheses are the number of oocytes used for each recep-
tor type. Statistical analysis was performed by comparing the log
EC
50
and n
H
of the mutant receptors to the WT nAChR (
a
p<0.001,
b
p<0.05) using one-way analysis of variance (ANOVA) followed by
Dunnett’s post-test to determine the level of significance.
c
p<0.001
compared with the aR55F receptor.
Receptor
Log EC
50

b,c
11.5 1.1 ± 0.07 0.47
cE93R-aR55F ) 4.89 ± 0.20 (3)
c
12.9 1.3 ± 0.1 0.53
Role of a–c subunit interface in nAChR function A. Kapur et al.
962 FEBS Journal 273 (2006) 960–970 ª 2006 The Authors Journal compilation ª 2006 FEBS
preperfusion with dTC produced a concentration
dependent inhibition of ACh-evoked currents charac-
terized by an apparent K
I
of $42 nm. dTC also inhib-
ited Ach-evoked currents in the receptors carrying the
aR55F and aR55W mutations with apparent K
I
values
of $52 and 34 nm, respectively (see Table 2). These
results suggest that mutations at position 55 of the
a-subunit do not affect either the binding affinity for
dTC or its ability to competitively inhibit ACh-evoked
currents. In these experiments, although dTC alone did
not elicit detectable whole cell currents, we observed
that low concentrations of dTC (1–3 nm) potentiated
ACh- evoked currents (by up to 25%) in both WT and
mutant receptors (Fig. 3B).
Expression levels and maximum amplitude
of WT and mutant nAChR
Fig. 4 compares the density of binding sites for
125
I-

ACh concentration used to induce responses was equivalent to its
EC
50
value for activation of that subtype. Similar data show a lack
of significant effect of dTC on the aR55W and R55E mutant recep-
tors (data not shown).
Table 2. Effects of PTMA and dTC on WT and mutant receptors.
Data were analyzed as described in the legend to Fig. 3. K
I
values
were determined as described in Experimental procedures. Each
experiment was repeated in 3–4 oocytes for each receptor sub-
type. No significant differences were observed between the WT
and mutant receptors.
Receptor
PTMA dTC
log IC
50
± SEM K
I
(lM) log IC
50
± SEM K
I
(nM)
WT ) 4.24 ± 0.06
a
57.0
a
) 7.07 ± 0.11 42.5

10
I
amx
(
µA)
Surface nAChR (fmol)
Fig. 4. Surface nAChR expression of WT and mutant receptors in
Xenopus oocytes. Maximum ACh-evoked currents (I
max
) were
determined using concentrations determined from concentration-
effect curves (as shown in Fig. 2). Surface receptor levels were
determined in the same oocytes by measuring
125
I-labelled a-BgTx
binding as described in Experimental procedures. The data repre-
sent the mean ±
SEM of 3–11 determinations from individual
oocytes and are presented in Table 3.
A. Kapur et al. Role of a–c subunit interface in nAChR function
FEBS Journal 273 (2006) 960–970 ª 2006 The Authors Journal compilation ª 2006 FEBS 963
the aR55F and aR55W mutants were reduced by
approximately three- to five-fold, respectively, com-
pared with the WT receptor (Table 3; see Discussion).
Overall, these results suggest that mutation of aR55,
which is predicted to lie at the a–c and a–b interfaces,
does not play a major role in receptor assembly or sur-
face expression.
Influence of aR55F and aR55W mutant receptors
on the binding of acetylcholine

As noted in the Introduction, it has been suggested
that the proximity of aR55 and c93E may be con-
ducive to an ion-pairing interaction (see Fig. 6).
Surprisingly, the cE89R mutation resulted in an
approximately eight-fold increase in the apparent
potency of ACh-induced activation. As shown in
Fig. 7A (see Table 1), the EC
50
for this mutant recep-
tor was reduced to 3 lm (Hill coefficient of 1.4) from
the value of 24 lm measured in the WT. In contrast,
the apparent affinity for the competitive antagonist,
dTC (as determined by inhibition of ACh-evoked cur-
rents in oocytes), for the cE93R mutant receptor was
unaltered as compared with the oocytes expressing WT
nAChR (K
I
$55 and 42 nm, respectively, see Fig. 7B,
Table 2). This figure also illustrates that, in contrast to
the WT receptor (see above), the potentiating effects
of low concentrations of dTC were abolished by the
cE93R mutation.
Effects of double mutations of aR55 and cE93
In order to investigate whether the aR55F and cE93R
mutations have an additive effect, we studied receptors
carrying the double mutations, aR55E-cE93R and
aR55F-cE93R. These double mutant receptors had
EC
50
values for ACh-induced activation of $11.5 and

max
⁄
WT I
max
)
WT 3.1 ± 0.8 (11) 3316 ± 537 1081 100
aR55E 9.2 ± 0.7 (3) 8680 ± 501 934.4 86.4
aR55K 8.0 ± 1.5 (5) 3652 ± 654 455.1 42.1
aR55F 3.8 ± 0.7 (6) 1445 ± 340 384.6 35.6
aR55W 5.9 ± 1.3 (7) 1154 ± 210 197.0 18.2
-10
-9
-8
-7
-6
-5
-4
0
20
40
60
80
100
120
log [ACh] (M)
[%
521
]I
α
-B xTgBiidngn

at an adjacent interface, i.e. residues aR55 (loop D)
and cE93 (loop A) which, in the muscle counterpart,
have been proposed to interact and to possibly play a
critical functional role in receptor properties [19].
Amino acid sequence alignments of loop D (see
Fig. 1A) reveal that the peripheral nAChR a-subunits
carry a unique amino acid at position 55, i.e. an argin-
ine residue rather that an aromatic amino acid that is
conserved in most other subunits of the Cys-loop
LGIC family. There is considerable evidence to suggest
that, in a number of subunits, the residue in the equiv-
alent position plays an important role(s) in modulating
agonist ⁄ antagonist sensitivity. Mutations of cW55 and
dW57 in the Torpedo nAChR have been shown to
affect the affinity for dTC and ACh [17,22] while the
W54 of the neuronal nicotinic a7 receptor has been
shown to contribute to the binding of agonists [23]. In
the GABA
A
receptor, the F64L mutation of the a1
subunit had a dramatic effect on GABA sensitivity
[24] and mutations of the F77 residue of the c2 subunit
significantly affected ligand affinity for the benzodi-
azepine binding site [25]. In addition, the GABA
A
receptor b2Y62 residue has been shown to be an
important determinant of high affinity agonist binding
[26]. In the 5-HT
3A
receptor, the homologous W89

a poor agonist on the WT nAChR, the lack of a
response could be attributable to either a greatly
reduced sensitivity of the receptor towards the ligand
or to a further reduction of conductance to undetecta-
ble levels. The lack of any response to PTMA was
exploited to differentiate between these possibilities
[28], and the results reveal that the effects of the muta-
tions are on PTMA efficacy rather than affinity. The
apparent K
I
for PTMA-inhibition of ACh-responses
mediated by the aR55F and aR55W mutant receptors
γ
γ
α
N
O
N
N
N
O
N
O
O
5.7 Å
γE93 αR55
DINNELVI
Human ε
DVNNELVI
Human γ

expression (nAÆfmol
)1
) the current responses were sub-
stantially lower than displayed by the WT receptor.
This may reflect a reduction in single channel conduct-
ance of the mutant receptors, a decreased efficacy of
ACh-mediated currents or the possibility that some of
the expressed receptors are nonfunctional. Distinction
between these possibilities requires further analysis at
the single channel level. We also observed a significant
reduction in the Hill slope of the activation curves in
the mutant receptors. While the interpretation of chan-
ges in Hill coefficients is controversial, the simplest
explanation is that these mutations reduce the level of
cooperativity between different agonist binding sites
[29].
Our present findings are consistent with previous
reports that mutations of the homologous residue
(W54) in the a7 nAChR W54 resulted in a reduction of
ACh potency without a disruption of a-BgTx binding
[23]. The present results point to a role of aR55 in the
transduction mechanism rather than in direct agonist
binding. However, there is some evidence in the litera-
ture that this region may also contribute to binding site
formation. A synthetic peptide equivalent to a55–74 of
Torpedo nAChR was shown to be able to bind a-BgTx
but this binding was inhibited by an R55G substitution
in the synthetic peptide [30]. However, since a synthetic
peptide is unlikely to have a similar conformation as
the equivalent domain in the native receptor, it is diffi-

ing effect in the mutant receptor is that the mutation
results in the loss of the ability of dTC to act as such
a ‘coagonist’.
-8 -7 -6 -5 -4 -3 -2
0
20
40
60
80
100
120
A
B
log [ACh] (M)
%M ixamumr esnopse
-9 -8 -7 -6 -5
0
20
40
60
80
100
120
log [dTC] (M)
I
CTd +AChEC50
I/
hCAEC05
Fig. 7. Effects of the cE93R mutation. (A) Concentration-effect
curves for ACh activation of WT (n), cE93R (,) and the double

50
for ACh activation. One possible explanation is
that this mutation facilitates the rotational movements
at intersubunit contact points that have been suggested
to occur during channel activation [20,21,32]. The
receptor carrying the double charge-reversal mutation
(cE93R-aR55E) was activated by ACh with an EC
50
that approached that of the WT receptor, although the
EC
50
values (Table 1) remained statistically different.
Taken together, these results suggest that, in the WT
receptor, an interaction between aR55 and cE93 is
unlikely to stabilize either the resting conformation (as
the mutation aR55E had little effect on activation) or
the activated state (as mutation cE93R increased ACh
potency). However, these residues lying at the c–a
interface do appear to play a role in receptor activa-
tion and ⁄ or the signal transduction mechanism.
In summary, we have identified a residue, R55 in
loop D of the extracellular ligand binding domain of
a-subunit that modulates ACh sensitivity and that lies
at some distance from the ‘classical’ high affinity bind-
ing sites for ACh. This residue has not previously been
implicated in nAChR function. However, our data
complement earlier work to suggest that loop D resi-
dues occurring in nonbinding domains may play
important roles in receptor function[see 26]. In addi-
tion, we show that E93 of the Torpedo nAChR c-sub-

In vitro transcription and site-directed
mutagenesis
The plasmid cDNAs were linearized by digestion with
either EcoRI (for the a-subunit), FspI (for the b-subunit)
or XbaI (for the c- and d-subunits). In vitro cRNA tran-
scription was performed using the methods described by
Goldin and Sumikawa [33]. Briefly, the linearized cDNA
templates (5 lg) were transcribed in vitro using SP6 RNA
polymerase (Promega) in the presence of ribonucleotide
triphosphate (NTP mix, Invitrogen) and RNA capping
analogue (NEB). The RNA transcripts were extracted
using 25 : 24 : 1 (v ⁄ v) phenol–chloroform–isoamyl alcohol.
Finally, the RNA pellets were resuspended in diethylpyro-
carbonate-treated water at a concentration of 1 lgÆlL
)1
.
The a-subunit mutants (R55F, R55W, R55K and R55E)
were constructed using Stratagene’s QuikChange site-direc-
ted mutagenesis protocol. Synthetic oligonucleotide muta-
genic primers were typically 23–34 base pairs long (with
10–15 base pairs lying on either side of the mismatch
region). A similar approach was undertaken to engineer the
cE93R mutation. Restriction endonuclease digestion and
DNA sequencing subsequently verified the presence of the
mutation.
Expression in Xenopus oocytes and
electrophysiology
Isolated, follicle-free oocytes were microinjected with 50 ng
of total subunit cRNAs in a ratio of 2a :1b :1c :1d.
Oocytes were maintained in ND96 buffer (96 mm NaCl,

full recovery from desensitization. For measuring the effects
of antagonists, oocytes were preperfused with various con-
centrations of antagonist in low Ca
2+
ND96 buffer for
2 min, before initiating the response by application of solu-
tion containing ACh (at a concentration eliciting 50% of
the maximum response, EC
50
) and including the same con-
centration of antagonist as used in the preperfusion.
Binding of
125
I-labelled a-BgTx to intact oocytes
Binding assays were performed on individual oocytes that
had previously been used in the electrophysiological experi-
ments. To measure the density of nAChR binding sites
expressed on the oocyte surface (fmol), oocytes were incu-
bated with 5 nm
125
I-labelled a-BgTx in a final volume of
100 lL of low Ca
2+
ND96 buffer containing 5 mgÆmL
)1
bovine serum albumin for 2 h at room temperature [36,37].
Excess unbound toxin was removed by washing the oocytes
three times with 1 mL of ice-cold low Ca
2+
ND96 buffer.

125
I-labelled a-BgTx binding was $30% of the
available a-BgTx-binding sites (data not shown). Non-speci-
fic binding was determined in the presence of 100 mm ACh.
Data and statistical analysis
Competition and concentration-effect curves for both elec-
trophysiological and radioligand binding experiments were
analyzed by nonlinear regression techniques using graph-
pad prism 3.0 software (GraphPad, San Diego, CA, USA).
Data from individual oocytes were normalized to the I
max
value obtained for that oocyte.
For receptor activation, concentration-effect curves for
agonist activation were analyzed using the following equa-
tion:
I ¼ I
Ã
max
½L
n
=ðEC
50
þ½LÞ
n
where I is the measured agonist-evoked current, [L] is the
agonist concentration, EC
50
is the agonist concentration
that evokes half the maximal current (I
max

I
¼ IC
50
=½1 þ½L=EC
50

where [L] is the ACh concentration used in the experiment
and EC
50
is the ACh concentration that evokes half the
maximal current.
Statistical analysis was performed using one-way analysis
of variance (anova, />anova.html) followed by Dunnett’s post-test to determine
the level of significance.
Acknowledgements
This work was supported by the Canadian Institutes
of Health Research. We are especially grateful to
Isabelle Paulsen for expert technical assistance.
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