-Conotoxins PnIA and [A10L]PnIA Stabilize Different States of the
7-L247T Nicotinic Acetylcholine Receptor*
Ron C. Hogg
,
Gene Hopping
,
Paul F. Alewood
,
David J. Adams
and
Daniel Bertrand
From the
Department of Physiology, CMU, 1 rue Michel Servet, CH-1211 Geneva 4,
Switzerland and the
School of Biomedical
Sciences, University of Queensland, Brisbane 4072, Australia
Received for publication, December 11, 2002
, and in revised form, April 28, 2003.
 |
ABSTRACT
|
---|
The effects of the native
-conotoxin PnIA, its synthetic derivative
[A10L]PnIA and alanine scan derivatives of [A10L]PnIA were investigated on
chick wild type
7 and
7-L247T mutant nicotinic acetylcholine
receptors (nAChRs) expressed in Xenopus oocytes. PnIA and [A10L]PnIA
inhibited acetylcholine (ACh)-activated currents at wt
7 receptors with
IC50 values of 349 and 168 nM, respectively. Rates of
onset of inhibition were similar for PnIA and [A10L]PnIA; however, the rate of
recovery was slower for [A10L]PnIA, indicating that the increased potency of
[A10L]PnIA at
7 receptors is conveyed by its slower rate of
dissociation from the receptors. All the alanine mutants of [A10L]PnIA
inhibited ACh-activated currents at wt
7 receptors. Insertion of an
alanine residue between position 5 and 13 and at position 15 significantly
reduced the ability of [A10L]PnIA to inhibit ACh-evoked currents. PnIA
inhibited the non-desensitizing ACh-activated currents at
7-L247T
receptors with an IC50 194 nM. In contrast, [A10L]PnIA
and the alanine mutants potentiated the ACh-activated current
7-L247T
receptors and in addition [A10L]PnIA acted as an agonist. PnIA stabilized the
receptor in a state that is non-conducting in both the wild type and mutant
receptors, whereas [A10L]PnIA stabilized a state that is non-conducting in the
wild type receptor and conducting in the
7-L247T mutant. These data
indicate that the change of a single amino acid side-chain, at position 10, is
sufficient to change the toxin specificity for receptor states in the
7-L247T mutant.
 |
INTRODUCTION
|
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Conotoxins are short peptides isolated from the venom of predatory marine
snails of the genus Conus. Many of these toxins are selective
inhibitors of ligand- and voltage-gated ion channels and are classified
according to the type of channel to which they bind. This high selectivity of
the conotoxins has lead to a great interest in the use of these molecules as
pharmacological tools and the design of novel therapeutics. Much of this work
has been summarized in several recent reviews
(1,
2). The
-conotoxins are
between 11 and 16 amino acids in length and are selective inhibitors of
nicotinic acetylcholine receptors
(nAChRs),1 see Refs.
3 and
4 for review. Native nAChRs are
composed of a number of distinct subunits, which combine to form functional
receptors each with distinct pharmacological properties. The
-conotoxins contain 4 cysteines, which in their natural conformation
form disulphide bridges giving the molecule a globular two-loop configuration
with sidechains projecting from a rigid backbone. The
-conotoxin PnIA,
isolated from the molluscivourous cone snail Conus pennaceus, is 16
amino acids long, and the x-ray crystal structure shows an
-helix
between residues 5 and 12 and a 310 helical turn at the N-terminal
end (5). PnIA has been
demonstrated to be a competitive inhibitor of native nAChRs in cultured
Aplysia neurons (6),
dissociated neurons from rat parasympathetic ganglia
(7), and recombinant nAChRs
expressed in Xenopus oocytes
(8) with poor selectivity
between receptor subtypes. A leucine for alanine substitution at position 10
makes the toxin a highly selective inhibitor of the
7 nAChR subtype
(7,
8), see
Table I. Since the A10L
mutation in PnIA changes the selectivity of the toxin for receptor subtypes,
it is also possible that the mechanism of inhibition differs between PnIA and
[A10L]PnIA, the A10L mutation may cause the toxins to have different
affinities for different states of the receptor.
The nAChRs have been presented as a prototype of allosteric membrane
protein (9) as described by the
Monod-Wyman-Changeux model of allosteric interactions
(10) in which the structure of
the molecule moves in concerted transitions between pre-existing
conformational states. The protein can exist in different states and undergoes
spontaneous conformational transitions, in the absence of a ligand the
equilibrium between these conformational states is in favor of the resting
(closed) state. Exposure to an agonist preferentially stabilizes the receptor
in the active (open) and desensitized (closed) states, whereas the binding of
an antagonist molecule binds to and stabilizes the molecule in a closed
(resting or desensitized) state (see Ref.
11). In such a model,
transition from one state to another depends upon both the presence of a
ligand and/or the isomerization coefficient, which governs changes between
states. Binding of a molecule at a site distinct from the agonist-binding site
may modify the isomerization coefficient, thus affecting agonist or antagonist
behavior. Molecules acting in this way are known as allosteric effectors
(10).
To test whether mutations of PnIA affected the inhibitory mechanism of the
toxin, we examined the effects of PnIA, [A10L]PnIA and alanine scan mutants of
[A10L]PnIA at wild type homomeric chick
7 receptors (wt
7) and at
chick
7 receptors with the L247T mutation (
7-L247T) expressed in
Xenopus oocytes.
7-L247T receptors display non-desensitizing
currents in response to agonist application
(1216).
Because it was proposed that the L247T mutation renders conductive one of the
desensitized states (12), this
mutant receptor can be used as a tool to determine whether the same closed
state of the receptor is stabilized by PnIA and [A10L]PnIA, allowing us to
probe states of the receptor which are otherwise electrophysiologically silent
in the wild type receptor. To test the contribution of projecting side-chains
at other positions to the activity of [A10L]PnIA, we have replaced each of the
residues in turn with an alanine to observe the effects of this mutation on
toxin activity. Circular dichroism spectra and 1H NMR experiments
have shown that substitution of individual residues in [A10L]PnIA with an
alanine do not result in perturbation of the global
fold,2 thus, any
changes in activity in these molecules can be directly correlated to the
interactions of the projecting side chains with the nAChR.
 |
EXPERIMENTAL PROCEDURES
|
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ElectrophysiologyXenopus oocytes were prepared and injected
as described previously (17).
Two-electrode voltage clamp recordings were made 23 days after cDNA
injection. During recordings the bath solution was OR2 medium containing (in
mM), NaCl, 82.5; KCl, 2.5; CaCl, 2.5; MgCl, 1; atropine, 0.5;
HEPES, 5, adjusted to pH 7.4 with NaOH. ACh was applied in a fast flowing
solution stream (
6 ml/min). Unless indicated, oocytes were incubated in
100 µM BAPTA-AM for at least 2 h prior to recording.
Two-electrode voltage clamp recordings were carried out using a GeneClamp 500B
amplifier (Axon Instruments Inc., Union City, CA), recording electrodes
contained 3 mM KCl and oocytes were clamped at 100 mV
throughout the experiments. Experiments were carried out at 18 °C.
Dose-response curves for toxin inhibition were fit with the equation
y = 1/1 + ([toxin]/(IC50)n), where
y is the normalized response, [toxin] is the toxin concentration, and
n is the Hill coefficient. Measurements of rates of toxin block and
recovery were carried out at toxin concentrations close to the
IC50. Data points for onset of block were fit with the equation
y = A
et/
+
B, where A = control amplitude normalized to 1, B =
current amplitude following block expressed as a fraction of the control
current, and t = time. Curves were fit using a
2
minimization fitting routine in Microcal origin 5.0 (Microcal software inc.).
Data are presented as mean ± S.E.
Toxin SynthesisThe 16-residue peptides were synthesized
manually on a 0.50-mmol scale using HBTU activation of
t-butoxycarbonylamino acids with in situ neutralization
chemistry as described previously
(18). The syntheses were
performed on p-methylbenzhydrylamine resin using standard amino acid
side chain protection. Each residue was coupled for 10 min and coupling
efficiencies determined by the quantitative ninhydrin reaction
(18). Prior to a standard HF
cleavage (10 ml of p-cresol:p-thiocresol:HF 1:1:8 (0 °C,
2 h) and workup (18), the
N-terminal t-butoxycarbonyl protecting group was removed (100%
trifluoroacetic acid), and the resin was successively washed with
dimethylformamide and dichloromethane. Air oxidations were carried out by
dissolving the lyophilized purified A10L[PnIA] analogues in 0.1 M
NH4HCO3/isopropyl alcohol (pH 8.25) with vigorous
stirring at room temperature overnight. Prior to purification the solution was
acidified to pH 3 with trifluoroacetic acid and analyzed by analytical C4 HPLC
using a linear gradient of 080% solvent B at 1%/min while monitoring by
UV absorbance at 214 nm and electronspray mass spectrometry. Oxidized material
was then purified by semipreparative HPLC using the same chromatographic
conditions.
 |
RESULTS
|
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PnIA and [A10L]PnIA Inhibit ACh-induced Currents at Chick
7 nAChR ReceptorsThe effects of PnIA were investigated
on chick
7 nAChR receptors expressed in Xenopus oocytes.
Membrane currents were activated with 200 µM ACh, which is close
to the EC50 (115 µM) for these receptors
(19). PnIA inhibited
ACh-induced currents in a concentration-dependent and reversible manner
(Fig. 1A). The
dose-response relationship is shown in Fig.
1B; the fitted curve had an IC50 of 349
nM and a slope of 1.8.
The position 10 mutant of PnIA, [A10L]PnIA, has previously been reported to
inhibit ACh-induced currents at rat
7 receptors expressed in
Xenopus oocytes (8)
and native
7 receptors in rat parasympathetic ganglion neurons
(7). [A10L]PnIA was twice as
potent as PnIA to inhibit ACh-activated currents at chick
7 receptors
(Fig. 1C).
Dose-response relationship was fit by a curve with an IC50 value of
168 nM and a slope of 1.55 (Fig
1D).
Rate of Block and RecoveryThe rate of onset and recovery
from block of ACh-activated currents at
7 receptors was measured for
PnIA and [A10L]PnIA. As PnIA competes with ACh at the binding site, ACh was
applied for 2 s, and it was assumed that these short applications of ACh did
not displace the toxin. Toxin concentrations used were close to the respective
IC50 values. The rate of block with PnIA (400 nM)
followed an exponential time course with a
onset of 11.3 s
(Fig. 2A).
An exponential rate of block and recovery would suggest that the reaction
is bimolecular. However, as the Hill coefficient for PnIA inhibition is
greater than unity (Fig.
1B), more than one toxin binding site must be present.
Since the homomeric
7 receptor contains five putative agonist binding
sites, we tried to fit the data using a scheme similar to MLA blockade of the
7 receptor (20). If the
occupation of a single binding site is sufficient to prevent activation of the
receptor by an agonist, then the onset of block will follow an exponential
time course. However, recovery from block requires that all of the binding
sites are unoccupied, causing an initial lag in the recovery. Palma et
al. (20) found that the
recovery of block from MLA was best fit by a five-site model. Since the time
course of recovery from MLA inhibition is
10-fold slower than recovery
from [A10L]PnIA and more than 20-fold slower than PnIA, this makes it likely
that any lag in the recovery would not be resolved at our sampling interval of
1, 5, and 10 s. Thus the recovery describes only the dissociation of the last
toxin molecule from the receptor. The time to half-recovery from PnIA
inhibition was in the region of 15 s. The onset of block of [A10L]PnIA (200
nM) also followed an exponential time course with a
onset of 12.0 s and had a slower half-recovery time in the
region of 70 s (Fig. 3, C and
D).
Fitting the data with a five site model (see Equation 3 from Ref.
20), using approximate rates
of Koff and Kon from
Fig. 2, A and
B, and Fig. 3,
A and B, did not describe the data better than
the Hill equations in Fig. 1, B
and D, indicating that this model is not appropriate to
describe the interaction of PnIA and [A10L]PnIA with the receptor. The
observation that brief (0.5 or 1 s) applications or low concentrations of PnIA
and [A10L]PnIA increased the peak current amplitude above control values
indicate that the scheme involving binding is complex, and this may affect the
fit to the data.
[A10L]PnIA Alanine MutantsTo investigate the contribution
of amino acid side chains in the [A10L]PnIA molecule to the inhibitory potency
at
7 receptors, we examined a series of alanine mutants of [A10L]PnIA.
All alanine mutants of [A10L]PnIA significantly inhibited currents at
wt
7 receptors (p < 0.05).
Fig. 4 shows a comparison of
the inhibitory effects of the [A10L]PnIA alanine mutants (200 nM)
on chick
7 nAChRs. Removal of the projecting side chains between
position 5 and 13 and at position 15, by the insertion of an alanine residue,
significantly reduced the potency of 200 nM [A10L]PnIA to inhibit
ACh-evoked currents (p < 0.05).

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FIG. 4. Relative inhibition of ACh-induced currents at wt 7 receptors by
PnIA, [A10L]PnIA, and alanine analogues. Control current amplitudes in the
absence of toxin have been normalized to 1.0. All toxins were applied at 200
nM, and currents were activated by 200 µM ACh. The
number of experiments is given in brackets, and asterisks
indicates values significantly different from [A10L]PnIA (p <
0.05).
|
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PnIA and [A10L]PnIA Have Different Effects on
7-L247T
ReceptorsThe ability of PnIA and [A10L]PnIA to inhibit currents
mediated by the wt
7 receptor indicates that they stabilize a state of
the receptor which is non-conducting. Since the A10L mutation changes the
selectivity of the toxin for receptor subtypes
(7,
8), it is possible that the
mechanism of inhibition may also be different. In the minimal hypothetical
gating scheme for a nAChR shown in Fig.
5, this could be either the resting or the desensitized state
(D), which are both non-conducting in the wt
7 receptor.

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FIG. 5. Hypothetical gating scheme for the nAChR. The nAChR is assumed to
exist in multiple interconvertible states, a closed resting state, an open
conducting state, and at least one desensitized state. In wild type receptors
the desensitized states are non-conducting; however, we propose that in the
7-L247T receptor one of the desensitized states (D*) of the
receptor is conducting.
|
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To investigate whether PnIA and [A10L]PnIA stabilize the same state of the
receptor, we used the chick
7 receptor containing the L247T mutation.
7-L247T receptors display non-desensitizing currents in response to
ACh, and it has been proposed that the L247T mutation causes one of the
desensitized states of the receptor to become conducting
(12); this desensitized open
state is represented as D* in Fig.
5 and could account for the non-desensitizing current. It has been
proposed that it is this state of the receptor that is stabilized by nAChR
antagonists, such as DH
E, which activate currents at this receptor
(14). Occasionally, slow
desensitization of
7-L247T receptors is observed, making it likely that
other desensitized non-conducting states of the receptor also exist. The
7-L247T receptors exhibited a slowly activating, non-desensitizing
current in response to ACh which reached a plateau within
10 s.
7-L247T receptors show an increased sensitivity to ACh compared with
wild type receptors with an EC50 of 0.62 µM
(14,
19). PnIA inhibited the
ACh-induced current through
7-L247T receptors in a
concentration-dependent and reversible manner
(Fig. 6A). The
dose-response relationship was fit by a curve with an IC50 of 194
nM and a slope of 0.9 (Fig.
6B).

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FIG. 6. PnIA inhibits the ACh-evoked current in 7-L247T mutant receptors,
whereas [A10L]PnIA and the alanine mutants facilitate ACh-induced
responses. A, PnIA inhibits non-desensitizing ACh-evoked currents
in 7-L247T receptors. B, concentration-response relationship
for inhibition had an IC50 of 194 nM, and ACh
concentration was 0.5 µM. C, PnIA, and [A10L]PnIA and
alanine mutants, have different effects on the ACh-evoked current at
7-L247T receptors; PnIA (200 nM) inhibits, while [A10L]PnIA
(200 nM) activates, currents in the same oocyte. S4A[A10L]PnIA also
activates a current, while PnIA inhibits the ACh-evoked current. The black
bar indicates the application of ACh (0.5 µM), and the
open bar indicates the toxin application. Currents have been
normalized and superimposed, and control current amplitude was 250 nA.
D, [A10L]PnIA (1 µM) activates a current at
7-L247T receptors.
|
|
A proportion of the
7-L247T receptors are spontaneously active
(14). PnIA also inhibited the
spontaneously active "leak" current
7-L247T receptors in a
concentration-dependent manner (data not shown). In contrast to the inhibitory
effect of PnIA at
7-L247T receptors, co-application of [A10L]PnIA
further activated the ACh-evoked current
(Fig. 6C), suggesting
that [A10L]PnIA and the alanine mutants stabilize a conducting state of the
receptor. All the alanine mutants of [A10L]PnIA had a similar activating
effect on the ACh-evoked currents at the
7-L247T receptors (see
Fig. 6C and
Table II). [A10L]PnIA (200
nM) increased the ACh-activated current to 1.79 ± 0.09 times
control (n = 9). The mean amplitude of the [A10L]PnIA-activated
current was 307 ± 196 nA (n = 9). [A10L]PnIA was also able to
evoke a current at
7-L247T receptors in the absence of ACh
(Fig. 6D), further
suggesting that [A10L]PnIA stabilizes an "open state" of the
receptor.
 |
DISCUSSION
|
---|
The aim of this study was to investigate the molecular interactions between
-conotoxins and the
7 nAChR and to determine what effect these
have on toxin activity. To this end, we have used a series of mutated toxins
and compared their effects on wild type and mutant nAChRs to determine whether
mutation of the native toxin changes the mechanism of inhibition, in
particular the affinity for different states of the receptor. We have examined
which amino acid side chains might be responsible for determining specificity
of binding to different receptor states.
PnIA and [A10L]PnIA both inhibit ACh-evoked currents at wt
7
receptors. [A10L]PnIA was approximately twice as potent as PnIA. The rates of
onset of block were similar for both toxins, whereas recovery from block was
slower for [A10L]PnIA. These data suggest that the greater potency of
[A10L]PnIA at
7 receptors is conveyed by a slower rate of dissociation
from the receptor. The rate of bath exchange was < 1 s, which is rapid
compared with the onset of inhibition, making it unlikely that the rate of
toxin binding is diffusion limited. A similar difference in off rates of PnIA
and [A10L]PnIA has been observed at cloned rat
7 receptors
(8). Attempts to describe toxin
blockade using the model proposed for MLA inhibition of
7 receptors
revealed that marked differences in the profile of recovery as well as
steady-state inhibition exist between
-conotoxin inhibition and MLA.
The absence of a lag phase at the onset of the recovery can be attributed to
either reflecting profound differences in the mechanism of inhibition or as an
inability to resolve the recovery phase with adequate time resolution.
Interestingly, Luo et al.
(8) have previously shown that
both the onset and recovery of PnIA and [A10L]PnIA blockade can be fitted with
a single exponential. However, the Hill coefficient greater than unity
observed for the steady-state dose-response inhibition indicates that the
toxin inhibition cannot be described by a simple bimolecular reaction.
NMR analysis has shown that the A10L substitution in PnIA does not affect
the backbone structure of the molecule or the angle of the projecting side
chains (7); therefore, this
increase in potency must be attributed to the longer aliphatic side chain of
the leucine at position 10. The circular dichroism spectra of the [A10L]PnIA
mutants were unchanged with regard to the native toxin, indicating that the
-helix is still present.2
All alanine mutants of [A10L]PnIA inhibited ACh-evoked currents at
wt
7 receptors. The potency profile of these toxins indicates that
removal of the projecting amino acid side chains between position 5 and 13 and
at position 15, by the insertion of an alanine residue, reduced the potency of
the [A10L]PnIA mutants to inhibit ACh-evoked currents at wt
7 receptors.
The pairwise interactions involved in the binding of PnIA and PnIB to the
human
7/5HT-3 receptor has been investigated by mutant cycle analysis
combined with a competitive binding assay
(21). The sequences of PnIB
and [A10L]PnIA are similar, differing only by a single serine in place of the
asparagine residue at position 11. The residues at positions 4, 5, 6, 7, 9,
and 10 were found to endow PnIB with an affinity for the
7/5HT-3
receptor. This study reported a dominant interaction between the leucine at
position 10 and Trp-149 in loop B of the positive face of the ACh binding
site, which anchors the toxin to the receptor, and also interactions between
the proline residues at positions 6 and 7 of the toxin with the receptor. Data
from the present study indicates that side chains projecting from the helical
central portion of the molecule contribute to the functional blocking activity
of [A10L]PnIA at
7 receptors. Since the previous study measured toxin
binding in the absence of agonist
(21) and the present study
functional activity, it is possible that the toxin may be binding to different
resting or desensitized states of the receptor. The modification of the
projecting side chains may change the affinity of the toxin to stabilize the
receptor in alternative closed states.
The presence of a longer aliphatic side chain at position 10 in PnIA has
been reported to increase the affinity of the toxin for
7 receptors
(21). Functional studies have
shown that A10L mutation in PnIA makes the resulting [A10L]PnIA toxin more
selective for
7 receptors
(7,
8). To determine whether this
mutation also changes the affinity of the toxin for different states of the
7 receptor, we used the
7-L247T mutant receptor. Two models have
been proposed to account for the non-desensitizing behavior of the L247T
mutant. One is that the desensitized state of the receptor becomes conducting,
and another proposes that the isomerization coefficient is altered favoring
the open state
(2224).
However, this second scheme does not adequately describe how antagonists of
the wt
7 receptor can act as agonists at the mutant receptor. If we
assume the first model, in which the toxin is stabilizing the desensitized
state of the receptor, we would expect to see a much reduced block at the
7-L247T receptor.
PnIA potently inhibited ACh-induced currents at
7-L247T receptors,
indicating that, as for MLA and
-bungarotoxin, it probably stabilizes
the receptor in the resting state. However, in contrast, [A10L]PnIA and the
[A10L]PnIA alanine scan mutants all potentiated the ACh-induced responses at
the
7-L247T receptors. In addition, [A10L]PnIA was able to activate a
current at
7-L247T receptors in the absence of ACh. Since [A10L]PnIA
potently inhibits currents at wt
7 receptors but potentiates currents at
the
7-L247T mutant, we conclude that [A10L]PnIA is stabilizing the
desensitized state of the receptor that is non-conducting in the wt
7
receptor but conducting in the
7-L247T mutant. PnIA stabilizes the
receptor in a state that is non-conducting in both receptors, which would
correspond to the resting state. The ability of all the toxins containing the
[A10L] mutation to potentiate the currents at
7-L247T receptors
indicates the presence of the longer lysine side chain at position 10 alone is
sufficient to change the specificity of the toxin to bind and stabilize the
7-L247T receptor in different states, and this is not affected by other
mutations. The observation that the amplitude of the ACh-induced current at
wt
7 receptors was increased following short 0.51-s applications
of PnIA and [A10L]PnIA (Figs.
2A and
3A) suggests both
toxins bind to the resting state of the receptor at low concentrations and
increase the ratio of receptors that are in the resting as compared with the
desensitized state. This effect supports the hypothesis that PnIA is
stabilizing the resting state of the receptor, but also suggests that at low
concentrations [A10L]PnIA may also have some affinity for a resting state of
the receptor. However, since the resting and desensitized states of the
wt
7 receptor are both non-conducting, there is no way to confirm that
[A10L]PnIA stabilizes the same state in the wt
7 and
7-L247T
receptor.
Fig. 4 illustrates that the
removal of side chains from residues between positions 5 and 13 reduce the
potency of [A10L]PnIA to inhibit currents at
7 receptors. However, it
is clear that that the removal of projecting side chains at positions other
than position 10 does not affect the ability of [A10L]PnIA to potentiate
responses at
7-L247T receptors.
While demonstrating unambiguously the ability of [A10L]PnIA to stabilize
the desensitized open state of the
7-L247T receptor, the rather complex
form of the evoked current indicates that a complex equilibrium must be
stabilized by the toxin. Since it is beyond the scope of this study to examine
these time courses in detail, no attempt was made to analyze the profile of
current decay.
This study highlights the usefulness of the
-conotoxins as research
tools, which can be subtly modified to probe the molecular interactions of
ligand binding to nAChRs. The stability of the peptide backbone to amino acid
substitutions also makes them ideal models to develop pharmacophores for nAChR
subtypes, which may lead to the development of non-peptide modulators of nAChR
function.
 |
FOOTNOTES
|
---|
* The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
To whom correspondence should be addressed: Dept. of Physiology, C.M.U. 1, rue
Michel Servet, CH-1211 Geneva 4, Switzerland. Tel.: 41-22-702-53-55; Fax:
41-22-702-54-02; E-mail:
hogg1{at}etu.unige.ch.
1 The abbreviations used are: ACh, acetylcholine; nAChR, nicotinic ACh
receptor; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',
N'-tetraacetic acid acetoxymethyl ester; HPLC, high performance
liquid chromatography; MLA, methyllycaconitine. 
2 R. C. Hogg, G. Hopping, P. F. Alewood, D. J. Adams, and D. Bertrand,
unpublished results. 
 |
REFERENCES
|
---|
- Adams, D. J., Alewood, P. F., Craik, D. J., Drinkwater, R., and
Lewis, R. J. (1998) Drug Dev. Res.
46,
219234[CrossRef]
- McIntosh, J. M., and Jones, R. M. (2001)
Toxicon 39,
14471451[CrossRef][Medline]
[Order article via Infotrieve]
- McIntosh, J. M., Santos, A. D., and Olivera, B. M.
(1999) Annu. Rev. Biochem.
68,
5988[CrossRef][Medline]
[Order article via Infotrieve]
- Arias, H. R., and Blanton, M. P. (2000)
Int. J. Biochem. Cell Biol.
32,
10171028[CrossRef][Medline]
[Order article via Infotrieve]
- Hu, S. H., Gehrmann, J., Guddat, L. W., Alewood, P. F., Craik, D.
J., and Martin, J. L. (1996) Structure
(Lond.) 4,
417423[Medline]
[Order article via Infotrieve]
- Fainzilber, M., Hasson, A., Oren, R., Burlingame, A. L., Gordon,
D., Spira, M. E., and Zlotkin, E. (1994)
Biochemistry 33,
95239529[Medline]
[Order article via Infotrieve]
- Hogg, R. C., Miranda, L. P., Craik, D. J., Lewis, R. J., Alewood,
P. F., and Adams, D. J. (1999) J. Biol.
Chem. 274,
3655936564[Abstract/Free Full Text]
- Luo, S., Nguyen, T. A., Cartier, G. E., Olivera, B. M., Yoshikami,
D., and McIntosh, J. M. (1999)
Biochemistry 38,
1454214548[CrossRef][Medline]
[Order article via Infotrieve]
- Changeux, J. P. (1990) Trends Pharmacol.
Sci. 11,
485492[CrossRef][Medline]
[Order article via Infotrieve]
- Monod, J., Wyman, J., and Changeux, J. P. (1965)
J. Mol. Biol. 12,
88118[Medline]
[Order article via Infotrieve]
- Edelstein, S. J., Schaad, O., Henry, E., Bertrand, D., and
Changeux, J. P. (1996) Biol. Cybern.
75,
361379[CrossRef][Medline]
[Order article via Infotrieve]
- Bertrand, D., Devillers-Thiery, A., Revah, F., Galzi, J. L., Hussy,
N., Mulle, C., Bertrand, S., Ballivet, M., and Changeux, J. P.
(1992) Proc. Natl. Acad. Sci. U. S. A.
89,
12611265[Abstract]
- Bertrand, D., Galzi, J. L., Devillers-Thiery, A., Bertrand, S., and
Changeux, J. P. (1993) Proc. Natl. Acad. Sci. U. S.
A. 90,
69716975[Abstract]
- Bertrand, S., Devillers-Thiery, A., Palma, E., Buisson, B.,
Edelstein, S. J., Corringer, P. J., Changeux, J. P., and Bertrand, D.
(1997) Neuroreport
8,
35913596[Medline]
[Order article via Infotrieve]
- Palma, E., Mileo, A. M., Eusebi, F., and Miledi, R.
(1996) Proc. Natl. Acad. Sci. U. S. A.
93,
1123111235[Abstract/Free Full Text]
- Palma, E., Maggi, L., Eusebi, F., and Miledi, R.
(1997) Proc. Natl. Acad. Sci. U. S. A.
94,
99159919[Abstract/Free Full Text]
- Bertrand, D., Cooper, E., Valera, S., Rungger, D., and Ballivet, M.
(1991) Methods Neurosci.
4,
174193
- Schnolzer, M., Alewood, P., Jones, A., Alewood, D., and Kent, S. B.
(1992) Int. J. Pept. Protein Res.
40,
180193[Medline]
[Order article via Infotrieve]
- Revah, F., Bertrand, D., Galzi, J. L., Devillers-Thiery, A., Mulle,
C., Hussy, N., Bertrand, S., Ballivet, M., and Changeux, J. P.
(1991) Nature
353,
846849[CrossRef][Medline]
[Order article via Infotrieve]
- Palma, E., Bertrand, S., Binzoni, T., and Bertrand, D.
(1996) J. Physiol. (Lond.)
491,
151161[Abstract]
- Quiram, P. A., McIntosh, J. M., and Sine, S. M. (2000)
J. Biol. Chem. 275,
48894896[Abstract/Free Full Text]
- Filatov, G. N., and White, M. M. (1995)
Mol. Pharmacol. 48,
379384[Abstract]
- Labarca, C., Nowak, M. W., Zhang, H., Tang, L., Deshpande, P., and
Lester, H. A. (1995) Nature
376,
514516[CrossRef][Medline]
[Order article via Infotrieve]
- Palma, E., Eusebi, F., and Miledi, R. (1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
15391543[Abstract/Free Full Text]