From the
Tryptophan 54 of the
Binding of ACh
The principal
component of the nicotinic binding site of muscle and neuronal
Neuronal
nicotinic receptor subunits
To combine
biochemical and electrophysiological approaches, we used the
Mutants were obtained as described previously
(19) and
subcloned in pMT
Chimeric cDNAs were
transfected into HEK 293 cells by calcium phosphate
precipitation
(23) . The level of expression was typically
5-20 fmol of
It
is noteworthy that this residue is highly conserved in all subunits
expected to contribute a complementary binding component: in muscle
Mutations of residue Gln-56, and to a smaller
extent that of residues Met-57 and Tyr-58, affect the apparent
affinities of ACh and nicotine. These positions are occupied by
different residues in Torpedo
Labeling and mutagenesis experiments have shown that residue Phe-65
of the
All values are shown as the mean and
standard deviation from triplicate experiments.
The normalized currents and binding data
are determined as described in Fig. 2. K
7 neuronal nicotinic homooligomeric
receptor is homologous to
-Trp-55 and
-Trp-57 of non-
subunits of Torpedo receptor labeled by
d-tubocurarine. This residue was mutated on the
7-V201-5-hydroxytryptamine (5HT)
homooligomeric
chimera, which displays
7 nicotinic pharmacology, and for which
both equilibrium binding studies and electrophysiological recordings
could be carried out in parallel. Replacement of Trp-54 by a Phe, Ala,
or His causes a progressive decrease both in binding affinity and in
responses (EC
or IC
) for acetylcholine,
nicotine, and dihydro-
-erythroidine, without significant
modification in
-Bgtx binding. Except for Gln-56, comparatively
small effects are observed when the other residues of the 52-58
region are mutated into alanine. These data support the participation
of Trp-54 to ligand binding, and provide evidence for a new
``complementary component'' of the
7 nicotinic binding
site, distinct from its three-loop ``principal component,''
and homologous to the ``non-
component'' present on
and
subunits.
(
)
to nicotinic
acetylcholine receptors causes channel opening and receptor
desensitization. Affinity labeling and expression experiments support
the notion that the two ACh binding sites of
muscle-type Torpedo receptor
result from the association of specific domains of the
subunits
(referred to as principal component of the site) with regions
of the non-
subunits (referred to as complementary component of the
site)
(1, 2, 3, 4, 5, 6) .
This notion has been extended to neuronal nicotinic receptors, whose
pharmacology varies with their
and
subunit
composition
(7, 8, 9) .
subunits comprises three separate regions from the large N-terminal
domain: loop A (Trp-86, Tyr-93), loop B (Trp-149), and loop C (Tyr-190,
Cys-192, Cys-193, and Tyr-198) (numbering according to the Torpedo receptor)
(10) . Some evidence supports the participation of
several residues of the
and
subunits of the Torpedo receptor to the complementary binding
component
(5, 11, 12, 13) . In
particular, sequencing experiments have shown that
d-tubocurarine labeling of non-
Torpedo receptor
subunits occurs on the homologous
-Trp-55 and
-Trp-57
residues
(3, 11) . Moreover, the
W55L mutation
results in a 8-fold decrease in ACh apparent affinity and
d-tubocurarine inhibitory potency from electrophysiological
recordings in the Xenopus oocytes
(14) .
7,
8, and
9 assemble as
functional homooligomers in the Xenopus oocytes
(15, 16, 17, 18) . Under
these conditions, the nicotinic binding sites form from neighboring
identical subunits, implying that each subunit potentially contains
both the principal component (which includes loops A, B, and C as
demonstrated for
7 receptor; Ref. 19) and the complementary
component. In this work, we investigate this second aspect of
7
contribution and mutate Trp-54, which is homologous to the
-Trp-55
and
-Trp-57 of Torpedo receptor.
7-V201-5HT
chimera (referred to as
wild-type)
(20) , which is efficiently transiently expressed in
HEK 293 cells whereas
7 is not. While the chimera appears somewhat
``artificial'' receptor, it possesses the extracellular
domain of
7. This confers to this construct a pharmacological
profile of activation nearly identical to that of
7 in the
Xenopus oocytes
(20) , supporting that the agonist
binding site of both receptors are structurally homologous. Here, the
pharmacological binding properties of the wild-type and mutant chimeras
are characterized by electrophysiological recording in the Xenopus oocytes and parallel binding measurements in HEK 293 cells.
vector for expression
(21) .
Electrophysiological recordings in the Xenopus oocytes were
performed as described previously
(22) .
I-
-Bgtx (Amersham Corp.) binding
sites/cm
of confluent cells. Receptor membranes were
prepared as described previously
(24) .
I-
-Bgtx binding measurements were performed at 18
°C. For
I-
-Bgtx association experiments,
receptor membranes (0.2-2 pmol of
I-
-Bgtx
binding sites) were diluted in 250 µl of HEPES buffer (10
mM HEPES, 2.5 mM CaCl
, 2.5 mM
MgCl
, 82.5 mM NaCl, pH 7.2).
I-
-Bgtx was added, and, after a selected time, the
sample was quickly diluted in 5 ml of phosphate-buffered saline buffer,
filtered through GF-C filter (Whatman), rinsed with 5 ml of
phosphate-buffered saline buffer, and counted. Nonspecific binding was
determined in the presence of 1 mML-nicotine.
Association experiments in the presence of competitors were performed
by preincubating the diluted membranes with the ligands for at least 10
min, and then measuring the initial rate of toxin binding after 5 min
incubation with 2.5 nM
I-
-Bgtx. We verified
that 3 µM nicotine equilibrated with the wild-type chimera
under the present conditions in less than 1 min (data not shown). ACh
was always used in combination with 0.1 mM eserine. For
I-
-Bgtx dissociation experiments, 2 ml (1-5
pmol) of diluted receptor membranes were preincubated with 5
nM
I-
-Bgtx for 30 min. 1 µM of
unlabeled
-Bgtx was then added, and 200 µl of the mixture was
filtered every 15 min to measure the decay of bound labeled
I-
-Bgtx. Nonspecific binding was measured by
preincubating membranes with 1 µM unlabeled
-Bgtx.
Kinetics and Equilibrium Binding in Vitro of
Since the kinetics of -Bgtx
and Nicotinic Ligands
-Bgtx
association and dissociation to the
7 receptor are slow, an
accurate measurement of the dissociation constant of nicotinic ligands
using
I-
-Bgtx as a labeled probe requires kinetic
measurements. We thus used the methodology described by Weber and
Changeux
(25, 26) to analyze the binding of
[
H]
-Bgtx to membranes from Torpedo electric organs. The time courses of association and dissociation
of
I-
-Bgtx with the wild-type chimera expressed in
HEK 293 cells can be fitted by single exponentials (Fig. 1). This
is consistent with a bimolecular mechanism of binding to an homogeneous
class of sites, giving the rate constants and K
values of
I-
-Bgtx (). As shown in
Fig. 2
, ACh, nicotine, and DH
E decrease the initial rates of
I-
-Bgtx binding on membranes expressing the
wild-type chimera. Fitting the competition data by the Hill equation
yielded protection constants K
(with which
Torpedo ACh receptor-rich membranes were shown to represent
actual dissociation constants; Ref. 26) and Hill coefficients. The
wild-type chimera displays high affinity (1.1 nM) for
I-
-Bgtx and micromolar affinities for ACh
(K
= 83 µM), nicotine
(K
= 3.6 µM) and DH
E
(K
= 2.5 µM) (Tables I and
II). These values are close to the K
values measured in binding experiments on
7 homomeric
receptors expressed in the Xenopus oocytes for
-Bgtx (1.6
nM), ACh (25 µM), and nicotine (0.5
µM)
(27) confirming that the wild-type chimera
exhibits a pharmacology closely related, if not identical, to that of
7.
Figure 1:
A, time
course of specific I-
-Bgtx binding to the wild-type
7-V201-5HT
chimera; the solidlines are the best fits obtained with the theoretical exponential.
B, time course of dissociation of
I-
-Bgtx
with the wild-type and mutant
7-V201-5HT
chimeras. For each construct, data from three separate
experiments were normalized to maximum values and then averaged. The
solidlines are the best fits obtained with the
theoretical single exponential.
Figure 2:
A-C, effect of ACh, nicotine, and
DHE on the initial rate of
I-
-Bgtx binding to
the
7-V201-5HT
wild-type and mutant chimeras.
Effect of these ligands are expressed as their capacity to slow down
the initial rate of specific
I-
-Bgtx binding
normalized to the maximum values. Each point represents the mean value
of three separate experiments. D-F, ACh and
nicotine dose-response relationship, and DH
E inhibition of
ACh-evoked responses, of
7-V201-5HT
wild-type
and mutant chimeras. Responses evoked by 3-s application of increasing
concentrations of ACh and nicotine were measured in 3-8 cells
from several batches held at -100 mV. In DH
E experiments,
several (3-7) oocytes were perfused with half-maximally effective
dose of ACh and increasing concentrations of DH
E. Peak currents
are normalized to the maximum values and averaged. In all cases, lines
are the best fits obtained with the empirical Hill
equation.
Effect of Mutations at Position 54 on the Pharmacological
Response to Nicotinic Effectors and on Ligand Binding in
Vitro
To investigate the role of the Trp side chain at position
54, we constructed mutants W54F, W54A and W54H. The time courses of the
currents evoked by ACh and nicotine on oocytes expressing each mutants
were similar to those observed for the wild-type chimera
(Fig. 3)
(20) . However, although both W54F and W54A
mutations yielded a 4-6-fold increase in ACh EC,
introduction of a histidine at this position caused a 190-fold increase
(Fig. 2, D-F). In contrast, W54F mutation had no
significant effect on nicotine elicited currents, while the W54A and
W54H mutations resulted, respectively, in 10- and 50-fold increases in
nicotine EC
. All mutations caused a comparable
10-20-fold increase in the IC
of the competitive
antagonist DH
E. Fig. 3also shows that the currents elicited
by saturating concentrations of ACh and nicotine were identical with
W54A and W54F, whereas in W54H ACh-evoked currents were only about 30%
of the nicotine-evoked currents.
Figure 3:
Current evoked by saturating
concentrations of nicotine (3 mM, trace a) and ACh
(30 mM, trace b) in the Xenopus oocytes
expressing 7-V201-5HT
W54F, W54A, and W54H
chimeras.
All constructs yielded
I-
-Bgtx binding sites when expressed in HEK 293
cells. Mutations of Trp-54 did not change the k
of
I-
-Bgtx, and only slightly increased the
k
of
I-
-Bgtx in the case of
the W54A and W54F (2- and 3-fold increase, respectively)
(). The mutations caused increases in the K
of ACh, nicotine, and DH
E, with no significant changes of
Hill coefficients (Fig. 2, A-C). The increases in
K
values for the three ligands were close to the
increases in apparent dissociation constants observed in
electrophysiological recordings (). For nicotine, the 1-,
10-, and 60-fold shifts in K
are almost identical
to the 1.5-, 10-, and 50-fold shifts in EC
, for mutants
W54F, W54A, and W54H, respectively. For ACh, the shifts in
K
(3.5-, 10-, and 44-fold) correlate reasonably
well with the shifts in EC
(4-, 6-, and 190-fold). All
mutations caused similar increases in DH
E K
(3-10-fold) and IC
(10-20-fold). W54H
mutation also resulted in a 10-fold increase in d-tubocurarine
K
(data not shown). Involvement of Residues at Positions 52-59 in the Agonist Binding
Site-We explored the possible contribution of amino acids
neighboring Trp-54 by mutating those present from position 52 to 59
into alanines (Fig. 4). These mutants yielded
I-
-Bgtx binding sites, except in the case of the
mutation of the canonical residue Trp-59, with which no toxin binding
nor electrophysiological response were observed. This feature can
result from either a poor expression of the receptor or a disruption of
the nicotinic binding site. The N52A, I53A, and L55A mutants appeared
identical to the wild-type chimera when tested for ACh and nicotine
inhibition of
I-
-Bgtx binding (data not shown). The
M57A and Y58A mutations caused small although significant effects on
receptor pharmacology, and the Q56A mutation caused a 3- and 20-fold
increase in K
and a 8- and 7- fold increase in
EC
, for ACh and nicotine, respectively ().
Figure 4:
Comparison of the amino acid sequences of
some nicotinic, GABA, 5HT
, and glycine
receptors (reviewed in Ref. 31) containing Trp-54 of chicken
7
nicotinic receptor (15).
DISCUSSION
Mutation of Trp-54 causes parallel increases in binding
affinity (K) and in apparent affinity for channel
activation (EC
or IC
) for ACh, nicotine, and
DH
E. This is compatible with the simple notion that mutations of
Trp-54 specifically alter the nicotinic binding site. In this respect,
the different efficacies observed for ACh and nicotine for the W54H
mutant can be interpreted either by a channel block of ACh, since the
recordings were performed in the presence of a high concentration of
agonist, or by the fact that the mutation affects to a different extent
the affinity for one of these agonists in the resting and active states
of the receptor. In contrast, the kinetics and equilibrium dissociation
constants of
I-
-Bgtx were not significantly altered,
showing that modifying the side chain of Trp-54 did not disrupt the
-Bgtx binding site and thus the general tertiary and quaternary
structure of the related part of the pentamer. Altogether, these
mutagenesis results are consistent with a local alteration of the ACh
binding domain. Moreover, the labeling of Torpedo
-Trp-55
and
-Trp-57 by d-tubocurarine supports the notion that
Trp-54 contributes directly to ligand binding. Consistent with this
view, we find that the presence of electronegative aromatic residues
(Trp and Phe as compared to Ala) favors agonist binding, while the
introduction of a positive charge with a histidine causes a dramatic
decrease in agonist affinity, suggesting a direct involvement of Trp-54
in the quaternary ammonium binding pocket of nicotinic ligands.
and
subunits and neuronal
2,
4,
8, and
9 subunits. However, it is absent in the corresponding regions of
muscle
and
subunits (Fig. 4). A tryptophan, however,
is present at a homologous position in the neuronal
2-4
subunits, which are not expected to contribute a complementary
component. A similar feature is seen for residues composing loops A and
B, which, while implicated in the principal binding component of muscle
and neuronal receptors, are also conserved in
2 and
4
subunits. This observation may point to a particular function of these
neuronal subunits.
and
subunits, and in
neuronal subunits (Fig. 4). This suggests that amino acids
located at these positions could be involved in the non-equivalence of
the two Torpedo receptor binding sites
(13, 28) and/or in the pharmacological diversity of neuronal
receptors distinct from the non-
subunits of muscle receptor.
1 subunit of GABA
receptors contributes to the
binding site of GABA
(29, 30) . This residue is
homologous to
7Trp-54, and
subunits of GABA
receptors appear to be functionally homologous to nicotinic
non-
subunits
(10) . The notion that residues homologous to
7Trp-54 contribute to a complementary component of the agonist
binding site in addition to its principal one may thus be extended to
other members of the superfamily of ligand-gated ion channels.
Table: Kinetic constants and calculated
dissociation constants ofI
-Bgtx for
7-V201-5HT
wild-type
and mutant chimeras
Table: Affinity constants of 7-V201-5HT
wild-type
and mutant chimeras
,
EC
, and IC
are presented as the mean and
standard deviation. ND, not determined.
-Bgtx,
-bungarotoxin; DH
E, dihydro-
-erythroidine; GABA,
-aminobutyric acid.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.