From the Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115
Received for publication, October 4, 2000, and in revised form, October 27, 2000
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ABSTRACT |
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Results of affinity-labeling studies and
mutational analyses provide evidence that the agonist binding sites of
the nicotinic acetylcholine receptor (nAChR) are located at the The nicotinic acetylcholine receptor
(nAChR)1 from
Torpedo electric organ and vertebrate skeletal muscle is a
pentameric transmembrane protein composed of four homologous subunits
with a stoichiometry of Several experimental approaches have identified residues in both In this report we examine the contributions of Materials--
ACh, dTC, tetramethylammonium,
phenyltrimethylammonium, suberyldicholine, nicotine, and gallamine were
obtained from Sigma. Epibatidine and dihydro- In Vitro Transcription and Expression in Xenopus
Oocytes--
SP64-based plasmids (pMXT) with cDNAs encoding
wild-type Binding of 125I- Biosynthetic Labeling and Immunoprecipitation of
nAChRs--
15-30 injected oocytes were incubated in 2 ml of low
Ca2+ ND96 (plus 50 µg/ml gentamicin) containing 0.1-0.5
mCi/ml [35S]methonine/cysteine (PerkinElmer Life
Sciences) in 24-well plates at 18 °C for at least 48 h before
use. Ten to fifteen healthy oocytes were selected and washed five times
with 2 ml of low Ca2+ ND96 to remove excess
[35S]Met/Cys before the addition of Sucrose Density Gradient Analysis of nAChRs in Oocytes--
To
characterize surface nAChRs, 15-30 injected intact oocytes were
incubated with 2.5 nM 125I- Electrophysiology--
Currents elicited by ACh were measured
using a standard two-electrode voltage clamp (Oocyte Clamp OC-725B,
Warner Instrument Corp.) at a holding potential of Data Analysis--
The concentration-dependent
inhibition of 125I-
For receptor activation, concentration-response curves for ACh and
other agonists were fit to the following equation.
The dose-dependent inhibition of ACh-induced currents by
antagonists was fit according to Equation 1 above. SigmaPlot (Jandel Scientific) was used for nonlinear least squares fit of the data, and
the S.E. of the parameter fits are indicated in the Tables.
Influence of
In contrast to what was observed for dTC binding,
The effects of the
The observed equilibrium binding reflects the binding affinity of the
ACh sites in the desensitized nAChR and the conformational equilibrium
between resting and desensitized states. To test whether the leucine
substitution had a predominant effect on the latter parameter, we
examined the effect of proadifen, a desensitizing noncompetitive
antagonist (24), on the ACh equilibrium binding function. For wild-type
and Composition and Assembly of Subunits in Mutant nAChRs--
To rule
out the possibility that the perturbation of ACh binding resulted from
nAChRs of altered subunit composition, we examined both the size and
subunit composition of the wild-type and mutant nAChRs formed in our
expression system. nAChR biosynthetic assembly intermediates can form
high affinity binding sites for 125I-
Sucrose density gradient analysis was carried out to determine the
size(s) of the receptors expressed in oocytes. To label surface nAChRs,
intact oocytes were incubated with 125I-
When the mutant
Subunit compositions of expressed wild-type and mutant nAChRs were
characterized by immunoprecipitation of
Fig. 5B shows a comparison of the number of
125I-
The Hill coefficients (nH) characterizing the ACh
dose-response relations were estimated from log-log plots of current amplitude (I) versus [ACh] at concentrations of
ACh producing currents less than 20% of maximal responses. This is a
reliable method of determining nH for ACh responses
without reference to the experimentally determined maximal currents,
which are limited by desensitization and/or channel block. For the
wild-type and dTC Potentiation of Agonist-induced Activation in
For the mutant receptor containing Influence of
The effects of the double mutation
( A wide body of evidence establishes that the two agonist binding
sites in muscle-type nAChRs are positioned at the interfaces of Although the leucine substitutions had no effect on dTC binding, on
nAChR subunit assembly as judged by sucrose density gradient velocity
sedimentation (Fig. 2) and immunoprecipitation (Fig. 3), or on the
level of surface expression of nAChRs (Fig. 5B), the
substitutions clearly altered agonist interactions as evidenced by the
perturbation of the ACh-induced currents (Figs. 4 and 5). The ACh
concentrations producing half-maximal currents
(Kap) were shifted to the right 8- and 20-fold
for the Although dTC binding affinity at the The leucine substitutions, especially the replacement of Replacement of Since the leucine substitutions had no effect on the equilibrium
binding of dTC, it was very surprising to observe for the Replacement of Although the presence of Our results establish the importance of Substitutions at positions equivalent to nAChR The results presented here demonstrate that for the Torpedo
nAChR a single mutation (-
and
-
subunit interfaces. For Torpedo nAChR,
photoaffinity-labeling studies with the competitive antagonist
d-[3H]tubocurarine (dTC) identified two
tryptophans,
Trp-55 and
Trp-57, as the primary sites of
photolabeling in the non-
subunits. To characterize the importance
of
Trp-55 and
Trp-57 to the interactions of agonists and
antagonists, Torpedo nAChRs were expressed in Xenopus oocytes, and equilibrium binding assays and
electrophysiological recordings were used to examine the functional
consequences when either or both tryptophans were mutated to leucine.
Neither substitution altered the equilibrium binding of dTC. However,
the
W57L and
W55L mutations decreased acetylcholine (ACh) binding
affinity by 20- and 7,000-fold respectively. For the wild-type,
W55L, and
W57L nAChRs, the concentration dependence of channel
activation was characterized by Hill coefficients of 1.8, 1.1, and 1.7. For the
W55L mutant, dTC binding at the
-
site acts not as a
competitive antagonist but as a coactivator or partial agonist. These
results establish that interactions with
Trp-55 of the
Torpedo nAChR play a crucial role in agonist binding and in
the agonist-induced conformational changes that lead to channel opening.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
(reviewed in Refs.
1-3). The nAChR contains two binding sites for agonists and
competitive antagonists, located at the
-
and
-
subunit
interfaces (4, 5). The two sites are nonequivalent, and many
competitive antagonists bind with high affinity to only one of the
sites (6, 7). Affinity labeling and mutational analyses provide
evidence that amino acids from three discrete regions of
subunit
primary structure and from three (or more) regions of the
(or
)
subunit contribute to the structure of the binding sites (reviewed in
Refs. 8 and 9).
and
subunits that contribute to the binding sites for agonists or
competitive antagonists. Photoaffinity labeling using d-[3H]tubocurarine (dTC), a competitive
antagonist, and [3H]nicotine, an agonist, established
that
Trp-55 is located near the agonist binding site in
Torpedo nAChR (10, 11), and [3H]dTC also
reacted with
Trp-57, the corresponding position in the
subunit,
as well as with
Tyr-111/
Tyr-117 (12). Substitution of
Trp-55
by leucine (
W55L) caused a 10-fold decrease in dTC potency as an
inhibitor of ACh-induced currents for Torpedo nAChRs expressed in Xenopus oocytes (13). A heterobifunctional
cross-linker ~9 Å in length cross-linked
Cys-192/
Cys-193 in
Torpedo nAChR to a residue in the
subunit identified as
Asp-180 (14, 15), and the mutation
D180N in the
subunit of
mouse muscle nAChR caused substantial decreases in agonist, but not
antagonist, binding affinities (16-18). Analysis of binding properties
of embryonic mouse nAChRs containing chimeras between
and
subunits led to the identification of three positions in the
subunit (Ile-116, Tyr-117, and Ser-161) and the corresponding residues
in the
subunit (Ser-118, Thr-119, and Lys-163), which can account
for the binding selectivity of the competitive antagonist
dimethyl-d-tubocurarine (metocurine (19)), whereas for the
agonist carbamylcholine, the primary determinants of site selectivity
were
Lys-34/
Ser-36 and
Phe-172/
Ile-178 (20). For the adult
mouse nAChR (
2
), a similar analysis of chimeric
-
subunits identified amino acids in another region of subunit
primary structure (
Ile-58/
His-60 and
Asp-59/
Ala-61) as
determinants of metocurine site selectivity (21).
Trp-55 and
Trp-57
as determinants of agonist binding and channel gating as well as
determinants of competitive antagonist function.
Trp-55 and
Trp-57 were mutated to leucine, and the interaction of agonists and
antagonists were examined using both binding assays and
electrophysiological recording. Concentration-dependent
inhibition of 125I-
-bungarotoxin (
-BgTx) binding by
agonists and antagonists was studied using wild-type and mutant
Torpedo nAChRs in membranes isolated from Xenopus
oocyte homogenates. In parallel experiments, we studied the activation
of wild-type and mutant nAChRs by ACh using a two-electrode voltage
clamp. Our results establish that the mutation
W55L has no effect on
dTC equilibrium binding affinity. However, the mutation has a profound
effect on ACh binding and on the gating of the ion channel.
Furthermore, for the
W55L mutant nAChR, dTC acts not as an
antagonist when bound to its high affinity site but as a coactivator
with ACh.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-erythroidine were
obtained from Sigma-RBI (Natick, MA), and pancuronium was from
Organon Inc. (W. Orange, NJ).
-BgTx was from Biotoxins Inc. (St.
Cloud, FL). 125I-
-BgTx (400-600 Ci/mmol) was prepared
using iodogen (Pierce) and Na[125I] as described (22).
Na[125I] and [35S]methionine/cysteine were
from PerkinElmer Life Sciences. Affinity-purified rabbit
antibodies against
-BgTx were kindly provided by Dr. Robert Sealock
(University of North Carolina), and metocurine and 13'-iodo-dTC were
donated by Dr. Steen E. Pedersen (23).
,
,
, and the
W55L subunits were gifts from Dr.
Michael M. White, and the cDNA (in plasmid SP64) encoding the
wild-type
subunit was from Dr. Henry Lester. Sequence analysis of
the
W57L mutant cDNA (from Dr. White) revealed a point deletion
353 bases 3' to the
W57L mutation point that resulted in a truncated
form of the subunit. Therefore, the full-length
W57L mutant was
prepared by subcloning a NheI (present in the vector) and
BstXI fragment that included the desired mutation (
W57L)
but excluded the point deletion into the wild-type
subunit cDNA
from which the corresponding region had been excised. cDNAs were
linearized with either XbaI (for wild-type
,
,
,
W55L, and
W57L mutant subunits) or FspI (for the
wild-type
subunit). In vitro transcription reactions were carried out in transcription buffer (Promega) containing 40 mM Tris (pH 7.5), 6 mM MgCl2, 2 mM spermidine, and 10 mM NaCl. Linear cDNAs
(5-10 µg) were incubated with 10 mM dithiothreitol, NTPs
(1 mM each except GTP, which was 0.2 mM), 0.6 mM diguanosine triphosphate (Amersham Pharmacia Biotech),
100 units of RNasin (Promega), and 40 units of SP6 RNA polymerase
(Promega) in transcription buffer at 37 °C for 1 h. An
additional 40 units of SP6 RNA polymerase was added after 1 h, and
the incubation was continued for another hour. In some experiments,
[3H]UTP was included in the reaction mixture as a tracer
for quantitation of reaction yields. RNAs were extracted with
phenol/chloroform and chloroform, precipitated from isopropanol, and
then resuspended in water at a concentration of
3 µg/µl.
Isolated, follicle-free oocytes were microinjected with 0.5-10 ng of
subunit-specific RNAs in a molar stoichiometry of
(
2
) for wild-type and mutant receptors. For
-less and
-less receptors, nAChR subunit RNAs were mixed in a
molar ratio of (
2
2) and
(
2
2), respectively. Oocytes were
injected with 10 ng of subunit RNA for assays of 125I-
-BgTx binding in intact oocytes or in membrane
fractions and for all electrophysiological assays with the exception of
full agonist-dose response relations for wild-type receptors. Because of the high currents produced by activation of wild-type receptors, maximal responses were determined for oocytes injected with 0.5 ng of
RNA. Oocytes were maintained in ND96 buffer containing 96 mM NaCl, 2 mM KCl, 1.8 mM
CaCl2, 1 mM MgCl2, 5 mM
HEPES (pH 7.6), and 50 µg/ml gentamicin for at least 48 h before use.
-BgTx to Intact Oocytes and to
Oocyte Membranes--
To measure binding to nAChRs expressed on the
surface, oocytes were incubated with 2.5 nM
125I-
-BgTx for 2 h in a final volume of 100 µl of
low Ca2+ ND96 buffer containing 96 mM NaCl, 2 mM KCl, 0.3 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES (pH 7.6).
Oocytes were washed 3 times with 1 ml of ice-cold low Ca2+
ND96 buffer containing 1% bovine serum albumin and counted in a
counter. Nonspecific binding was determined using uninjected oocytes.
Oocyte membranes were prepared by homogenizing oocytes in ice-cold
homogenization buffer (HB, 0.1 ml/oocyte) containing 140 mM
NaCl, 20 mM sodium phosphate, 1 mM EDTA, 1 mM EGTA, 1 mM phenylmethysulfonylfluoride, and
0.1 units of aprotinin/ml (pH 7.6). Membranes were isolated as
described (12) by differential and sucrose density centrifugation.
Membranes were resuspended in HB at 5 µl per oocyte, frozen in liquid
nitrogen, and stored at
80 °C. A centrifugation assay (12) was
used to measure the initial rate of 125I-
-BgTx binding
to oocyte membranes in low Ca2+ ND96 assay buffer
(supplemented with the cholinesterase inhibitor diisopropylphosphofluoridate (0.1 mM) for ACh binding
experiments). Binding assays were initiated by adding
125I-
-BgTx (a final concentration of 2.5 nM)
to triplicate samples (2-3 oocytes/sample) in a total volume of 100 µl. Membranes were preequilibrated for 15 min with appropriate
concentrations of dTC, ACh, or other ligands before the addition of
125I-
-BgTx, and then after a 20-min incubation, the
reactions were stopped by adding 1 µM unlabeled
-BgTx.
Under these conditions, in the absence of competing ligands
125I-
-BgTx binding was
30% of equilibrium.
Nonspecific binding of 125I-
-BgTx to wild-type and
mutant receptors was determined either in the presence of 100 µM dTC (24 ± 6% of total binding,
n = 8 experiments) or 100 mM ACh (27 ± 15% of total binding, n = 8 experiments) for dTC
and ACh inhibition, respectively.
-BgTx (2.5-5
nM) to label surface receptors in a final volume of 1 ml.
After a 2-h incubation, unbound
-BgTx was removed by washing oocytes
three times with 1 ml of low Ca2+ ND96. The oocytes were
then used to prepare membranes containing
-BgTx-labeled surface
receptors. To characterize internal receptors, membranes containing
nonradioactive
-BgTx-prelabeled surface nAChRs were incubated with
-BgTx (2.5-5 nM) for another 2 h followed by
washing. The membranes prepared in this manner contain both
-BgTx-labeled surface and internal nAChRs and are designated as
"total receptors." The membranes containing either surface or total
receptors were then resuspended in HB buffer supplemented with 1%
Triton X-100. After solubilization for 30 min, the extracts were
treated with 1% Immunoprecipitin (Life Technologies, Inc.) at 4 °C
for 20 min and centrifuged for 1 min in an Eppendorf microcentrifuge. Immunoprecipitin-pretreated supernatants were then collected and incubated with an excess amount of rabbit anti-
-BgTx antibody at
4 °C overnight. After overnight incubation, Immunoprecipitin (1%)
was added to the nAChR-antibody complexes and incubated at 4 °C for 30 min. The samples were then spun in a
microcentrifuge for 1 min, and the supernatants were discarded.
The immunoprecipitate was washed four times with HB buffer containing
1% Triton X-100, 0.1% SDS, and 0.5% bovine serum albumin and once
with HB buffer containing 0.1% SDS and 0.05% Triton X-100 (without
bovine serum albumin). After the last wash, the pellets were
resuspended in 50 µl of SDS-polyacrylamide gel electrophoresis sample
buffer and incubated at room temperature for 20-30 min. The samples
were electrophoresed on an 8% SDS-polyacrylamide gel. After staining with Coomassie Blue and destaining, the gels were soaked in Amplify (Amersham Pharmacia Biotech) for 30 min, dried at 65 °C for 2 h, and exposed to x-ray film at
80 °C for 24 h.
-BgTx for 2 h followed by a wash, and oocyte membranes were then prepared as
described above. The membranes were then solubilized with 1% Triton
X-100 in a final volume of 200 µl of HB buffer and centrifuged in a
Ti 42.2 rotor at 35,000 rpm for 20 min. The supernatants (
180 µl)
were saved and used for sedimentation analysis. For total nAChRs,
oocyte membranes containing surface nAChRs prelabeled by
125I-
-BgTx were then incubated with 2.5 nM
125I-
-BgTx for another 2 h. After a 2-h incubation,
membranes were washed and solubilized as described above. 180-µl
extracts from 10-15 oocytes containing either surface or total
receptors were sedimented on a 10-ml 3-30% sucrose gradient (150 mM NaCl, 5 mM EDTA, 50 mM Tris pH
7.6, 1% Triton X-100, and 1 mM dithiothreitol) at 40,000 rpm in a SW 45 rotor (Beckman) for 22 h at 4 °C. 200-µl extracts from Torpedo membranes prelabeled with
125I-
-BgTx were used as the control for monomeric (9.5 S) and dimeric nAChR (13 S), and free 125I-
-BgTx (1.7 S), alkaline phosphatase (6.1 S), and
-galactosidase (15.9 S) were
used as standards for sedimentation coefficients. Fractions (200 µl)
were collected and counted on a
counter.
70 mV. Electrodes
were filled with 3 M KCl and had resistances of 0.5-1.5
megaohms. The recording chamber (about 150 µl in volume) was perfused
continually by gravity with low Ca2+ ND96 (plus 1 µM atropine, pH 7.6). Appropriate concentrations of ACh
(or other agonists) in the absence or presence of antagonists were
applied through solenoid valves into the recording chamber for 3-5 s.
For some experiments, oocytes were preincubated with dTC by perfusing
the oocytes for ~1 min with dTC in low Ca2+ ND96 before
application for 5 s of solution containing ACh with the same
concentration of dTC.
-BgTx binding by agonists and
antagonists was fit according to two models as follows.
where [X] is the concentration of inhibitor, n is
the Hill coefficient, and IC50 is the inhibitor
concentration reducing the initial rate of 125I-
(Eq. 1)
-BgTx
binding by 50% and
where [X] is the concentration of competing ligand,
KH and KL are the ligand
affinities for the high and low affinity binding sites, respectively.
This equation is based on the assumption that
(Eq. 2)
-BgTx binds at equal
rates to the two sites.
where I and Imax are the
currents at a given concentration of ACh and the maximal value,
respectively, and Kap is the concentration of
ACh required for half-maximal current. Because high concentrations of
ACh do not result in a concentration-independent maximal response (due
to desensitization and/or channel block), the Hill coefficients (nH) for the agonist dose-response relations were
estimated from the slope of plots of log I versus
log[agonist] at currents less than 20% of the maximal response for
each agonist.
(Eq. 3)
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
W55L and
W57L on the Binding of d-Tubocurarine
and Acetylcholine--
Torpedo nAChRs were expressed in
Xenopus oocytes by injection of cRNAs encoding
,
,
, and
subunits. We characterized the equilibrium binding of
antagonists and agonists by their inhibition of
125I-
-BgTx binding to Torpedo nAChRs in
membranes isolated from oocyte homogenates. dTC inhibited
125I-
-BgTx binding to wild-type
(
2
) nAChRs in a
concentration-dependent manner with an IC50 of
440 ± 30 nM (Hill coefficient, nH = 0.50 ± 0.01) (Fig. 1A,
see "Experimental Procedures"). These data were well fit by a
two-site model with KH = 50 nM (high affinity site) and KL = 4 µM
(low affinity site) (see Table I), and
the data were consistent with the equilibrium binding of
[3H]dTC to nAChRs in Torpedo membranes (5, 6)
and with the assumption that 125I-
-BgTx binds to the two
agonist sites with equal association rate constants. dTC binds to
-less (
2
2) receptors with an IC50 of 140 nM and to
-less
(
2
2) receptors with an
IC50 of 8 µM, consistent with the notion that
for dTC the high and low affinity sites are formed at the
-
and
-
interfaces, respectively (Fig. 1A, inset,
and Table I). We determined the effects of the
W55L and
W57L
mutations on 125I-
-BgTx binding and on dTC competition
with 125I-
-BgTx binding. Control experiments indicated
that
W55L and
W57L had no effect on 125I-
-BgTx
binding affinity (data not shown). Despite the decrease of dTC potency
as an inhibitor of nAChRs containing
W55L (Ref. 13 and see below),
nAChRs containing either a
W55L (
m), a
W57L
(
m) subunit, or both mutant subunits
(
2
m
m) had the same
binding affinities for dTC as wild-type receptors (Fig. 1A).
The concentration dependence for dTC inhibition of 125I-
-BgTx binding to mutant receptors was clearly
different from that observed for either
-less or
-less nAChRs
(Fig. 1, inset), which indicates that the lack of effect of
the mutations on dTC binding is not due to omission of either
m or
m subunit. Thus, substitution of
Trp-55 or
Trp-57 by leucine has no effect on dTC binding
affinity. These tryptophan residues are, however, located near the
agonist binding sites because they can be affinity-photolabeled by
[3H]dTC and [3H]nicotine (10, 11). The
discrepancy between binding and functional antagonism will be explained
below.
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Fig. 1.
Effects of W55L
and
W57L mutations on the equilibrium binding
of dTC (A) and ACh (B) to
Torpedo nAChRs. Equilibrium binding was
determined by the inhibition of 125I-
-BgTx binding to
Torpedo nAChRs in membranes isolated from oocyte homogenates
for wild-type nAChR (
,
2
) or mutant
receptors containing
W55L (
,
2
m
),
W57L (
2
m), or both mutant subunits (
2
m
m). For ACh, binding
was also determined for
2
(
m)2 (
) and
2
(
m)2 (
) mutant nAChRs
as well as for native Torpedo membranes (
). Assay
aliquots (100 µl) of Torpedo membranes contained 50 fmol
of nAChR and membranes from 3 uninjected oocytes and were treated with
100 µM diisopropylphosphofluoridate to inactivate
cholinesterase. Insets, dTC (A) and ACh
(B) binding to
-less (
,
2
2) and
-less (
,
2
2) nAChRs compared with wild-type
(
). For each data set, the data points represent the mean ± S.D. of triplicate samples from a single experiment representative of
2-4 experiments. Solid curves are calculated from the
parameters of Table I for the two-site model ("Experimental
Procedures," Equation 2).
Equilibrium binding parameters for dTC and ACh
W55L and
W57L
dramatically increased the IC50 for ACh competition against 125I-
-BgTx binding to nAChRs. ACh inhibited
125I-
-BgTx binding to the wild-type Torpedo
nAChR expressed in oocytes in a concentration-dependent
manner with an IC50 of 340 nM and a Hill
coefficient of 0.6 (Fig. 1B).
W55L and
W57L increased the IC50 for ACh binding by 62- and 8-fold, respectively,
and for the double mutant (
2
m
m), the IC50 was 530-fold higher than for
wild-type receptor (Fig. 1B). The IC50 values
for ACh binding to either
2
(
m)2 or
2
(
m)2 receptors were each
increased by about 1,300-fold (Fig. 1B). The concentration
dependence of ACh inhibition of 125I-
-BgTx binding
reflects the binding of both ACh and 125I-
-BgTx to two
agonist binding sites. For the wild-type receptor this concentration
dependence was well fit by a two-site model with KH
of 55 nM and KL of 2.7 µM (Table I). ACh bound to the
W55L-containing
receptor with KH of 750 nM and KL of 360 µM. For the
W57L-containing receptor, ACh bound with KH
of 140 nM and KL of 66 µM.
W55L and
W57L mutations on ACh binding were
clearly different than that observed for nAChRs lacking either
subunit (
-less,
2
2,
IC50 = 0.8 µM) or
subunit (
-less,
2
2, IC50 = 0.3 µM), which were characterized by inhibition curves
similar to wild-type (Fig. 1B, inset and Table
I). As judged by inhibition of 125I-
-BgTx binding, ACh
binding to Torpedo nAChRs expressed in oocytes also differed
from the binding to native nAChRs in Torpedo membranes (IC50 = 25 nM and a Hill coefficient of 0.9. (Fig. 1B)).
2
m
m, proadifen
produced only a modest (less than 3-fold) left shift of the ACh
equilibrium binding function (data not shown), as it does for nAChRs in
membranes from Torpedo electric organ (24), but in contrast
to the 100-fold enhancement of ACh affinity seen for mouse muscle nAChR
in the presence of proadifen (20).
-BgTx (
subunit
alone) or agonists and competitive antagonists (
/
or
/
subunit pairs) (4). The binding studies described above were performed
with membranes isolated from oocyte homogenates that contain both
surface receptors and receptors from intracellular membranes. This
internal pool may contain agonist binding sites that are very different
than those found in the pentameric nAChRs, since the internal
-BgTx
binding sites may include nAChR assembly intermediates. We therefore
compared surface and internal receptors in oocyte membranes by
examining both the size(s) of the 125I-
-BgTx binding
components by sedimentation analysis and the subunit composition by
biosynthetic labeling and immunoprecipitation.
-BgTx before
isolation of oocyte membranes, and isolated membranes were reincubated
in 125I-
-BgTx to label all sites made accessible after
homogenization of the oocytes (total receptors). Membranes were
extracted in 1% Triton X-100, and the sedimentation properties of
these nAChRs were compared (Fig. 2) with
125I-
-BgTx-labeled native Torpedo nAChRs
(monomer, 9.5 S; dimer, 13 S) (Fig. 2A). In the membranes
isolated from oocyte homogenates, there were 3-5 times more total
125I-
-BgTx sites (Fig. 2, B-E, closed
circles) than surface sites (Fig. 2, B-E, open
circles). However, the major populations of both surface and total
125I-
-BgTx binding sites in oocyte membranes were
pentameric nAChRs, as revealed by a characteristic large peak of
125I at 9.5S for wild-type as well as mutant nAChRs
(
m,
m,
m
m,
Fig. 2, B-E). In some experiments (Figs. 2, B
and D), total receptors in oocyte membranes also yielded a
much smaller peak of 125I at 5.0 S, which has been shown
previously to be subunit pairs of either
or
subunits
(25). The peak at 1.7 S in all our experiments represented free
125I-
-BgTx. Thus, as for wild-type nAChRs, binding of
125I-
-BgTx to each of these mutants in membrane
homogenates will reflect binding to assembled, pentameric nAChRs, and
partial assembly cannot account for the altered ACh binding seen for
the mutant nAChRs.
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Fig. 2.
Sucrose density gradient characterization of
nAChRs containing W55L and/or
W57L mutant subunits. Membranes were prepared
from oocytes preincubated with 125I-
-BgTx to label
surface nAChRs (
), and isolated membranes were further incubated
with 125I-
-BgTx to label surface and internal
-BgTx
binding sites (
, total receptors). Triton X-100 extracts of labeled
membranes prepared from pools of 10-15 injected oocytes were
sedimented on 3-30% sucrose gradients. Fractions (200 µl) were
collected from the top of the gradient and counted in a
counter.
125I-
-BgTx-labeled native Torpedo nAChRs in
Triton X-100 extracts, which sedimented primarily as pentameric monomer
(9.5 S) along with 13 S dimers, were used as controls (Panel
A). For nAChRs expressed in oocytes (panels B-H), the
total receptors (
) were 3-5 times more abundant than surface
receptors (
) for
2
(B),
2
m
(C),
2
m (D),
2
m
m (E),
2
2 (G), or
2
(
m)2 (H). When
the mutant
subunit was coexpressed with wild-type
and
subunits without the
subunit
(
2
(
m)2, panel
F), as seen for wild-type
-less receptor
(
2
2) (data not shown), no stable
subunit complexes were detected in Triton X-100. For nAChRs containing
four different subunits, including either
m,
m, or
m
m, total as well as
surface receptors sedimented as 9.5 S pentamers (panels
C-E), whereas for receptors lacking the
subunit but
containing either
or
m, a 5.0 S peak of
125I-
-BgTx binding was also prominent (panels
G and H), consistent with the expected sedimentation of
dimeric forms containing
-
subunits. The peak of 125I
found at 1.7 S in all experiments resulted from the presence of free
125I-
-BgTx.
subunit was coexpressed with wild-type
and
subunits without the
subunit
(
2
(
m)2, Fig.
2F), stable assembly of nAChR subunits was not observed in
Triton X-100. This result, which was also seen for wild-type
-less
receptor (
2
2, data not shown), is
consistent with previous observations (25, 26). For the mutant
subunit coexpressed with wild-type
and
subunits without the
subunit
2
(
m)2, in addition
to the 9.5 S peak of 125I, there was a prominent 5 S
component in the total homogenate, as was seen for wild-type
-less
receptor (
2
2) (Figs. 2, G and H). These results indicated that partial assembly
intermediates are not prominent for the mutant nAChRs containing all
four subunits, but they may be more significant for wild-type or mutant
nAChRs lacking the
or
subunit.
-BgTx-labeled receptors
(surface and total) extracted from oocyte homogenates after
biosynthetic labeling with [35S]Met/Cys. Fig.
3 shows that for both surface
(A) and total receptors (B), the presence of
W55L,
W57L, or both mutant subunits had no effect on subunit
assembly, and the mutations had no effect on subunit glycosylation as
judged by the mobilities of mutant compared with wild-type subunits.
Furthermore, the mobilities of these subunits in nAChRs expressed in
Xenopus oocytes were the same as the native
Torpedo nAChR subunits (not shown). However, for nAChRs
lacking the
subunit, mutant
subunit had the same mobility as
wild type, but both had slightly higher mobility than
subunits in
nAChRs also containing the
subunit. Similarly, omission of the
subunit resulted in
subunits of enhanced mobility (data not
shown).
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Fig. 3.
Biosynthetic labeling and immunoprecipitation
analyses of the subunit composition of nAChRs containing
W55L and/or
W57L mutant
subunits. 10-15 injected oocytes were incubated in ND96 (plus 50 µg/ml gentamicin) containing [35S]methionine/cysteine
(0.1-0.5 mCi/ml) for 48 h. Surface (A) and total
(B) receptors were labeled with nonradioactive
-BgTx (see
"Experimental Procedures"). Triton X-100 extracts containing either
surface or total receptors labeled by
-BgTx were incubated with
affinity-purified rabbit antibody against
-BgTx and
immunoprecipitated with 1% Immunoprecipitin. Immunoprecipitates were
extracted in sample buffer and analyzed by SDS-polyacrylamide gel
electrophoresis. Gels were stained with Coomassie Blue, destained, and
prepared for fluorography (36-h exposure). For both surface
(A) and total (B) receptors (1,
2
; 2,
2
m
; 3,
2
m; 4,
2
m
m; 5,
uninjected oocytes)
W55L and
W57L mutant subunits were expressed
and assembled with other wild-type subunits as efficiently as seen for
wild-type subunits.
W55L and
W57L Alter nAChR Activation--
We examined the
effects of
W55L and
W57L on the activation of nAChRs expressed in
oocytes using a two-electrode voltage clamp. When holding the oocyte
membrane potential at
70 mV, there was a
concentration-dependent activation of inward current upon 5-s application of ACh for wild-type (Fig.
4A) and mutant nAChRs (
2
m
, Fig. 4B). For
wild-type nAChR, preincubation with dTC produced a
dose-dependent inhibition of ACh currents characterized by
an IC50 of 40 nM (Figs. 4, C and
D), as expected when binding to its high affinity site
results in functional antagonism. However, as reported previously (13)
for nAChRs containing
W55L, dTC inhibited ACh currents only at
higher concentrations (IC50 = 4 µM, Fig.
4D), despite the presence of the high affinity dTC binding site (KH = 40 nM, Fig. 1A).
Based upon the peak transient current observed for each ACh
concentration, the ACh concentration for half-maximal response
(Kap) for wild-type nAChRs was 20 ± 2 µM, whereas the Kap values for
nAChRs containing
W55L or
W57L subunits were 170 ± 30 µM and 86 ± 2 µM, respectively (Fig.
5A). The double mutant
receptor (
2
m
m) was
activated by ACh with a Kap of 340 ± 40 µM, about 15-fold higher than the wild-type receptor. The
consequences of the leucine substitutions were clearly different from
that seen for
or
subunit omission, since for
-less
(
2
2), Kap = 21 ± 4 µM and for
-less
(
2
2), Kap = 14 ± 1 µM (not shown).
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Fig. 4.
Electrophysiological recordings from
wild-type and mutant nAChRs expressed in oocytes. A,
inward currents evoked by 5 s applications of ACh to an oocyte
expressing wild-type nAChRs. The oocyte was injected with diluted RNA
(2.5 ng, a molar ratio of 2:1:1:1 :
:
:
) to limit the current
amplitudes at high ACh concentrations. The recordings were made 48 h after injection, and the oocyte membrane potential was held at
70
mV. B, concentration-dependent activation of
W55L mutant by ACh. The oocyte was injected with 100 ng RNA (2:1:1:1
:
:
m:
), which resulted in currents of similar
magnitude as seen for wild-type nAChR after injection of lower amounts
of RNA. C, dTC inhibition of ACh-induced currents for
wild-type nAChR. Currents evoked by 3 µM ACh (3 s) from
an oocyte injected with 10 ng of RNA were inhibited by preincubation
with various concentrations of dTC. D, upon preincubation,
dTC inhibited 3 µM ACh-induced currents from wild-type
(WT) nAChRs (
) with an IC50 of 40 ± 4 nM (nH = 0.9 ± 0.1). For
W55L
mutant receptors (
) after preincubation, dTC inhibited 100 µM ACh-induced currents with an IC50 of
3.7 ± 0.6 µM (nH = 0.9 ± 0.1). The data represent the mean ± S.D. of three
measurements.
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Fig. 5.
ACh dose-response relations for
W55L and
W57L mutant
nAChRs. A, for wild-type (W.T.) nAChRs (
,
2
) the ACh dose-response was fit by
Kap = 20 ± 2 µM
(n = 6 oocytes). For the
W55L mutant (
2
m
) Kap = 165 ± 30 µM (n = 4 oocytes), and
for the
W57L mutant (
,
2
m)
Kap = 86 ± 2 µM
(n = 3 oocytes). For the double mutant (
,
2
m
m),
Kap = 340 ± 40 µM
(n = 4 oocytes). Dose-response relations for wild-type
receptors were determined for oocytes injected with 0.4 ng of subunit
RNAs, whereas for mutant receptors, 10 ng were injected. The data
represent the means ± S.D. B, oocytes were injected
with 10 ng of subunit cRNAs. Mutant receptors were expressed on the
oocyte surface at the same level as wild-type receptors as determined
by 125I-
-BgTx binding (open bars,
n = 10 oocytes). The maximal currents for the
W57L
mutant were twice as large as the currents seen for wild-type receptors
at 3 µM ACh (hatched bar), a concentration
producing only 5% of maximal currents. The maximal currents
(closed bars) induced by saturating concentrations of ACh
for
W55L (
2
m
, 1.5 ± 0.3 µA, n = 6 oocytes) and for
W55L/
W57L
(
2
m
m, 0.16 ± 0.04 µA, n = 6 oocytes) mutants were only 6 and 0.6% that
seen for the
W57L mutant (
2
m,
25 ± 4 µA, n = 5 oocytes). The data represent
the means ± S.D. C, Hill coefficients for ACh
activation of wild-type and mutant nAChRs, determined from the
concentration-response relationship at concentrations of ACh producing
less than 20% maximal currents. ACh-induced currents (µA) recorded
from wild-type (
),
W55L (
), and
W57L (
) receptors were
plotted logarithmically against the concentration of ACh, with each
data point the mean ± S.D. of three recordings. The data for a
single oocyte were fit by linear regression, and the slope of the line
(nH) for the wild-type receptor (
) was 1.85, with
the same analysis for data from three oocytes characterized by a slope
of 1.8 ± 0.1. For the
W55L mutant (
), the slope for this
representative experiment was 1.04, and the average slope from three
experiments was 1.10 ± 0.05. The
W57L mutation, however, had
no effect on the slope of the concentration-response relationship of
ACh for receptor activation. The slope of the line for the
W57L
mutant (
) presented here was 1.71, and the average value of the
slope from three experiments was 1.66 ± 0.14.
-BgTx binding sites on the oocyte surface and the
current amplitudes for mutant AChRs activated by saturating
concentrations of ACh. nAChRs containing either
W55L,
W57L, or
both mutant subunits were expressed at nearly the same levels as the
wild-type nAChR, as indicated by the number of surface
125I-
-BgTx binding sites. In contrast, the maximal
currents for the mutant nAChRs were much lower than for wild type. When
equal amounts of subunit cRNAs were injected for wild-type and mutant nAChRs, the maximal currents for the
W57L mutant were twice as large
as the currents seen for wild-type nAChR at 3 µM ACh, a concentration producing ~5% of maximal currents. Thus, for nAChRs containing the
W57L subunit, the maximal currents were ~10% that of wild-type, whereas for receptors containing
W55L or both mutant subunits, the maximal currents were only 6 ± 1 and 0.6 ± 0.2% that seen for
W57L. These results indicate that mutation of
Trp-55 and
Trp-57 alters the activation of these channels by
ACh.
W57L mutant receptors, the concentration-response
relationship had slope values (nH) of 1.8 ± 0.1 and 1.7 ± 0.1, respectively (Fig. 5C). In
contrast, the slope for the
W55L mutant was one (nH = 1.1 ± 0.1) (Fig. 5C).
W55L
nAChRs--
For the wild-type nAChR, upon preincubation, dTC acted as
a competitive antagonist characterized by an IC50 of 40 nM (Fig. 4D), and without preincubation, when
coapplied with 3 µM ACh for 5 s, dTC inhibited with
an IC50 of 250 nM (Fig.
6C). Similar results were
obtained for nAChRs containing
W57L (data not shown). For nAChRs
containing
W55L, despite the fact that dTC binds with high affinity
(KH = 40 nM) to one of the sites (Fig. 1A), upon preincubation, dTC inhibited ACh currents only at
high concentrations (IC50 = 4 µM, Fig.
4D), as reported by O'Leary et al. (13). We
therefore examined in greater detail the interactions of dTC with
nAChRs containing
W55L. When applied alone, dTC produced no
detectable whole cell currents (<10 nA, data not shown). However, we
observed that at low concentrations dTC (10 nM to 1 µM) actually potentiated currents activated by ACh
(10-100 µM) when both ligands were applied
simultaneously to individual oocytes (Fig. 6). The magnitude of the
potentiation was dependent on the concentration of both dTC and ACh
(Fig. 6C), with the concentration dependence of potentiation
by dTC for 10 µM ACh consistent with dTC binding to its
high affinity binding site. The largest dTC potentiation was observed
at low concentrations of ACh, and the magnitude of the potentiation
decreased with higher concentrations of dTC. Concentrations above 10 µM dTC only inhibited currents activated by any
concentration of ACh (Fig. 6C). For
W55L nAChRs
preincubated with dTC, no potentiation was ever seen for responses to
ACh at concentrations between 0.3 and 100 µM.
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Fig. 6.
For the W55L mutant,
dTC potentiates responses to ACh. For an oocyte expressing
W55L
mutant receptors, responses to 10 µM (A) and
30 µM (B) ACh were recorded alone or with the
simultaneous application of 1 µM dTC. C,
concentration-dependent potentiation of ACh-induced
currents by dTC. Concentrations of dTC were applied to oocytes
simultaneously with fixed concentrations of ACh for 5 s. Control
responses (100%) to different concentrations of ACh were obtained in
the absence of dTC. For wild-type nAChR (
), dTC inhibited the
response to 3 µM ACh with an IC50 of 250 nM (the data represent the mean ± S.D. of three
measurements). In contrast, for
W55L mutant receptors, low
concentrations of dTC (30 nM-1 µM)
potentiated currents evoked by ACh at 10 (
) and 30 (
)
µM. For 10 µM ACh, dTC at 40 nM
produced half-maximal potentiation, whereas at higher concentrations
dTC inhibited the response with an IC50
3 µM. At 100 µM ACh (
), low concentrations
of dTC caused little or no potentiation, and at 300 µM
ACh (
), dTC inhibited responses with an IC50 of 3 µM. The data represent the mean ± S.D. from 3-5
experiments.
W55L, potentiation by dTC was
also observed for responses to two other agonists, carbamylcholine and
suberyldicholine (Fig. 7). As for ACh,
for these agonists the Hill coefficient (nH)
characterizing the dose-response relation was reduced from 1.6 for
wild-type nAChRs to ~1 for the mutant receptor (Fig. 7A).
dTC at concentrations between 10 nM and 1 µM
produced a dose-dependent enhancement of responses for agonist concentrations producing submaximal responses. Higher dTC
concentrations produced a progressive inhibition of the currents. For
carbamylcholine, dTC enhanced currents as much as 3-fold (Fig. 7B), whereas for suberyldicholine, currents were increased
by about 2-fold (Fig. 7C). For
2
m
, potentiation of agonist-induced
currents was not limited to dTC, since metocurine, the
7',12'-dimethoxy-2-methyl dTC analog, also potentiated activation of
the mutant receptor by ACh (Fig. 7D), However, neither
13'-iodo-dTC nor two other competitive antagonists, pancuronium and
gallamine, potentiated ACh responses (Fig. 7D). These
results establish that when dTC (or metocurine) first binds to nAChRs
containing
W55L, it does not act as an antagonist and, in contrast,
acts as a coactivator or weak partial agonist, as evidenced by the
potentiation of responses seen when agonists bind to the site at the
-
subunit interface.
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Fig. 7.
For the W55L mutant
nAChR, dTC potentiates currents elicited by other agonists, and other
antagonists potentiate ACh-induced currents. Panel A,
the
W55L mutation alters the cooperativity of nAChR activation by
the agonists suberyldicholine (Sub;
,
) and
carbamylcholine (Carb;
,
). In the individual
experiments presented here, the Hill coefficient
(nH) of suberyldicholine for wild-type nAChRs was
1.70 (
), whereas for
W55L, nH = 1.05 (
).
For carbamylcholine and wild-type nAChR (
), nH = 1.60, and for
W55L, nH = 1.02 (
). For each
case the data are for responses determined in a single oocyte and are
means ± S.D. for three measurements at each concentration. For
activation of the mutant receptor by suberyldicholine or by
carbamylcholine, the average value of nH from three
experiments was 1.1 ± 0.1. Suberyldicholine activated wild-type
and
W55L nAChRs with Kap values = 1.9 ± 0.1 µM and 7 ± 2 µM,
respectively, and Carb activated these receptors with
Kap values = 350 ± 10 µM and 750 ± 125 µM, respectively (3 oocytes each). Panels B and C, for
W55L mutant
nAChR, dTC potentiates currents induced by carbamylcholine
(Carb, B) and suberyldicholine (Sub,
C). Panel D, effects of competitive antagonists
on responses to 30 µM ACh for
W55L. The dTC analog
metocurine (
) at concentrations between 0.1 and 10 µM
potentiated responses, whereas no potentiation was seen for
13'-iodo-dTC (
), gallamine (
), or pancuronium (
). The
IC50 values for iodo-dTC (IdTC) , gallamine, and
pancuronium (Pan) were 44 µM, 5 µM, and 27 nM, respectively. The data
represent the means ± S.D. of three recordings made in single
oocytes, and the experiments were repeated with at least three
oocytes.
W55L/
W57L on the Binding of Nicotinic Agonists
and Antagonists--
We also examined the effects of the double
mutation (
2
m
m) on the
binding affinities of several agonists and antagonists to learn more
about the effects of these substitutions on the binding of structurally
diverse agonists and antagonists (Fig. 8). Most of the agonists tested,
including tetramethylammonium, phenyltrimethylammonium,
suberyldicholine, and epibatidine, inhibited 125I-
-BgTx
binding to double mutant receptors with IC50 values that were ~50-500-fold larger than for the wild-type receptor. Thus, mutation of
Trp-55 and
Trp-57 influences the binding of most nicotinic agonists, even very small agonists like tetramethylammonium. It is interesting that the agonists with highest affinity for wild-type
receptors were affected the most by the double mutation. One exception
to the general pattern was nicotine, which bound to the wild-type and
double mutant receptors with very similar affinity (Fig.
8F).
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Fig. 8.
The effects of the double mutation
( 2
m
m)
on the equilibrium binding affinities of nicotinic agonists and
antagonists. Inhibition of 125I-
-BgTx binding to
wild-type receptors (
,
2
) and to
W55L/
W57L (
,
2
m
m) was determined for
the agonists ACh (A), suberyldicholine (Sub,
B), tetramethylammonium (TMA, C),
phenyltrimethylammonium (PTA, D), epibatidine
(Epi, E), and nicotine (Nic,
F) and for the competitive antagonists gallamine
(Gal, G) and pancuronium (Pan,
H). The double mutation
W55L/
W57L (
,
2
m
m) had no effect on
nicotine binding (F) but increased the IC50 of
the other agonists by 50-500 fold (see Table II). The double mutation
had no effect on gallamine (G) binding but decreased the
binding affinity of pancuronium (H) at the high affinity
site. The data represent the mean ± S.D. (three
samples/point).
2
m
m) on the binding
affinities of competitive antagonists are more diverse and complex than
that seen for the agonists. The double mutation had no effect on the
binding affinity of gallamine (Fig. 8G), similar to what was
observed with dTC (Fig. 1). Pancuronium was bound with 10,000-fold selectivity by wild-type nAChRs (KH = 3 nM, KL = 20 µM), with binding at the high affinity site weakened by 100-fold in the
double mutant and binding at the low affinity site weakened by less
than 3-fold (Table II). The binding
affinity of dihydro-
-erythroidine was decreased by about 10-fold at
each site by the double mutation. The results of the binding
experiments shown in Fig. 8 are summarized in Table II.
Equilibrium binding parameters for agonists and antagonists
E, dihydro-
-erythroidine.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-
and
-
subunits, with amino acids contributed from three distinct
regions of
subunit primary structure and at least three regions of
(or
) subunit (reviewed in Refs. 3, 8, and 9). We set out to
study the functional contribution of two tryptophans,
Trp-55 and
Trp-57, at homologous positions of the
and
subunits.
Tryptophans
Trp-55 and
Trp-57 are within or near the
agonist/competitive antagonist binding sites, since they are the
principle sites of specific photoincorporation by [3H]dTC
within those subunits (10), and
Trp-55 is the amino acid in the
subunit specifically photolabeled by [3H]nicotine (11).
In addition, they appeared likely to contribute to dTC binding
affinity, because replacement of
Trp-55 by leucine resulted in a
decrease of dTC potency as an inhibitor of ACh-induced currents for
Torpedo nAChRs expressed in Xenopus oocytes (13). Although we confirmed the observation that for the
W55L mutant nAChR, the IC50 for dTC inhibition was increased 100-fold
compared with wild type (Fig. 4), we were surprised to find that the
mutation of either or both tryptophans had no effect on dTC equilibrium binding affinity, based upon the inhibition of binding of
125I-
-BgTx (Fig. 1). Instead, our results indicate that
the shift in dTC potency as an inhibitor of ACh-induced currents was a
secondary consequence of a dramatic reduction in the affinity of ACh
binding at the
-
site.
W55L and for
W55L/
W57L mutant nAChRs, and although
there were no significant changes in the levels of surface expression,
the maximal currents were only 0.5 and 0.05%, respectively, that seen
for wild-type nAChR. In addition, for the
W55L mutant, there was a
clear shift in the concentration dependence of the response compared
with the wild-type or the
W57L mutant receptor. For the
W55L
mutant the observed Hill coefficient (nH),
determined at ACh concentrations producing <20% maximal responses,
was close to 1 (nH = 1.1 ± 0.1), whereas it
was close to 2 for wild-type (nH = 1.8 ± 0.1)
and for the
W57L mutant (nH = 1.7 ± 0.1).
-
site was unaltered by the
replacement of
Trp-55 by leucine, concentrations of dTC sufficient
to occupy the
-
site did not inhibit ACh-induced currents. After
pre-equilibration with nAChRs, dTC did not inhibit the ACh response
until present at concentrations sufficient to occupy the
-
site
(IC50 = 4 µM, Fig. 4D). When
applied simultaneously with ACh, dTC at concentrations sufficient to
occupy the
-
site actually potentiated the ACh responses seen at
concentrations less than 100 µM (Fig. 6). Therefore,
replacement of
Trp-55 by leucine allows dTC to function as a partial
agonist or coactivator; dTC binding to the
-
site in the absence
of ACh did not produce measurable currents, but when coapplied with
ACh, dTC potentiated the ACh response by as much as 5-fold. These
functional consequences of the replacement of
Trp-55 by leucine were
not limited to ACh, since the concentration dependence for the current
responses seen for carbamylcholine and suberyldicholine were also
characterized by Hill coefficients of 1, and those responses were
potentiated by dTC (Fig. 7). Not all competitive antagonists act as ACh
coactivators of the
W55L mutant nAChR. Although potentiation was
seen for dTC and its close structural analog metocurine, pancuronium
and gallamine remained antagonists (Fig. 7D).
Trp-55,
caused a major perturbation of the equilibrium binding of ACh, as
deduced from the inhibition of the initial rate of
125I-
-BgTx binding (Fig. 1B). The equilibrium
binding function reflects both the affinity of binding to the
desensitized state of the nAChR and the conformational equilibrium
between resting and desensitized states. Before considering the effects
of the substitutions, it is important to note several aspects of the
observed binding to wild-type Torpedo nAChRs expressed in
oocytes. The equilibrium binding of ACh by wild-type Torpedo
nAChRs expressed in oocytes was well fit by a two-site model with an
equal number of high and low affinity sites (KH = 55 nM, KL = 3 µM).
The parameters for ACh binding Torpedo
2
are similar to those seen for embryonic mouse
nAChR expressed in oocytes (16), but they were quite different than
those seen for ACh binding to Torpedo nAChR-rich membranes, which was characterized by high affinity (K = 25 nM) binding to a single site (Fig. 1B). We do
not know the source of this difference, but it does not appear to
result from a shift of the preexisting equilibrium between resting and
desensitized states, since the desensitizing noncompetitive antagonist
proadifen had similar effects on ACh binding to either the native or
expressed Torpedo nAChR. A noteworthy distinction between
ACh interactions with Torpedo and embryonic mouse nAChR
(
2
) is that our data indicate that in the
Torpedo nAChR, ACh binds with higher affinity to the
-
than to the
-
site, whereas it binds with higher affinity at the
-
site for the mouse nAChR (16, 27). The latter conclusion was
based upon the observation that mouse
2
2 binds ACh with 15-fold higher
affinity than
2
2. For
Torpedo nAChRs, ACh binds nonequivalently to the two sites
even for receptors lacking the
or
subunit, but ACh binds with
higher affinity to
2
2 than to
2
2 (Fig. 1B, Table I). For
mouse nAChRs, preferential agonist binding to the
-
site is not a
general rule. For the adult nAChR containing an
subunit in place of
the
subunit, ACh binds nonselectively at the two sites, whereas
epibatidine binds with higher affinity at the
-
(or
-
) site
than at the
-
site, and for carbamylcholine the rank order is
-
~
-
>
-
(28, 29).
Trp-55 by leucine had a much larger effect on ACh
equilibrium binding than did replacement of
Trp-57 (Fig. 1 and Table
I). Although the high and low affinity sites characteristic of the ACh
equilibrium binding to wild-type nAChRs cannot be assigned unambiguously to binding at
-
and
-
sites, the observed
binding by wild-type and mutant nAChRs is consistent with a simple
interpretation if the high affinity ACh binding is at the
-
site
in wild-type nAChR, and the effects of the mutations are greatest at
the binding site containing the mutation. Then ACh binds with high
affinity (KH = 50 nM) to the
-
site in wild-type nAChR, and that binding is 7,000-20,000-fold weaker
in
2
m
(KL = 360 µM) and
2
m
m
(KL = 1 mM) but shifted less
than 3-fold in
2
m
(KH = 140 nM). ACh binds with low
affinity (KL = 3 µM) at the
-
site in wild-type nAChR, and that binding is preserved in
2
m
(KH = 0.8 µM) but weakened by 10-20 fold in
2
m (KL = 66 µM) and
2
m
m
(KH = 23 µM). Thus the
W55L
mutation disrupts ACh binding at the
-
m site without
causing a structural change that substantially perturbs ACh binding at
the
-
site. Within the
-
m site, the change in
structure must be limited, because the mutation does not alter dTC
binding. If the selective disruption of ACh binding at equilibrium seen
at the
-
site (and not the
-
site) in
2
m
also occurs in the resting
state, then the
-
site will become the higher affinity site in
the
2
m
nAChR. Further studies at
the level of single channel analysis will be required to determine the
extent to which the leucine substitution alters the initial binding
step or the conformational equilibria related to channel gating.
W55L nAChR
that dTC binding to the high affinity (
-
) site no longer acted as
an antagonist. Rather, inhibition of ACh responses was seen only when
dTC was bound to the
-
site, and dTC actually acted as a partial
agonist or coagonist when it bound to the mutated
-
site. These
observations appear to suggest paradoxically that the
W55L
substitution converted dTC from an antagonist into a partial agonist
without having any effect on the energetics of its binding. However, an
alternative explanation is that the mutation alters ACh binding only,
with a secondary consequence that this mutation reveals the functional
consequences of dTC occupancy of its high affinity site when ACh is
binding to the
-
site. Two lines of reasoning support this
interpretation. First, the experimental results can be accounted for by
a three-state allosteric model (resting, open, desensitized) that
assumes that ACh binding to the
-
site as well as dTC binding to
both sites is unaltered between wild-type and
W55L mutant AChR. By
adjusting parameters only for ACh binding at the
-
site in the
three states, it is possible to account for the observed shift of ACh
Kap and the Hill coefficient, the equilibrium
binding of ACh and dTC to wild-type and mutant nAChRs, and the observed
concentration dependence of dTC potentiation of the ACh responses in
the mutant nAChR.2 Secondly,
although we have not found experimental conditions where dTC acts as a
coactivator for wild-type Torpedo nAChR, dTC and metocurine
(but not pancuronium) act as weak partial agonists potentiating the
responses seen at low ACh concentrations for rat and mouse muscle
embryonic nAChRs (
2
) (30-33). In contrast, they all act as competitive antagonists at adult nAChRs
(
2
). Despite the difference in pharmacology,
dTC binds with the same high affinity to the mouse
-
site as to
the
-
site, but as we discussed above, ACh binds with higher
affinity to the
-
site than to the
-
site in the embryonic
nAChR, whereas it binds nonselectively to the
-
and
-
sites
(29).
Trp-55 by leucine weakens ACh equilibrium binding at
the
-
site by 7,000-fold, a decrease of affinity substantially larger than the ~20-500-fold shift of affinity seen for
substitutions of the conserved tyrosines within the
subunit binding
loops (34) or for charge-neutralizing mutations (
D152Q,
D174N,
D180N) in mouse nAChR (16, 18, 35). For nAChRs containing
D174N/
D180N as well as for nAChRs containing
D152Q the
mutations appear to affect ligand binding directly rather than the
conformational equilibria between resting and desensitized states (18).
However, for the simplest agonist, tetramethylammonium, equilibrium
binding is weakened by 100-fold for the
W55L mutant, for
D174N
(16), and also by substitutions at
Tyr-93,
Tyr-190, and
Tyr-198 (34). It is unlikely that tetramethylammonium is
simultaneously in contact with all of these side chains.
Trp-55 within the agonist binding site was
identified on the basis of its photolabeling by [3H]dTC,
leucine substitution has no effect on dTC binding affinity. It is
likely that replacement by other amino acids will alter dTC binding
affinity, because even the leucine substitution weakens the binding of
the competitive antagonists dihydro-
-erythroidine and pancuronium by
10- and 70-fold, respectively. In addition, substitution of the
corresponding tryptophan in homooligomeric
7 nAChRs (
7Trp54) by
histidine weakened dihydro-
-erythroidine binding by 10-fold
(36).
Trp-55 for agonist binding
and channel gating, but they do not establish that ACh interacts
directly with this tryptophan. In studies of mutant Torpedo
nAChRs containing cysteines within the binding site, there was no
evidence that
W55C was accessible for reaction with cationic methylthiosulfonates, whereas an adjacent position (
E57C) as well as
Y93C and
Y198C were all accessible for modification (37). This
result, in conjunction with the fact that
Trp-55 is photolabeled by
[3H]dTC and [3H]nicotine, suggests that
Trp-55 may be within a hydrophobic subdomain of the binding site.
Trp-55 also have
important functional consequences for other members of the superfamily
of ligand-gated ion channels related to the nAChRs. For
1
2
2
-aminobutyric acid type A receptors, replacement of
1Phe-64 by leucine results in 200-fold decrease of
-aminobutyric acid potency, whereas substitution of the equivalent
positions in the other subunits had no effect on agonist potency (38). In contrast, substitution at the equivalent position in
2 subunit (
2Phe-77) selectively alters binding of ligands at the
benzodiazepine site (39). The serotonin 5-HT3 receptor
also contains a tryptophan (Trp-89) at the equivalent position, and
substitutions at the position reduce antagonist but not agonist
affinity (40).
W55L) within a part of the agonist binding site contributed from the
subunit can effectively prevent ACh binding at that site without altering ACh binding at the
-
site. In addition, for this mutation, dTC binding to the
-
site acts as
a partial agonist or coactivator of ACh responses. Since dTC binds at
equilibrium to the
-
site in the mutant nAChR with the same high
affinity as in wild type, it is most likely that the mutation does not
alter dTC binding to any conformational state. Instead, by altering the
interactions of ACh with the
-
site, the mutation reveals an
aspect of dTC interaction with wild-type Torpedo nAChR that
is not normally seen because of the relative affinities of dTC and ACh
for the binding sites.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Stuart J. Edelstein for developing the three-state allosteric model used to interpret the data and Drs. Kenton J. Swartz, Deirdre Sullivan Stewart, and Gerald D. Fischbach for many helpful comments concerning this manuscript.
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FOOTNOTES |
---|
* This research was supported in part by United States Public Health Service Grant NS 19522.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
A Harvard Mahoney Neuroscience Institute Fellow. Present address:
Millenium Pharmaceuticals, Inc., Cambridge, MA 02139.
§ To whom correspondence should be addressed: Dept. of Neurobiology, Harvard Medical School, 220 Longwood Ave., Boston, MA 02115. Tel.: 617-432-1728; Fax: 617-734-7557; E-mail: jonathan_cohen@hms.harvard.edu.
Published, JBC Papers in Press, October 30, 2000, DOI 10.1074/jbc.M009085200
2 S. J. Edelstein and J. B. Cohen, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
ACh, acetylcholine;
nAChR, nicotinic acetylcholine receptor;
-BgTx,
-bungarotoxin;
dTC, d-tubocurarine;
HB, homogenization buffer.
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