Water-soluble models of ligand-gated ion channels
would be advantageous for structural studies. We investigated the
suitability of three versions of the N-terminal extracellular domain
(ECD) of the
7 subunit of the nicotinic acetylcholine receptor
(AChR) family for this purpose by examining their ligand-binding and assembly properties. Two versions included the first transmembrane domain and were solubilized with detergent after expression in Xenopus oocytes. The third was truncated before the first
transmembrane domain and was soluble without detergent. For all three,
their equilibrium binding affinities for
-bungarotoxin, nicotine,
and acetylcholine, combined with their velocity sedimentation profiles, were consistent with the formation of native-like AChRs. These characteristics imply that the
7 ECD can form a water-soluble AChR
that is a model of the ECD of the full-length
7 AChR.
 |
INTRODUCTION |
Nicotinic acetylcholine receptors
(AChRs)1 are
integral-membrane, pentameric ion channels in the central and
peripheral nervous systems that participate in signal transmission
associated with the release of acetylcholine (ACh). A considerable
collection of studies of their cell biology, electrophysiology, and
structure makes them the best characterized family of a superfamily of
homologous neurotransmitter-gated channels that includes glycine,
-aminobutyric acidA, and
5-hydroxytryptamine3 receptors (1-3). Muscle-type AChRs
are composed of four different subunits with the subunit composition
(
1)2(
1)
(
or
) and bind the snake venom toxin
-bungarotoxin (
Bgt). Neuronal AChRs that do not bind
Bgt are formed from combinations of
2,
3,
4, or
6 subunits with
2,
3,
4, and/or
5 subunits. Neuronal AChRs that do bind
Bgt are formed from
7,
8, and
9 subunits, perhaps in
combination with unknown subunits. When heterologously expressed,
7,
8, and
9 form functional homomeric AChRs that appear to contain
five identical subunits.
AChRs are composed of five homologous membrane-spanning subunits that
are ordered around a central, cation-selective channel. The topology of
AChRs that is predicted by hydrophobicity plots has received
substantial experimental support (4, 5). The approximately 200 residues
at the N-terminal half of each AChR subunit are extracellular, are
N-glycosylated, contain sites for agonist and antagonist
binding, and form the vestibule through which cations reach the
transmembrane channel. Relatively little of the remainder of the
primary sequence is extracellular. Three of the four transmembrane
domains (M1-M3) that form the channel are grouped together following
the N-terminal extracellular domain (ECD) and are separated from M4 by
a large cytoplasmic loop. For the muscle-type AChR, three distinct
regions of the primary sequence around amino acid residues 86-93, 149, and 190-198 of
1 subunits and peptide loops around residues 34, 55-59, 113-119, and 174-180 of the
or
subunit contribute to
the ACh binding sites at the interfaces between
and
and between
and
(or
) subunits, based on photoaffinity labeling and
site-directed mutagenesis (6, 7). Because of primary sequence and
topological similarity, homologous residues at subunit-subunit
interfaces in other neuronal AChR subunits are expected to have similar
roles in the agonist binding site. For example, residues of the
7
subunit homologous to those of
1 and to those around
55 and
57
have been shown to contribute to the agonist binding in homomeric
7
AChRs (8, 9).
Our knowledge of the molecular structure of AChRs, however, is far from
complete. Electron diffraction methods using two-dimensional, tubular
arrays of AChRs from Torpedo californica have successfully yielded structural details at 9 Å resolution in three dimensions (10-12) and at 7.5 Å in two-dimensional projection (13). Achieving higher resolution, however, has been elusive with membrane-bound or
detergent-solubilized AChRs. No member of this superfamily of
integral-membrane receptors has been crystallized, and the intact AChRs
are too large (more than 200 kDa) for nuclear magnetic resonance
spectroscopy.
An AChR formed from the ECD may be a suitable structural model of a
full-length AChR, if the ECD can both fold and oligomerize in the
absence of the remainder of the subunit. Several lines of evidence
suggest that the ECD meets these requirements. First, the specific
interactions between subunits that are important in assembly of the
muscle-type AChR and for formation of mature acetylcholine binding
sites appear to depend primarily on the ECD (14-21). The long
cytoplasmic loop of
1 participates in assembly subsequent to the
formation of heterodimers (22). Second, membrane-tethered ECDs of mouse
muscle
1 and
form heterodimers with ligand binding sites that
reflect properties of a full-length AChR (23-25). Third, sequences of
7 that affect homomeric assembly also have been localized to the
first half of the ECD and an area around M1 based on chimeras of
7
and
3 (26). According to this report, the long cytoplasmic loop of
7 is not essential for oligomerization.
To determine whether AChRs formed from the ECD (residues 1-208) of
7 subunits are water-soluble structural models for the ECD of the
full-length
7 AChR, we expressed two constructs of the ECD of the
7 subunit with M1 retained and one construct of the ECD without M1
in Xenopus oocytes. The constructs with M1 were included to
explore the feasibility of removing M1 by in vitro
processing subsequent to in vivo synthesis. We examined ligand binding properties and velocity sedimentation profiles as
indicators of global structure and local structure at the agonist binding site of the resulting AChRs, which we have designated as
7
ECD AChRs. We found that each construct, including the water-soluble one without M1, assembles into an AChR with affinities for
125I-labeled
Bgt (125I-
Bgt), nicotine,
and ACh that match those of the full-length
7 AChR. These properties
demonstrate that the
7 ECD forms a water-soluble
7 ECD AChR that
can be a starting point for structural studies of this superfamily of
ion channels.
 |
EXPERIMENTAL PROCEDURES |
Design of
7M1,
7 Enterokinase (
7EK), and
7
Water-soluble (
7WS) Plasmids--
The full-length cDNA sequence
of chicken
7 (27) previously was cloned into a modified SP64T
expression vector (28, 29). For
7M1, which encodes the ECD of
7
up to the start of M2, the
7 coding sequence from the beginning of
M2 to past the native stop codon was removed by digestion with
BglII and BsmI. It was replaced by a
double-stranded oligonucleotide cassette coding in-frame for the
19-amino acid sequence SQVTGEVIFQTPLIKNPRV and a stop signal. This
sequence contained the epitope of mAb 142 from residues 2 to 17 (5),
followed by a MluI restriction site in the DNA sequence. The
last native residue from
7 was Ile240, according to the
numbering scheme of the mature chicken
7 AChR subunit (27). The mAb
142 epitope was introduced for immunoblotting and for binding the
subunit protein to mAb 142-coated plastic wells for solid-phase assays
(5). The MluI site was included so that the epitope insert
could be extended with additional residues after digestion with
MluI and BsmI. It has been shown that such extension may be necessary for accessibility of the epitope by the
antibody (5).
The other construct that included M1 was
7EK, which was similar to
7M1 except for the inclusion of a peptide spacer between the end of
the ECD and the beginning of M1. To prepare
7EK, a double-stranded
oligonucleotide cassette coding for the 38-amino acid residue sequence
TMRRRTGTVSISPESDRPDLSTFTSDDDDKILERRRTL was ligated in frame between the
proximal BsmAI and distal HgaI sites that are
nearly juxtaposed to the 5
side of M1 in
7M1. Residues 1-6
reconstructed the end of the N-terminal extracellular domain from
residues Thr203 to Thr208; the DNA sequence for
residues 7-8 introduced a KpnI site; residues 9-23 were
the epitope for mAb 236 (5); the DNA sequence for residues 24-25
introduced a SpeI site; residues 26-31 (DDDDKI) were the
specificity sequence for EK that is modeled after its site of
proteolysis on trypsinogen (30, 31); the DNA sequence for residues
32-33 introduced a XhoI site; and residues 34-38 reconstructed the native sequence of Arg205 to
Leu209 at the distal end of the insert. Beginning with
Tyr210 (native numbering), the remainder of the sequence
was identical to
7M1. The KpnI, XhoI, and
SpeI restriction sites were included so that the mAb 236 epitope and the target for protease digestion easily could be modified
readily. A total of 27 amino acid residues were inserted between the
proximal copy of Thr208 and the distal copy of
Arg205.
The third construct,
7WS, was truncated at the end of the ECD of
7 and did not include M1. To prepare
7WS, the full-length sequence of
7 was cut at the BglII site between M1 and
M2. The resulting N-terminal domain sequence was cut at the
BsmAI site proximal to the start of the M1 coding sequence.
A double-stranded oligonucleotide cassette coding for the 22-amino acid
residue sequence TMRRRTQVTGEVIFQTPLIKNP followed by a stop codon was
ligated between the BsmAI site of the N-terminal sequence
and the EcoRI site of a modified SP64T expression vector.
Residues 1-6 of this sequence reconstructed the native sequence
Arg203 to Thr208; residues 7-22 constituted
the epitope for mAb 142. The oligonucleotides were synthesized
using the phosphoramidite method on a MilliGen oligonucleotide DNA
synthesizer.
Protein Expression in Xenopus oocytes--
DNA from each of the
three plasmids was purified on a CsCl gradient. cRNA was synthesized
using an SP6 mMessage mMachineTM kit (Ambion, Austin, TX) and
linearized DNA. The cytoplasm of each oocyte was injected with
approximately 50 ng of cRNA and incubated at 18 °C for 3-5 days in
50% Leibovitz's L-15 medium (Life Technologies, Inc.) in 10 mM HEPES, pH 7.5, containing 10 units/ml penicillin and 10 µg/ml streptomycin.
For
7M1 and
7EK, extraction of membrane-bound subunit protein
with a buffer containing Triton X-100 was accomplished with a
previously reported procedure (29). Oocytes were homogenized by hand in
ice-cold buffer A (50 mM sodium phosphate, 50 mM NaCl, 5 mM EDTA, 5 mM EGTA, 5 mM benzamidine, 15 mM iodoacetamide, pH 7.5).
The membrane-containing fraction was separated by centrifugation, was
washed twice with buffer A, and then was extracted with buffer B (40 mM sodium phosphate, 40 mM NaCl, 4 mM EDTA, 4 mM EGTA, 4 mM
benzamidine, 12 mM iodoacetamide, 2% Triton) during gentle agitation for 2 hours at 4 °C. The soluble fraction from this detergent extraction step was separated by centrifugation and was used
for both Western blotting and assays of ligand binding.
For
7WS, the secreted fraction was defined as the subunit protein
present in the L-15 medium incubating injected oocytes. Particulates in
this fraction were removed by centrifugation. The cytoplasmic fraction
of
7WS was defined as the subunit protein present in the soluble
fraction following homogenization of the oocytes by hand in buffer A
and centrifugation to sediment the insoluble, membranous component. The
Triton-extracted fraction of
7WS was defined as the subunit protein
present in the solvent following extraction in buffer B of the
membranous component from the homogenization step during gentle
agitation for 2 h at 4 °C.
Immunoblotting--
Triton-extracted
7M1 and
7EK or
secreted
7WS was incubated overnight at 4 °C with mAb 142 that
had been coupled to Sepharose CL-4B (Pharmacia) with CNBr (32). After
this concentration step, the protein was eluted at 55 °C with 2%
SDS and 20 mM
-mercaptoethanol. Proteins were
deglycosylated for 18 h at 37 °C with 1 unit of a mixture of
endoglycosidase F and glycopeptidase F according to instructions of the
manufacturer (Boehringer Mannheim). A sample without enzyme was run in
parallel as the negative control.
Protein was denatured and reduced at 55 °C in SDS-polyacrylamide gel
electrophoresis sample buffer containing 2% SDS, separated on a 13%
acrylamide SDS-polyacrylamide gel electrophoresis gel, and transferred
to an Immobilon-P polyvinylidene difluoride membrane (Millipore). After
being blocked in 5% powdered milk in phosphate-buffered saline (PBS,
100 mM NaCl, 10 mM sodium phosphate, pH 7.5)
containing 0.5% Triton, the membrane was incubated with 2 nM 125I-mAb 142. Specific activities of the
labeled antibodies ranged from 1017 to 1018
cpm/mol. Labeling was visualized by autoradiography.
Ligand Affinities--
Immulon 4 plastic microwells (Dynatech
Laboratories, Chantilly, VA) were coated with mAb 142 or mAb 236 for
solid-phase assays (5). The wells were blocked with 3% bovine serum
albumin in PBS. For
7M1 and
7EK, a volume of the
Triton-solubilized protein from the equivalent of from one to three
oocytes that had been injected with cRNA was added to each microwell.
For the measurement of
Bgt affinity, 125I-
Bgt was
added to the Triton extract and incubated overnight at 4 °C. Total
volume in each well was 100 µl. The wells were washed three times
with ice-cold PBS containing 0.5% Triton, and the amount of
radioactivity was measured using a
counter. Each data point was
measured in duplicate. For the competitive inhibition assays, the wells
were washed free of the Triton solution after 24 h and loaded with
125I-
Bgt at 4 nM in the presence of
L-nicotine or ACh. The wells were incubated 8 h at
4 °C before washing and then measuring the amount of radioactivity.
Each data point was measured in duplicate. Nonspecific binding was
measured with Triton-solubilized protein extracts from uninjected
oocytes and generally was less than 5% of the specific binding.
For
7WS, the volume of L-15 medium above about six oocytes was added
to mAb 142-coated microwells and incubated overnight at 4 °C for
capture of the secreted
7WS. The L-15 was washed away with PBS. For
the measurement of
Bgt affinity in 0% Triton, 125I-
Bgt was added to buffer C (same composition as
buffer B, except without Triton) and incubated overnight at 4 °C.
Total volume in each well was 100 µl. The wells were washed three
times with ice-cold PBS, and the amount of radioactivity was measured
using a
counter. Inhibition by nicotine and ACh in 0% Triton was
measured after capture of the secreted
7WS, washing of L-15, and
loading of each well with 0.6 nM 125I-
Bgt
and the appropriate amount of inhibitor in buffer C. Total volume in
each well was 100 µl. The wells were incubated 8 h at 4 °C
before washing and measuring the amount of radioactivity. Data points
were measured in duplicate. Nonspecific binding was measured with L-15
medium that was used to incubate uninjected oocytes and generally was
less than 10% of the specific binding. Buffer B was substituted for
buffer C at the step of loading the wells with 125I-
Bgt
or with 125I-
Bgt and nicotine or ACh for the ligand
affinity measurements of
7WS in 2% Triton.
The equilibrium dissociation constants Kd for
125I-
Bgt were determined by least-squares, nonlinear
fitting to a Hill-type equation (Equation 1) using the graphing
software KaleidaGraph (Synergy Software)
|
(Eq. 1)
|
where C is the measured signal (counts/min),
C0 is the maximal signal (which corresponds in
this case to the maximum number of 125I-
Bgt binding
sites), L is the concentration of 125I-
Bgt,
n is the Hill coefficient. The half-maximal inhibition constants, IC50, for nicotine and ACh in the presence of
125I-
Bgt were determined by nonlinear fitting to
Equation 2, where L is the concentration of nicotine or ACh,
C0 is the maximal signal, and
C1 is a constant that represents signal that is
not displaced by high concentrations of agonist. We used the
Cheng-Prusoff equation (Equation 3) to estimate equilibrium
dissociation constants from IC50 values
(33),
|
(Eq. 2)
|
|
(Eq. 3)
|
although other equations also have been described for that
purpose (34). The Kd values shown in Table I are the average and standard error of at least three independent assays of
ligand affinity unless otherwise noted. Uncertainties shown in figures
are standard errors.
Sucrose Gradient Sedimentation--
Membranes from 10-20
oocytes that had been injected with the
7M1,
7EK, or full-length
7 cRNA were extracted in buffer B. AChRs in membrane vesicles from
T. californica and AChRs from the TE671 cell line (35) were
solubilized in buffer B. About 200-µl aliquots of solubilized protein
containing from 20 to 100 fmol of 125I-
Bgt binding sites
were layered onto 5-ml sucrose gradients (5-20% (w/v)) in 0.5%
Triton solution of 100 mM NaCl, 10 mM sodium phosphate, 5 mM EGTA, 5 mM EDTA, and 1 mM NaN3 at pH 7.5. The gradients were
centrifuged 75 min at 70,000 rpm (approximately 340,000 × g) and 4 °C in a Beckman NVT90 rotor. For determining a
ligand binding profile, aliquots of 11 drops each (approximately 130 µl) from the gradient were collected into Immulon 4 plastic microwells coated with mAb 142 for
7M1, mAb 236 for
7EK, or mAb
318 (a rat monoclonal antibody against an epitope in the cytoplasmic domain) (27) for the full-length
7. The entire gradient was collected in 40 fractions. After 24 h at 4 °C, the microwells were washed and filled with from 4 to 12 nM
125I-
Bgt in buffer B for 6 h at 4 °C, followed
by washing and quantitation of bound 125I-
Bgt by
counting.
Processing of
7WS was slightly more involved because of the low
concentration of the secreted protein in the incubation medium. The
protein was concentrated by binding overnight at 4 °C to
-toxin that had been isolated from the venom of Naja naja siamensis
with ion exchange chromatography (36) and then coupled to Sepharose CL-4B with CNBr (32). The bound protein was eluted from the
-toxin
with 200 µl of 100 mM nicotine and was layered onto 5-ml sucrose gradients (5-20%) without Triton in centrifuge tubes. The
gradient was centrifuged 75 min at 70,000 rpm (approximately 340,000 × g) and 4 °C, as was done for
7M1 and
7EK. The entire gradient was collected in 39 fractions of 6 drops
each, because the drop size was larger in the absence of detergent.
After 24 h at 4 °C, the microwells were washed and filled with
1 nM 125I-
Bgt in buffer C for 6 h at
4 °C, followed by washing and quantitation of bound
125I-
Bgt by
counting.
Comparison of AChR Yields--
The yield was defined as the
theoretical maximal amount of bound 125I-
Bgt, which was
the value of C0 in Equation 1 from
125I-
Bgt-binding assays. The AChR yield was calculated
in terms of 125I-
Bgt binding sites per oocyte.
 |
RESULTS |
Expression of
7 ECD Proteins in Xenopus Oocytes--
The three
variations of the ECD sequence of the
7 subunit were studied (Fig.
1). The first construct,
7M1, includes
the N-terminal ECD, M1, and the portion of
7 between M1 and M2 up to
residue Ile240. It was intended to demonstrate the
pharmacological properties and oligomerization behavior of the
7
subunit proximal to M2 and tethered to the membrane through M1. The
second construct, designated
7EK, contains a peptide spacer of 27 amino acid residues that is spliced between Thr208 and
Leu209 at the junction of the ECD and M1.
7EK also
contains M1 and terminates at Ile240, like
7M1. The
position of an RRR motif from 205 to 207 at the junction of the ECD
with M1 suggests a structural role for these positively-charge
residues; therefore, the native sequence Arg205 to
Thr208 was repeated at the C-terminal end of the interposed
segment before M1. The name "
7EK" is derived from the DDDDKI
sequence that was included in the peptide spacer for site-specific
proteolysis by EK.
7EK was intended to test the feasibility of
removing M1 from the ECD by in vitro enzymatic proteolysis
after synthesis. The third variation,
7WS, was intended to be a
direct route to a water-soluble
7 ECD AChR. It is truncated at
Thr208, just before M1 and contains no transmembrane
domain. Epitopes for monoclonal antibodies mAb 142 and mAb 236 were
included in the truncated constructs so that the proteins could be
detected by immunoblotting and tethered through the antibodies to
plastic wells for solid-phase ligand-binding assays (5).

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Fig. 1.
Designs of the three 7 ECD subunit
proteins 7M1, 7EK, and 7WS. The ECD, which extends from
residues 1 to 208 of the mature chicken 7 AChR (27) is included in
each protein. In 7M1 and 7EK, the first transmembrane domain, M1,
extends from residues 209 to 233. The 7 sequence from residues 234 to 240, which extends to the beginning of M2, is included after M1 in
these two proteins. The segment labeled 142t on the
C-terminal side of M1 is the epitope tag for mAb 142. Besides the mAb
142 epitope, 7EK also contains an epitope tag for mAb 236, designated 236t, as well as an EK-specific protease
site between the extracellular domain and M1. In contrast to the two
membrane-tethered proteins, 7WS does not contain M1; the mAb 142 epitope tag of the 7WS protein follows directly after the ECD.
|
|
Immunoblotting of Triton X-100 extracts from oocytes injected with
7M1 and
7EK cRNA confirmed the expression of these proteins (Fig.
2).
7M1 before deglycosylation
migrated at an apparent mass of 37 kDa;
7EK migrated at 40 kDa.
These apparent masses were about 7 kDa higher than the values of 30 kDa
for
7M1 and 33.5 kDa for
7EK that were calculated from amino acid
compositions. The detection of predominately a single band before
deglycosylation suggested that post-translational modifications were
comparatively uniform on all molecules. Deglycosylation shifted each
band to approximately the molecular mass of the protein calculated
without modifications.

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Fig. 2.
Protein expression and glycosylation of
7M1, 7EK, and 7WS demonstrated by immunoblotting with
125I-mAb 142. Deglycosylated protein is compared with
control protein that was processed in parallel except without
glycosidase. The presence (+) or absence ( ) of glycosidase is noted
above each lane. Each lane of 7M1 and 7EK contains the
protein from about two oocytes; each lane of 7WS contains the
protein secreted from about 24 oocytes. Although 7WS has the lowest
molecular weight as calculated from amino acid sequences, it is the
most heavily glycosylated and migrates at the largest apparent
molecular weight. Triton extracts and incubation medium from uninjected oocytes were used as negative controls and showed no binding of 125I-mAb142. The positions of molecular mass markers are
indicated on the left.
|
|
Without M1,
7WS was secreted in soluble form into the incubation
medium by oocytes that had been injected with
7WS cRNA. The secreted
protein displayed an apparent mass of about 41 kDa by immunoblotting,
compared with a calculated mass of 26 kDa (Fig. 2). The detection of a
diffuse pattern of bands before deglycosylation suggested more
heterogeneous glycosylation than was present on
7M1 and
7EK.
Deglycosylation shifted the apparent mass down to about 26 kDa,
confirming that
7WS had the most extensive glycosylation of the
three proteins. The larger shift in apparent mass compared with
7M1
and
7EK also suggested that the pattern of glycosylation differed
between membrane-tethered and soluble forms of the ECD. An additional
observation from the immunoblot is that the yield of secreted
7WS
was about 10-fold less than the yields of
7M1 and
7EK from
Triton-solubilized membrane fractions, based on a comparison of the
numbers of injected oocytes needed to produce approximately equal
signals on the immunoblot.
High Affinity Binding of 125I-
Bgt to
7 ECD
AChRs--
Because high affinity binding of
Bgt is a hallmark of
the
7 AChR, measurements of binding affinity with
125I-
Bgt were appropriate starting points for analyzing
the functional and structural properties of the three
7 ECD
constructs. The equilibrium dissociation constant,
Kd, of 125I-
Bgt was 1.6 nM for
7M1 and 1.9 nM for
7EK when
measured in solid phase assays in which the Triton-solubilized proteins
were bound in mAb 142-coated wells (Fig.
3 and Table
I). These values compare favorably with
the published Kd of 1.6 nM for 125I-
Bgt binding to Triton-solubilized full-length
7
AChR and for
7-containing AChR from chick brain (37). Binding of
125I-
Bgt was measured for
7EK by also using the mAb
236 epitope tag to bind the protein to mAb 236-coated wells. The
Kd was 2.3 nM, demonstrating that the
mAb 236 epitope tag at the C-terminal end of the ECD did not interfere
significantly with the function of the
Bgt binding site.

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Fig. 3.
Equilibrium binding of
125I- Bgt to 7M1 and 7EK AChRs. The
Triton-solubilized AChRs were bound to mAb 142-coated microwells. Assays were performed in 2% Triton X-100. The curves are the best fits
of Equation 1 to the data. Each data point is the mean of duplicate
determinations. Scatchard analysis of the data is shown in the
insets. The Kd values are taken from
Table I. Data from representative single experiments are shown.
|
|
The affinity of secreted
7WS for 125I-
Bgt was
measured first without Triton (Fig. 4 and
Table I). The Kd value of 0.4 nM was
about 4-fold smaller than the values for Triton-solubilized
7M1,
7EK, and full-length
7. The presence of 2% Triton during the
binding assay of
7WS for 125I-
Bgt increased the value
of Kd to 1.7 ± 0.1 nM (Fig. 4),
which is in the range measured for the Triton-solubilized AChRs.
Triton, however, did not significantly affect the total number of
125I-
Bgt binding sites. The ratio of the number of
binding sites calculated from Equation 1 in the absence of Triton
compared with the number of binding sites in the presence of Triton
from otherwise identical pools of secreted
7WS was 0.99 ± 0.13 (n = 2). Only the secreted fraction of
7WS bound
125I-
Bgt to a measurable extent, despite the significant
amount of
7WS protein that was retained intracellularly. The total
amount of
7WS protein per oocyte that was detected in the
cytoplasmic and the Triton-solubilized membrane fractions by
immunoblotting was approximately equal to the amount of
Triton-solubilized
7M1 or
7EK protein in similarly-injected
oocytes. Hence all three constructs apparently were synthesized and
accumulated to an approximately equal extent. The intracellular pool of
7WS, however, showed no significant binding of
125I-
Bgt.

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Fig. 4.
Effect of Triton X-100 on the equilibrium
binding of 125I- Bgt to 7WS. 7WS was bound to
mAb 142-coated microwells. The binding assays with
125I- Bgt were performed without Triton X-100
(upper panel) or with 2% Triton X-100 (lower
panel). The curves are the best fits of Equation 1 to the data.
Each data point is the mean of duplicate determinations. Scatchard
analysis of the data is shown in the insets. The
Kd values are taken from Table I. In the absence of
Triton, Kd was about 4-fold smaller than the values
measured for 7M1 and 7EK. In the presence of Triton, however, the
value for 7WS shifted into the range for the M1-containing AChRs.
Data from representative single experiments are shown.
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|
The yield of 125I-
Bgt binding sites from each of the
three ECD proteins was measured by the binding of
125I-
Bgt to AChR on mAb 142-coated wells (Fig.
5). These measurements assumed that the
mAb 142 epitope in each protein was equally accessible to the antibody.
The yield of 8 ± 1 fmol of 125I-
Bgt binding sites
per oocyte with
7M1 AChR was the largest among the three proteins.
This value is near the value of 15 fmol per oocyte that was reported
for Triton-solubilized AChR from the full-length
7 subunit expressed
in oocytes (29). Compared with
7M1, the addition of the peptide
spacer in
7EK decreased the yield of AChR by about three-quarters to
1.9 ± 0.6 fmol of 125I-
Bgt binding sites per
oocyte. The yield of secreted
7WS AChR was the smallest of the three
at 0.21 ± 0.02 fmol of 125I-
Bgt binding sites per
oocyte. These comparisons indicate that insertion of residues before M1
or the absence of M1 decreases the yield of ECD AChRs.

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Fig. 5.
Comparison of yields of 7 ECD AChRs from
7M1, 7EK, and 7WS. The yield was defined as the number of
125I- Bgt binding sites per oocyte. This value was the
maximal amount of bound 125I- Bgt that was estimated from
solid phase binding assays on mAb 142-coated microwells using Equation 1 (calculated from the value of C0). At least
three independent measurements were performed for each type of AChR.
From 20 to 50 oocytes were pooled for the assays of 7M1 and 7EK.
The incubation medium from 80 to 160 oocytes was the source of AChRs
for the assays of 7WS.
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High affinity for 125I-
Bgt was the first step in
demonstrating the structural similarity of
7M1,
7EK, and
7WS
AChRs to the full-length
7 AChR. It was only an initial test of the
properties that we hoped to find in a model AChR. Specifically, we were
seeking a model that also was a pentamer and that had native-like
affinity for agonists like nicotine and ACh. High affinity for
Bgt
does not necessarily imply either of these properties. Muscle-type AChR
illustrates this point. The human
1 subunit in the absence of other
muscle-type subunits binds
Bgt relatively tightly, with Kd of 0.6 nM compared with
Kd of the pentameric muscle-type AChR of 0.1 nM (35). Instead of a pentamer, however, it exists as a
monomer according to its velocity sedimentation profile relative to the
profiles of fully assembled muscle-type AChR and 
and 
dimers (38, 39). Moreover, it does not bind agonists with native-like
affinity. In light of these characteristics for
1, other experiments
besides binding of 125I-
Bgt were required for assessing
the oligomerization of the
7 ECD constructs.
Velocity Sedimentation Implies
7M1,
7EK, and
7WS Form
Multimeric AChRs, Probably Pentamers--
Oligomerization of
7M1,
7EK, and
7WS proteins was examined first with velocity
sedimentation profiles in sucrose gradients. The proteins were detected
by binding of 125I-
Bgt in solid-phase assays. These
profiles were compared with full-length AChRs and monomeric
1
subunits as sedimentation standards. Fully assembled, full-length
7
AChR (calculated molecular mass of 272 kDa from amino acid sequence)
were pentameric standards (37). Muscle-type AChR from T. californica were pentameric (i.e. a monomeric AChR with
calculated molecular mass of 268 kDa) and decameric standards
(i.e. a dimeric AChR with calculated molecular mass 536 kDa)
(40). The monomeric
1 subunit (calculated molecular mass of 52 kDa)
from the human rhabdomyosarcoma cell line TE671 was the monomeric
standard (35). As expected because of their much larger molecular
masses, the pentameric AChR standards sedimented faster than monomeric
1 subunit (Fig. 6).

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Fig. 6.
Velocity sedimentation profiles of 7 ECD
AChRs and full-length 7 AChRs. The profiles of
125I- Bgt binding to full-length 7 AChR, 7M1 AChR,
and 7EK AChR that were sedimented on a 5-20% sucrose gradient with
0.5% Triton X-100 are shown in the upper panel. Two of the
arrows indicate the peaks of 125I- Bgt binding
to Torpedo AChR monomers (five subunits) and dimers (10 subunits) (40) that were observed in a parallel experiment. The peak of
125I- Bgt binding to the human 1 monomeric subunit
from the TE671 cell line from a parallel experiment is indicated by a
third arrow. These three proteins served as oligomerization
standards for the 7 ECD AChRs. The profiles for 7M1 and 7EK
are inconsistent with monomers as the high affinity species but are
consistent with pentamers. The lower panel shows the profile
of the 7WS AChR on a 5-20% sucrose gradient without Triton. The
profile again is inconsistent with a monomer as the high affinity
species but is consistent with a pentamer.
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The single 125I-
Bgt binding peak from
7M1 and from
7EK also sedimented much faster than the monomeric
1 subunit and
slightly slower than the full-length AChRs (Fig. 6). Two conclusions
can be drawn from these velocity sedimentation data in the absence of
measurements of diffusion and partial specific volume. First, the
position of the 125I-
Bgt binding peak was distant from
the
1 monomer standard, strongly suggesting that a multimeric
species was responsible for the 125I-
Bgt binding.
Second, this multimeric species probably was a pentamer. This
conclusion about subunit stoichiometry was deduced from both the single
peak and its relatively narrow distribution. The single peak implied
that a multimer with a single distinct stoichiometry was the dominant
species that bound 125I-
Bgt with nanomolar affinity. In
contrast, if multiple stoichiometries such as dimers and trimers and
tetramers had significant representation in the high affinity
population, then one would have expected either several discrete peaks
or a low, broad profile. Because of the pentameric structure of the
full-length
7 AChR (41), the most likely stoichiometry for
7M1
and
7EK AChRs also was pentameric, although minor fractions of
smaller oligomers may also have been present. Moreover, it has been
reported that only pentameric
7 subunits bind
125I-
Bgt with nanomolar affinity, whereas monomeric
full-length subunits and aggregated or incompletely assembled forms do
not (37).
Velocity sedimentation of the secreted
7WS in sucrose gradients also
revealed a single 125I-
Bgt binding peak (Fig. 6). It
sedimented slightly slower than the peaks for
7M1 and
7EK. As
with
7M1 and
7EK, the position of the single, relatively narrow
peak that was distant from the peak of the
1 subunit did not suggest
a monomeric form for
7WS AChR and was consistent with the conclusion
that the dominant form of
7WS AChR was a pentamer.
High Affinity Binding of Nicotine and ACh to
7 ECD
AChRs--
High affinity binding for agonists was the second test of
oligomerization and the last test of structural equivalence between the
7 ECD AChRs and the full-length
7 AChR. Interpretation of this
test was based on an analogy with characteristics of the
1 subunit
of the muscle-type AChR. High affinity for agonists develops for
1
only after it assembles with
or
(39, 42), because the agonist
binding site consists of structural components from both subunits at

and 
interfaces (3). Similarly, if
7M1,
7EK, and
7WS AChRs indeed were pentamers and structural models of the
full-length
7 AChR, then we expected native-like affinities for
nicotine and ACh.
The affinities of
7M1 and
7EK for the small ligands nicotine and
ACh were measured by competitive inhibition of the binding of
125I-
Bgt (Fig. 7 and Table
I). The equilibrium dissociation constants, Kd, of
7M1 and
7EK were 1.0 µM for nicotine and 50 µM for ACh. In the case of
7EK, tethering in the solid
phase assays via the mAb236 epitope tag slightly increased the values of Kd for nicotine and ACh to 1.4 µM
and 60 µM, respectively. For both proteins, the
Kd values are similar to those for
Triton-solubilized, full-length
7 and for
7-containing AChRs from
chick brain (Table I). Competitive binding with each ligand eliminated
almost all binding of 125I-
Bgt, implying that the
majority of the 125I-
Bgt binding sites also bound
nicotine and ACh.

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Fig. 7.
Competitive inhibition by nicotine and ACh of
125I- Bgt binding to Triton-solubilized 7M1 and 7EK
AChRs. The AChRs first were bound to mAb 142-coated microwells.
After washing, the microwells were loaded with 4 nM
125I- Bgt in 2% Triton X-100 and varying concentrations
of either nicotine or ACh. The curves are the best fits of Equation 2
to the data. Each data point is the mean of duplicate determinations. Kd values are taken from Table I. Similar inhibition assays were performed with 7EK using mAb 236-coated microwells (Table I). Data from representative single experiments are shown.
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In inhibition assays with 125I-
Bgt and
7WS, the
values of Kd for nicotine binding in the absence of
Triton were 10-fold smaller than the values for nicotine binding to
7M1 and
7EK in the presence of Triton (Fig.
8 and Table I). The values for ACh
binding to
7WS without Triton were 40-fold smaller than the values
for ACh binding to
7M1 and
7EK with Triton. The water-soluble
7WS AChR without Triton, therefore, bound to small ligands more tightly than did the full-length
7 AChR in Triton. The presence of
2% Triton in the inhibition assays increased the values of Kd by about 3-fold (Fig. 8). As with
7M1 and
7EK, the majority of the 125I-
Bgt binding sites of
7WS AChR also bound nicotine and ACh, as shown by the elimination of
almost all binding of 125I-
Bgt at high concentrations of
agonists in either the presence or absence of Triton.

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Fig. 8.
Effect of Triton X-100 on the inhibition by
nicotine and ACh of 125I- Bgt binding to 7WS
AChR. The 7WS AChR first was bound to mAb 142-coated microwells
in the absence of Triton X-100. After washing, the microwells were
loaded with a fixed concentration of 125I- Bgt (0.6 nM with 0% Triton or 4 nM with 2% Triton) and
with varying concentrations of either nicotine or ACh. The curves are the best fits of Equation 2 to the data. Each data point is the mean of
duplicate determinations. Kd values are taken from
Table I. Data from representative single experiments are shown.
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In light of the velocity sedimentation data, these results confirm that
the
7M1,
7EK, and
7WS AChRs are oliogomers and probably are
pentamers. These results demonstrate that the
7 ECD with or without
M1 is sufficient for the expression of a
7 ECD AChR with native-like
affinities for
Bgt and nicotinic agonists.
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DISCUSSION |
Structural Implications of the Ligand Binding Properties and
Oligomerization of ECD AChRs--
We have shown that the N-terminal
ECD of the
7 AChR subunit with or without M1 forms an AChR whose
ligand-binding and sedimentation properties imply that it is a
structural model of the full-length AChR. In particular, the
7WS
AChR is water-soluble. The ECD, therefore, constitutes an autonomous
unit capable of folding and assembling in the absence of the
transmembrane, cytoplasmic, and other extracellular portions of the
7 subunit sequence. We examined three characteristics that implied
retention of the native-like structure in the three ECD AChRs: 1) high
affinity binding of
Bgt, which is a hallmark of
7 AChRs; 2)
velocity sedimentation, which is most consistent with assembly of each
of the three
7 ECD subunits into pentamers; and 3) native-like
affinities for nicotine and ACh.
First, the affinity for 125I-
Bgt of each of the three
7 ECD AChRs was equal to or higher than that of the fully assembled, full-length
7 AChR. Because the
Bgt binding site incorporates noncontiguous regions of primary sequence, these affinities suggest that the global structure of each
7 ECD AChR models the structure of
the ECD in the full-length
7 AChR. The moderately higher affinity of
the
7WS AChR for 125I-
Bgt reverted back into the
range of Kd values observed with
7M1 and
7EK
AChRs when Triton was included in the assay. Triton could have exerted
its effects by modifying the interaction of
Bgt with either the AChR
or with the solvent environment, since both interactions can affect
Kd. In other words, Triton may have caused
structural perturbations of the AChR that affected the binding site, or
125I-
Bgt may have partitioned differently in
Triton-containing solvent compared with detergent-free solvent.
Second, velocity sedimentation data suggested that the
7 ECD
subunits had assembled into multimers that probably were pentamers. Other AChRs or AChR subunits were used as oligomerization standards for
estimating the extent of oligomerization of the
7 ECD subunits. The
single 125I-
Bgt binding peak for each of the ECD AChRs
migrated on the sucrose gradient slightly slower than full-length,
pentameric
7 AChR and Torpedo AChR and much faster than
the peak of monomeric
1 subunit. Although differences in the amount
of bound detergent (40) and in molecular shape (43, 44) will affect the
sedimentation of AChRs, we do not think that these effects alone can
account for the differences between the position of the monomeric
subunit standard and the positions of
7 ECD AChRs on the gradients.
A reasonable interpretation of the position of the
125I-
Bgt binding peaks relative to the monomeric
1
subunit peak was that these
7 ECD subunits were not monomeric.
Multimeric stoichiometry also is consistent with their native-like
affinity for agonists. Given that full-length
7 AChR are pentamers
(41), the single, relatively narrow 125I-
Bgt binding
peak from each
7 ECD AChR probably arose from pentamers.
Third, affinities of the three
7 ECD AChRs for nicotine and ACh also
matched or were slightly higher than those of the full-length
7
AChR. In the case of
7WS AChR, Triton reduced the affinity of
nicotine and ACh compared with that observed without Triton. The
differences in affinity for agonists of
7WS AChR compared with
7M1 and
7EK AChRs were not caused solely by Triton and may have
been contributed in part by M1. Overall, these results imply that the
local structure of the ACh binding site of each of the
7 ECD AChRs
closely matches that of the full-length
7 AChR. By analogy with the
behavior of the muscle-type AChR (39, 42), the binding site for small
ligands is thought to form at interfaces between subunits. Therefore,
native-like affinities for agonists by the
7 ECD AChRs, combined
with the results from velocity sedimentation, confirmed that the
7
ECD subunits had assembled into multimers.
The
7EK subunit demonstrated another structural property of the ECD
AChRs. Splicing a peptide spacer of 27 amino acid residues between
these two domains did not significantly alter ligand affinity. Therefore, strict continuity of the native primary amino acid sequence
between the ECD and M1 is not required for formation of the agonist
binding site or for oligomerization. In other words, an ECD that is
displaced away from M1 by a peptide spacer retains its assembly and
binding properties. This flexibility in design raises the possibility
of first expressing a membrane-bound ECD AChR, followed by release of a
water-soluble ECD AChR from M1 by enzymatic proteolysis in
vitro.
Elimination of transmembrane domains either by recombinant techniques
or by proteolysis of native protein has been the starting point for
structural studies of other integral membrane proteins. In the family
of ionotropic glutamate receptors, the soluble agonist binding domain
of GluR-B and GluR-D was successfully designed by fusion of
discontinuous sections of the primary sequence that apparently are
separated by two transmembrane domains (45, 46). This strategy also has
been successful for the x-ray crystallography of many integral membrane
proteins including growth hormone receptor (47), prolactin receptor
(48), tissue factor (49), interferon-
receptor (50), human class II
histocompatibility antigen (51), insulin receptor protein-tyrosine
kinase domain (52),
and
chains of the T cell receptor (53, 54),
neuraminidase (55), hemagglutinin (56), and bacterial aspartate
receptor (57).
Yield of ECD AChRs--
The protein sequence between the beginning
of M1 and the start of M2 (residues Leu209 to
Ile240) was needed for production of AChRs from the ECD at
a level comparable to that of the full-length AChR. M1 probably was the
key component of this region. Compared with the yield of AChRs from
7M1 AChR, the yield was smaller from
7EK and even smaller from
7WS. The differences were unlikely to be caused by reduced
translation efficiencies of
7EK and
7WS, because of the extent of
sequence identity among all three designs and because the amounts
7EK and intracellular
7WS proteins per oocyte that were detected by immunoblotting were approximately equal to the amount of
7M1. Instead, the difference in the case of
7EK AChR suggests some adverse effects on folding and assembly caused by the alteration of the
primary sequence at the ECD/membrane interface.
The low yield in the case of
7WS AChR highlighted an important role
for M1. M1 has been shown to function as a tether to the ER membrane
during the dimerization of truncated
1 and
subunits in the ER
(23). It was successfully replaced in mouse
1 by unrelated
transmembrane domains with unrelated primary sequences or with a
glycosylphosphatidylinositol (GPI) moiety (24). A similar role for M1
probably applies to the production of
7 ECD AChRs. M1 constrains the
subunits to a membrane surface, which may favor orientations between
subunits that are required for assembly and may help retain them in the
endoplasmic reticulum (ER) and Golgi apparatus for folding, subunit
assembly, and post-translational modifications. In contrast, failure to
retain the subunits on the ER and Golgi membranes removes such
constraints and decreases the efficiency of these processes. In
particular, M1 may enhance folding and assembly by increasing the local
concentration of subunits relative to folding cofactors, such as
calnexin, that are found in the ER membrane (58, 59).
Attaching the ECD to a membrane may not be the sole function of M1 for
7M1 and
7EK, however. Instead, the yield of AChRs may depend on
particular properties of M1. Chimeras of
7/
3 (26) and point
mutations in M1 of
7 (60) demonstrate that the specific sequence
used in the first transmembrane domain affects the yield of surface
AChRs. Similarly, the total amount of bound 125I-
Bgt per
oocyte varied when unrelated transmembrane domains replaced any one of
the regions M1-M4 of
1 in recombinantly expressed Torpedo AChRs (61).
Strategy for Structural Studies of
7 ECD AChRs--
What is the
optimum design of an
7 ECD AChR for structural studies? The complete
ECD of the
7 subunit incorporating all of the amino acid sequence up
to the start of M1 probably is necessary to satisfactorily reproduce
the ligand-binding and assembly properties of the full-length
7
AChR. Although we did not attempt to truncate the
7 ECD N-terminal
to Thr208, results from the mouse
1 subunit suggest that
there is little flexibility for moving the truncation point closer to
the N terminus and into residues known to contribute to the
Bgt
binding site (23). Truncation of mouse
1 after Pro211,
which is homologous with our truncation point in
7WS, did not disrupt the formation of a high affinity binding site for
Bgt. In
contrast, truncation of the mouse
1 subunit after Met207
(mouse
1 numbering) caused a loss of affinity for
Bgt.
More extreme truncations of the ECD of
1 have been explored in
attempts to bypass the difficulties presented by AChRs for structural
studies. None, however, appears as successful in duplicating properties
of full-length AChRs as the
7 ECD AChRs described here. For example,
peptide sequences from the region around residues 170-200 of the
1
subunit have been used as potential mimics of the
Bgt binding site
for NMR and ligand-binding studies (62, 63). The affinity for the
binding of
Bgt, however, typically is three orders of magnitude less
than with full-length AChRs (64). In addition, peptide models from the
1 subunit are incapable of oligomerization and do not show
significant affinity for small ligands.
Three conclusions from our investigation are relevant to a strategy for
obtaining
7 ECD AChRs for structural studies. First, the
extracellular domain constitutes a stable, fully water-soluble AChR
that quantitatively retains essential ligand-binding properties of the
full-length AChR and eliminates confounding factors of detergent
solubilization that hamper the crystallography of membrane proteins.
Second, M1 in the
7M1 and
7EK constructs significantly enhances
the yield of ECD AChRs compared with the yield from
7WS. Although
membrane tethering of the AChR subunit proteins was essential for
association of the ECD of the mouse muscle-type
1 subunit and the
full-length
subunit when expressed in COS cells (24), it was
advantageous but not essential for assembly of
7WS AChR in oocytes.
Third, the
7 ECD still can form an AChR even when a peptide spacer
separates it from M1 and the adjacent membrane surface.
These findings suggest that a water-soluble
7 ECD AChR more likely
will be produced in large amounts in two stages via a membrane-bound
intermediate than directly from a water-soluble design of the ECD. The
first stage is in vivo synthesis of a membrane-bound AChR
that subsequently will be removed from its membrane tether by in
vitro processing. The second stage is enzymatic removal of the
membrane tether in vitro.
7EK demonstrates that an AChR substrate can be designed for specific proteolysis within a peptide spacer between the ECD and M1. Our attempts to separate an ECD AChR
from the M1 domain of
7EK using EK, however, resulted in substantial
nonspecific proteolysis. An alternative method that allows truncated
1 subunits to dimerize with full-length
is substitution of M1
with a GPI moiety (24, 65). A GPI tether also leads to the expression
of membrane-bound extracellular domains of chick
7 on the surface of
oocytes (66). A GPI tether may be preferred at the stage of enzymatic
processing, because the carbohydrate portion of the anchor at the C
terminus of the protein can be separated from the more distal,
membrane-embedded fatty acid portion with phosphatidylinositol-specific
phospholipase C (67, 68). This enzyme is expected to eliminate the risk of nonspecific enzymatic proteolysis.
In addition to
7, we anticipate that ECD receptors of other members
of the family of AChR subunits and other subunits of this entire
superfamily of neurotransmitter-gated ion channels will mimic the
structure and function of their respective full-length receptors.
Demonstrations that ECD sequences are important in the assembly of both
glycine receptors (69) and
-aminobutyric acidA receptors
(70) suggest that the ECD of other members of the superfamily also will
fold and assemble autonomously. The ultimately successful design of an
ECD AChR for high level expression may require other design
modifications. Moreover, structural aspects of the gating and ion
permeation functions of transmembrane domains will have to be explored
with different strategies. A water-soluble, recombinantly generated ECD
AChR, however, appears to be a promising foundation for structural
studies of this superfamily of ion channels.