(Received for publication, November 17, 1994)
From the
Although 10 genes have been cloned encoding putative subunits of
neuronal nicotinic acetylcholine receptors, little is known about the
variety or subunit composition of such receptors expressed by
individual neurons. Chick ciliary ganglion neurons express five of the
known genes and assemble a class of synaptic-type receptors
collectively containing gene products from three of them: 3,
4, and
5. Using subunit-specific monoclonal antibodies, we
show here that all of the synaptic-type acetylcholine receptors having
3 also have
4 subunits and vice versa. In addition, most, if
not all, of the
5 gene product present in fully assembled
receptors is associated with both
3 and
4 subunits. Although
the receptors may be homogeneous in these respects, only about 20% of
them also contain the fourth gene product,
2, newly identified in
the ganglion; essentially all of the neurons express the
2 gene.
No
2 subunits are found coassembled with the fifth acetylcholine
receptor gene product expressed by the neurons,
7, which has been
shown previously to comprise a class of abundant, nonsynaptic receptors
on the cells. The identification of three acetylcholine receptor
subtypes distinguished by subunit composition on the same neurons
provokes questions about their individual physiological roles.
Neurotransmitter receptors, because of their central role in mediating chemical synaptic transmission in the nervous system, have been the subject of intensive investigation. Most thoroughly characterized have been ligand-gated ion channels where molecular cloning has isolated entire gene families encoding putative subunits of receptors for each of the known neurotransmitters. The complexity of the families has raised questions both about the rules governing subunit assembly and about the purpose of multiple receptor subtypes responding to the same neurotransmitter. Expression studies in Xenopus oocytes and in transfected cell lines have been used to determine the gene combinations that produce functional receptors and to examine the properties of the receptors that result. The types and subunit composition of native receptors, however, are only beginning to be understood.
Nicotinic acetylcholine receptors
(AChRs) ()represent a family of ligand-gated ion channels
that is widely expressed in the vertebrate nervous system(1) .
Ten genes have been cloned to date encoding putative subunits of
neuronal AChRs. Seven (
2-
8) were originally thought to
encode ligand-binding subunits, and three (
2-
4) were
considered more structural in purpose. Increasing evidence now
indicates that both classes of gene products can influence the
pharmacological properties of the receptors. Expressing the genes in Xenopus oocytes has shown that any one of the
2,
3,
and
4 genes in combination with either the
2 or
4 gene
can produce a functional AChR. More remarkably, the
7 and
8
genes can do so alone. No function has been revealed by this approach
for the
5,
6, and
3 gene products.
Physiological analysis reveals complex ACh responses from individual neurons. Several time constants can be required for a proper fit of the desensitization, and several kinds of nicotine-induced single channel events can be detected in the same cell. One explanation is that multiple receptor species may contribute to the response. Recently, AChR transcript analysis has confirmed the expression of as many as six AChR genes in a defined population of neurons such as sympathetic or parasympathetic ganglionic cells(2, 3, 4) . How many receptor subtypes can be produced from the gene products and whether all of the subtypes are found in the same neurons remain unanswered questions.
A system in which these questions can be addressed in some detail is
the chick ciliary ganglion. The ganglion contains only two kinds of
neurons: choroid neurons, which innervate smooth muscle in the
vasculature of the choroid layer, and ciliary neurons, which innervate
striated muscle in the iris and ciliary body. All of the neurons are
cholinergic, and all receive cholinergic input from preganglionic
terminals as their primary source of chemical synaptic transmission.
Previous studies have shown that the neurons express two major classes
of AChRs. One class, termed mAb 35-AChRs, binds the monoclonal antibody
(mAb) 35 but not -bungarotoxin (
-Bgt). They are largely
concentrated in the synaptic membrane and mediate chemical transmission
through the ganglion(5, 6, 7, 8) .
The other class, termed
-Bgt-AChRs, binds
-Bgt but not mAb
35. They are located primarily in nonsynaptic regions and have no
identified role in the ganglion though they function as ligand-gated
ion channels and are nearly an order of magnitude more abundant than
mAb 35-AChRs(9, 10, 11) .
Of the 10 known
neuronal AChR genes, RNase protections demonstrate that five are
expressed in ciliary ganglion neurons: 3,
5,
7,
2,
and
4(3) . Analysis of subunit composition using
subunit-specific mAbs has shown that mAb 35-AChRs collectively contain
the
3,
5, and
4 gene products but not the
7 gene
product; the opposite is true of
-Bgt-AChRs(12, 13) . Although some mAb 35-AChRs
were known to contain all three gene products (
3,
5, and
4), it was not clear whether the receptors are homogeneous,
particularly with respect to
4 composition(13) . More
importantly, no information was available on the possible distribution
of
2 subunits among AChR subtypes in the ganglion. Those issues
are resolved here.
mAb A3-1, which is
specific for the 3 gene product, and mAbs B4-1 and B4-2, which are
specific for the
4 gene product, were raised against fusion
proteins containing putative cytoplasmic domains of their respective
gene products and have been described previously (13) . Unless
otherwise indicated these mAbs were diluted from ascites fluid for use
or were purified using protein G-Sepharose 4 Fast Flow (Pharmacia
Biotech Inc.). Purified mAbs 35, 313, 318, A3-1, B4-1, and B4-2, and
normal IgG were individually coupled to Actigel (Sterogene
Bioseparations) at 3-6 mg/ml according to the
manufacturer's specifications.
mAb B2-1, which has not been
described previously, was raised against a fusion protein encoding a
portion of the 2 gene product which includes amino acids
326-396(13) . The fusion protein was expressed in
bacteria, purified, and used as an immunogen for mAb production as
described previously(12) . Hybridomas were screened for their
ability to precipitate AChRs in chick brain extracts that were labeled
with
I-mAb 270. mAb B2-1 was found to be an IgG1 and was
used as diluted ascites fluid and was purified and coupled to Actigel
as described above.
Solid phase
immunoprecipitation assays were used to quantify AChRs present in
detergent extracts or sucrose gradient fractions by tethering the
receptors in Immulon 2 Removawells (Dynatech Laboratories, Inc.) with
subunit-specific mAbs and using a radiolabeled mAb to determine the
amount of receptor bound. The wells were coated with mAb by first
incubating the wells overnight at 4 °C on a shaker with affinity
purified rabbit anti-mouse antibodies (Jackson ImmunoResearch Inc.) at
a concentration of 20 µg/ml in 0.15 M sodium chloride and
0.01 M sodium phosphate, pH 7.4 (phosphate-buffered saline,
PBS) containing 0.02% azide. The wells were then washed three times
with PBS containing 0.5% Triton X-100 (PBS-TX) and incubated on a
shaker overnight at 4 °C with 50 µl of anti-AChR mAb diluted in
buffer. To analyze AChRs containing 3 or
4 subunits, mAbs
A3-1 and B4-1 were combined and used at dilutions of 1:200 to 1:400
from ascites fluid. After incubating for 6 h at 37 °C or overnight
at 4 °C, the wells were rinsed three times with PBS-TX and
incubated overnight with the detergent extracts or gradient samples.
The samples were then removed, and the wells were washed four times
with PBS-TX. Normal rat serum plus normal mouse serum at 2% (v/v) each
in PBS-TX were incubated in the wells for 30 min at 37 °C followed
by incubation with 5 nM
I-mAb 35 in 2% (v/v)
normal rat and normal mouse serum solution for 2 h at 37 °C.
Unbound
I-mAb 35 was removed with four washes of PBS-TX,
and bound radioactivity was determined by gamma counting individual
wells. Nonspecific binding was determined by including an excess of
unlabeled mAb 35 (1 µM) with the labeled mAb 35 or by
substituting buffer for the sample and was subtracted in all cases.
Combining mAbs A3-1 and B4-1 for tethering receptors from sucrose
gradient fractions yielded the same results as using either of the mAbs
individually.
AChRs containing 2 subunits were assayed by
adsorbing the receptors to anti-
2 mAb-Actigel and measuring the
amount bound with radiolabeled mAb. Ganglion extracts or sucrose
gradient fractions were incubated with 10 µl of mAb B2-1-Actigel
and 5 nM
I-mAb 35 in the presence and absence of
1 µM unlabeled mAb 35 in a total volume of 80 µl.
After an overnight incubation at 4 °C on a shaker, the mAb-Actigel
was centrifuged and washed four times with PBS-TX. The resin was
recovered and counted in a gamma counter to quantify bound
I-mAb 35. AChRs containing
4 subunits were assayed
using mAb B4-1-Actigel. Nonspecific immunoprecipitation was assessed by
substituting normal rat IgG-Actigel for the mAb-Actigels. A similar
immunoprecipitation assay was used to test for
2 subunits in
-Bgt-AChRs, substituting 10 nM
I-
-Bgt
for the labeled mAb 35 and using 100 µM nicotine to
determine nonspecific binding. The resin was incubated, washed, and
counted as above. mAb 318-Actigel was substituted for the B2-1-Actigel
to quantify the
7-containing
-Bgt-AChRs as a positive
control.
Aliquots of Torpedo electric organ extract, prepared as described previously (23) and containing the AChR 13 s dimer and the 9.5 s monomer were included in all sucrose gradients for calibration. In separate gradients, Torpedo AChRs were centrifuged together with catalase (11.1 s), human IgG (7.2 s), bovine hemoglobin (4.3 s), and soybean trypsin inhibitor (2.3 s) to generate standard curves for sedimentation velocity.
The solid phase assay detected a
large number of mAb 35-AChRs in the 10 s region of the
gradient when ciliary ganglion extracts were mock depleted with
IgG-Actigel prior to sucrose gradient sedimentation (Fig. 1).
mAbs specific for 3 subunits (A3-1) and for
4 subunits (B4-1)
were combined to tether receptors in the assay so that mAb 35-AChRs
having either or both kinds of subunits would be scored. Depleting the
extracts with mAbs specific for either
3 or
4 subunits prior
to sucrose gradient sedimentation removed essentially all of the mAb
35-AChRs that could be detected subsequently in gradient fractions with
the solid phase assay. The results demonstrate that mAb 35-AChRs
containing
3 subunits also contain
4 subunits and vice versa.
Figure 1:
Sucrose density gradient analysis
showing that mAb 35-AChRs sedimenting at 10 s contain both
3 and
4 subunits. Ciliary ganglion extracts were fractionated
by sucrose gradient sedimentation, and the fractions were assayed for
mAb 35-AChRs using the solid phase immunoprecipitation assay with mAbs
A3-1 (anti-
3) and B4-1 (anti-
4) combined to tether receptors
and
I-mAb 35 to quantify them. Depleting the extracts
either with anti-
4 mAbs (triangles) or anti-
3 mAbs (circles) prior to sedimentation removed essentially all of
the
I-mAb 35 binding activity. Extract depleted with
normal mouse IgG (squares) served as controls for loss of mAb
35-AChRs through nonspecific adsorption. The gradients were calibrated
by including Torpedo AChRs in the IgG-depleted samples; the 13 s dimer and the 9.5 s monomer peaked in fractions 4
and 8, respectively.
Ciliary ganglion extracts mock
depleted with IgG-Actigel were fractionated by sucrose gradient
sedimentation, and the fractions were analyzed by probing immunoblots
with the 5-specific mAb 268 (Fig. 2A; positive
control). Substantial amounts of
5 protein were detected in the 10 s region of the gradient, implying assembly into pentameric
receptors. More than half of the
5 protein was recovered as
smaller weight material, indicating that a significant amount of the
protein, at least in embryonic neurons, is apparently not in fully
assembled receptor. Similar patterns were observed for the
distributions of
3 and
4 protein on the gradients (data not
shown). Immunodepleting the extracts with mAbs specific for either the
3 or
4 subunits removed nearly all of the
5 protein
present in the 10 s region of the gradient (Fig. 2A; fractions 5-7). Quantifying the results
with laser densitometry indicated that more than 80% was removed by the
anti-
4 mAbs and more than 90% by the anti-
3 mAbs (Fig. 2B).
Figure 2:
Immunoblot analysis showing that 5
protein sedimenting at 10 s is associated with
3 and
4 subunits. Panel A, ciliary ganglion extracts were
immunodepleted with IgG-Actigel as a positive control (top) or
with mAbs A3-1- and 313-Actigels to remove
3-containing receptors (middle) or with mAbs B4-1- and B4-2-Actigels to remove
4-containing receptors (bottom) and then fractionated by
sucrose gradient sedimentation. The indicated fractions were analyzed
on immunoblots probed with the anti-
5 mAb 268 followed by goat
anti-rat IgG coupled to horseradish peroxidase and enhanced
chemiluminescence. The molecular mass markers (top to bottom) are 97.4, 66.2, 45, and 31 kDa. Panel B,
laser densitometry was used to quantify the
5 signals obtained in panel A. Control experiments demonstrated that the
densitometer signals were within the linear range of the immunoblot
assay. The results are taken from the same experiment shown in Fig. 1, permitting direct comparison of receptor and subunit
positions on the gradients. Integrating the areas under the curves indicates that the anti-
3 and
4 mAbs each depleted
80-90% of the
5 protein in the 10 s region of the
gradient, suggesting that essentially all of the
5 protein present
in receptor is coassembled with both
3 and
4 subunits.
Similar results were obtained in two other
experiments.
Some of 5 protein in the 6-8 s region of the gradient (Fig. 2B, fractions
8-11) was also immunodepleted by mAbs to
3 and
4
subunits, indicating that it was coassembled with one or both of the
two kinds of subunits even though the sedimenting species were not
fully assembled receptor, judging by their sedimentation rates. The
material may represent assembly intermediates or inappropriately folded
or assembled protein destined for degradation. Despite its coassembly
with
3 and/or
4 protein,
5-containing material in this
region of the gradient did not score as mAb 35 binding in the solid
phase immunoprecipitation assay presumably because the conformation or
access of the relevant epitope(s) was not appropriate. The
5
protein in the 5 s region does not appear to be assembled with
either
3 or
4 protein (Fig. 2B, fractions
12-14) and may be monomeric.
Since the mAbs used for the
immunodepletions are subunit-specific(13) , the results permit
the conclusion that essentially all of the fully assembled 5
protein is present in mAb 35-AChRs containing both
3 and
4
subunits. It is possible the reverse is also true, namely that all
ciliary ganglion receptors containing
3 and
4 subunits also
contain
5, but the mAbs available do not allow the proposition to
be tested.
mAb 270-Actigel was used to immunopurify 2 protein from ciliary
ganglion extracts. The recovered material was analyzed on immunoblots
probed with mAb B2-1 (Fig. 3A). A doublet was detected
with the lower band being the more prominent of the two. Similar
results were obtained when mAb B2-1-Actigel was substituted for mAb
270-Actigel and the blots were probed with either mAb B2-1 or 270, as
well as when mAb 270 was used to probe blots of material immunopurified
with mAb 270-Actigel (data not shown). From experiments in which mAb
B2-1 was used on immunoblots, the calculated molecular mass for the
upper component was 55.5 ± 0.6 kDa (n = 4), and
the lower component was 53.7 ± 0.8 kDa (n = 4).
In two experiments, only a single band was detected. Whether the
doublet arises from partial degradation or differential processing of
the
2 protein is unclear and has not been investigated further.
The size of the
2 protein determined here is slightly larger than
the 49-50 kDa reported previously for the
2 subunit in brain
AChRs (18) and in cells transfected with the
2
gene(25) , but it is close in size to the molecular mass of 54
kDa deduced from the nucleotide sequence of the
2
gene(26) .
Figure 3:
Immunoblots identifying 2 protein in
ciliary ganglion extracts and showing its association with other AChR
gene products. Panel A, mAb 270-Actigel was used to adsorb
2-containing material from ciliary ganglion extracts, and the
adsorbed material was eluted and analyzed by immunoblot probed with mAb
B2-1. A doublet containing components of about 55 and 53 kDa was
detected (lane 2) when the bound antibody was visualized with
secondary antibody and chemiluminescence as in Fig. 2. The
labeling was specific since it was not detected when the primary mAb
was omitted (lane 1); the adsorption to mAb 270-Actigel was
specific since it did not occur with IgG-Actigel (lane 3). Panel B, the
2-containing material recovered from mAb
B2-1-Actigel (lanes 5-8) and from mAb 270-Actigel (lanes 9-12) was analyzed on immunoblots probed with
mAbs specific for the other AChR gene products known to be expressed in
ciliary ganglion neurons. All three gene products previously identified
in mAb 35-AChRs (
3,
4, and
5) were found to be present
in both preparations of
2-containing material when probed with
mAbs 313, B4-2, and 268, respectively. The immunopurifications were
specific because the components were not obtained when IgG-Actigel was
substituted for the mAb-Actigels (lanes 1-4). No
7
protein was detected by mAb 318 in the material immunopurified with
anti-
2 mAbs (lanes 8 and 12). The molecular mass
markers are as in Fig. 2.
The components were not obtained when IgG-Actigel
was substituted for mAb 270-Actigel in the immunopurification and were
not detected when the primary mAb was omitted from the blot analysis (Fig. 3A). mAb 270, which was previously shown to
recognize the 2 gene product
specifically(12, 13, 18, 19) , binds
to an extracellular epitope, whereas mAb B2-1 is presumed to bind to an
intracellular epitope because the mAb was raised against a fusion
protein containing the putative cytoplasmic domain of the
2 gene
product. Like mAb 270, mAb B2-1 is specific for the
2 protein. It
recognized the
2 fusion protein on immunoblots but not fusion
proteins corresponding to similar regions of the
3,
5, or
4 subunit. In addition, mAb B2-1 recognized the
2 protein
translated in vitro and analyzed by immunoblots but not the
full-length
3,
4,
5, or
4 gene products translated in vitro and analyzed similarly (data not shown). The
specificity and the distinct epitopes recognized by the two mAbs
provide assurance that the components identified are indeed
2
protein. Neither mAb B2-1 nor mAb 270 was sufficiently sensitive to
detect the small amounts of
2 protein in ganglion extracts by
immunoblot analysis unless the protein was first concentrated by
immunopurification.
Coassembly of the 2 protein with other AChR
gene products known to be present in mAb 35-AChRs was revealed by
immunoblot analysis. mAbs specific for
3 subunits (mAb A3-1),
4 subunits (mAb B4-2), and
5 subunits (mAb 268) each detected
their corresponding antigen in material immunopurified either with mAb
270 or mAb B2-1 (Fig. 3B). Since mAbs 270 and B2-1 are
specific for the
2 subunit, the detection of the
3,
4,
and
5 protein in the immunoprecipitated material can only be
attributed to the coassembly of these subunits with
2 protein. mAb
318 did not detect a component in the immunopurified material,
indicating that few, if any,
7 subunits are coassembled with
2 protein. The immunopurifications were specific in that
IgG-Actigel did not substitute for mAb 270-Actigel or mAb B2-1-Actigel
in recovering the AChR proteins (Fig. 3B).
Corroboration that the 2 gene product is coassembled with the
AChR gene products found in mAb 35-AChRs was provided by
immunopurifying material with mAb 35-Actigel and probing the
immunoblots with subunit-specific mAbs. In addition to
3,
4,
and
5 protein, which were expected to be present, the antibodies
also revealed
2 protein in the recovered material (Fig. 4).
The immunopurification of
2 protein by mAb 35-Actigel was specific
in that it could not be duplicated by IgG-Actigel (Fig. 3A).
Figure 4:
Immunoblot analysis showing that mAb
35-purified material includes 2 protein. Ciliary ganglion extracts
were incubated with mAb 35-Actigel, and the adsorbed material was
eluted and analyzed on immunoblots probed with no primary mAb (np) as a negative control (lane 1), mAb 313 for
3 (lane 2), mAb B4-2 for
4 (lane 3), mAb
268 for
5 (lane 4), and mAb B2-1 for
2 protein (lane 5). Bound mAbs were detected as in Fig. 2except
that the secondary antibody included both anti-rat and anti-mouse IgG.
The small components seen in lanes 2-4 most likely
represent degradation products; they were detectable only when the
immunoblots were deliberately overexposed, as done here to demonstrate
2 protein. The molecular mass markers are as in Fig. 2.
Figure 5:
Sucrose gradient analysis showing that the
2-containing component recognized by mAb 35 has the size expected
for a fully assembled AChR. Ciliary ganglion extracts were fractionated
by sucrose gradient sedimentation, and the fractions were assayed for
I-mAb 35-binding components tethered in the solid phase
assay with the anti-
2 mAb B2-1 (filled squares) or the
anti-
4 mAb B4-1 (open triangles). To compensate for the
small amounts of
2-containing material present, five times more
fraction volume was used with B2-1 in the assays than with B4-1.
Nonspecific binding, which has been subtracted from each point, was
assessed either by replacing the gradient fraction with buffer in the
assay or by including an excess of unlabeled mAb 35 along with the
I-mAb 35. The
2-containing species that binds mAb 35
cosediments with mAb 35-AChRs shown above to contain
3 and
4
subunits coassembled. Similar results were obtained in a second
experiment.
The number of
2-containing AChRs recognized by mAb 35 appeared to be
substantially smaller than the number of mAb 35-AChRs containing
3
and
4 subunits, judging from the relative amounts of mAb 35
binding in the sucrose gradient analysis. The reduced amount of mAb 35
binding obtained in the solid phase assay when mAb B2-1 was used to
tether receptors could not be attributed to poor antibody affinity
because a second pass of the extract through the assay yielded only
one-third as much as the first time (see below). To provide additional
quantification of the relative amounts of the two kinds of mAb
35-binding components, solid phase assays were carried out in parallel
on ciliary ganglion extracts using mAb B4-1 to tether mAb 35-AChRs
containing
4 subunits and mAb B2-1 to tether
2-containing
AChRs.
I-mAb 35 was used to measure the number of binding
sites retained in each case. By this criterion,
4-containing mAb
35-AChRs are nearly five times more abundant than
2-containing
AChRs (Fig. 6A). Very little mAb 35 binding was
detected in material adsorbed nonspecifically to IgG-Actigel as a
negative control.
Figure 6:
Immunoprecipitations quantifying the
amounts of 2-containing components binding mAb 35 or
-Bgt. Panel A, the amount of
2-containing material that binds
mAb 35 in ciliary ganglion extracts was determined using a solid phase
assay with mAb B2-1 to tether the material and
I-mAb 35
to measure it. For comparison, mAb B4-1 was used to tether mAb 35-AChRs
in similar assays. In both cases nonspecific binding was determined as
described in Fig. 5and was subtracted from all points. Normal
IgG was substituted for the mAbs to assess nonspecific retention of mAb
35 binding sites in the assay. Values represent the mean ± S.E.
from at least six separate experiments and are reported as the number
of mAb 35 binding sites tethered per ciliary ganglion (CG).
The number of mAb 35 binding sites associated with
2-containing
material is about one-fifth of that associated with
4-containing
mAb 35-AChRs. Panel B, the amount of
2-containing
material that binds
-Bgt was determined in a similar manner,
substituting
I-
-Bgt for labeled mAb 35. For
comparison, mAb 318 was substituted for mAb B4-1 in the assay to tether
7-containing
-Bgt-AChRs. Normal IgG was used to assess
nonspecific retention of
-Bgt-binding material in the solid phase
assay. Values represent the mean ± S.E. of three determinations
and are reported as the number of
-Bgt binding sites tethered per
ciliary ganglion. No significant
-Bgt binding was detected in
2-containing material.
No detectable 2 protein is present in
-Bgt-AChRs (Fig. 6B). The number of
-Bgt
binding sites tethered in the solid phase assay by mAb B2-1 was no
greater than that obtained when IgG was substituted for the mAb. In
contrast, the number of
-Bgt binding sites was substantial when
mAb 318 was used to tether
7-containing receptors in the assay, as
shown previously by other means(13) .
Are the
2-containing mAb 35-AChRs a subset of the mAb 35-AChRs containing
3 and
4 subunits? This question was addressed by
immunodepleting AChRs with mAbs to either
3,
2, or
4
subunits and then measuring the number of remaining mAb 35 binding
sites in each case which could be tethered by mAb B2-1 or mAb B4-1 in
the solid phase assay. IgG was used as a negative control. The
anti-
2 mAb B2-1 was reasonably efficient at removing
2-containing AChRs, depleting about 70% of the signal detected in
the solid phase assay (Fig. 7). The mAb removed little of the
4-containing receptors, consistent with
2 subunits being
present in only a small fraction of mAb 35-AChRs. Anti-
3 mAbs
depleted about 80% of both
2- and
4-containing receptors
defined by mAb 35 binding in the solid phase assay (Fig. 7).
This provided strong evidence that the
2-containing mAb 35-AChRs
are a subset of those containing
3 and
4 subunits. The
depletion of the
2-containing AChRs by the anti-
4 mAb B4-1,
however, was variable. In six experiments, the remaining
2-containing mAb 35-AChRs ranged from 11 to 150% (69 ± 23%;
mean ± S.E.) of the IgG-depleted control extracts even when mAb
B4-1 efficiently removed 90 ± 3% (n = 4) of the
4-containing mAb 35-AChRs. The reasons for the variation with B4-1
are not clear. Nonetheless, the fact that most mAb 35-AChRs contain
both
3 and
4 subunits and the fact that the anti-
3 mAbs
are efficient at depleting
2-containing components indicate that
the latter are a subset of mAb 35-AChRs. This interpretation is also
consistent with the immunoblot results presented above showing that
2 is associated with all three gene products known to be present
in mAb 35-AChRs. In fact, taken together, the immunoprecipitation and
immunoblot analyses strongly suggest that at least a portion of mAb
35-AChRs contains four kinds of subunits coassembled:
3,
5,
2, and
4.
Figure 7:
Immunodepletions showing that the
2-containing material recognized by mAb 35 is a subset of mAb
35-AChRs containing
3 and
4 subunits. Ciliary ganglion
extracts were immunodepleted as described in Fig. 2to remove
components containing either
2 (mAb B2-1) or
3 (mAbs A3-1 or
313) protein. Nonspecific depletion was assessed by substituting
IgG-Actigel for the mAbActigels. The depleted extracts were then
measured for the number of remaining
I-mAb 35 binding
sites that could be tethered with either mAb B2-1 or mAb B4-1 in the
solid phase assay. Values are expressed as a percent of those obtained
with extracts depleted only by IgG-Actigel (positive control) and
represent the mean ± S.E. of six separate experiments for mAb
B2-1 and two experiments each for mAbs A3-1 and 313. mAbs A3-1
and 313 immunoprecipitated about 80% of the mAb 35 binding sites
associated either with
2- or
4-containing components,
supporting the contention that
2-containing components that
sediment at 10 s and bind mAb 35 are a subset of the mAb
35-AChRs known to contain
3 and
4
subunits.
Figure 8:
Immunocytochemistry with mAb 270
demonstrating the presence in situ of 2 protein in most
ciliary ganglion neurons. Cryostat sections of 18-day embryonic ciliary
ganglia were incubated with mAb 270, mAb 35, or normal rat IgG followed
by a biotinylated anti-rat IgG antibody and an
avidin-biotin-horseradish peroxidase complex. The sections were
developed for peroxidase activity and photographed with bright-field
microscopy. Comparable numbers of neurons were intensely stained both
with mAb 270 (panel A), which recognizes the
2 subunit,
and with mAb 35 (panel B), which has previously been shown to
recognize antigen in most ciliary ganglion neurons(24) . Only
light background staining was seen when normal IgG was substituted for
the mAbs in treating the sections (panel C). Similar results
were obtained in adjacent sections as well as sections taken from other
regions of the ganglion; a second experiment produced the same outcome.
The results indicate that
2 gene expression is not confined to a
small subset of neurons in the ganglion. Calibration bar, 20
µm.
The main findings reported here are that a single population
of neurons can express at least three classes of AChRs based on subunit
composition and that the heterogeneity observed cannot be attributed to
differences in gene expression among cells within the population. The
results also strongly suggest that some neuronal AChRs may be as
complex in subunit composition as muscle and electric organ AChRs,
having four types of subunits: 3,
5,
2, and
4. The
association of
2 with
4 subunits as well as
2 with both
3 and
5 subunits together represent new combinations not
previously reported for native AChRs.
Ciliary ganglion AChRs that
bind mAb 35 but not -Bgt contain both
3 and
4 subunits.
Previous studies had suggested that the receptors were heterogeneous
with respect to
4 composition, based on the inability of mAb B4-1
to immunoprecipitate all of the mAb 35-AChRs as measured(13) .
Those results may have been confounded by the inclusion of assembly
intermediates or other components detected in the column assay used to
quantify mAb 35-AChRs. The more sensitive and direct solid phase assay
used here, together with sucrose gradient fractionation to eliminate
small molecular weight material, found that essentially all mAb
35-AChRs containing
3 subunit also contained
4 subunit and
vice versa. Available mAbs did not permit a conclusion as to whether
all of the mAb 35-AChRs also contain
5 subunit. It is clear,
however, that most, if not all, of the
5 subunit assembled into 10 s receptor is associated with
3 and
4 subunits.
Conceivably mAb 35-AChRs are homogeneous with respect to containing
3,
4, and
5.
The conclusion that 2 subunits are
present in a portion of ciliary ganglion mAb 35-AChRs is supported by
several lines of evidence. Identification of
2 protein on ciliary
ganglion immunoblots was unambiguous since it made use of two
2-specific mAbs recognizing different epitopes.
Immunoprecipitation of
2 protein using mAbs specific for the
2 gene product depleted a species that bound mAb 35 and
cosedimented with mAb 35-AChRs. It represented about one-fifth of the
mAb 35 binding associated with
3- and
4-containing mAb
35-AChRs. Immunoblot analysis of the material immunoprecipitated by
2specific mAbs revealed all three gene products known to be
present in mAb 35-AChRs. Reciprocally, immunopurification of receptors
with mAb 35 yielded
2 protein. The
2-containing species that
binds mAb 35 was efficiently precipitated by anti-
3 mAbs as
expected if it represents a subset of the mAb 35-AChRs containing
3 and
4 subunits. An anti-
4 mAb was less reliable in
immunoprecipitating the
2-containing component that binds mAb 35,
but there can be little doubt that
2 subunits are also associated
with
4 subunits in the receptors. Essentially all mAb 35-AChRs
that contain
3 also contain
4. Moreover, immunoprecipitation
of
2 protein with specific mAbs coprecipitates
4 protein.
Much of the 2 gene product in brain is assembled with
4
subunits to make up the major receptor species binding nicotine with
high affinity(17, 18, 27) . Analysis of the
4 and
2 gene products expressed either in Xenopus oocytes or in the stably transfected fibroblast cell line M10
indicates that pentameric receptors are produced which contain two
4 subunits and three
2
subunits(19, 25, 28) . In ciliary ganglion
neurons little, if any,
4 gene product is present(3) .
Instead, the results clearly demonstrate that the
2 subunit is
coassembled with
3,
4, and
5 subunits in the ganglion,
producing almost certainly at least one neuronal AChR as complex as the
muscle AChR with its four kinds of subunits. The alternative
possibility, namely that the data might be explained by a mixture of
several different receptor subtypes having no more than three kinds of
subunits each, is difficult to reconcile with the observation that
essentially all of the mAb 35-AChRs contain both
3 and
4
subunits, at least some contain
5 subunits, and
2 is
coassembled with a portion of each of the three gene products in the
mAb 35-AChR pool.
The fact that the vast majority of ciliary
ganglion neurons express 2 protein indicates that the incomplete
penetration of
2 subunits into the mAb 35-AChR population does not
represent an all-or-none exclusion of the protein among neurons. It is
possible that the
2 gene product confers special properties on the
receptors and therefore is carefully regulated in abundance by the
neurons. One such property might be receptor location in the plasma
membrane. If the
2 subunit targets mAb 35-AChRs to a presynaptic
location on axon terminals for example, it might explain why relatively
few of the receptors are present in dissected ganglia that lack axons
although the
2 transcript is as abundant as
4 transcripts
that contribute subunits to all mAb 35-AChRs in the
neurons(3) . The mechanism of receptor targeting in the
membrane and the functional significance of possible presynaptic
receptors are subjects of considerable interest.
An alternative
possibility is that the 2 subunit imposes regulatory constraints
on mAb 35-AChRs such as making them cyclic AMP-dependent. The nicotinic
response of ciliary ganglion neurons acquires a partial dependence on
cyclic AMP during development both in vivo and in cell
culture(29, 30) . The molecular basis for the cyclic
AMP effect is unknown. It will be important to determine what
proportion of mAb 35-AChRs on the cell surface contains
2 subunits
and whether the proportion changes with conditions that alter the
cyclic AMP dependence of the nicotinic response.
Nicotinic responses
from neurons can be complex both in kinetics and single channel
events(1) . The present results suggest that the complexity may
in part reflect heterogeneity in subunit composition among AChRs
produced. Ciliary ganglion neurons have -Bgt-AChRs and two classes
of mAb 35-AChRs, namely those with and without
2 subunit. In
situ hybridizations and immunohistochemical results presented both
here and previously (3, 6, 9, 31) make it clear that
most, if not all, neurons in the ganglion express all of the gene
products known to comprise the three receptor classes. Accordingly, it
is highly likely that all ciliary ganglion neurons, both choroid and
ciliary, express each of the three classes. The multiplicity of AChR
subtypes distinguished in a single population of neurons calls
attention to functional and regulatory features that might be uniquely
associated with individual receptor subtypes. Differences in the
conditions of activation, receptor location, ion permeability, duration
of response, and regulatory control would seem to be among the most
likely reasons accounting for comaintenance of multiple receptor
species by the same neuron.