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Vol. 53, Issue 3, 392-401, March 1998
4, 
2, and 5 Gene
Products
Department of Biology, University of California, San Diego, San Diego, California 92093-0357
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Summary |
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Summary
Introduction
Procedures
Results
Discussion
References
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Increasing evidence suggests nicotinic receptors regulate developmental
events in the nervous system. We used [3H]epibatidine and
125I-
-bungarotoxin, together with subunit-specific
monoclonal antibodies, to distinguish and quantify nicotinic receptor
subtypes in developing chick brain. The results show that more than
three fourths of the epibatidine-binding receptors at both early and
late embryonic stages contain 4 and 
2 subunits, representing
receptors previously distinguished by high affinity nicotine binding. A
fraction of these also contain the 5 gene product, which is
consistent with studies on transfected cells showing that the 4,
2, and 5 gene products coassemble to produce epibatidine-binding
receptors. A small portion of the receptors contain 3 and 4
subunits, assembled in part with either 4 or 2 subunits. The most
abundant nicotinic receptors, however, at both early and late embryonic
stages are those having high affinity for -bungarotoxin rather than
epibatidine. Most contain 7 subunits, whereas about half contain
8 subunits as well. The sharpest developmental increase between
embryonic days 8 and 17/18 occurs with receptors containing 5
subunits, whereas receptors containing 3 or 4 subunits undergo no
specific increase. The three major receptor species (containing 4
and 2 but not 5 subunits; 7 subunits; or 7 and 8
subunits) each increase 
3-fold during the same period. The results
indicate greater receptor complexity than appreciated previously; they provide information about the rules governing subunit assembly in
neuronal nicotinic receptors and draw attention to the role of 5
subunits in late development.
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Introduction |
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Top
Summary
Introduction
Procedures
Results
Discussion
References
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AChRs
in the vertebrate nervous system are ligand-gated ion channels that
depolarize neurons by permitting cations to flow across the plasma
membrane. Like their counterparts in vertebrate skeletal muscle, the
receptors are thought to be pentameric transmembrane proteins encoded
by one or more members of a multigene family (Karlin and Akabas, 1996
).
It is clear that neuronal AChRs can participate in a variety of
functions. Presynaptically, the receptors can modulate neurotransmitter
release, whereas postsynaptically, they can generate synaptic currents
from both synaptic and perisynaptic locations (Role and Berg, 1996;
Zhang et al., 1996; Wonnacott, 1997). Some classes of AChRs
have a high relative permeability to calcium and can influence
calcium-dependent events in neurons, including activation of second
messenger cascades.
Neuronal AChRs may play important developmental roles as well. The
receptors are expressed early during embryogenesis, as is the enzyme
responsible for synthesis of ACh (Zoli et al., 1995; Role
and Berg, 1996). AChRs can be found on the tips of growing neurites in
cell culture and, when activated, can influence the pattern of neurite
growth. Presynaptic AChRs also can enhance neurotransmitter release at
newly formed neuromuscular synapses in culture (Fu and Liu, 1996), and
they may contribute to the early stages of synaptogenesis.
It is not clear which AChR subtypes are likely to be most important
during development, or, in fact, how many AChR subtypes exist. Nine
genes encoding neuronal AChR subunits (2-7; 2-4) have been
isolated from both chick and rat (Role and Berg, 1996). In addition,
8 has been isolated uniquely from chick, and 9 has been isolated
uniquely from rat. Two major AChR subtypes have been identified in
brain from both species: one is a receptor with 4 and 2 subunits
that binds nicotine with high affinity, and the other is a receptor
with 7 subunits that binds -Bgt with high affinity (Lindstrom,
1996). Receptor analyses with subunit-specific mAbs also have
identified other AChR subunit combinations in brain (Flores et
al., 1996; Lindstrom, 1996; Forsayeth and Kobrin, 1997).
Recently, the alkaloid epibatidine emerged as a broad-spectrum
cholinergic ligand for neuronal AChRs.
[3H]Epibatidine seems to bind with high
affinity to all AChR species examined to date, except for some that
bind -Bgt (Gerzanich et al., 1995; Houghtling et
al., 1995; Wang et al., 1996). As a result, [3H]epibatidine, together with
125I--Bgt and subunit-specific mAbs, offers a
means of identifying and quantifying additional AChR subtypes in brain.
In the current report, we used this strategy to address four questions:
Do the major AChR species identified in brain contain other gene
products as well? Do individual AChR genes often contribute subunits to different AChR subtypes in the central versus peripheral nervous system? Can new AChR subtypes be detected? Do the relative amounts of
individual AChR subtypes change substantially during development? Answers to these questions should provide information about the rules
governing subunit assembly in neuronal AChRs and may help identify the
receptor species most important at early developmental stages.
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Experimental Procedures |
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Summary
Introduction
Procedures
Results
Discussion
References
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mAbs.
All subunit-specific mAbs used in the current study
have been characterized and described previously (see references in
Vernallis et al., 1993; Conroy and Berg, 1995). Regarding
specificities with chicken neuronal AChR gene products, mAbs 270, 289, 308, 313, and B4-1 both immunoprecipitate and immunoblot selectively the 2, 4, 8, 3, and 4 gene products, respectively,
whereas mAbs 318 and 319 do the same with the 7 gene product. mAb
268 selectively immunoblots (but does not immunoprecipitate) the 5 gene product, whereas mAb 35 immunoprecipitates and immunoblots the
5 gene product (it was raised against electric organ AChRs and
recognizes the 1 subunit therein). Experiments with transfected cells indicate that mAb 35 also immunoprecipitates receptors with 3
subunits but does not recognize the 4, 7, 8, 2, or 4
gene products (Conroy et al., 1992; W. Conroy and D. Berg,
unpublished observations). Unless otherwise indicated, the mAbs were
diluted from concentrated stocks of hybridoma culture supernatants, or mouse ascites fluid (B4-1 only) for use or were purified using Protein
G-Sepharose-4-Fast-Flow (Pharmacia, Piscataway, NJ) or size exclusion
chromatography (mAb 289). Purified mAbs 35, 270, 289, 313, and B4-1
and normal IgG were coupled individually to Actigel (Sterogene
Bioseparations, Arcadia, CA) at 2-4 mg/ml according to the
manufacturer's specifications.
Binding assays.
Whole brains were dissected from embryonic
day (E) 8-18 chicks and stored frozen at 
70° until use. Triton
X-100 (Pierce Chemical, Rockford, IL) extracts of the brains were
prepared as described previously (Conroy et al., 1992) using
two to four volumes of 2% (w/v) Triton X-100 extraction buffer/g of
tissue. For filter binding assays, 2-4 nM
[3H]epibatidine or 5-10 nM
125I--Bgt was incubated with 25 µl of brain
extract in a total volume of 0.1 ml for 2 hr at room temperature. After
the incubation, 4 ml of wash buffer (10 mM Tris, pH 7.5, containing 0.05% Triton X-100) was added, and the solution was
filtered immediately through Whatman (Clifton, NJ) GF/B filters
presoaked in 0.5% polyethyleneimine. The filters were washed two
additional times with 4 ml of wash buffer and counted either by liquid
scintillation counting in Ecolite (ICN, Costa Mesa, CA) for
[3H]epibatidine or by 
-counting for
125I--Bgt. Nonspecific binding was determined
by incubation in the presence of 200 µM nicotine. Protein
in the extracts was quantified by the BCA Protein Assay (Pierce) using
bovine serum albumin as standard.
) and the
mAbs 35 (3, 5), 270 (2), 289 (4), 313 (3), and B4-1
(4) to tether AChRs. Immulon 2 Removawells (Dynatech Laboratories,
Chantilly, VA) were coated with mAb by first incubating the wells
overnight at 4° on a shaker with affinity-purified rabbit anti-mouse
antibodies (Jackson Immunoresearch, West Grove, PA) at a concentration
of 20 µg/ml in PBS (1× = 0.15 M sodium chloride and 0.01 M sodium phosphate, pH 7.4) containing 0.02% azide. The
wells then were washed three times with PBS-TX and incubated on a
shaker overnight at 4° with 50 µl of anti-AChR mAb diluted in
buffer. The wells were rinsed three times with PBS-TX and incubated
overnight with the detergent extracts. The extracts were removed, and
the wells were washed four times with PBS-TX.
[3H]Epibatidine (100-200 µl) was added at
2-4 nM in the presence and absence of 200 µM
nicotine. The wells were washed four times with PBS-TX and placed in 5 ml of Ecolite (ICN) for liquid scintillation counting.
When competition binding experiments were performed with
[3H]epibatidine, the incubations included the
indicated compounds with 1 nM
[3H]epibatidine and were carried out for 4 hr
at room temperature. Plots of competition curves were generated, and
IC50 values were determined by a nonlinear
least-squares method using Prism (GraphPAD Software, San Diego, CA)
assuming a single class of sites in each case.
KD values for epibatidine were
determined from saturation binding reactions in which the receptor
concentration did not exceed 60 pM. Because
ligand depletion may nevertheless have had some effect on the
KD determinations (Kenakin, 1993),
the values are considered approximate (apparent
KD values).
Immunodepletions. Extracts were depleted of AChR subtypes by incubation of extracts (0.2 ml) with 40 µl of anti-AChR mAb coupled to Actigel beads. After an overnight incubation, the Actigel beads with bound material were removed by centrifugation. [3H]Epibatidine binding sites then were quantified in the depleted extracts using the filter binding assay.
AChRs also were depleted by incubation of extracts in Immulon 2 Removawells (Dynatech Laboratories) coated with mAbs 270 or 289, as described above for the solid-phase immunoprecipitation assay, to remove AChRs containing2 and 4 subunits, respectively. After
recovery of the depleted extracts from the wells, they were used in a
solid-phase assay with [3H]epibatidine to
quantify the remaining AChRs. Similarly, a combination of mAbs 318 and
319 was used to deplete AChRs with 7 subunits, and mAb 308 was used
to deplete AChRs with 8 subunits. 125I--Bgt
binding sites remaining in the depleted extracts then were quantified
by the filter binding assay. In sequential depletion experiments,
recovered extracts from one round of depletion were subjected to a
second round of depletion in a new well containing either the same mAb
as in the first round or an mAb to another subunit. In all depletion
experiments, normal rat IgG was used in place of the anti-AChR mAbs as
a negative control.
Immunoprecipitations and immunoblots.
AChRs containing 4,
2, and 5 subunits were analyzed by monitoring the coprecipitation
of 5 subunits with AChRs containing both 4 and 2 subunits.
Similar analyses have been used to show the association of 3, 5,
and 4 subunits in ganglia (Vernallis et al., 1993; Conroy
and Berg, 1995). E8 and E18 brain extracts containing equal amounts of
protein (0.7 mg) were incubated on Immulon 2 Removawells (Dynatech
Laboratories) coated with mAb 289, as described above for the
solid-phase assay, to remove AChRs containing 4 subunits.
Removawells coated with normal rat IgM served as the negative control.
Extracts recovered from these wells then were incubated overnight with
20 µl of mAb 270-Actigel to bind AChRs containing 2 subunits. The
gel was washed three times with PBS-TX and 2 times with PBS-TX
containing 1 M NaCl. Bound material was eluted with
SDS-PAGE sample buffer, subjected to SDS-PAGE, electroblotted to
nitrocellulose, and probed with mAbs 268 for 5 and 289 for 4 as
described previously (Vernallis et al., 1993; Conroy and
Berg, 1995). Horseradish peroxidase coupled to goat anti-rat IgG
(Jackson Immunoresearch) was used to detect bound mAbs. Signals were
visualized by enhanced chemiluminescence (Amersham, Arlington Heights,
IL). Molecular mass markers for the blots included phosphorylase B
(97.4 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), and
carbonic anhydrase (31 kDa) (low range; BioRad).
Transfections.
Chicken AChR gene constructs were used for
the transfections. The expression constructs pCDM8-Ch 23.1 and pCDM8-Ch
26.1 containing the 2 and 4 cDNAs, respectively, under CMV
promoters (Whiting et al., 1991) were kindly provided by Dr.
Paul Whiting (Merk, Sharp, & Dohme Laboratories, Essex, England). An
5 cDNA kindly provided by M. Ballivet (University of Geneva, Geneva)
was modified to delete untranslated 3
and 5 sequences and to add the
3 leader sequence, which has an efficient Kozak consensus sequence
for translation initiation. The modified 5 construct was subcloned into the RSV.An vector (obtained from D. Donahue, University of California, San Diego) under an RSV promoter.
). Transfections usually
contained 1 µg of 4, 0.2 µg of 2, and/or 0.5 µg of 5
cDNA/dish; pBluescript SK
DNA (Stratagene, La
Jolla, CA) was added as needed to adjust the total to 2 µg in each
case. The ratio of 4 to 2 cDNA was chosen to optimize the amount
of [3H]epibatidine binding expressed by the
cells (see below). The presence of 5 DNA did not significantly alter
the amount of [3H]epibatidine binding expressed
by cells transfected with 4 and 2 cDNA.
After 18-24 hr of transfection, the cells were washed with fresh
medium, incubated an additional 24 hr, and then harvested by scraping
in 0.5 ml of solubilization buffer containing 50 mM sodium
phosphate, pH 7.4, 1% Triton X-100, and the protease inhibitors iodoacetamide (0.4 mM), benzamidine (5 mM),
phosphoramidon (5 µg/ml), soybean trypsin inhibitor (10 µg/ml),
leupeptin (10 µg/ml), pepstatin A (20 µg/ml), EDTA (5 mM), EGTA (5 mM), aprotinin (2 µg/ml), and
phenylmethylsulfonyl fluoride (1 mM). Insoluble material was removed by centrifugation for 15 min in a microfuge at 4°. [3H]Epibatidine binding sites were quantified
using the solid-phase assay. Immunoblot analysis was used to confirm
the expression of transfected genes.
Materials.
White Leghorn embryonated chick eggs were
obtained locally and maintained at 39° in a humidified incubator as
described by Conroy et al. (1992). mAb 35 was purified and
radioiodinated as described by Smith et al. (1985). -Bgt
was purchased from Biotoxins (St. Cloud, FL) and radioiodinated to a
specific activity of 0.5-0.7 × 1018
cpm/mol using chloramine T. [3H]Epibatidine
(56.5 Ci/mmol) was a generous gift from DuPont-New England Nuclear
(Boston, MA). Unlabeled epibatidine was purchased from Research
Biochemicals (Natick, MA). mAbs 268, 270, 289, 308, 313, 318, and 319 were generously supplied by Dr. Jon Lindstrom (University of
Pennsylvania, Philadelphia, PA). All compounds were purchased from
Sigma Chemical (St. Louis, MO) unless otherwise indicated. Dr. Paul
Whiting (Merck, Sharp, & Dohme Laboratories) generously provided the
chicken AChR 4 and 2 gene constructs, and Dr. Marc Ballivet
(University of Geneva, Geneva, Switzerland) generously provided the
chicken AChR 5 cDNA. Dr. Jeffrey Wahl (Salk Institute, La Jolla, CA)
provided the HEK 293 cells, and Dr. Daniel Donoghue (University of
California, San Diego) provided the RSV.An vector.
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Results |
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Top
Summary
Introduction
Procedures
Results
Discussion
References
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Epibatidine-binding AChRs in brain.
Filter binding assays were
used to measure the total number of epibatidine binding sites present
in chick brain extracts. A mean value of 109 ± 28 fmol/mg of
protein (mean ± standard error; three experiments) was obtained
with 2 nM [3H]epibatidine for
extracts prepared from E17/18 chick brain. No additional sites were
revealed by increasing the concentration of epibatidine (data not
shown), which is consistent with previous studies showing that 2 nM epibatidine is sufficient to saturate epibatidine
binding sites on neuronal AChRs (Gerzanich et al., 1995;
Houghtling et al., 1995; Wang et al., 1996).
4 mAb 289 immunodepleted 75%
of the epibatidine binding capacity of E17/18 brain extracts, whereas
anti-2 mAb 270 immunodepleted 85% (Fig.
1A). No significant depletion was caused
by either anti-3 mAb 313 or anti-4 mAb B4-1. mAb 35, which
recognizes the neuronal 3 and 5 gene products, immunodepleted
10% of the binding. The results are consistent with the major
epibatidine-binding AChR species in E17/18-brain containing the 4
and 2 gene products. Few AChRs at this stage contain either the 3
or 4 gene products, but a portion are likely to contain the 5.
This latter conclusion comes from the different amounts of
immunodepletion achieved by mAbs 313 and 35.
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4 and 2 gene products (4/2-AChRs; Fig. 1B). The
anti-4 mAb 289 immunoprecipitated 86 ± 13% (three
experiments), whereas the anti-2 mAb 270 immunoprecipitated 93 ± 12% of the total [3H]epibatidine binding
measured with the filter assay. mAb 289 is specific under these
conditions; it does not, for example, retain in the two-site assay
chick ciliary ganglion AChRs that contain the 3, 4, and 5
subunits coassembled (Vernallis et al., 1993) and bind
epibatidine in E17/18 extracts (data not shown).
Only small numbers of binding sites were retained by either the
anti-3 mAb 313 or the anti-4 mAb B4-1 in E17/18 brain extracts (Fig. 1B). mAb 35, in contrast, immunotethered approximately one sixth
of the total number of sites. Control experiments performed with E17/18
ciliary ganglion extracts demonstrated that mAb 35 is 2.0 ± 0.1-fold (mean ± standard error; three experiments) more efficient than mAb 313 in the two-site assay at capturing AChRs containing the appropriate subunit (summing two sequential
immunoprecipitations for each mAb). Identical experiments performed on
E17/18 brain extracts indicated that mAb 35 was able to capture
12.1 ± 4.6-fold (three experiments) more receptors than mAb 35 (again summing two sequential passes with each mAb). The results are
consistent with those in Fig. 1B and suggest that substantially more
brain AChRs contain 5 subunits than 3 subunits. This, together
with the other immunoprecipitation data, is consistent with most of the
brain receptors recognized by mAb 35 being 4/2/5-AChRs.
Subunits coassembled with the 4 and 2 gene products.
The
ability of the 4 and 2 gene products to form receptors that bind
nicotine with high affinity is well documented (Whiting et
al., 1991; Lindstrom, 1996). Less clear is the extent to which the
two kinds of gene products are associated with other kinds of subunits
in brain AChRs. This was tested by using a combination of
immunodepletions and solid-phase assays. One mAb was used to immunodeplete all receptors containing a given gene product, whereas a
second mAb was used to immunotether the remaining receptors containing
a different gene product. The tethered receptors then were quantified
with [3H]epibatidine binding in the solid-phase
assay. By comparing the binding values obtained with and without the
immunodepletion step, it was possible to assess the proportion of
receptors containing both gene products.
4 mAb 289 was used for immunodepletion, approximately
three fourths of the epibatidine-binding receptors containing the 2
gene product were removed from the extract (Fig.
2A). In addition, more than half of the
receptors containing either the 3 or 4 gene products were
removed. More than half of the receptors recognized by mAb 35 also were
removed by the depletion with mAb 289. To test the efficiency of the
immunodepletion step, the depleted extracts were tested for residual
receptors containing the 4 gene product; approximately one fifth of
the control value remained. Taking into account the immunodepletion
efficiency determined in this manner, the results indicate that most of
the brain AChRs containing the 2 gene product and more than half of
the receptors containing the 3 or 4 gene products also contain
the 4 gene product. The same is likely to be true of receptors
containing the 5 gene product and recognized by mAb 35, namely, that
they also contain the 4 gene product.
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2 mAb 270 removed >80% of the 4-
and 2-containing receptors, corroborating the results obtained with
mAb 289 (Fig. 2B). Between one half and three fourths of the 3 and
4-containing receptors were removed, as was more than three fourths
of the receptors recognized by mAb 35. More than three fourths of the
2-containing receptors also were removed by the immunodepletion,
demonstrating the efficiency of the antibody. The results support
previous findings indicating that a substantial portion of the 4 and
2 gene products are assembled into 4/2-AChRs with no other
apparent subunits. The results also show, however, that some 4 and
2 subunits combine with additional kinds of subunits to make up a
heterogeneous population of epibatidine-binding AChRs in the CNS.
Coassembly of 4, 2, and 5 subunits in transfected
cells.
The fact that mAb 35 immunoprecipitates many more
epibatidine-binding sites from brain extracts than does the
3-specific mAb 313 suggests that the receptors contain 5 subunits
instead of 3 subunits. These could represent 4/2/5-AChRs
and constitute a significant fraction of all brain receptors containing
the 4 and 2 gene products at the end of embryogenesis. Recently,
the 5 gene product has been shown capable of assembling with 4
and 2 when coexpressed in Xenopus laevis oocytes
(Ramirez-Latorre et al., 1996). We examined this issue in
cells that were not oocytes.
4, 2, and 5 cDNAs. Two days later, cell extracts were
prepared and examined in solid-phase assays for receptors that could
bind [3H]epibatidine and be immunotethered by
anti-AChR mAbs. The 4 and 2 gene combination produced a large
number of receptors tethered either by anti-4 mAb 289 or by
anti-2 mAb 270, but none were recognized by mAb 35 (Fig.
3). Inclusion of 5 cDNA in the
transfection with 4 and 2 had little effect on the total number
of epibatidine-binding receptors tethered by mAbs 289 or 270 but
generated a significant number of receptors recognized by mAb 35. Transfection with 5 alone, 5 plus 4, or 5 plus 2 yielded
no significant epibatidine binding retained by any of the mAbs. The
results clearly indicate that the 5 gene product can combine with
the 4 and 2 gene products to produce epibatidine-binding
4/2/5-AChRs.
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Ligand binding properties of AChR subtypes.
In X. laevis oocytes, heterologous expression of the chick AChR 4 and
2 genes with and without the 5 gene shows that
4/2/5-AChRs have a 100-fold higher EC50
value for activation by ACh than that of 4/2-AChRs (20). In
contrast, expression of the human 5 gene in oocytes with the 3
and 4 genes has no effect on the ACh dose-response curve, whereas
expression of 5 with the 3 and 2 genes shifts the
EC50 value to smaller values (Wang et
al., 1996).
5 subunits to the
pharmacology of native AChRs containing the 4 and 2 gene product, we first used mAb 35 to isolate a population of receptors from brain
extract that included those with 5 subunits. mAb 289 then was used
on the same mAb 35-depleted extracts to isolate a receptor population
containing 4 (and presumably 2) but not 5 subunits. Binding
assays revealed no significant difference in the affinity of the two
receptor populations for [3H]epibatidine. The
presumed 4/2/5-AChRs had an apparent
KD value of 2.8 ± 0.7 pM (five experiments), whereas 4/2-AChRs had an apparent KD value of 5.4 ± 2.6 pM (three experiments). Similar binding
assays with 4/2/5-AChRs isolated by mAb 35 from HEK 293 cells
transfected with 4, 2, and 5 cDNA or with 4/2-AChRs
isolated by mAb 289 from cells transfected with 4 and 2 cDNA
yielded apparent KD values of
2.3 ± 0.5 pM (seven experiments) and
2.6 ± 0.9 pM (four experiments),
respectively. Thus, receptors of known subunit composition from
transfected cells corroborated the results with native AChRs in showing
no difference in epibatidine affinity that could be ascribed to the presence of 5 subunits.
Competition binding studies with
[3H]epibatidine were used to compare the
binding affinities of receptor subtypes for other nicotinic ligands
(Fig. 4). A comparison of IC50 values indicated at most a nominal 5-fold difference in affinity between putative 4/2/5- and 4/2-AChRs isolated from brain extract as
described above (Table 1). The
IC50 values for 4/2/5- and
4/2-AChRs from transfected cells were essentially
indistinguishable from their brain counterparts. The results are
consistent with the inferred subunit composition of the brain AChR
subtypes and indicate little effect of 5 subunits on ligand affinity
in binding assays.
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Changing populations of epibatidine-binding AChRs during
development.
The relative contributions of individual AChR gene
products to epibatidine-binding AChR subtypes in brain were measured
during development. The goal was to determine whether different species predominated at early versus late times. Solid-phase assays revealed three patterns. Of the epibatidine-binding AChRs, receptors containing either the 4 or the 2 gene product were the most abundant at all
times examined and increased developmentally 3-fold between E8 and
E17/18 (Fig. 5A). This was over and above
increases due to cell growth as reflected in the amount of total
protein. Receptors containing either the 3 or 4 gene products
were the least abundant at all times examined and showed no
developmental increase between E8 and E17/18; the small increase in
binding sites represented net growth because no change was apparent
when the values were normalized for protein. Most striking was the
change in the number of epibatidine-binding receptors recognized by mAb
35. These increased 7-fold between E8 and E17/18 when normalized for
protein (Fig. 5B). Because no developmental increase was observed in
the number of receptors containing the 3 gene product over the same
time period, much of the increase is likely to involve receptors with 5 subunits.
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5 subunits. Brain
AChRs were concentrated by immunoprecipitation, eluted, gel
electrophoresed, and analyzed on blots probed with subunit-specific
mAbs. All five AChR gene products inferred above to be present in brain
AChRs were detected on the blots (Fig. 6A). The relative amounts of 5 protein
assembled with 4 and 2 protein at early and late developmental
stages then was assessed by using the anti-2 mAb 270 to
immunoprecipitate the receptors. The material was eluted,
electrophoresed, and analyzed on blots probed with mAb 289 to detect
4 protein and mAb 268 to detect 5 protein. Both 4 and 5
protein could be detected, and both increased substantially between E8
and E17/18 (Fig. 6B). Immunodepletion with the anti-4 mAb 289 before
immunoprecipitation with mAb 270 removed large amounts of the 4 and
5 proteins subsequently detected on the blots. The results confirm
the conclusions that a portion of the native AChRs containing 4 and
2 subunits also contain 5 subunits and that the amount of this
species increases developmentally. Interestingly, a portion of the 5
protein also seems to be associated with a different receptor species
late in development because some of the 5 gene product associated
with 2 (and immunoprecipitated by the anti-2 mAb 270) cannot be
immunodepleted by the anti-4 mAb 289 (Fig. 6B).
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Developmental changes in the population of brain AChRs that bind
-Bgt.
The total number of -Bgt-binding AChRs detected in
brain extracts using 125I--Bgt in the filter
assay was substantially higher at all stages examined than was the
total number of epibatidine-binding AChRs (Fig.
7A). A developmental increase of
3-fold occurred in the number of -Bgt-binding receptors between
E8 and E17/18. Immunodepletion with the anti-7 mAbs 318/319
indicated that most of the -Bgt-binding receptors at both early and
late times in embryonic brain contain the 7 gene product, whereas
approximately half also contain the 8 gene product (Fig. 7B).
Sequential immunoprecipitations were required to collect all of the
receptors containing the 8 gene product, presumably because of the
inefficiency of the antibody. A small fraction of the total
-Bgt-binding AChRs at both early and late times may contain gene
products other than 7 and 8 gene because some -Bgt-binding
species remained in solution even after repeated immunoprecipitations.
A similar fraction of -Bgt-binding species remained in E17/18
ciliary ganglion extracts after sequential immunoprecipitations in the
two-site assay with mAbs 318/319 (data not shown), and a portion of
this has been attributed previously to an AChR species lacking the 7
gene product (Pugh et al., 1995). Much of the remainder,
however, could represent receptor in which the relevant epitopes are
blocked or degraded. No significant change was observed between E8 and
E17/18 in the ratios of the -Bgt-binding species in brain.
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Discussion |
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Top
Summary
Introduction
Procedures
Results
Discussion
References
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Epibatidine and -Bgt together as probes distinguish several
major classes of AChRs in brain. Most abundant are receptors binding
-Bgt and containing either 7 or 7 plus 8 subunits. Together
they make up at least three fourths of the total -Bgt-binding species at all developmental stages examined and represent nearly two
thirds of the total AChRs distinguished in the combined filter assays.
The findings are consistent with earlier reports of abundant -Bgt-binding AChRs containing 7 and 8 subunits in chick brain (Gotti et al., 1994a; Lindstrom, 1996). The current study
shows that only a small fraction of the total -Bgt binding in brain at either early or late developmental times is a candidate for receptor
species lacking both the 7 and 8 gene products. Gotti et
al. (1994b) reported that approximately half of the
-Bgt-binding receptors in chick brain between E7 and E13 could not
be immunoprecipitated by antisera against either 7 or 8 peptides.
The reason for the discrepancy between the earlier results and those
reported here is not clear but may be due to differences in the
efficiency of receptor immunoprecipitation in the two studies.
The next most abundant species of brain AChRs is one that binds
epibatidine and contains the 4 and 2 gene products. These constitute >80% of the total epibatidine-binding receptors at all
developmental stages examined and represent 15% of the total brain
AChRs detected in filter assays with epibatidine and -Bgt. The same
AChR subtype was identified previously as a major species capable of
binding nicotine with high affinity in rat and chick brain (Lindstrom,
1996).
Another AChR species distinguished here in brain is one that binds
epibatidine and contains not only the 4 and 2 gene products but
also gene products recognized by mAb 35. Previous studies in chick have
shown that mAb 35 recognizes the 3 and 5 neuronal AChR gene
products but not the 4, 7, 8, 2, or 4 gene products (Conroy et al., 1992; Vernallis et al., 1993;
Lindstrom, 1996). Because the anti-3 mAb 313 is unable to
immunoprecipitate the vast majority of such receptors, they are
unlikely to contain 3 subunits. Instead, sequential
immunoprecipitations with other mAbs followed by immunoblot analysis
with the anti-5 mAb 268 confirm that a portion of the AChRs in brain
containing 4 and 2 subunits also contain 5 subunits and seem
to be 4/2/5-AChRs.
Heterologous coexpression of 4, 2, and 5 constructs in both
X. laevis oocytes (Ramirez-Latorre et al., 1996)
and HEK 293 cells produces 4/2/5-AChRs. An
electrophysiological comparison of 4/2- and 4/2/5-AChR
in oocytes shows that the 5 subunit reduces receptor affinity for
ACh as judged by dose-response curves (Ramirez-Latorre et
al., 1996). Binding studies on receptors from HEK 293 cells,
however, reveal no difference between 4/2- and 4/2/5-AChRs in affinity for several nicotinic ligands. A
likely reason for the different results is that the
electrophysiological analysis measured ligand affinity of
nondesensitized receptors, whereas the equilibrium binding studies used
for the current report almost certainly measured ligand affinity of
desensitized receptors. An alternative possibility is that oocytes
and HEK 293 cells influence receptor pharmacology differently through
the kinds of post-translational modifications they provide. A similar
argument has been advanced to account for differences in the
single-channel events seen for AChRs produced by oocytes versus neurons
(Sivilotti et al., 1997). Consistent with current results,
previous researchers (Lindstrom, 1996) have found no difference in the
binding affinities of nicotine, cytisine, or epibatidine for chick
brain AChRs immunoprecipitated by either mAb 35 or anti-4 mAbs. The
results underscore the need for caution in using pharmacology alone to
draw conclusions about subunit composition, but they do not conflict
with the conclusion based on immunoprecipitations and immunoblot
analysis showing that the 5 gene product coassembles with 4 and
2 in brain.
In situ hybridization analyses demonstrate that 4 and
2 transcripts are widely expressed in both rat and chick brain (Wada et al., 1989; Morris et al., 1990). Although the
distribution of 5 mRNA has not been examined in chick, in
situ hybridization studies in rat brain show extensive overlap
between regions expressing 5 transcripts and those expressing 4
and 2 (Wada et al., 1990). Overlap also is found in both
rat and chick between brain regions expressing 3 and those
expressing 4 and 2 (Wada et al., 1989; Morris et
al., 1990; Lobron et al., 1995). The same is true for 4 expression in rat brain (Dineley-Miller et al., 1992).
Three remaining neuronal AChR genes in chick, 2, 6, and 3,
were not included in the current study because subunit-specific antibodies were not available. Least relevant is 3 because in situ hybridization analysis indicates that expression of the gene is largely confined to the retina and trigeminal ganglion in chick (Hernandez et al., 1995). Both the 2 and 6 genes are
expressed in chick brain (Wada et al., 1989; Daubas et
al., 1990; Gerzanich et al., 1997) and may contribute
subunits to receptors currently thought to be either 4/2-AChRs or
4/2/5-AChRs. Neither 2 nor 6 is likely to define a large
class of entirely separate brain AChRs, however, because most
epibatidine-binding receptors can be immunoprecipitated by both
anti-4 and anti-2 mAbs. The 2 and 6 gene products could, in
principle, account for some of the mAb 35 binding to brain AChRs.
Preliminary results opposing this for the 2 gene product come from
immunoprecipitations with mAb 321, which was raised against an
2-specific peptide sequence (R. Schoepfer, W. Conroy, and J. Lindstrom, unpublished observations); mAb 321 immunoprecipitates only
1-2% of the total epibatidine-binding receptors from E17/18 chick
brain extracts (W. Conroy and D. Berg, unpublished observations). The
6 gene product has yet to be tested for mAb 35 binding but is not a
likely candidate because it lacks a critical moiety in the domain
recognized by the antibody (Saedi et al., 1990; Gerzanich
et al., 1997). Nevertheless, the subunit heterogeneity of
AChRs that bind mAb 35 and contain 4 and 2 gene products could be
even greater than shown here.
Previous studies indicated that neuronal AChRs can contain more than
one kind of -type subunit, as well as more than one kind of -type
subunit (Conroy et al., 1992; Vernallis et al., 1993; Conroy and Berg, 1995; Lindstrom, 1996; Ramirez-Latorre et
al., 1996; Wang et al., 1996; Forsayeth and Kobrin,
1997). An individual AChR gene product also can enter into different subunit associations depending on the partners available. The current
findings extend this pattern, showing that AChR gene products common in
the peripheral nervous system also are expressed in the central nervous
system but largely assemble with different partners in the two cases.
Thus, the 5 gene product is assembled with 3, 2, and 4 in
autonomic neurons but usually not with 4 (Listerud et
al., 1991; Vernallis et al., 1993; Mandelzys et al., 1994; Conroy and Berg, 1995; Flores et al., 1996),
whereas in brain, 5 subunits are assembled in considerable measure
with 4 and 2. Similarly, the 3 and 4 gene products are
assembled with 5 and to some extent 2 in autonomic neurons but
usually not with 4 (Listerud et al., 1991; Vernallis
et al., 1993; Mandelzys et al., 1994; Conroy and
Berg, 1995; Flores et al., 1996), whereas in brain 3 and
4 are assembled mostly with 4 and 2.
Changing AChR patterns during brain development suggest that receptors
containing the 3 and 4 gene products are likely to be most
important at early times and less so at later stages because their
levels undergo no developmental increase beyond that present at E8.
This view is consistent with in situ hybridization studies in rat showing that 3 and 4 mRNA appear early in development and
then virtually disappear from the brain and spinal cord at later times
(Zoli et al., 1995). Transcripts for 7 and for 4 and
2 in rat brain undergo more sustained increases throughout development (Broide et al., 1995; Ostermann et
al., 1995; Zoli et al., 1995; del Toro et
al., 1997), which is consistent with the sustained developmental
increases in AChRs containing the gene products reported here. An
interesting exception is the case of 2 gene expression in chick
optic tectum, which is transient and dependent on innervation from the
eye (Matter et al., 1990).
The largest developmental increase observed in the current study was
that of AChRs recognized by mAb 35. They undergo a 7-fold increase
between E8 and E17/18 over and above that expected from net growth and
represent approximately one sixth of all epibatidine-binding receptors
at the end of embryogenesis. Immunoblot analysis confirmed a
developmental increase in 5 protein associated with 4 and 2
subunits in such receptors. Some of the increase in epibatidine-binding AChRs recognized by mAb 35 also may represent the appearance of receptors containing 5 subunits coassembled with different AChR gene
products, however, because not all of the receptors could be
immunoprecipitated by anti-4 mAbs. In fact, AChRs that bind mAb 35 constitute approximately one half of the receptors capable of high
affinity nicotine binding in adult chicken brain and can be
immunoprecipitated by the anti-2 mAb 270 but not by the anti-4 mAb 285 (Lindstrom, 1996). The subunit composition of this species includes components of 49 and 58 kDa (Whiting and Lindstrom, 1986), with the former being in the size range expected for both the 5 and
2 proteins (Conroy et al., 1992).
Differences in subunit composition are likely to influence many aspects of receptor function. Among these are the rates of receptor activation and desensitization, agonist sensitivity, calcium permeability, second messenger regulation, and possibly location on the cell surface. Considered broadly, the fact that a given AChR gene product is assembled with different subunit partners suggests the receptors perform different physiological functions but share a need for the specific features conferred by individual subunits. Identification of unique features contributed by individual subunits is a challenge for subsequent studies.
| |
Acknowledgments |
|---|
We thank Dr. Jon Lindstrom (University of Pennsylvania, Philadelphia, PA) for generously providing the indicated mAbs and Dr. Paul Whiting (Merk, Sharp, & Dohme Laboratories, Essex, England) and Dr. Marc Ballivet (University of Geneva, Geneva, Switzerland) for generously providing cDNA constructs. Lynn Ogden provided expert technical assistance.
| |
Footnotes |
|---|
Received August 5, 1997; Accepted November 23, 1997
This work was supported by grants from the National Institutes of Health (NS12601 and NS35469), Muscular Dystrophy Association, Council for Tobacco Research (4191), and Tobacco-Related Disease Research Program.
Send reprint requests to: Dr. Darwin K. Berg, Department of Biology, 0357, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0357. E-mail: dberg{at}ucsd.edu.
| |
Abbreviations |
|---|
ACh, acetylcholine;
AChR, nicotinic
acetylcholine receptor;
-Bgt, -bungarotoxin;
DHBE, dihydro--erythroidine;
HEK, human embryonic kidney;
mAb, monoclonal
antibody;
SDS, sodium dodecyl sulfate;
PAGE, polyacrylamide gel
electrophoresis;
PBS, phosphate-buffered saline;
PBS-TX, phosphate-buffered saline containing 0.5% (w/v) Triton X-100;
EGTA, ethylene glycol bis(-aminoethyl
ether)-N,N,N,N-tetraacetic
acid.
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References |
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Top
Summary
Introduction
Procedures
Results
Discussion
References
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75%. B, Receptors containing 
), 
), 
), 
). A, Absolute amounts; binding is expressed per mg of
protein. B, Relative increases; binding is normalized to that present
at E8. Values represent the mean ± standard error from three or
four experiments conducted on two or more preparations of extract.
) or, for comparison,
[3H]epibatidine binding sites (