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Volume 17, Number 21,
Issue of November 1, 1997
pp. 8201-8212
Copyright ©1997 Society for Neuroscience
Neuronal  -Bungarotoxin Receptors Differ Structurally from
Other Nicotinic Acetylcholine Receptors
Fatima Rangwala,
Renaldo C. Drisdel,
Sergey Rakhilin,
Elizabeth Ko,
Pramod Atluri,
Amy B. Harkins,
Aaron P. Fox, and
Suleiman B. Salman, and William N. Green
Department of Pharmacological and Physiological Sciences,
University of Chicago, Chicago, IL 60637
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
We have characterized the  -bungarotoxin receptors (BgtRs)
found on the cell surface of undifferentiated pheochromocytoma (PC12)
cells. The PC12 cells express a homogeneous population of
7-containing receptors that bind -Bgt with high affinity (Kd = 94 pM). The BgtRs mediate
most of the response elicited by nicotine, because the BgtR-specific
antagonists methyllycaconitine and -Bgt block ~90% of the
whole-cell current. The binding of nicotinic agonists to cell-surface
BgtRs was highly cooperative with four different agonists showing Hill
coefficients in the range of 2.3-2.4. A similar agonist binding
cooperativity was observed for BgtR homomers formed from chimeric
7/5HT3 subunits expressed in tsA 201 cells. Two classes of agonist
binding sites, in the ratio of 4:1 for PC12 cell BgtRs and 3:1 for
7/5HT3 BgtRs, were revealed by bromoacetylcholine alkylation of the
reduced sites on both PC12 BgtRs and 7/5HT3 BgtRs. We conclude from
this data that PC12 BgtRs and 7/5HT3 homomers contain at least three distinguishable agonist binding sites and thus are different from other
nicotinic receptors.
Key words:
nicotine;
-bungarotoxin;
pheochromocytoma;
nicotinic
receptors;
electrophysiology;
pharmacology
INTRODUCTION
Neuronal nicotinic acetylcholine
receptors (AChRs) are members of a large family of
neurotransmitter-gated ion channels that includes muscle-type AChRs,
GABAA, glycine, and serotonin (5HT3) receptors (for
reviews, see Barnard, 1992 ; Unwin, 1993; Karlin and Akabas, 1995).
Muscle-type AChRs are composed of four homologous subunits (1,  1,
 , and  ) assembled into a pentamer with a stoichiometry of
121. Neurons, in contrast to muscle, express a
wide array of AChR subtypes, and this heterogeneity has made neuronal
AChR isolation and characterization more difficult. Recent molecular cloning studies have identified AChR genes encoding eight ACh binding
subunits (2-9) and three structural subunits (2-4) (Role,
1992; Sargent, 1993). These genes have distinct, overlapping patterns
of expression in the brain, sensory end organs, and peripheral ganglia,
and the different and subunits contribute to the pharmacologic
and functional diversity of neuronal AChRs (Lindstrom et al., 1995;
McGehee and Role, 1995; Role and Berg, 1996). Structurally, the best
characterized neuronal AChRs are the high-affinity nicotine binding
sites found in brain, which are composed predominantly of 4 and 2
subunits with a stoichiometry of 4223
(Anand et al., 1991; Cooper et al., 1991).
Another neuronal AChR subtype is the high-affinity -bungarotoxin
receptor (BgtR) (Clarke, 1992; Lindstrom et al., 1995). Recently, BgtRs
have been shown to be functional (Alkondon and Albuquerque, 1993; Zhang
et al., 1994). They are found at presynaptic terminals of several
synapses where they appear to regulate synaptic vesicle release
(McGehee et al., 1995; Alkondon et al., 1996; Gray et al., 1996). A
common feature of most BgtRs is that they contain 7 subunits
(Schoepfer et al., 1990; Vernallis et al., 1993; Gotti et al., 1994,
1995). 7 subunits when expressed in Xenopus oocytes form
functional, Bgt binding homomers (Couturier et al., 1990; Seguela et
al., 1993; Peng et al., 1994), raising the possibility that native
BgtRs are 7 homomers. However, BgtRs purified from chicken and rat
brain preparations appear to consist of one to four different
polypeptide subunits (Conti et al., 1985; Kemp et al., 1985; Whiting
and Lindstrom, 1987; Hermans-Borgmeyer et al., 1988; Schoepfer et al.,
1990; Gotti et al., 1994) with a pharmacology different from the 7
homomers expressed in oocytes (Anand et al., 1993). These data are
consistent with BgtRs composed of more than one subunit subtype. By
analogy to the muscle-type AChR and the neuronal high-affinity nicotine
binding site, this suggested a subunit stoichiometry of two 7
subunits to three structural subunits (Schoepfer et al., 1990; Anand et
al., 1993).
The data presented in this paper are not consistent with a BgtR subunit
composition of two ligand binding and three structural subunits. BgtRs
expressed in PC12 cells were studied and the agonist binding sites of
these receptors characterized. To compare the properties of the PC12
cell BgtR with a BgtR of known subunit composition, a heterologously
expressed BgtR homomer was also studied. We find that there are at
least three, and more likely four or five, distinguishable agonist
binding sites on both BgtRs expressed in PC12 cells and the BgtR
homomers, and conclude that these neuronal BgtRs differ structurally
from other characterized AChRs that have two binding sites.
MATERIALS AND METHODS
cDNAs. The human 7 cDNA was the gift of Dr. J. Lindstrom (University of Pennsylvania), and the rat 7 cDNA was the
gift of Dr. J. Boulter (UCLA). Both constructs were subcloned into the pCB 6 expression vector, the gift of Dr. M. Roth (University of Texas,
Southwest), into the KpN-Xba sites. Chick 7 and chick 7/mouse
5HT3 cDNA were the gift of Dr. J.-L. Eisele (Pasteur Institute). These
cDNAs were subcloned into the pMT3 expression vector (Swick et al.,
1992), which contains the same cytomeglavirus promoter as pCB 6, in the
NotI-XhoI sites.
Cell lines and transfection. The N21 variant of the
PC12 cell line was the gift of Dr. R. Burry (Ohio State University).
The cells were cultured in DMEM supplemented with 5% heat-inactivated horse serum, 5% fetal bovine serum (Hyclone, Logan, UT), 10,000 U/ml
of penicillin and streptomycin (Life Technologies, Gaithersburg, MD)
maintained at 37°C in the presence of 5% CO2. The cell
line stably expressing Torpedo 2
AChRs (Claudio et al., 1987) and the cell line stably expressing
2 AChRs (Green and Claudio, 1993) were
established as described previously.
The cells expressing 7/5HT3 BgtRs were established by the stable
transfection of the 7/5HT3 cDNA in pMT3 into tsA 201 cells (Margolskee et al., 1993), which were the gift of Dr. J. Kyle (University of Chicago). The 7/5HT3 cDNA was transfected along with
the hygromycin resistance gene for selection using a calcium phosphate
protocol as described previously (Claudio, 1992). The resultant cell
line was maintained in DMEM supplemented with 10% calf serum (Hyclone)
and 1000 U/ml of hygromycin (Calbiochem, La Jolla, CA). The 7 and
7/5HT3 cDNAs were also expressed by transiently transfecting the
cDNAs into tsA 201 cells using the same calcium phosphate protocol.
In vitro translation. The rat 7 cDNA was
subcloned into pBluescript (Stratagene, La Jolla, CA) in the T7
orientation and linearized with XbaI (New England Biolabs,
Beverly, MA). Complementary RNA was transcribed in vitro
with T7 polymerase (Boehringer Mannheim, Indianapolis, IN) at 37°C
for 1 hr. The size of the RNA was analyzed on a 0.7% formaldehyde
agarose gel before translation. A total of 0.1 mg of the RNA was
translated at 30°C for 1 hr in an RNA-free rabbit reticulocyte lysate
(Promega, Madison, WI) in the presence of 35S-methionine
(Amersham, Arlington Heights, IL; 1000 Ci/mmol) and RNasin (Boehringer
Mannheim). Post-translational processing of polypeptides was observed
using 3 µl canine pancreas microsomes (Promega) per 50 µl of
translation reaction volume. Translated proteins were analyzed by 10%
SDS-PAGE.
Cell-surface 125I-Bgt binding. A
125I-Bgt binding assay was used to measure the number of
cells surface Bgt binding sites. Under our culture conditions in which
the cell were not grown on any substrate, both the PC12 and the
transfected tsA201 cells were poorly adherent. This property allowed
the cells to be removed from confluent cultures simply by applying a
gentle fluid stream from a Pasteur pipet. Cells removed from the plates
were washed with PBS and incubated at room temperature in PBS
containing 10 nM 125I-Bgt (140-170 cpm/fmol;
NEN) for 1.5-2 hr. This incubation time was sufficient to saturate
binding. The cells were then washed three times with PBS and counted in
a Wallac 1470 automatic gamma counter. Nonspecific binding was
estimated by the addition of the indicated competitive ligand during
the incubation. To estimate the toxin association rate,
125I-Bgt was bound to the cell surface at 4°C for the
incubation times indicated, and the number of cell-surface Bgt binding
sites were determined. To estimate the dissociation rate, cell
cultures, first bound with 125I-Bgt under saturating
conditions, were further incubated at 4°C for the indicated times in
PBS in the absence of 125I-Bgt, and the number of
cell-surface Bgt binding sites were determined.
In experiments for which cell-surface 125I-Bgt binding was
inhibited by several different ligands, only the initial rate of
125I-Bgt binding was measured. Cells were first
preincubated for 15-30 min with the indicated concentration of ligand,
after which the cells were incubated with 4 nM
125I-Bgt for 15-30 min. The increase in the Bgt
association rate during the 15-30 min incubation time was linear (see
Figs. 1C and
3B).
Fig. 1.
The PC12 BgtR contains 7 subunits and binds
Bgt with high affinity. A, The cell surface of PC12
cells was bound with 125I-Bgt in the absence or presence of
10 mM carbachol or 1 mM Bgt. The ligands were
added to estimate nonspecific binding. In the absence of ligand, the
maximum binding was 0.45 ± 0.05 pmol (mean ± SD) obtained
from four 6 cm cultures containing on average 7 × 106 cells. Based on these data, there are ~40,000
Bgt binding sites per cell. B,
125I-Bgt-bound, cell-surface BgtRs from PC12 cells are
specifically immunoprecipitated by 7-specific mAb 319, but not by
other AChR subunit-specific antibodies. PC12 cells were cell-surface
bound with 125I-Bgt and precipitated with mAbs specific for
7 subunit, mAb 319 (Schoepfer et al., 1990), 3 and 5 subunit,
mAb 35 (Whiting and Lindstrom, 1986), or 2 subunit mAb 270 (Whiting
et al., 1987). Approximately 40% of the total cell-surface-labeled
receptors (6000 ± 400 cpm; mean ± SD from from three 6 cm
cultures) were precipitated by mAb 319. The counts precipitated by the
mAbs specific for the 3, 5, or 2 subunits were
indistinguishable from background. C, Estimation of the
Bgt association rate. PC12 cells were incubated with 10 nM
125I-Bgt at 4°C for the indicated times.
Cell-surface-bound 125I-Bgt (in femtomoles) is shown
plotted against time of incubation. Each point
represents the mean of two 10 cm cultures. The line through the points represents a least-squares fit to the data of the
equation: 125I-Bgt bound = 1 exp(k+1t[Bgt]), where
k+1 is the association rate. The results for
all experiments are summarized in Table 1. D, Estimation
of the Bgt dissociation rate. After PC12 cells were
cell-surface-labeled with 125I-Bgt, cultures were incubated
in the absence of 125I-Bgt at 4°C for the indicated
times. Cell-surface bound 125I-Bgt divided by the value at
0 hr (fraction of maximum bound) is shown plotted against time of
incubation. Each point represents the mean of two 10 cm
cultures. The line through the points represents a
least-squares fit to the data of the equation: fraction of maximum bound = exp (k 1t),
where k1 is the dissociation rate. The
results for all experiments are summarized in Table 1.
[View Larger Version of this Image (31K GIF file)]
Fig. 3.
The Bgt binding site of the 7/5HT3
homomer. A, Cells stably expressing the 7/5HT3
subunit were cell-surface labeled with 125I-Bgt in the
absence or presence of 100 µM nicotine. Nicotine was
added to estimate nonspecific binding. In the absence of nicotine, the
maximum binding was 2.5 ± 0.1 pmol (mean ± SD) obtained
from four 6 cm cultures containing on average 3 × 106 cells. Based on these data, there are ~500,000
Bgt sites per cell. B, Estimation of the Bgt association
rate. Cells stably expressing the 7/5HT3 subunit were incubated with
20 nM 125I-Bgt at 4°C for the indicated
times. Cell-surface-bound 125I-Bgt (in femtomoles) is shown
plotted versus time of incubation. Each point represents
the mean of two 6 cm cultures. The line through the
points represents a least-squares fit to the data of the equation:
125I-Bgt bound = 1exp(k+1t[Bgt]), where
k+1 is the association rate. The results for
all experiments are summarized in Table 1. C, Estimation
of the Bgt dissociation rate. After cells stably expressing the
7/5HT3 subunit were cell-surface labeled with 125I-Bgt,
cultures were incubated in the absence of 125I-Bgt at 4°C
for the indicated times. Cell-surface-bound 125I-Bgt
divided by the value at 0 hr (fraction of maximum bound) is shown
plotted against time of incubation. Each point
represents the mean of two 6 cm cultures. The line
through the points represents a least-squares fit to the data of the
equation: fraction of maximum bound = exp(k1t), where
k1 is the dissociation rate. The results
for all experiments are summarized in Table 1.
[View Larger Version of this Image (25K GIF file)]
In experiments for which cell-surface 125I-Bgt binding
sites were immunoprecipitated, 125I-Bgt-bound cells were
solubilized in 150 mM NaCl, 5 mM EDTA, 50 mM Tris, pH 7.4, 1% Triton X-100, 0.02% NaN3 (lysis
buffer) supplemented with the protease inhibitors 2 mM
phenylmethylsulfonyl fluoride, 2 mM
N-ethylmaleimide, and 10 µg/ml each chymotrypsin, leupeptin, pepstatin, and tosyl-lysine chloromethyl ketone. Extracts were clarified by centrifugation at 10,000 × g for 30 min. Antibodies were added to the supernatants, and samples were
rotated overnight at 4°C followed by precipitation with protein
G-Sepharose (Pharmacia, Piscataway, NJ). Tubes were centrifuged for 30 sec at 8000 × g and the pellets washed three times
with lysis buffer before counting.
Alkylation with bromoACh. After being harvested and
washed, cells were resuspended in PBS containing 0.5 mM
dithiothreitol (DTT). Tubes were rotated at room temperature for 30 min, after which cells were washed rapidly three times with PBS. Cells
were then rapidly exposed to the indicated concentrations of bromoACh in PBS, incubated for 30 min, washed three times, and subjected to
cell-surface 125I-Bgt binding under saturating conditions.
Similar results were obtained by performing the DTT incubation in the
same buffer with 0.3 mM DTT or in a different buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris, pH
8.0) with 0.5 mM DTT (data not shown).
In experiments for which the DTT concentration was varied, cells were
washed in 150 mM NaCl, 5 mM EDTA, 50 mM Tris, pH 8.0, and then the DTT incubation was performed
in the same buffer with the indicated DTT concentrations. The bromoACh
incubation was performed in 150 mM NaCl, 5 mM
EDTA, 50 mM Tris, pH 7.0, with 300 µM
bromoACh for the PC12 cells and 10 µM bromoACh for the cells expressing the muscle-type AChRs.
Sucrose gradient sedimentation. To distinguish
cell-surface PC12 and 7/5HT3 BgtRs on the basis of their size, the
receptors, bound with 125I-Bgt and solubilized as described
above, were sedimented on sucrose gradients. For this procedure,
lysates were layered on a 5 ml 5-20% linear sucrose gradient prepared
in the lysis buffer. Gradients were centrifuged in a Beckman SW 50.1 rotor at 40,000 rpm for 14.25 hr ( 2t = 9.0 × 1011). Three-hundred microliter fractions were
collected from the top of the gradient and counted. The linearity of
the gradient was confirmed by measuring the osmolality of each
fraction.
Cross-linking of the 125I-Bgt-bound BgtRs.
BgtRs on the cell surface of PC12 cells and cells expressing
7/5HT3 BgtRs were bound with 125I-Bgt. Cells,
resuspended in PBS, were exposed to 2.5 mM 3,3 dithiobissulfosuccinimidylproprionate (DTSSP; Pierce, Rockford, IL) at
room temperature for 10 min. This DTSSP concentration and time of
incubation were found to saturate the cross-linking between 125I-Bgt and the receptor. The reaction was stopped by
centrifuging cells for 30 sec at 8000 × g and washing
once with PBS. The washed pellets were resuspended in lysis buffer and
solubilized overnight at 4°C, followed by centrifugation for 30 min
at 10,000 × g to pellet the unsolubilized debris. To
resolve the cross-linked receptor subunits on a gel, a denaturing
SDS-polyacrylamide borate buffer system (Gething et al., 1989) was
used. Solubilized samples in a 87.3 mM borate, 87.3 mM sodium acetate, pH 8.5, 0.1% SDS buffer were loaded
onto a 3.5% polyacrylamide gel in the same buffer system.
Whole-cell electrophysiology. Undifferentiated PC12
cells were replated to collagen-coated glass coverslips 6 to 18 hr
before recording was begun. A single coverslip of cells was removed
from the culture media, set into a recording chamber, and perfused with
an external Na solution that contained (in mM): 135 NaCl, 2 KCl, 1 MgCl2, 5 CaCl2, 12 HEPES,
and 10 glucose, pH = 7.3 (osmolality ~295 mOsm). Electrodes were
coated with Sylgard, fire-polished, and filled with an internal
solution that contained (in mM): 120 CsAsp, 5 MgCl2, 0.1 EGTA, 40 HEPES, 2 ATP, and 0.3 GTP,
pH = 7.3 (osmolality ~310 mOsm). A single PC12 cell was
patch-clamped in the whole-cell configuration (Hamill et al., 1981)
with an Axopatch-1C amplifier (Axon Instruments, Foster City, CA) at a
holding potential of 80 mV. The data were collected at a 500 µsec
sampling rate and filtered at 2 kHz. For rapid application of nicotine
or antagonists, the outlet pipe from a fast perfusion system (Adams and
List, Westbury, NY) was situated directly adjacent (~250 µm) to the cell and rapidly switched (10-150 msec) between different external solutions. For application of nicotine, the perfusion system was primed
for 50 msec to clear the small dead volume ~20-30 sec before the
test application. Nicotine (50 µM; Sigma, St. Louis, MO)
was added to the Na solution (described above) or to an Na solution that also contained either -Bgt (0.5 µM) or
methyllycaconitine citrate (MLA, 1 µM). All experiments
were performed at room temperature (22-24°C). Statistical analysis
of the data are expressed as mean (×) ± SEM, and an independent
Student's t test was performed to test statistical
significance (p = 0.05).
RESULTS
Bgt binding sites on the surface of PC12 cells
In this study, we have characterized BgtRs found on the cell
surface of undifferentiated cells of the rat PC12 line variant N21
(Burry, 1993). Using 125I-Bgt to measure the number of
Bgt-sensitive AChRs (Fig. 1A), each culture expressed
0.45 pmol of Bgt binding sites or 40,000 surface 125I-Bgt
binding sites per cell, because a 6 cm culture contained on average
7 × 106 cells. Nonspecific binding was
determined by the addition of carbachol or unlabeled Bgt, both of which
blocked 125I-Bgt binding almost completely (Fig.
1A).
PC12 cells have been shown previously to express 3, 5, 7,
2, and 4 AChR subunits (Rogers et al., 1992; Boyd, 1994;
Henderson et al., 1994), which assemble into at least two AChR
subtypes. One AChR subtype is the BgtR (Patrick and Stallcup, 1977),
but the exact subunit composition remains unknown. The other subtype contains at minimum 3 subunits (Rogers et al., 1992) and 2
subunits (Whiting et al., 1987). The 32 subtype is expressed
poorly in undifferentiated PC12 cells; its expression is upregulated by treating PC12 cells with NGF (Whiting et al., 1987; Henderson et al.,
1994). In contrast, approximately equal numbers of BgtRs are expressed
in undifferentiated and NGF-treated PC12 cells (Whiting et al., 1987).
To minimize the expression of the 32 subtype, the PC12 cells were
cultured without NGF. BgtRs on the surface of these cells were
specifically immunoprecipitated by the 7-specific monoclonal
antibody (mAb) mAb 319 (Schoepfer et al., 1990) but were not recognized
by mAbs specific for the AChR subunits 3, 5, or 2 (Fig.
1B). Thus, PC12 BgtRs contain 7 subunits, but do
not appear to contain the 3, 5, or 2 AChR subunits. BgtRs have
been shown to contain 8 and 9 subunits (Schoepfer et al., 1990;
Elgoyhen et al., 1994). However, 8 subunits appear to be absent from
mammalian preparations, and 9 subunits do not form high-affinity
BgtRs (Elgoyhen et al., 1994) such as those found in the PC12 cells
(see below).
To determine the BgtR affinity for Bgt, we measured the kinetics of
125I-Bgt binding to intact PC12 cells. The time course of
125I-Bgt association and dissociation was determined at
4°C to minimize any effects of receptor turnover on these
measurements. Although very slow, both 125I-Bgt association
and dissociation are well fit by single exponentials (Fig.
1C,D) providing estimates for the association and
dissociation rates of 2.5 ± 0.4 × 104
M1·sec1 and 2.3 ± 0.1 × 106·sec1,
respectively, and a dissociation constant
(Kd) of 94 pM. The results
are summarized in Table 1.
Heterologous expression of BgtR homomers
To determine the subunit composition of the PC12 cell BgtRs, we
attempted to compare them with BgtRs of known subunit composition expressed in a similar environment. Specifically, 7 subunits were
transfected into cultured cell lines to express cell surface BgtRs.
Unfortunately, our efforts met with limited success. Figure 2A graphs the number of
Bgt binding sites found on the cell surface in an experiment in which
chicken, rat, and human 7 cDNAs, cloned into appropriate expression
vectors, were transfected transiently into tsA 201 cells. Transfection
of the chicken and human 7 subunit reproducibly yielded Bgt sites
only two- to three-fold higher than background, whereas the rat 7
subunit transfection was indistinguishable from background. In other
experiments, 7 cDNAs were subcloned into several different
expression vectors and were transfected, either transiently or stably,
into a variety of different cell lines including National Institutes of
Health 3T3 cells, mouse L cells, QT-6 cells, and HEK 293 cells. Again,
no significant expression was observed (data not shown). The lack of
rat 7 expression cannot be explained by an error within the open
reading frame (ORF), because this subunit, when expressed by in
vitro translation techniques, resulted in a product of appropriate
size. The translated subunit was smaller, ~48 kDa, when the
35S-methionine-labeled rat 7 subunit was translated in
the absence (Fig. 2B, lane A) of
microsomes in comparison with the larger, ~54 kDa subunit obtained in
the presence of microsomes (Fig. 2B, lane
B). The larger size of the subunit translated in the presence of
microsomes indicates that the subunit is N-linked
glycosylated.
Fig. 2.
The failure to express 7 subunit homomers.
A, tsA201 cells were transfected with 5 µg of chick,
rat, or human 7 cDNAs or the 7/5HT3 chimeric cDNA, all in
cytomegalovirus-based vectors. Two days after transfection, cells were
assayed for cell-surface 125I-Bgt binding. Only cells
transfected with the 7/5HT3 construct showed a sizable increase in
cell-surface BgtR expression. Nonspecific 125I-Bgt binding
was measured by 125I-Bgt binding to sham transfected tsA201
cells. B, Rat 7 cDNA was translated in
vitro with 35S-methionine and labeled in the
absence (lane A) or presence (lane B) of
canine pancreatic microsomes and analyzed on 10% SDS-PAGE.
[View Larger Version of this Image (27K GIF file)]
In marked contrast to the low level of expression obtained by
transfecting native 7 subunits, BgtR expression levels 100-fold larger were observed after transiently transfecting an 7/5HT3 chimeric subunit cDNA (Fig. 2A) consisting of the
N-terminal half of the chicken 7 subunit and the C-terminal half of
the mouse 5HT3 receptor subunit (Eisele et al., 1993). Previous studies of the 7/5HT3 chimera indicate that this BgtR displays pharmacologic properties similar to those of either native BgtRs or to BgtRs resulting from the expression of 7 subunits in Xenopus
oocytes (Eisele et al., 1993; Corringer et al., 1995). We used the
7/5HT3 BgtR homomer in our study to compare the properties of PC12
BgtRs with a BgtR of known subunit composition. A tsA201-derived cell line was established that stably expresses 7/5HT3 homomers.
Confluent cultures, which contained on average 3 × 106 cells per culture, expressed 2.5 pmol of surface
Bgt binding sites under saturating conditions (Fig.
3A) corresponding to 500,000 surface 125I-Bgt binding sites per cell.
The affinity of the 7/5HT3 homomer for Bgt was determined by
measuring the kinetics of 125I-Bgt binding to intact cells.
The estimated association and dissociation rates are 3.9 ± 1.1 × 104
M1·sec1 and 2.8 ± 0.4 × 106·sec1,
respectively (Fig. 3B,C), with a
Kd of 74 pM. The results are summarized in Table 1. The affinity of the 7/5HT3 homomer for Bgt is
almost identical to that of the native PC12 BgtR.
PC12 BgtRs and 7/5HT3 homomers are similar in size
Two different experiments were performed to determine structural
features of the PC12 BgtRs and the 7/5HT3 homomers. First, the
cell-surface, 125I-Bgt-bound receptors were solubilized and
subjected to density gradient sedimentation. Both receptors migrate as
single peaks on the sucrose gradients, with the PC12 cell BgtRs
sedimenting at 10 S and the 7/5HT3 homomers at 9 S (Fig.
4A). The sedimentation of 7/5HT3 homomers was indistinguishable from that for cell-surface Torpedo AChRs, which sediment in a 9 S peak and were run in
a parallel gradient as a standard. The data indicate that the 7/5HT3 homomers are pentamers, approximately the same size as the
Torpedo AChRs, which have an estimated molecular weight of
~270 kDa (Popot and Changeux, 1984). The molecular weight of an
7/5HT3 subunit pentamer is virtually identical, 269 kDa, as
estimated from the 7/5HT3 subunit ORF (53.8 kDa).
Fig. 4.
The size of PC12 and 7/5HT3 BgtRs.
A, Sucrose gradient sedimentation profiles of
cell-surface PC12 (open circles) and 7/5HT3 (open squares) BgtRs. Solubilized cell-surface receptors
bound with 125I-Bgt were subjected to sucrose gradient
sedimentation (5-20%). Both receptors migrate as single peaks, 10 S
for the PC12 BgtRs and 9 S for the 7/5HT3 BgtRs. The amount of
125I-Bgt binding was normalized by dividing by the cpm in
the peak fraction, 10,387 cpm for the PC12 BgtRs (fraction 12) and
33,269 cpm for the 7/5HT3 BgtRs (fraction 11). The fraction of the
maximum 125I-Bgt binding (left
y-axis) for the two receptors is plotted for each gradient fraction. Also plotted are the S values (right
y-axis) of the peak fractions
(x-axis) of each of the three standards (solid
diamonds): alkaline phosphatase (5.4S),
cell-surface Torpedo AChRs (9S), and
catalase (11S), which were run on gradients in parallel
with the BgtRs. The peak fraction S values were
determined from the plotted linear regression line through the
S values of standards. B, Cell-surface
PC12 and 7/5HT3 BgtR subunits cross-linked together and to
125I-Bgt. Cell-surface receptors bound with
125I-Bgt were cross-linked by DTSSP so that both receptor
subunits and 125I-Bgt were covalently linked. Cross-linked
complexes were run on SDS-PAGE in a borate-acetate buffer system.
Standards were cross-linked complexes of hemocyanine: monomer, 70 kDa;
dimer, 140 kDa; trimer, 210 kDa; tetramer, 280 kDa. Relative to the
standards shown, the estimated molecular weight of the PC12 BgtR is 300 kDa and of the 7/5HT3 BgtR is 260 kDa.
[View Larger Version of this Image (17K GIF file)]
Second, the molecular weights of the PC12 BgtR and the 7/5HT3
homomer were estimated. Cell-surface receptor subunits were cross-linked together and with 125I-Bgt using 3,3-DTSSP.
The cross-linked complexes were run on a 3.5% borate-acetate gel to
resolve the high molecular weight complexes (Fig.
4B). At saturating concentrations of DTSSP, single bands were observed on the gel for both 125I-Bgt
cross-linked receptors. The cross-linking was very efficient, as shown
by the small amount of unincorporated 125I-Bgt (8 kDa) in
the gel dye front relative to cross-linked 125I-Bgt. The
observation that PC12 BgtRs produced a single band on the gel when
labeled and cross-linked provides good evidence for a single
homogeneous population of receptors. Based on the size of the standards
(Fig. 4B), the PC12 BgtR has a molecular weight of
~300 kDa and the 7/5HT3 homomer a molecular weight of ~260 kDa,
in agreement with the density gradient sedimentation measurements.
Figure 4B provides additional evidence that the 7/5HT3 homomer exists in pentameric form, because the measured molecular weight is similar to the estimated molecular weight of the
7/5HT3 pentamer (269 kDa). If the PC12 BgtR is also a homomeric
pentamer, its calculated molecular weight predicts a subunit in the
range of ~60 kDa, which is similar to previous estimates of the
molecular weight of mature 7 subunits (Schoepfer et al., 1990; Anand
et al., 1993; Gotti et al., 1994). The molecular weight of the PC12
BgtR is consistent with it being an 7 homomer, but does not exclude
the possibility that it contains other subunits.
PC12 BgtR current response to nicotine
To test whether the PC12 BgtRs are functional, the whole-cell
current response to nicotine was measured. Rapid application of
nicotine to undifferentiated PC12 cells elicited a current that
activated rapidly and then desensitized (Fig.
5). Figure 5A shows the
current response observed during a 10 sec application of nicotine (50 µM) to a cell that was voltage-cell-clamped at 80 mV.
The rate of desensitization was approximately comparable with that
observed for rat BgtRs in cultured hippocampal neurons (Alkondon and
Albuquerque, 1993), but was much slower than for chick ciliary ganglion
BgtRs (Zhang et al., 1994). Two BgtR-specific antagonists, Bgt and MLA
(Ward et al., 1990; Alkondon et al., 1992), were used to block the
whole-cell currents to determine whether the currents observed resulted
from the activation of BgtRs. Bgt blocked most of the nicotine-induced
current response (Fig. 5B). The mean block of the
nicotine-induced current response by 500 nM Bgt was 91%
(Fig. 5C) and was never observed to reverse, which is
consistent with the slow dissociation rate measured by 125I-Bgt binding (Fig. 1D). Although Bgt
binds to PC12 cells with a very high affinity (94 pM), the
association rate is extremely slow (Fig. 1C). Based on our
measurement of the association rate from the
125I-Bgt-binding measurements (Table 1), inhibition of the
current response by 500 nM Bgt should take several minutes.
The time-dependent decrease of the current response after the
application of 500 nM Bgt is consistent with this
prediction (Fig. 5D). The Bgt association rate constant
calculated from the exponential decay of the current response after
application of Bgt is 0.7 × 104
M1 sec1. That the rate
constant is somewhat slower than that obtained from binding
measurements may be attributable to competition with the nicotine used
to elicit the response (Fig. 6). MLA also
blocked most of the nicotine-induced current response (88%), and its
block was at least partially reversible (Fig.
5E,F). The block by both Bgt
and MLA demonstrates that the PC12 BgtRs are functional channels and
mediate most of the nicotine-induced current response in these undifferentiated cells.
Fig. 5.
Whole-cell currents elicited by
nicotine application in undifferentiated PC12 cells. A,
The current is shown in response to rapid application of 50 µM nicotine, which was applied at the arrow and continued for 10 sec. The cell was
voltage-clamped at a holding potential of 80 mV. B,
The current response to 50 µM nicotine before and after
application of 500 nM Bgt for 12 min is shown. Nicotine was
applied for 200 msec in each trace. C, The fraction of
peak current, elicited by nicotine, is plotted, which remains after
application of 500 nM Bgt for 10-20 min. On average, the
peak current was reduced to 8.8 ± 1.2% of the current measured
in the absence of Bgt (Control) for 5 cells.
D, The fraction of peak current that remains after
application of 500 nM Bgt as a function of time is plotted.
Currents were normalized in amplitude to the first application of
nicotine (inset currents). The dotted line represents the fit of a single exponential function to the data with a time constant of 5 min. E, The current
response to 50 µM nicotine before and after application
of 1 µM MLA for 5 min is shown. Nicotine was applied for
200 msec in each trace. There was partial recovery of the current
response after 7 min of wash. F, The fraction of peak
current that remains after application of 1 µM MLA for 5 to 7 min is plotted. The peak current was reduced to 12.3 ± 1.1%
of the current measured in the absence of MLA
(Control) for four cells. The peak current
reverses to 30.8% of the control current after 7-10 min of wash after
the MLA application.
[View Larger Version of this Image (20K GIF file)]
Fig. 6.
Inhibition of cell-surface Bgt binding by
different ligands. A, Inhibition of the initial rate of
125I-Bgt binding to cell-surface PC12 BgtRs by the agonists
ACh (solid circle), carbachol (solid
square), nicotine (solid diamond), cytisine (solid triangle), and the antagonists MLA (open
triangle) and dTC (open square). Plotted in
A and B is the fraction of maximum 125I-Bgt bound, i.e., 125I-Bgt bound in the
presence of ligand divided by 125I-Bgt bound in the absence
of ligand, as a function of ligand concentration. The
line through the points represents a least-squares fit
to the data of the equation: fraction of maximum 125I-Bgt
bound = (Max.)/(1 + ([ligand]/IC50))n, where n
is the Hill coefficient and Max. is the maximum fitted
value. The results for all experiments are summarized in Table 2.
B, Inhibition of the initial rate of
125I-Bgt binding to cell-surface 7/5HT3 homomers by the
agonists ACh (solid circle), carbachol (solid
square), nicotine (solid diamond), cytisine
(solid triangle), and the antagonists MLA (open triangle) and dTC (open square).
[View Larger Version of this Image (38K GIF file)]
Agonist binding to BgtRs indicates three or more sites
per BgtR
To explore more fully the pharmacologic properties of the BgtRs,
we characterized the competition between different cholinergic agonists
and 125I-Bgt binding by measuring the inhibition of the
initial rate of 125I-Bgt binding to PC12 BgtRs (Fig.
6A) and 7/5HT3 homomers (Fig. 6B). Based on the measured IC50 values
(Table 2), the order of relative
affinities of the PC12 BgtR for the different agonists is nicotine > cytisine > carbachol > ACh. The agonist rank order for
the 7/5HT3 homomers, nicotine > cytisine > ACh > carbachol, was different than that for the PC12 BgtRs. In particular,
the IC50 value for carbachol was 30-fold smaller for PC12
BgtRs than for 7/5HT3 homomers.
Hill coefficients provide a measure of the degree of cooperativity
between different agonist binding sites. Surprisingly, the BgtR found
in PC12 cells exhibited Hill coefficients for the agonist ligands
tested between 2.3 and 2.4 (Table 2). The 7/5HT3 homomer Hill
coefficients were similar and fell in the range of 2.1 to 2.6 (Table
2). When the same protocol was performed on cell-surface muscle-type
AChRs, which have two ligand binding sites, the Hill coefficient for
carbachol was 1.5 (Table 2), similar to previous measurements (Claudio
et al., 1987; Sine and Claudio, 1991). A Hill coefficient >2 indicates
that both PC12 BgtRs and 7/5HT3 homomers have at least three agonist
binding sites per receptor.
To characterize further the number of agonist binding sites per
receptor, we searched for probes that could distinguish among the
different BgtR sites. Previously, antagonists have been used to
distinguish among AChR ligand binding sites. In particular, curare
binds to the two muscle-type AChR sites with an ~100-fold difference
in affinity (Neubig and Cohen, 1979; Sine and Taylor, 1981). Using the
same protocol described above for the agonists, the competition between
D-tubocurarine (dTC) and 125I-Bgt was measured.
dTC binds to the different sites on both receptors with the same
affinity, because inhibition by dTC of the 125I-Bgt binding
is well fit to a binding isotherm with a Hill coefficient of 1 (Fig.
6; Table 2). Similar results were
obtained for the inhibition of 125I-Bgt binding to
cell-surface PC12 cell BgtRs and 7/5HT3 homomers by MLA (Fig. 6;
Table 2). Based on these results, we conclude that the antagonists bind
to the different sites on the PC12 cell BgtRs and 7/5HT3 homomers
with the same affinity.
Fig. 7.
BromoACh alkylation of PC12 BgtRs and 7/5HT3
homomers. A, BromoACh alkylation of PC12 BgtRs. Before
cell-surface 125I-Bgt binding, PC12 cells were first
treated with 0.5 mM DTT followed by the indicated
concentration of bromoACh. The fraction of maximum 125I-Bgt
bound as a function of bromoACh concentration is plotted in the figure.
Shown are the data from five separate experiments in which each
data point is the mean of two determinations. The data
could not be fit by a single-site binding isotherm. The
line through the points represents a least-squares fit
to the equation: fraction of maximum 125I-Bgt bound = A(1/(1 + ([bromoACh]/IC501)) + B(1/(1 + ([bromoACh]/IC502)), where
IC501 and IC502 are IC50 values for
two separate binding sites, and A and B
are the fraction of total sites for each of the two sites. From the fit
to the data, 80% of the surface toxin binding sites are alkylated by
increasing concentrations of bromoACh with an IC50 of
3.5 × 106 M. Twenty percent of the sites are
relatively insensitive to reduction and alkylation. B,
BromoACh alkylation of 7/5HT3 homomers. Cells expressing 7/5HT3
homomers were treated as in A to alkylate the 7/5HT3
homomers with bromoACh. The fraction of maximum 125I-Bgt
bound as a function of bromoACh concentration is plotted in the figure.
Data from three separate experiments are shown in which each
data point is the mean of two determinations. Like the
PC12 cells, the data could not be fit by a single-site binding isotherm. The line through the points represents a
least-squares fit to the same equation as in A. From the
fit to the data, 75% of the 125I-Bgt binding sites were
alkylated by bromoACh with an IC50 of 2.5 × 105 M, whereas 25% were insensitive.
C, BromoACh alkylation of muscle-type AChRs. Cells
stably expressing the 122 AChR subtype were
treated as in A and B to alkylate the
AChR with bromoACh. The fraction of maximum 125I-Bgt bound
as a function of bromoACh concentration is plotted in the figure. Each
data point is the mean of two determinations. Unlike
PC12 BgtRs and 7/5HT3 homomers, all muscle AChR Bgt binding sites
were equally sensitive to bromoACh alkylation. The data were well fit
by the equation: fraction of maximum 125I-Bgt bound = Max.(1/(1 + ([bromoACh]/IC50)) with
a fitted IC50 value of 3.1 × 108
M. D, The DTT dependence of bromoACh alkylation. For
both the PC12 cells and the cells expressing muscle-type AChRs, the
concentration of DTT was varied over the indicated concentration range
before the bromoACh alkylation and 125I-Bgt binding assay.
After incubation in the indicated DTT concentration, the PC12 cells
were incubated with 300 µM bromoACh and the cells expressing muscle-type AChRs with 10 µM. The fraction of
maximum 125I-Bgt bound as a function of DTT concentration
is plotted in the figure. Each data point is the mean of
two determinations.
[View Larger Version of this Image (33K GIF file)]
PC12 and homomeric BgtR agonist binding sites
are distinguishable
The two agonist binding sites on the Torpedo AChR were
shown to be distinguishable by reduction of the receptor and specific alkylation of free sulfhydryl at the agonist binding site with an
agonist such as bromoACh (Damle et al., 1978; Moore and Raftery, 1979;
Wolosin et al., 1980). To test whether agonist binding sites on PC12
BgtRs and 7/5HT3 homomers could be distinguished, we reduced and
alkylated BgtR agonist binding sites. PC12 BgtRs were reduced with 0.5 mM DTT and then exposed to increasing concentrations of
bromoACh. Only ~80% of the 125I-Bgt binding sites could
be alkylated and blocked by bromoACh with an IC50 of
3.5 × 106 M, whereas 20% of the sites
retained the ability to bind 125I-Bgt (Fig. 7A).
Approximately 75% of the 125I-Bgt sites on 7/5HT3
homomers were alkylated and blocked by bromoACh with an
IC50 of 2.5 × 105 M, whereas
25% of the sites retained the ability to bind 125I-Bgt
(Fig. 7B). Thus, thiol modification of PC12 cell BgtRs and 7/5HT3 homomers reveals two classes of agonist binding sites on both
receptors. If there were only two agonist sites per BgtR, there should
be a 1:1 ratio of alkylated to unalkylated sites, as observed for the
Torpedo AChR. We find a ratio of 4:1 for the PC12 BgtR and
3:1 for the 7/5HT3 homomer, which provides additional evidence that
there are at least three agonist sites per receptor and is more
consistent with four or five agonist sites per receptor.
Because the population of 125I-Bgt sites that retained the
ability to bind 125I-Bgt after exposure to bromoACh was
relatively small, additional experiments were performed to test this
result. In contrast to Torpedo AChRs, both agonist sites on
skeletal muscle AChRs can be alkylated by agonists after reduction
(Froehner et al., 1977). Using an identical protocol with mouse muscle
121 AChRs expressed in a mouse fibroblast cell
line, we found that both sites on these AChRs could be alkylated, i.e.,
~100% of the 125I-Bgt sites were alkylated and blocked
by bromoACh [IC50 of 3.1 × 108
M (Fig. 7C)]. This result indicates that the unalkylated
agonist sites on the PC12 and 7/5HT3 cells were not artifacts of our protocol attributable to such potential problems as the reoxidation of
the disulfide bond after removal of the DTT. Another concern was that
the concentration of DTT was insufficient to reduce all of the PC12
cell BgtR sites. This possibility was tested by determining the
fraction of sites alkylated by bromoACh after reduction with varying
DTT concentrations (Fig. 7D). For the PC12 BgtRs, ~20% of
the agonist sites could not be alkylated by bromoACh, even at DTT
concentrations as high as 10 mM. For the mouse AChRs,
virtually all sites were alkylated by bromoACh at 0.3 mM.
DISCUSSION
Characteristics of PC12 BgtRs
In this study, we have characterized cell-surface BgtRs from the
PC12 cell variant N21 (Burry, 1993). Most of the previous characterizations of neuronal BgtRs used brain preparations,
principally chick brain preparation. Although these preparations are
rich sources of BgtRs, they express at least two BgtR subtypes
(Schoepfer et al., 1990; Gotti et al., 1992, 1994). Furthermore in
these earlier studies, cells were solubilized with detergent, and the preparation contained both intracellular and cell-surface BgtRs. The
study of BgtRs in the N21-PC12 cell line avoids these problems. The
PC12 cell line appears to express a homogeneous class of BgtRs, which
enabled us to perform our studies on intact living cells with the
receptors in their native conformation. Thus, the pharmacologic measurements, the 125I-Bgt and bromoACh cross-linking
experiments, and the electrophysiologic measurements were all performed
on cell-surface receptors from the same PC12 cell line.
The conclusion that the PC12 cells express a single BgtR subtype is
based on several observations. The best evidence was provided by the
experiments in which cross-linking of the cell-surface BgtRs resulted
in a single band on a gel (Fig. 4B), the size of which corresponded well with the measured sedimentation coefficient of
the receptor (Fig. 4A). Additional evidence for a
single subtype came from competition experiments between different
cholinergic ligands and Bgt for the receptor (Fig. 6). These
experiments were consistent with a single set of sites and, therefore,
a single BgtR subtype.
Characterization of PC12 cell BgtRs demonstrates that these receptors
are functional nicotinic AChRs, containing 7 subunits, similar to
chick BgtRs from ciliary ganglion cells (Vernallis et al., 1993; Zhang
et al., 1994) and human BgtRs from Imr32 neuroblastoma cells (Gotti et
al., 1995). The whole-cell response to nicotine is consistent with a
single BgtR subtype, because the current is blocked almost completely
by BgtR antagonists. The 10% of the current elicited by nicotine that
was not blocked by either Bgt or MLA may represent the 32 AChR
subtype, which is expressed at low levels in undifferentiated PC12
cells (Whiting et al., 1987; Henderson et al., 1994), but see Rogers et
al. (1992) for an exception.
Failure to express 7 homomers
Expression of 7 subunits in Xenopus oocytes results
in the assembly and cell-surface delivery of functional BgtRs
(Couturier et al., 1990; Seguela et al., 1993; Peng et al., 1994). As
assayed by 125I-Bgt binding, transfection of 7 cDNAs
from three different species resulted in little to no cell-surface
expression of BgtRs (Fig. 2A). One possible
explanation for our failure to express significant amounts of
7-containing BgtRs is that the cell lines lacked one or more
subunits needed for proper assembly in the more stringent mammalian
expression system. Recent studies that reported successful expression
of 7 subunits are at odds with this possibility. In two studies, the
7 cDNA was transfected into a human neuroblastoma cell line or PC12
cells, resulting in a sizable increase in the number of cell-surface
BgtRs (Puchacz et al., 1994; Cooper and Millar, 1997). Although it is
possible that the cells express other BgtR subunits, only a single
subunit band corresponding to the 7 subunit was observed on SDS-PAGE
gels, indicating that most BgtRs were 7 homomers. It seems more
likely that PC12 and neuroblastoma cells have additional elements
needed to properly process 7 subunits into homomers. In another
study, 7 homomers were expressed when human 7 subunits were
stably expressed in HEK 293 cells (Gopalakrishnan et al., 1995). The
difference between that study and our attempts to express human 7
subunits is that they screened and selected among a number of stably
transfected clonally isolated cells. Our poor expression levels can be
explained if only a few of the cells properly processed 7 subunits
into homomers.
The agonist binding sites
Marked differences were observed in the apparent affinities of
different agonist for PC12 cell BgtRs and 7/5HT3 homomers (Table 2).
Given the difference in the amino acid sequences of the rat 7
subunit and the chicken 7/mouse 5HT3 chimeric subunit, this result
is not surprising even assuming that the PC12 BgtR is an 7 subunit
homomer. What is surprising was the finding that the Hill coefficients
fall in the range of 2.1-2.6. The data indicate that PC12 cell BgtRs
and 7/5HT3 homomers contain the same number of agonist binding sites
and that the number of sites is at least three. Changes in the Hill
coefficient can track the number of ligand binding sites accurately.
Glycine receptors composed of only the ligand binding subunits had
a Hill coefficient of 4.2, consistent with a pentameric structure and
five subunits (Bormann et al., 1993). Different glycine receptors,
composed of ligand binding and structural subunits, had a Hill
coefficient of 2.5, consistent with three ligand binding sites and the
presumed stoichiometry of 32 (Langosch et al., 1988). We would
expect similar differences in the Hill coefficient of PC12 cell BgtRs and 7/5HT3 homomers if they had a different number of ligand binding
subunits.
Additional evidence about the number of agonist binding sites on PC12
cell BgtRs and 7/5HT3 homomers was obtained by alkylation of the
receptors with bromoACh. BromoACh was unable to alkylate all of the
sites on each receptor: the ratio of alkylated to unalkylated sites was
4:1 for the PC12 cell BgtR and 3:1 for the 7/5HT3 homomer. These
data are consistent with four or five agonist sites per receptor. Our
results with the 7/5HT3 homomer are also consistent with
electrophysiologic data indicating that 7 homomers expressed in
Xenopus oocytes have five MLA binding sites (Palma et al., 1996).
Previous work on Torpedo membrane preparations has
characterized in detail the alkylation of the AChR binding sites by
different ligands. The residues alkylated by the affinity labeling were identified as adjacent disulfide-linked cysteine residues Cys 192 and
193 (Kao et al., 1984; Kao and Karlin, 1986). An adjacent pair of
cysteines, homologous to those on Torpedo 1 subunits, is
found on the 7 subunits and are presumably the residues on the PC12
cell BgtRs and 7/5HT3 homomers that are labeled by bromoACh. Additional evidence indicating that the BgtR alkylated sites are similar to that of the muscle-type AChR is that the DTT dependence of
the alkylation is almost identical for the two receptor types (Fig.
7D).
In this paper, we have demonstrated that the PC12 cell BgtR contains
7 subunits (Fig. 1B), sediments at 10S on sucrose
gradients (Fig. 4A) and has a molecular weight of
~300 kDa (Fig. 4B). These results together with our
data showing that the PC12 cell BgtR contains at least three, and more
likely four or five, ligand binding sites suggest that the PC12 BgtR is
a pentamer of 7 subunits. This picture of neuronal BgtRs contradicts
a number of studies in which BgtRs, purified primarily from chicken
preparations, appear to contain two to four different polypeptide
subunits (Conti et al., 1985; Kemp et al., 1985; Whiting and Lindstrom,
1987; Hermans-Borgmeyer et al., 1988; Schoepfer et al., 1990; Gotti et
al., 1994). Several possibilities may explain the differences. First,
BgtRs in the chicken brain are heterogenous (Schoepfer et al., 1990;
Gotti et al., 1994). The chick brain expresses the 8 subunit, which
is highly homologous to the 7 subunit isoform but has a higher
molecular weight (Schoepfer et al., 1990). 8 subunits can assemble
into BgtRs with or without 7 subunits in a tissue-specific manner
(Keyser et al., 1993). Thus, one simple explanation of multiple subunit
bands found in chick brain may be the presence of multiple BgtR
subtypes in the preparation. Second, the N terminus of a molecular band
smaller than the mature 7 subunit was shown to be identical to the N
terminus of the 7 subunit (Conti et al., 1985). This raises the
possibility that the BgtR preparations from brain were contaminated by
proteolytic fragments of the 7 subunit. Finally, partially processed
as well as fully processed 7 subunits, i.e., 7 subunits of
different molecular weights, may have been purified from the
solubilized brain preparation, because there was no effort to isolate
cell-surface from intracellular BgtRs. Evidence that the 7 subunit
is processed post-translationally is that it has three potential
glycosylation sites and that it binds to both concanavalin A (Gotti et
al., 1992) and wheat germ agglutinin (Hermans-Borgmeyer et al., 1988). Therefore, previous data characterizing the subunit composition of
BgtRs do not exclude the possibility that the BgtR is a pentamer of
7 subunits.
How does a homomeric receptor form distinguishable agonist
binding sites?
Our finding that bromoACh was unable to alkylate all of the BgtR
binding sites indicates that the agonist binding sites are structurally
different. Similar results on muscle-type AChRs has led to the
conclusion that the differences observed for the two agonist binding
sites on these receptors arise because the two 1 subunits interface
with different subunits, either the or subunits (Blount and
Merlie, 1989; Pedersen and Cohen, 1990; Sine and Claudio, 1991).
Clearly, a different mechanism must cause any structural differences in
the agonist binding sites of the 7/5HT3 homomer, because all five
subunits are identical. One possibility is that a subset of 7/5HT3
subunits undergoes different post-translational processing. Possible
post-translational modification of these subunits includes N-linked
glycosylation, disulfide bond formation, proline isomerization, and
phosphorylation. Any differential processing of the subunits could
produce two classes of 7/5HT3 subunits, which in turn could lead to
distinguishable ligand binding sites. Alternatively, two classes of
7/5HT3 subunits could be created because of differences in subunit
folding. If the biogenesis of 7 subunits is similar to that of 1
subunits, then the Bgt and agonist binding sites form after their
synthesis (Merlie and Lindstrom, 1983) and during assembly of the
pentamer (Green and Claudio, 1993). Perhaps these sites are forming on
only some of the 7 subunits, and both ligand binding and structural
subunits could be generated from the same gene product.
FOOTNOTES
Received May 30, 1997; revised Aug. 13, 1997; accepted Aug. 21, 1997.
This work was supported by grants from National Institutes of Health
(W.N.G., A.P.F.) and Brain Research Foundation (W.N.G.), a Council for
Tobacco Research Scholar Award (W.N.G.), and an American Heart
Association (Chicago, IL) Senior Fellowship (S.R.). We thank Dr.
Richard Burry for the PC12 N21 cell line; Drs. J.-L. Eisele, J. Boulter, and J. Lindstrom for the 7 and 7/5HT3 cDNAs; and J. Lindstrom for MAb 319. MAb 35 and 270, also developed by J. Lindstrom,
were obtained from the Developmental Studies Bank (Johns Hopkins
University and University of Iowa). We also thank Christian Wanamaker
for help with some of the experiments.
Correspondence should be addressed to Dr. Green, 947 East 58th Street,
Chicago, IL 60637.
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Compound via MeSH
