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Vol. 56, Issue 1, 11-19, July 1999
6-Containing Nicotinic Receptors Are Present in
Chick Retina
Consiglio Nazionale delle Ricerche (CNR) Cellular and Molecular Pharmacology Center, Department of Medical Pharmacology, University of Milan, Milan, Italy (S.V., A.B., Ba.B., M.M., F.C., C.G.); Institute of Zoophysiology, Hohenheim University, Stuttgart, Germany (W.H.); Department of Experimental Medicine and Pathology, Università di Roma "La Sapienza," Rome, Italy (Be.B.); and CNR Center of Hormone Chemistry, Milan, Italy (R.L.)
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Summary |
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 Top
 Summary
 Introduction
Materials and Methods
Results
Discussion
References
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Despite the fact that the neuronal chick 
6 subunit was first cloned
several years ago and recently has been shown to form acetylcholine
(ACh)-activated channels in heterologous systems, no information is yet
available concerning the structure and function of the 6-containing
nicotinic receptors in neuronal tissues. Using subunit-specific
antibodies directed against two different epitopes of the chick 6
subunit, we performed immunoprecipitation experiments on immunopurified
6-containing receptors radiolabeled with the nicotinic agonist
[3H]epibatidine (Epi): almost all of the 6 receptors
contained the 
4 subunit, 51% the 3 subunit, 42% the 3
subunit, and 7.5% the 2 subunit. Western blot analyses of the
purified receptors confirmed the presence of the 3, 3, 2, and
4 subunits, and the absence of the 4, 5, and 7 subunits.
The 6-containing receptors bind [3H]Epi
(Kd = 35 pM) and a number of other
nicotinic agonists with very high affinity, the rank order being Epi
cytisine > nicotine > 1,1-dimethyl-4-phenylpiperazinium > acetylcholine > carbamylcholine. The 6 receptors also have a distinct antagonist
pharmacological profile with a rank order of potency of -conotoxin
MII > methyllycaconitine > dihydro--erythroydine > MG624 > d-tubocurarine > decamethonium > hexamethonium. When reconstituted in lipid bilayers, the
6-containing receptors form functional cationic channels with a main
conductance state of 48 pS. These channels are activated by nicotinic
agonists in a dose-dependent manner, and blocked by the nicotinic
antagonist d-tubocurarine.
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Introduction |
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Summary
Introduction
Materials and Methods
Results
Discussion
References
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Neuronal
nicotinic acetylcholine receptors (nAChRs) are a family of ligand-gated
ion channels that play a role in central and peripheral nervous systems
under both normal and pathological conditions (Dani and Heinemann,
1996
; Gotti et al., 1997a; Léna and Changeux, 1997;
Wonnacott, 1997). Eleven genes coding for the nAChR subunits
2 to 9 and 2 to 4 have so far been identified. Although
there are many subtypes consisting of different subunit combinations,
two main classes of nicotinic receptors can be identified on the basis
of their function and pharmacology: homomeric channels, that are all
blocked by the competitive antagonist -bungarotoxin (Bgtx), and heteromeric channels that are completely insensitive to
it (reviewed in Sargent, 1993; Role and Berg, 1996). Heterologous expression studies have demonstrated that homomeric channels can only
be formed by the 7, 8, and 9 subunits, whereas the heteromeric channels can be formed by the 2, 3, or 4 subunits combined with the 2 or 4 subunits, as well by the coexpression of 5 or
3 with another and subunit (other than the subunits forming homomeric channels) (McGehee and Role, 1995; Ramirez-Latorre et
al., 1996; Groot-Kormelink et al., 1998).
Although heterologously expressed nAChR subtypes have provided a lot of
information concerning the functional diversity of nAChR subtypes,
recent data have clearly shown that channels with different
pharmacological and biophysical properties can be obtained depending on
the expression system used (Fucile et al., 1997; Lewis et al., 1997),
and biochemical and immunological experiments in vertebrate brain and
ganglia have suggested that native nAChRs may be more complex than
previously thought (Conroy and Berg, 1995; Forsayeth and Kobrin, 1997).
Among the nAChR subunits cloned so far, 6 (Lamar et al., 1990) has
long been considered an "orphan" subunit, because it was impossible
to obtain any 6-containing functional channels. The amino acid
sequence of the chick 6 subunit is most closely related to that of
3 subunit (66.5% identity). In rat brain, 6 mRNA is selectively
concentrated in catecholaminergic nuclei where it is colocalized with
3 mRNA (Le Novère et al., 1996); in the chick nervous system,
6 mRNA is abundantly expressed in neuroretina (where it coexists
with 3, 2, and 4 mRNAs) but not in the optic tectum or
peripheral ganglia (Fucile et al., 1998).
It has only very recently been demonstrated that the chick 6 subunit
forms a functional channel when expressed with the human 4 subunit
in oocytes (Gerzanich et al., 1997) and can form functional receptors
in human BOSC 23 cells when it is coexpressed with either the
chick 2 or 4 subunits or with both the 4 and 3 subunits together (Fucile et al., 1998).
Because no information is available concerning the structure and
function of the 6-containing channels present in neuronal tissues,
we decided to immunopurify this receptor subtype from chick retina.
This article reports the purification and characterization of this
receptor, its subunit composition and pharmacology, and its functional
behavior after reconstitution in planar lipid bilayers.
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Materials and Methods |
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Summary
Introduction
Materials and Methods
Results
Discussion
References
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Antibody Production and Characterization
The monoclonal antibody (mAb) 270 raised against chicken brain
nAChR and directed against the 2 subunit (Whiting et al., 1987) was
a generous gift of Dr. J. Lindstrom, University of Pennsylvania, Philadelphia, PA. The mAb 35 raised against the muscle-type AChR recognizes the 1 subunit and cross-reacts with the 5 and 3 subunits (Conroy et al., 1992, 1998), was purified from hybridoma cell
line obtained from the American Type Culture Collection (Rockville, MD). The mAb 299, raised against rat brain nAChR and directed against
the 4 subunit (Whiting and Lindstrom, 1988), as well as the mAb 313 raised against the fusion protein containing the putative cytoplasmic
of the 3 subunit (Whiting et al., 1991), were both purchased from
Research Biochemicals Inc. (Natick, MA).
The polyclonal Abs (Abs) against the 3, 4, 5, 6, 7,
8, 2, and 4 peptides were raised as previously described
(Gotti et al., 1994); their peptide sequences are shown in Table
1. Two different peptides were chosen for
all of the subunits: one located in the cytoplasmic loop between M3 and
M4 (CYT), and the other located at the COOH terminal (COOH). The
antibodies raised against the peptides were purified on an affinity
column made by coupling the corresponding peptide to cyanogen
bromide-activated Sepharose 4B (Pharmacia Inc., Uppsala, Sweden)
according to the manufacturer's instructions.
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For the 3 protein, a polyclonal antiserum was raised in rabbit using
a fusion protein (the generous gift of Dr. Marc Ballivet, University of
Geneva, Geneva, Switzerland) located in the extracellular part
of the 3 subunit between S105 and I180.
The antipeptide serum titers were evaluated by means of enzyme-linked
immunoassay and Western blots of the purified subtypes (Gotti et al.,
1998; Maggi et al., 1999). The serum antibodies were specific
only for their respective immunizing peptide in enzyme-linked
immunoassay, and each immunoprecipitation and immunolabeling was
specifically inhibited only by the peptide used for the immunization.
In addition, to exclude cross-reactivity between the anti-3 and
anti-6 Abs, the anti-3 COOH, and anti-3 CYT polyclonal antibodies were also respectively passed through columns with bound
6 COOH or 6 CYT peptides, and the anti-6 COOH and
anti-6 CYT Abs through columns with bound 3 COOH or 3 CYT peptides.
6 Subtype Immunopurification
The chick optic lobe (COL) and retina were dissected from
1-day-old chicks, immediately frozen in liquid nitrogen, and then stored at 
80°C. The retina and COL extracts were prepared as previously described (Gotti et al., 1991, 1997b). To bind the 6-containing receptors, the retina extract was incubated three to
four times with 5 ml of Sepharose-4B bound to anti-6 Abs. The bound
receptors were eluted with 0.2 M glycine (pH 2.2) or 100 µM of the
corresponding peptide used for Ab production as previously described
(Gotti et al., 1991, 1997b).
Recovery was determined by means of both
[3H]epibatidine (Epi) binding and quantitative
immunoprecipitation of the receptors present in the solution before and
after each immunopurification step as previously described (Gotti et
al., 1994, 1997b).
Binding Assay and Pharmacological Experiments on Immunoimmobilized
6 Receptors
The affinity-purified anti-6 Abs were bound to microwells
(Maxi-Sorp; Nunc, Naperville, IL) by means of overnight incubation at
4°C at a concentration of 10 µg/ml in 50 mM phosphate buffer, pH
7.5. On the following day, the wells were washed to remove the excess
of unbound Abs, and then incubated overnight at 4°C with 200 µl of
2% Triton X-100 retina membrane extract containing 100 to 200 fmol of
[3H]Epi binding sites.
[3H]Epi Binding to Solubilized Receptor.
Like
Gerzanich et al. (1995), we found that [3H]Epi
binds with high affinity to the 2- and 4-containing subtypes
(picomolar affinity), but it also binds to the 7 subtypes with a low
nanomolar affinity and to the 8-containing receptor with picomolar
affinity. To ensure that the 7 and 8 subtypes did not contribute
to [3H]Epi binding during the 6 receptors
purification, all the binding and immunoprecipitation experiments were
performed in the presence of 2 µM Bgtx, which specifically binds
to the 7 and 8 subtypes and blocks
[3H]Epi binding.
Immunoimmobilized Subtype. The receptors immobilized by the corresponding subunit-specific Abs were incubated overnight at 20°C with 300 µl of [3H]Epi at concentrations ranging from 0.005 to 5 nM. All of the incubations were performed in a buffer containing 50 mM Tris-HCl, pH 7, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2.5 mM CaCl2, 2 mg/ml BSA, and 0.05% Tween 20. Specifically labeled ligand binding was defined as total binding minus the binding in the presence of 100 nM cold Epi. The inhibition of [3H]Epi binding to the immobilized subtypes induced by the cholinergic ligands was measured by preincubating the indicated concentrations of the compounds for 30 min at room temperature, followed by overnight incubation with 0.1 nM [3H]Epi.
After incubation, the wells were washed seven times with ice-cold PBS containing 0.05% Tween 20, and the bound radioactivity recovered by incubation with 200 µl of 2N NaOH for 2 h. The bound radioactivity was then determined by means of liquid scintillation counting in a beta counter for [3H]Epi.Data Analysis.
The experimental data obtained from the
saturation binding experiments performed in membrane or solubilized
receptors were analyzed by means of a nonlinear least square procedure
using the LIGAND program as described by Munson and Rodbard (1980). The
calculated binding parameters were obtained by simultaneously fitting
10 independent experiments.
) .
The Ki values of all of the tested drugs
were also determined by means of the LIGAND program using the data
obtained from three independent competition experiments and compared by
means of the F test as described above.
Expression of Chick 64 and 34 Subtypes in BOSC 23 Cells
Calcium phosphate-mediated transient tranfections of the
34 and 64 subtypes in the human BOSC23 cell line were
carried out as previously described by Ragozzino et al. (1997) and
Fucile et al. (1998).
The presence of the expressed subtypes was measured by means of
[3H]Epi saturation binding experiments to cell
homogenates as described in Gotti et al. (1998); the
Kd and Bmax
were 98 pM (CV = 14%) and 51 fmol/mg of protein for the 34
subtype, and 46 pM (CV = 13%) and 42 fmol/mg of protein for the
64 subtype.
Bilayer Formation and Subtype Insertion
The purified 6 subtype eluted from the corresponding
immunoaffinity columns were dialyzed, concentrated, and stored at
20°C until used.
The purified receptors were incorporated in asolecithin liposomes (Sigma Chemical Co., St. Louis, MO) by means of dialysis, and then fused with preformed bilayers as previously described. All of the point-amplitude histograms were constructed from 5-s current fluctuation traces digitized at a sampling rate of 2000 samples/s. The current-voltage curve was constructed from all of the histograms, and the channel conductance was calculated from the linear portion of the curve. In addition, the global open-state probability (Po) was calculated from the areas under the peaks of the histograms.
Preliminary experiments were performed by adding carbamylcholine (Carb) 1 mM to the trans or cis side of the bilayer to identify the orientation of the channels. In the reported experiments, the agonists dissolved in 150 mM NaCl and 5 mM Tris-HCl (at the concentrations given in Results) were applied to the side of the bilayer in which the channels have been correctly incorporated.
The 50% activation value (EC50) was calculated from the plot of Po versus [agonist] as the value of the agonist concentration necessary to obtain a level of activity midway between spontaneous activity and maximum Po.
Further details of the experimental procedures have been previously
described (Gotti et al., 1991, 1994, 1997).
Materials
Antiprotease inhibitors, asolecithin type IIS, cholinergic
ligands, Triton X-100, and anti-rabbit and anti-rat antisera were purchased from Sigma; nonradioactive Epi was purchased from RBI; cyanogen bromide-activated Sepharose 4BCL was purchased from
Pharmacia, Sweden; [(±)-3H]Epi,
125I-labeled Bgtx,
125I-labeled protein A, and
[3H]Epi were purchased from Amersham
(Buckinghamshire, UK); and the reagents for gel electrophoresis were
purchased from Bio-Rad Labs (Hercules, CA).
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Results |
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Top
Summary
Introduction
Materials and Methods
Results
Discussion
References
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Specificity of Antisubunit Antibodies Against nAChR Subtypes Present in COL and Retina. For each subunit, we raised polyclonal antibodies against a peptide located in the cytoplasmic loop, which is the most divergent region of the otherwise homologous nAChR subunits; to have an additional control for each subunit, we also raised Abs directed against a peptide located on the COOH terminal region (see Table 1).
Given the high degree of identity between the3 and 6 subunits
(66.5%), special care was taken to detect the specificity of the
anti-3 and anti-6 Abs. To this end, the antipeptide Abs were
tested on Western blots of human BOSC 23 cells tranfected with the
34 and 64 subtypes. Homogenates of 34 (Fig. 1, lane
1) and 64 (Fig. 1, lane 2) tranfected and/or untranfected BOSC 23 cells (Fig. 1, lane 3) were run on 7.5% SDS-polyacrylamide gel
electrophoresis and then electrotransferred to nitrocellulose and
tested with the anti-3 COOH (Fig. 1A),
anti-3 CYT (Fig. 1B), anti-6 COOH (Fig. 1C), and anti-6 CYT
(Fig. 1D). Both anti-3 Abs only recognized the tranfected 3 and
not the 6 subunits, and the anti-6 Abs only recognized the
tranfected 6 and not the 3 subunits.
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Bgtx to block the binding of [3H]Epi to the
7 and 8 subtypes, and then labeled with
[3H]Epi and immunoprecipitated by the
antibodies directed against the 3, 4, 5, 6, 2, 3, and
4 subunits. The Abs immunoprecipitated the receptors to a different
extent in the two tissues, but the percentage of immunoprecipitation
was very similar for the Abs directed against the same subunit and, in
the case of the 3, 4, 5, and 2 subunits, also similar to
that obtained using commercially available mAbs (Table
2).
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2 subunit, which is mainly associated with the 4 subunit but can also
be found with the 5 and/or the 3 subunits. In chick retina, the
majority of the [3H]Epi binding receptors
had the 4 subunit and a subpopulation also contains the 2
subunit, but they are very heterogeneous in terms of their subunit
expression. Both types of Abs directed against the 6 and 3
subunits recognized [3H]Epi- labeled receptors
in the retina to a similar and much greater extent than that determined
in the chick optic tectum. This finding is in line with previously
reported data that the 6 and 3 subunit mRNAs are detectable in
the retina and not in the optic tectum (Hernandez et al., 1995; Fucile
et al., 1998).
COL and retina also have receptors containing the 7, 8 and
7-8 subunits that bind 125I-labeled
Bgtx. To have a complete profile of these Abs, we tested them for
their ability to immunoprecipitate 125I-labeled
Bgtx receptors, but never detected any specific immunoprecipitation (data not shown).
Subunit Composition of Purified 6 Subtype.
Our
immunoprecipitation experiments suggested that retina would be a
suitable tissue from which to purify the 6-containing receptors
selectively. To this end, we thrice passed the retina extract on an
affinity column with bound anti-6 cytisine (Cyt) Abs and, after the
passage, monitored depletion by means of immunoprecipitation. Selective
immunodepletion of the 6-containing receptors was demonstrated by
the fact that their number decreased from 34 ± 4% (retina
extract) to 5 ± 3% in the final flow through of the
immunoaffinity column.
6 subtype, we
used immunoprecipitation to analyze the receptor eluted from the
affinity column by the corresponding 6 peptides (Table 3). The 4 Abs immunoprecipitated
almost all of the [3H]Epi- labeled
6-receptors, thus indicating that almost all of the 6 receptors
contain the 4 subunit; half were immunoprecipitated by anti-3
Abs, 42% by anti-3 Abs, and 7.5% by anti-2 Abs. Because the
anti-4 and 5 Abs were able to immunoprecipitate these receptors only to a very limited extent, we think that these subunits are not
coassembled with the 6 and 4 subunits.
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6 receptors was also
analyzed on Western blots using the same panel of Abs as that used for
the immunoprecipitation experiments (see Fig.
2). Both anti-6 Abs recognized a
peptide of molecular mass 57.1 ± 0.9 kDa (anti-6 COOH
lane 5 and anti-6 CYT lane 6), the anti-4 Abs recognized a single
band of molecular mass 52.3 ± 0.5 kDa (anti-4 COOH lane
11 and anti-4 CYT lane 12), the anti-2 COOH recognized a faint
band of 54 kDa (lanes 8), and the anti-3 recognized a peptide of
molecular mass 56.3 ± 0.3 kDa (anti-3 COOH lane 1 and
anti-3 CYT lane 2). The anti-3 Abs directed against the COOH
peptide did not recognize any peptide on the purified receptors, whereas the anti-3 CYT recognized a band of molecular mass 55 ± 1 kDa (lane 10) that was also recognized by a serum obtained from
rabbit immunized with the fusion protein containing the S105-I180 sequence of the extracellular 3 chick subunit (lane 9). We also tested the purified 6 receptors for the possible presence of 4
(lane 3), 5 (lane 4), and 7 (lane 7) but could not detect any
labeling using subunit specific Abs. This absence of recognition is due
to a lack of proteins for the 4 and 5 subunits since the same Abs
were able to recognize the appropriate subunits in receptors purified
from COL using anti-2 Abs, and the anti-7 Abs recognized the
appropriate peptide in receptors purified by affinity on
Sepharose-Bgtx (data not shown).
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6, 3, and 4 subunits, determined
by Western blot are given as the mean ± S.E.M. obtained from three to four experiments, and corresponded to the expected sizes deduced from their cDNA sequences. The Mr
of the 3 subunit was slightly higher, but this may be due to
glycosylation of the subunit because it has two potential glycosylation sites.
Pharmacological Experiments on 6 Subtype.
The
pharmacological experiments were all carried out on receptor
immobilized by the corresponding anti-6 Cyt-specific Abs as
described in Materials and Methods.
6 receptors bind [3H]Epi with high
affinity; the Kd value calculated from 10 separate experiments was 35 pM [percentage of coefficient of variation
(CV) = 18%].
Figure 3 shows a typical saturation curve
of the total and nonspecific binding of [3H]Epi
to the immunoimmobilized subtype. The interaction of
[3H]Epi with the 6-receptors was consistent
with the presence of a single class of high-affinity binding sites, and
the Scatchard plot of the saturation curve also shown in Fig. 3
indicates the presence of a single class of high- affinity sites in
these receptors.
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6 receptors was further
characterized by testing the relative efficacies by which various cholinergic agonists and antagonists inhibit the binding of 0.1 mM
[3H]Epi at equilibrium.
Figure 4 shows the inhibition curves of
cholinergic agonists (Fig. 4A) and antagonists (Fig. 4B) for the
binding of [3H]Epi to the immunoimmobilized
subtype. The Ki values of the inhibition curves shown in Table 4 were obtained by
simultaneously fitting the data from three to four separate
experiments.
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Cyt > nicotine (Nic) > 1,1-dimethyl-4-phenylpiperazinium (DMPP) > ACh > Carb, and
except for Carb, all of them had relatively low
Ki values (in the low nanomolar range). The
rank order of antagonist potencies was -conotoxin MII (MII) > methyllycaconitine (MLA) > dihydro--erythroidine (DHE) > MG624 > d-tubocurarine (d-TC) > decamethonium > hexamethonium. We found that the toxin MII, a
compound described as a antagonist of the rat 32 subtype, is the
most potent drug (Ki = 66 nM) in competing
for 6 receptors, and has an affinity that is respectively 20 and 40 times higher than that of MLA (Ki 1.35 µM) and DHE (Ki 2.8 µM).
To exclude possible interference by the immunoimmobiliating Abs on the
pharmacology of the 6 receptors, we also tested the binding of
[3H]Epi and ACh in receptors immunoimmobilized
on the anti-6 COOH Abs. The results were qualitatively the same with
a Kd of 30 pM (CV = 15%) for
[3H]Epi and a Ki of
80 nM (CV = 20%) for ACh.
Functional Reconstitution in Lipid Bilayers.
To study the
biophysical properties of the 6 receptors, the immunopurified
receptors were reconstituted in lipid bilayers and their properties
studied after agonist activation. In our experiments, traces with more
than one channels were rare and are disregarded in the analysis.
6 receptor
activated by 10 µM ACh. Figure 5B shows an amplitude histogram
derived from the recording shown in Fig. 5A and Fig. 5C the
distribution of the open- and closed-state lifetime evaluations. The
open-channel lifetime followed a single exponential distribution of the
channel with a mean open lifetime of 1.9 ms, whereas the closed time
followed a double exponential distribution with a mean closed
lifetime of 0.9 ms within bursts and 9 ms between bursts. This channel
had a conductance of about 50 picosiemens and, in addition to
the main open-state channel, also showed some other conductances,
although their frequency of occurrence was very low.
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6 receptors obtained from three separated immunopurifications.
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6 channels activated by nicotinic agonists were blocked by the
nicotinic antagonist d-TC at 100 µM.
Figure 7 shows the current-voltage
relationship for the 6 subtype, which can be fitted by a straight
line with a main conductance of 48 pS. The
Po of the channels was found to be voltage
dependent (Fig. 7B) and was tested in 10 separate experiments.
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Discussion |
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Top
Summary
Introduction
Materials and Methods
Results
Discussion
References
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The only previously available data about the 6 subunit were its
cloning, its mRNA distribution in rat brain and some areas of the chick
central nervous system, and the electrophysiological properties of 6
receptors heterologously expressed in oocytes and mammalian cells; no
data were available concerning the structure and function of native
6-containing receptors. However, it has recently been shown that the
functional and pharmacological properties of heterologous subtypes may
be influenced by the kind of cell expressing them, which means that the
results need to be interpreted very carefully (Fucile et al., 1997;
Lewis et al., 1997).
One approach to identifying the subunit composition of native receptors is to purify them using subunit-specific Abs. The reliability of the present work depends on the specificity of the Abs, which is why we raised them against two distinct epitopes of the same subunit and tested them all in immunoprecipitation experiments using detergent-solubilized receptors of two neuronal tissues that are known to have different nAChR subtype expressions.
Although there was a difference in the absolute percentages of
receptors immunoprecipitated by the anti-3 and anti-6 Abs in COL
and retina, the percentage of receptors immunoprecipitated by the Abs
were similar in the individual tissues (see Table 2). In order to
exclude any cross-reactivity between the anti-3 and anti-6 Abs,
the specificity of this Abs was confirmed on human cell lines
expressing the chick 34 or 64 subtypes.
Using these Abs, in agreement with other published data (Morris et al.,
1990; Whiting et al., 1991; Vernallis et al., 1993; Conroy et al.,
1995; Conroy and Berg, 1997), we found that almost all of the receptors
in chick brain contain the 4 and 2 subunits, and only a minority
contain the 5, 3 and 4 subunits.
Fucile et al. (1998) and Hernandez et al. (1995) reported that there is
a high level of 6 and 3 subunit mRNA in chick retina, whereas
these mRNAs are undetectable in the optic tectum. In agreement with
their results, we found that both COOH and CYT Abs were able to
immunoprecipitate a consistent amount of
[3H]Epi-labeled receptors in retina but not in
the chick optic tectum (less than 6%), and so we used the former for
the purification of the native receptor containing the 6 subunit.
Immunoprecipitation and Western blot analyses of the purified
6-containing receptors showed that almost all of them also contained
the 4 subunit, one-half contained the 3 subunit, 42% the 3
subunit, and 7.5% the 2 subunit. These results show that our
purified receptors are a mixture of different populations of
6-containing receptors (surely 64; probably 643,
364, and/or 3634; and possibly a minor
subpopulation containing the 2 subunit) but we do not know whether
the 3 subunit coexists with the 3 and/or 2 subunit. Binding
analyses of the immunoimmobilized 6 receptors showed the presence of
a single population of high-affinity [3H]Epi
binding sites, and the competition experiments with nicotinic agonists
and antagonists did not reveal any binding site heterogeneity. The
electrophysiological studies showed that the reconstituted receptors
form a channel with a main conductance state of 48 pS, with other
conductance levels being much less frequent. The homogeneity in the
pharmacological and biophysical properties of 6 receptors may be
surprising, but could be due to the fact that the presence of the 3
or/and 3 subunits does not modify their affinity for nicotinic drugs
but regulates some other channel properties and/or confers other
properties unrelated to channel function (e.g., localization), as has
been recently demonstrated for the intracellular loop of the 3
subunit (Williams et al., 1998). Another possibility is that the
affinities in the different subtypes are so close that we cannot
discriminate them by binding experiments. The fact that the presence of
the 3 subunits does not seem to change the affinity for nicotinic
ligands is in agreement with the data reported by
Groot-Kormelink et al. (1998), who found no detectable shift in
the ACh concentration-response curve in human 34 receptors expressed in oocytes alone or together with the 3 subunit. However, it seems that the presence of the 3 subunit (together with the 6
and 4 subunits) in human transfected cells leads to a 3-fold decrease in the affinity for ACh (Fucile et al., 1998).
The order of potency of nicotinic agonists is Epi > Cyt > Nic > DMPP > ACh > Carb, which, at least for
Epi > Nic > ACh > Carb, is in line with our findings
in reconstituted receptors, and very similar to the rank order in
oocyte-expressed chick-human 64 receptors.
The pharmacological profile of the rat forebrain 42 subtype has
recently been compared with that of the human 34 subtype expressed in human embryonic kidney cells (Xiao et al., 1998). These
two subtypes have a similar rank order for agonists but markedly
different Ki values: the
Ki values that we determined for the 6
receptors are closer to those of the 42 than to those of the
34 subtype.
The most interesting finding in the antagonist pharmacological profile
is the extremely high affinity for the toxin MII
(Ki = 66 nM). This toxin is very selective
for the oocyte-expressed rat 32 subtype
(IC50 3.5 nM; Cartier et al., 1996), and further work is necessary to establish whether this high affinity for 6
receptors is due to species and/or subtype differences. Interestingly, MLA inhibited [3H]Epi binding with a
Ki of 1.3 µM, whereas it is known to
inhibit the binding to the chick 42 subtype with a much higher
Ki (>50 µM; Maggi et al., 1999); this
low Ki is in agreement with the finding of
Fucile et al. (1998), who found that 10 µM MLA blocks induced
responses in the oocyte-expressed 64 subtype. Furthermore, the
Ki of 2.8 µM of DHE (a drug that acts
on multiple subtypes and discriminates their different subunit
composition), is intermediate between its very low
Ki for the 42 rat subtype (29 nM) and
its very high Ki for the rat 34
subtype (>200 µM) (Cartier et al., 1996).
Finally, MG624 (a selective antagonist for the chick 7 homomeric
receptor) binds the chick 7 subtype with a
Ki of 106 nM (Gotti et al., 1998; Maggi et
al., 1999), but the Ki of its binding to
native 6 receptors is 40 times higher.
In conclusion, our binding data show that the presence of the 6
subunit gives AChRs a pharmacological profile that is different from
that of both the homomeric subtype and the heteromeric 42 and
34 subtypes, and also confers a high affinity for nicotinic agonists. When reconstituted in lipid bilayers, the purified 6 receptors formed functional channels that were activated by agonists in
a dose-dependent manner and blocked by d-TC. The main
conductance of these channels is 48 pS, although channels with other
conductances are less frequently present. The single channel has an
ohmic behavior, but its Po is voltage
dependent. Depending on physiological conditions and voltage sign
definition, this may account for some channel rectification in
agreement with the results of as Gerzanich et al. (1997), who found a
strong inward current rectification of the 64 subtype expressed
in oocytes at both positive and negative potentials. The
EC50 values of the agonists on the reconstituted receptors were 1000 times greater than their
Ki values determined in our binding
experiments. This discrepancy could be due to the fact that binding
studies are performed on desensitized receptors that have a high
affinity for nicotinic agonists, or that reconstituted receptors do not
mimic all of the properties of their "in situ" native receptors.
The fact that the EC50 value (100 µM) of ACh in
reconstituted receptors is very similar to those obtained for the chick
64 subtype in transfected cells (EC50 105 µM), and lower but always in the same range as that obtained with the
643 subtypes (EC50 204 µM) (Fucile et
al., 1998), strongly supports the first hypothesis.
Like other neuronal subtypes purified and reconstituted using the same
technique (Gotti et al., 1994, 1997), the 6 receptors do not show
any desensitization at high agonist concentrations. We believe that
this may be due to: 1) a lack of certain lipids in lipid bilayers that
could be important for receptor activation and fast desensitization, as
has recently been demonstrated in the case of cholesterol and torpedo
receptor (Rankin et al., 1997); 2) the purification and/or
reconstitution procedures may favor an open conformation of the
desensitized receptor, as has been shown to occur in the homomeric 7
channel, in which the mutation of a single site in the M2 region (Leu
247 to Thr) yields an additional higher conductance channel (80 pS)
that desensitizes very slowly (Revah et al., 1991); and/or 3) the
removal of certain regulatory factors or peptides that are possibly
present in native membrane that may play a role in channel properties.
Another point that emerges from our data is that the
64-containing receptors are heterogeneous and can contain other
and subunits, whereas our pharmacological and functional
studies revealed only a single class of receptors. This may be
explained by one of two different hypotheses: 1) the 64 subunits
are the major determinants of the characteristics of the binding sites and channel properties, or 2) the other receptor subtypes are a
minority and their properties cannot be detected by our assays.
| |
Acknowledgments |
|---|
We thank Dr. Ballivet and Dr. McIntosh for their generous gifts
of 3 fusion protein and toxin MII and Mr. Kevin Smart and Ms. Ida
Ruffoni for their aid with the manuscript.
| |
Footnotes |
|---|
Received January 11, 1999; Accepted April 15, 1999
This work was supported in part by grants from Fabriques de Tabac Réunies, Neuchâtel, Switzerland, the Italian Ministry of University and Scientific and Technological Research, the European Program "Training and Mobility of Researchers," Contract ERB4061PL97-0790 and the Telethon Grant 1047 to C.G.
Send reprint requests to: Dr. Cecilia Gotti, Consiglio Nazionale della Ricerche, Cellular Molecular Pharmacology Center, Via Vanvitelli 32, 20129 Milano, Italy. E-mail: Gotti{at}Farma4.csfic.mi.cnr.it
| |
Abbreviations |
|---|
nAChR, neuronal nicotinic acetylcholine
receptor;
Bgtx, -bungarotoxin;
ACh, acetylcholine, Carb,
carbamylcholine;
COOH, subunit COOH peptide;
Cyt, cytisine;
CYT, subunit cytoplasmic peptide;
Epi, epibatidine;
DMPP, 1,1-dimethyl-4-phenylpiperazinium;
Nic, nicotine;
MLA, methyllycaconitine;
DHE, dihydro--erythroidine;
MII, -conotoxin MII;
d-TC, d-tubocurarine;
COL, chick optic lobe;
Abs, polyclonal antibodies;
mAbs, monoclonal
antibodies.
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References |
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Top
Summary
Introduction
Materials and Methods
Results
Discussion
References
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-Conotoxin which targets 32 nicotinic acetylcholine receptors.
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322-333This article has been cited by other articles:
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) and nonspecific binding (
)
shown is that obtained from a representative experiment; the
Kd value of 35 pM (c.v. = 18%) was
calculated by simultaneously fitting 10 separate experiments. Scatchard
plot of the saturation curve shows the presence of a single class of
high-affinity sites.
