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Vol. 57, Issue 5, 913-925, May 2000
-Conotoxin MII Identifies a Novel Nicotinic
Acetylcholine Receptor Population in Mouse Brain
Institute for Behavioral Genetics, University of Colorado, Boulder, Colorado (P.W., A.C.C., M.J.W.), and Departments of Biology (J.M.M., S.L.) and Psychiatry (J.M.M.), University of Utah, Salt Lake City, Utah.
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Abstract |
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 Top
 Abstract
 Introduction
Experimental Procedures
Results
Discussion
References
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-Conotoxin MII (CtxMII), a peptide toxin from the venom of the
predatory cone snail Conus magus, displays an unusual
nicotinic pharmacology. Specific binding of a radioiodinated derivative (125I--CtxMII) was identified in brain region
homogenates and tissue sections. Quantitative autoradiography indicated
that 125I--CtxMII binding sites have an unique
pharmacological profile and distribution in mouse brain, being largely
confined to the superficial layers of the superior colliculus,
nigrostriatal pathway, optic tract, olivary pretectal, and mediolateral
and dorsolateral geniculate nuclei. Expression of -CtxMII binding
sites in the nigrostriatal pathway, combined with evidence for
-CtxMII-sensitivity of nicotine-induced
[3H]dopamine release in rodent striatal preparations
indicates that 125I--CtxMII binding nicotinic
acetylcholine receptors are likely to be physiologically important.
Unlabeled -CtxMII potently (Ki < 3 nM) competed for a subset of [3H]epibatidine binding
sites in mouse brain homogenates, but weakly (IC50 > 10 µM) interacted with 125I--bungarotoxin and
(
)-[3H]nicotine binding sites, confirming this
compound's novel nicotinic pharmacology. Quantitative autoradiography
revealed that -CtxMII binds with high affinity at a subset of
[3H]epibatidine binding sites with relatively low
cytisine affinity ("cytisine-resistant" sites), resolving
[3H]epibatidine binding into three different populations,
each probably corresponding to a receptor subtype. The majority
population seems to correspond to that which binds nicotine and
cytisine with high affinity ("cytisine-sensitive" sites).
Comparison of the cytisine-resistant population's distribution with
that of 3 subunit mRNA expression suggests that the fractions both
more and less sensitive to -CtxMII probably contain the 3
subunit, perhaps in combination with different 
subunits.
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Introduction |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Molecular
cloning approaches have revealed the expression of 10 nicotinic
acetylcholine receptor (nAChR) subunits [2-7, 9 (to date
8 has been identified only in avian neurons), and 2-4] in
mammalian neuronal tissue (Lindstrom et al., 1996
). Each of the
subunits' mRNA has a distinct pattern of expression, suggesting the
possibility that they may mediate different processes. In the central
nervous system, many of these neuronal nAChRs seem to be presynaptic,
where they modulate the release of neurotransmitters such as dopamine,
norepinephrine, acetylcholine, and 
-aminobutyric acid with
differential pharmacologies (Wonnacott, 1997).
Heterologous expression of neuronal nAChR subunits has shown different
subunit combinations (or expression of 7-9 alone) result in
functional nAChR subtypes with differing pharmacological and
biophysical properties (Lindstrom et al., 1996). The number of neuronal
nAChR subunits known theoretically allows the possibility of vast
numbers of potential subunit combinations and receptor subtypes.
However, efforts to discover which nAChR subtypes exist in the central
nervous system (and their locations) have been hindered by the lack of
subtype-specific pharmacological probes to identify individual subtypes
within the mixed native nAChR population. Indeed, only two subtypes of
neuronal nAChRs, those which correspond to
125I--Bgt and `high-affinity agonist
binding' sites, have been thoroughly characterized so far. In
mammalian neuronal systems, 125I--Bgt is
believed to bind largely (if not exclusively) to nAChRs containing the
7 subunit (Schoepfer et al., 1990; Seguela et al., 1992), whereas
()-[3H]nicotine,
[3H]cytisine,
[3H]acetylcholine, and
[3H]methylcarbamylcholine binding is only
detectable at the 42 combination of subunits (Whiting and
Lindstrom, 1987; Flores et al., 1992). Neuronal bungarotoxin (Bgt), a
minor component of the venom of the Taiwanese banded krait
Bungarus multicinctus (also named 
-Bgt, toxin F, and Bgt
3.1) showed initial promise as a selective antagonist of 32
subtype nAChRs (Luetje et al., 1990). However, problems of availability
(B. multicinctus is a protected species), complex kinetics
of interaction at multiple nAChR subtypes (Papke et al., 1993), and the
difficulty of ensuring the toxin's purity have severely restricted its
usefulness. More recently, it has become apparent that the agonist
ligand [3H]epibatidine binds with detectable
affinity to other neuronal nAChRs in addition to the 42 subtype
(Perry and Kellar, 1995; Marks et al., 1998; Parker et al., 1998),
raising hopes of identifying further native nAChR populations.
-CtxMII was identified in the venom of Conus magus by
sequential fractionation and testing of the isolates for inhibition of
32 nAChRs expressed in Xenopus laevis oocytes (Cartier
et al., 1996). Experiments in native systems have demonstrated potent (nanomolar IC50 values) and selective blockade of
nAChR subpopulations in rodent striatal (Grady et al., 1997; Kulak et
al., 1997; Kaiser et al., 1998) and avian ciliary ganglion (Ullian et
al., 1997) preparations. These functional studies provided strong
evidence that -CtxMII was a highly selective antagonist of a novel
native receptor population. The high affinity and novel subtype
selectivity of -CtxMII make it a potentially useful ligand,
particularly in light of its proven ability to interact with native
neuronal nAChRs and the present paucity of selective pharmacological tools.
In this study, a radiolabeled version of -CtxMII
([125I]CtxMII) was used to identify, locate,
and enumerate -CtxMII binding nAChRs in mouse brain. The
distribution and pharmacology of these receptors differs from those
previously characterized, indicating that they represent a novel
population. Using unlabeled -CtxMII in combination with existing
nicotinic ligands allowed identification of -CtxMII binding nAChRs
as part of the set of high-affinity [3H]epibatidine binding nAChRs, but distinct
from the traditionally recognized "high-affinity agonist binding"
42 subtype. The pharmacological characteristics and distribution
of -CtxMII binding nAChRs indicate that they are likely to be
composed of (at least) 3 and 2 subunits.
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Experimental Procedures |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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Animals. Male mice (C57BL/6J, 60-90 days old) were used throughout this study. Mice were bred at the Institute for Behavioral Genetics and housed five per cage. The vivarium was maintained on a 12-h/12-h light/dark cycle (lights on 7 AM to 7 PM), and mice were given free access to food and water. The Animal Care and Utilization Committee of the University of Colorado, Boulder, approved all procedures used in this study.
Materials.
[3H]Epibatidine (specific
activity, 33.8 Ci/mmol), ()-[3H]nicotine
(specific activity, 81.5 Ci/mmol), Na125I
(specific activity, 2200 Ci/mmol), and uridine triphosphate (-35S; initial specific activity, 800 Ci/mmol)
were obtained from DuPont NEN (Boston, MA).
125I--Bgt (initial specific activity, 230 Ci/mmol), Hyperfilm -max, and Hyperfilm-3H
were purchased from Amersham (Mt. Prospect, IL). NaCl, NaOH, KCl,
MgCl2, MgSO4,
CaCl2, chloramine T, ammonium acetate, lysozyme, Tris · HCl, sodium carbonate, sodium bicarbonate, polyethylenimine (PEI; 50% w/v solution), yeast tRNA, triethanolamine, sodium citrate, dithiothreitol, Denhardt's solution, acetic anhydride,
diethylpyrocarbonate, sodium phosphate, gelatin,
poly-L-lysine, chromium aluminum sulfate, BSA (Fraction V),
phenylmethylsulfonyl fluoride, EDTA, EGTA, aprotinin, leupeptin
trifluoroacetate, and pepstatin A were obtained from Sigma Chemical Co.
(St. Louis, MO). ()-nicotine bitartrate and DPX mountant were bought
from BDH Chemicals (Poole, UK). Glass fiber filters Type A/E were
obtained from Gelman Sciences (Ann Arbor, MI) and Type GB from MFS
(Dublin, CA). Budget Solve scintillation fluid was purchased from RPI
(Arlington Heights, IL). ATP, CTP, GTP, and RNase A were purchased from
Boehringer-Mannheim (Indianapolis, IN). The enzymes SP6 RNA polymerase
and HindIII, were obtained from Promega (Madison, WI).
Dextran sulfate was purchased from Pharmacia (Uppsala, Sweden), and
formamide from Fluka Chemical Corp. (Ronkonkoma, NY).
Preparation of -CtxMII and
Y0--CtxMII and Iodination of
Y0--CtxMII.
Unmodified -CtxMII was
synthesized as described previously (Cartier et al., 1996). To provide
an iodination site, -CtxMII was synthesized with the addition of a
tyrosine at the N terminus (Y0--CtxMII).
Although the two histidine residues present in native MII also provide
potential iodination sites, structure-function studies suggested that
modification of these sites would lead to unacceptable levels of
decreased toxin potency (G. E. Cartier, unpublished
observations). Synthesis of Y0--CtxMII was
achieved by methods described previously (Cartier et al., 1996).
-CtxMII
was dissolved in 25 µl of H2O. To this was
added 40 µl of 0.3 M NH4Ac, pH 5.3. Approximately 10 mCi of Na125I (volume ~22
µl) was added. The iodination reaction was initiated by the addition
of 40 µl of freshly prepared 0.4 mM chloramine T. The reaction
proceeded at room temperature (~22°C) for 10 min and was then
terminated by the addition of 65 µl of 0.5 M ascorbic acid. The pH of
the reaction mix was further lowered by the addition of 0.8 ml of 0.1%
trifluoroacetic acid (TFA). Mono- and di-iodinated peptides were
separated from unmodified peptides by reversed-phase HPLC using an
analytical Vydac C18 column. Buffer A was 0.1% TFA, buffer B was
0.09% TFA, 60% acetonitrile, and the loading loop size was 5 ml. The
gradient was 25%-75% B over 50 min. Flow rate was 1 ml/min and
absorbance was monitored at 220 nM. A solution of sodium thiosulfate
(2.5%) and potassium iodide (0.2%) in 1 N NaOH was added to the waste
collection beaker to trap unreacted 125I.
Fractions containing peptide were collected in polypropylene tubes
containing 10 µl of 20 mg/ml lysozyme to decrease adsorption to the
tubes. After collection, peptide material was lyophilized and
resuspended in 100 µl of 40% MeOH. Under the above chromatographic conditions, the unmodified peptide elutes at approximately 27 min with
monoiodo peptide eluting at approximately 29 min. Final yield
(estimated from counting an aliquot in a gamma counter) was
approximately 2 nmol of monoiodo peptide. Monoiodination of the
N-terminal tyrosine was verified by chemical sequencing and mass spectrometry.
Quantitative Autoradiography of 125I--CtxMII and
[3H]Epibatidine Binding.
Quantitative
autoradiography procedures were similar to those described previously
(Pauly et al., 1989; Marks et al., 1998). Three C57BL/6J mice were
sacrificed by cervical dislocation. The brains were removed from the
skulls and rapidly frozen by immersion in isopentane (35°C, 10 s). The frozen brains were wrapped in aluminum foil, packed in ice, and
stored at 70°C until sectioning. Tissue sections (14 µm thick)
prepared using an IEC Minotome Cryostat refrigerated to 16°C were
thaw mounted onto subbed microscope slides (Richard Allen, Richland,
MI). Slides were subbed by incubation with gelatin (1% w/v)/chromium
aluminum sulfate (0.1% w/v) for 2 min at 37°C, drying overnight at
37°C, incubation at 37°C for 30 min in 0.1% (w/v)
poly-L-lysine in 25 mM Tris, pH 8.0, and drying at 37°C
overnight. Mounted sections were stored desiccated at 70°C until
use. Eight series of sections were collected from each mouse brain.
-CtxMII, three
adjacent series of sections from each mouse were incubated in binding
buffer (144 mM NaCl, 1.5 mM KCl, 2 mM CaCl2, 1 mM
MgSO4, 20 mM HEPES, 0.1% BSA (w/v), pH 7.5) + phenylmethylsulfonyl fluoride (1 mM, to inactivate endogenous serine
proteases) at 22°C for 15 min. For all
125I--CtxMII binding reactions, the standard
binding buffer was supplemented with BSA [0.1% (w/v)], 5 mM EDTA, 5 mM EGTA, and 10 µg/ml each of aprotinin, leupeptin trifluoroacetate,
and pepstatin A to protect the ligand from endogenous proteases. The
sections were then incubated with 0.5 nM
125I--CtxMII for 2 h at 22°C. The first
series of sections was used to determine total
125I--CtxMII binding (no competing ligands),
the second to measure cytisine-resistant
125I--CtxMII binding (in the presence of 20 nM
unlabeled cytisine), whereas the third series of sections from each
mouse was used to determine nonspecific
125I--CtxMII binding (in the presence of 1 µM unlabeled epibatidine). 125I--CtxMII
binding was further investigated by coincubation of sections with
varying concentrations of unlabeled ligands (cytisine, 1-300 nM;
epibatidine, 10-1000 pM; -Bgt, 1 µM; ()-nicotine, 30-3000 nM).
After incubation with 125I--CtxMII, the slides
were washed as follows: 30 sec in binding buffer + 0.1% (w/v) BSA
(22°C), 30 sec in binding buffer + 0.1% (w/v) BSA (0°C), 5 sec in
0.1× binding buffer + 0.01% (w/v) BSA (twice at 0°C), and twice at
0°C for 5 sec in 5 mM HEPES (pH 7.5).
Sections for use in [3H]epibatidine binding
were rehydrated in binding buffer at 22°C for 15 min, followed by
incubation with 500 pM [3H]epibatidine for
2 h at 22°C. Four series of adjacent sections were used from
each mouse to measure total [3H]epibatidine
binding (no competing ligand), [3H]epibatidine
binding in the presence of 100 nM unlabeled cytisine, [3H]epibatidine binding in the presence of 100 nM unlabeled cytisine + 50 nM unlabeled -CtxMII, and nonspecific
[3H]epibatidine binding [in the presence of 1 mM unlabeled ()-nicotine]. Concentrations of unlabeled cytisine and
-CtxMII were chosen on the basis of results obtained in this study
from [3H]epibatidine inhibition binding studies
in membrane preparations (Fig. 4, right). Slides were washed by
sequential incubation in the following buffers (all steps at 0°C): 5 sec in binding buffer (twice), 5 sec in 0.1× binding buffer (twice),
and 5 sec in 5 mM HEPES, pH 7.5 (twice).
Sections were initially dried with a stream of air, then by overnight
storage (22°C) under vacuum. Mounted, desiccated sections were
apposed to film (1-3 days, Amersham Hyperfilm -Max film for
125I-labeled sections; 8 weeks, Amersham
Hyperfilm-3H for 3H-labeled
sections). To allow quantification, each film was also exposed to
tissue paste standards of defined specific activity (Geary et al.,
1985). For tritium, specific activities were 0.05 to 50 nCi/mg (wet
weight), whereas for 125I, specific activities
were 0.25 to 60 nCi/mg (wet weight). The exact specific activities of
the tissue paste standards were determined by measuring radioactivity
in weighed aliquots.
After the films had been exposed to the sections for an appropriate
length of time, they were developed and signal intensity in selected
brain regions was measured by digital image analysis. Films were
illuminated using a Northern Light light box, and autoradiographic images of the sections and tissue paste standards were captured using a
CCD imager camera. Signal intensity was determined using NIH-Image
1.61. Where possible, six independent measurements from different
tissue sections were made for each brain region, under each incubation
condition, for each mouse. The absorbance measurements for each
brain area were averaged, and the mean absorbance was used to calculate
the degree of labeling by reference to the relevant standard curve.
Membrane Preparation.
Each C57BL/6J mouse was sacrificed by
cervical dislocation. The brain was removed from the skull and placed
on an ice-cold platform. Brains were either dissected into 12 regions
[olfactory bulbs, cerebellum, hindbrain (pons-medulla), hypothalamus,
hippocampus, striatum, cerebral cortex, thalamus, midbrain,
interpeduncular nucleus, superior colliculus, and inferior colliculus]
or the hindbrain, cerebellum, and olfactory bulbs were discarded
without further dissection ("whole brain" preparation). Samples
were homogenized in ice-cold hypotonic buffer (14.4 mM NaCl, 0.2 mM
KCl, 0.2 mM CaCl2, 0.1 mM
MgSO4, 2 mM HEPES, pH 7.5) using a glass-Teflon tissue grinder. The particulate fractions were obtained by
centrifugation at 20,000g (15 min, 4°C; Sorval RC-2B
centrifuge). The pellets were resuspended in fresh homogenization
buffer, incubated at 37°C for 10 min, then harvested by
centrifugation as before. Each pellet was washed twice more by
resuspension/centrifugation, then stored (in pellet form under
homogenization buffer) at 70°C until used. Protein concentrations
in the membrane preparations were measured using the method of Lowry et
al. (1951), using BSA as the standard.
()-[3H]Nicotine Binding to Membranes.
The
binding of ()-[3H]nicotine was measured using
the method of Marks et al. (1986), modified for use with a 96-well
plate washer. Membrane samples (200 µg of whole brain preparation)
were incubated in 96-well polystyrene plates with 20 nM
()-[3H]nicotine in 100 µl of binding buffer
for 30 min at 22°C. Binding reactions were terminated by filtration
of samples onto PEI-soaked (0.5% w/v in binding buffer) glass fiber
filters (types GFA/E and GB) using an Inotech Cell Harvester (Inotech,
East Lansing, MI). Samples were subsequently washed six times with
ice-cold binding buffer. Total and nonspecific [in the presence of 1 mM ()-nicotine tartrate] binding were determined in triplicate. Where inhibition binding was being measured, various concentrations of
competing ligands were included in triplicate wells.
125I--Bgt Binding to Membranes.
Binding of
125I--Bgt to membrane preparations was
performed using procedures similar to those used with
()-[3H]nicotine, except that incubation times
were extended to 5 h and samples contained 1 nM
125I--Bgt instead of 20 nM
()-[3H]nicotine. The GFA/E filters were
treated with 0.5% PEI and the GFB filters were treated with 5% (w/v)
nonfat dry milk before filtration.
[3H]Epibatidine Binding to Membranes.
Binding
of [3H]epibatidine was quantified as described
previously (Marks et al., 1998). Incubations were performed in 1-ml polypropylene tubes in a 96-well format, using 50 to 200 µg of membrane protein per tube (depending on brain region). A 500-µl reaction volume was used to minimize problems of ligand depletion, and
all incubations progressed for 2 h at 22°C. The concentration of
[3H]epibatidine (500 pM) used in inhibition
binding experiments was chosen to maintain binding of ligand to the
tissue at 5% or less of total ligand added. Saturation binding
experiments were performed for membrane preparations from each brain
region, using ligand concentrations in the range 10 to 800 pM. At the
lower concentrations, a significant proportion of ligand bound to the tissue. Free [3H]epibatidine concentrations
were estimated by correcting for the amount of ligand bound to tissue,
and these corrected concentrations were used to calculate
Kd values for
[3H]epibatidine binding in each brain region
and, thus, the Ki values for each compound
versus [3H]epibatidine binding.
125I--CtxMII Binding to Membranes.
Large
amounts of nonspecific binding were seen when using
125I--Ctx (0.2-32 nM) in filtration binding
assays. Best assay performance was produced using the following
modifications to the ()-[3H]nicotine binding
procedure. Incubation times were extended to 2 h, and incubation
buffer was supplemented with BSA [0.1% (w/v)], 5 mM EDTA, 5 mM EGTA,
and 10 µg/ml each of aprotinin, leupeptin trifluoroacetate, and
pepstatin A to protect the ligand from endogenous proteases. The glass
fiber filters were treated with 5% (w/v) nonfat dry milk before filtration.
In Situ RNA Hybridization.
The method used for in situ
hybridization using riboprobes was identical with that used by Simmons
et al. (1989) and Marks et al. (1992). Probes were prepared by in vitro
transcription, using -35S-UTP as the sole
source of UTP. The 3 probe was prepared from clone pPCA48E(4) cloned
in pSP65, linearized using HindIII, and synthesized using
SP6 RNA polymerase. The synthesis was designed to yield full-length
antisense transcript. Immediately before hybridization, the probe was
subjected to alkaline hydrolysis using the method of Cox et al. (1984)
to yield products with average sizes of 500 bases.
-Max film (10 days).
To allow 3 hybridization to be quantified, the film was also exposed
to a set of dot-blotted 35S standards. Serial
dilutions of the [35S]cRNA were made in 5×
standard saline citrate (SSC; 1× SSC, 150 mM NaCl, 15 mM trisodium
citrate, pH 7.0, with HCl), and 400 µl of each dilution was
applied to a prewetted (5× SSC) nylon membrane (New England Nuclear,
Beverly, MA) by vacuum filtration through a 96-well manifold (Life
Technologies, Bethesda, MD). The samples were washed three times with
5× SSC and allowed to dry at room temperature. One set of the dilution
series was cut from the membrane and counted on a liquid scintillation
counter to determine exact counts per unit area. Standards ranged from
0.1 to 60 pCi/mm2. After exposure to the sections and
standards, the films were developed. Autoradiographic images were
captured and hybridization densities quantified as described above for
autoradiographic analysis of ligand binding.
Calculations.
Results for saturation binding experiments
were calculated using the Hill equation: B = Bmax Ln /
(Ln + Kdn), where B
is the binding at the free ligand concentration L, Bmax is the maximum number of binding
sites, Kd is the equilibrium binding
constant, and n is the Hill coefficient. Values of
Bmax, Kd, and
n were calculated using the nonlinear least-squares fitting algorithm of Sigma Plot version 5.0 (Jandel Scientific, San Rafael, CA). Results for inhibition of
()-[3H]nicotine and
125I--Bgt binding were calculated using a
one-site fit: B = Bo / [1 + (I / IC50)] where B is ligand bound
at inhibitor concentration I, Bo is the
binding in the absence of inhibitor, and IC50 is the concentration of inhibitor required to reduce binding to 50% of
Bo. Results for inhibition of ligand
binding were calculated using the formulae for either one (as above) or
two binding sites: B = B1
/ [1 + (I / IC50-1)] + B2 / [1 + (I /
IC50-2)], where B is ligand bound at
inhibitor concentration I, and B1 and
B2 are binding sites sensitive to
inhibition with IC50-1 and
IC50-2, respectively. Values for
Ki (inhibition binding constant) were
derived by the method of Cheng and Prusoff (1973):
Ki = IC50 / 1 + (L /
Kd).
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Results |
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Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
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125I--CtxMII Autoradiography, Mouse Brain
Sections.
Preliminary experiments (using monoiodinated but
nonradioactive Y0--CtxMII) showed that it
remained a potent inhibitor at 32 nAChRs expressed in X. laevis oocytes (Kd = 1.9 nM, compared with 0.35 nM for the native toxin; data not shown). In light of these
data, attempts were made to identify specific, nicotinic -CtxMII
binding using the monoradioiodinated version of this ligand
(125I--CtxMII) in mouse brain sections.
-CtxMII was used in autoradiography
experiments. Under these conditions, a small amount of tissue mediated
nonspecific binding of 125I--CtxMII binding
(defined in the presence of 1 µM unlabeled epibatidine) was seen.
However, specific 125I--CtxMII binding could
be clearly distinguished over the tissue background (Fig.
1). Minor variations in nonspecific
binding were noted, making it necessary to measure nonspecific binding
in each individual region to ensure accurate quantification of specific signal strength. Subsequent experiments showed that using higher 125I--CtxMII concentrations produced
unacceptably high levels of nonspecific binding. Monoiodination
produced radiolabeled toxin of high specific activity (2200 Ci/mmol),
allowing short film exposures (24-72 h). Toxin was used through one
half-life without any detectable increase in nonspecific signal or
decrease in specific signal strength.
|
-CtxMII
binding [>5 fmol/mg (wet weight)] were detected in the dorsolateral
and ventrolateral geniculate nuclei, olivary pretectal nucleus, and the
zonal layer of the superior colliculus. High levels (4-5 fmol/mg) of
125I--CtxMII binding were also detected in the
superficial gray of the superior colliculus and in the oculomotor
nerve. Outside these highly labeled regions, binding densities were
lower and sites were mainly found in nigrostriatal and
optic-tract-associated regions, as summarized in Table
1.
|
-CtxMII
binding resembled a subset of the cytisine-resistant high affinity
[3H]epibatidine binding population reported by
Marks et al. (1998). The abilities of unlabeled epibatidine, cytisine,
-Bgt, and nicotine to compete for
125I--CtxMII-binding sites were measured by
quantitative autoradiography. Competition binding was assessed in
striatum and the superficial gray of the superior colliculus, as most
of the other regions containing 125I--CtxMII
binding are too small to allow sufficient tissue slices to be
collected. Even high concentrations of -Bgt (1 µM) had no effect
on 125I--CtxMII-binding. In contrast, all
three of the remaining ligands competed effectively for
125I--CtxMII-binding in a monophasic manner
(Fig. 2). Assuming a Kd value of 1.9 nM for
125I--CtxMII-binding (obtained in competition
binding experiments using nonradioactive, monoiodinated
Y0--CtxMII, as mentioned previously),
Ki values in the superficial gray of the
superior colliculus were epibatidine, 81 ± 32 pM; cytisine,
14 ± 6 nM; and ()-nicotine, 381 ± 43 nM, whereas the
corresponding values in the striatum were epibatidine, 89 ± 30 pM; cytisine, 18 ± 4 nM; and ()-nicotine, 276 ± 67 nM.
Hill coefficients were not significantly different from 1 for each drug
in each region (Fig. 2).
|
-CtxMII binding
sites nAChRs are uniformly cytisine resistant, the cytisine sensitivity
of 125I--CtxMII binding was assessed by
addition of 20 nM cytisine to the binding buffer (a concentration that
the previous workers' data indicated would abolish binding to
cytisine-sensitive [3H]epibatidine binding
nAChRs). Binding of 125I--CtxMII (0.5 nM) was
noticeably diminished by coincubation with cytisine (20 nM) but was not
reduced to background levels (Fig. 1; Table 1). Comparison of
125I--CtxMII binding densities in the presence
and absence of cytisine showed that across brain regions, addition of
20 nM cytisine reduced 125I--CtxMII binding by
an average of 40% (Table 1, right column; Fig.
3). In all regions where it was
detectable, specific 125I--CtxMII binding
displayed the same cytisine sensitivity (correlation analysis showed
r = 0.96; Fig. 3).
|
-CtxMII binding using
125I--CtxMII in regionally dissected brain
tissue. However, high nonspecific binding (presumably to the brain
homogenates) was observed even under optimized conditions (see
Experimental Procedures; approximately 2% of the ligand
added was retained as nonspecific binding when using 100 µg of
protein/well of membranes). The autoradiography data suggested that
superior colliculus membranes should contain the highest density of
125I--CtxMII binding sites; indeed, evidence
for specific, nicotinic binding of
125I--CtxMII (displaceable by 1 µM
epibatidine) was observed in superior colliculus membrane preparations.
Interassay variation in total and nonspecific binding was substantial,
but intra-assay variation was more manageable and each individual
experiment using superior colliculus membranes provided evidence for
specific binding above the nonspecific binding recorded. Fitting to a
Hill binding curve indicated a Bmax of
60 ± 21 fmol/mg of protein, a Kd of 4.9 ± 3.3 nM, and a Hill coefficient of 1.09 ± 0.34 (mean ± S.E.M. of five separate determinations). No evidence for
specific 125I--CtxMII binding could be
obtained in the other brain regions tested, presumably because of the
lower densities of binding sites in these regions.
()-[3H]Nicotine, 125I--Bgt and
[3H]Epibatidine Binding, Mouse Brain Membrane
Preparations.
To expand on the data provided by
125I--CtxMII competition binding experiments,
the ability of -CtxMII to displace
()-[3H]nicotine,
125I--Bgt, and
[3H]epibatidine binding to mouse brain membrane
preparations was assessed. For comparison, the ability of the nicotinic
agonist cytisine to inhibit binding of the same ligands was also measured.
)-[3H]nicotine (20 nM) and
125I--Bgt (1 nM) binding (to 42 and 7
nAChRs, respectively) was measured in whole-brain membrane
preparations. Cytisine produced a monophasic inhibition of both
()-[3H]nicotine and
125I--Bgt binding (Fig.
4) but was much more potent in its
interaction with the former (Ki = 0.36 ± 0.04 nM and 1.1 ± 0.5 µM, respectively). In contrast,
-CtxMII was only a weak inhibitor of
()-[3H]nicotine and
125I--Bgt binding
(Ki > 10 µM in each case).
|
-CtxMII
inhibition of [3H]epibatidine binding in
membrane preparations from 12 brain regions. Levels of
[3H]epibatidine binding varied widely among
brain regions (Table 2). Levels were
particularly high in the interpeduncular nucleus, with large amounts of
[3H]epibatidine binding also detected in
superior colliculus and thalamic membranes. The lowest amounts of
binding were found in the cerebellum and olfactory bulbs. As reported
previously (Marks et al., 1998), saturation binding analysis provided
[3H]epibatidine binding
Kd values of 20 to 40 pM in each region investigated, with no evidence for multiphasic ligand binding (data not
shown). Also matching the results of Marks et al. (1998), cytisine
inhibition of [3H]epibatidine binding exhibits
two phases (with KI = 0.27 ± 0.05 nM
and 32 ± 6 nM: "cytisine-sensitive" and "cytisine
resistant," respectively), indicating that
[3H]epibatidine binds to receptor populations
with differential cytisine affinity (but similar affinities for
[3H]epibatidine, according to the saturation
binding profiles).
|
)-[3H]nicotine binding site (Marks et al.,
1998)] predominated. However, in olfactory bulb and interpeduncular
nucleus preparations, the density of binding sites less sensitive to
cytisine inhibition equaled or exceeded that of the cytisine-sensitive
fraction (50% and 62% of
[3H]epibatidine binding in the two regions,
respectively). Hippocampal membranes contained only cytisine-sensitive
[3H]epibatidine binding. Binding of
[3H]epibatidine was not detectably inhibited by
-CtxMII in interpeduncular nucleus, hindbrain, olfactory bulb,
hippocampal, or cerebellar membranes. However, it is important to note
that these region's small size (interpeduncular nucleus) or low
overall receptor densities (hippocampus, hindbrain, olfactory bulb, and
cerebellum) resulted in low numbers of total
[3H]epibatidine counts being retained, so minor
populations of -CtxMII-sensitive [3H]epibatidine binding sites may have been
overlooked. In contrast, -CtxMII potently
[Ki = 2.7 ± 1.3 nM (mean ± S.E.M. of values in the seven regions where -CtxMII competition was
observed); Table 2] inhibited a fraction of
[3H]epibatidine binding in the remaining brain
regions studied. At the concentrations studied (30 pM-300 nM),
-CtxMII inhibition of [3H]epibatidine
binding appeared monophasic. The -CtxMII-sensitive fraction of
[3H]epibatidine binding was approximately equal
to the cytisine-resistant portion in the superior colliculus, striatum,
and olfactory tubercles but was smaller in the remaining brain regions.
Statistical analysis showed no significant differences in
Ki values between regions for -CtxMII
inhibition of [3H]epibatidine binding (one-way
ANOVA; F(6,13) = 2.64, P > .05). Displacement of [3H]epibatidine binding to
superior colliculus membranes by cytisine and -CtxMII is shown in
Fig. 4, right, whereas the regional distribution of total,
cytisine-resistant, and -CtxMII-sensitive specific [3H]epibatidine binding is summarized in Table
2.
[3H]Epibatidine Autoradiography, Mouse Brain
Sections.
Competition binding experiments demonstrated that
-CtxMII was able to potently displace a fraction of
[3H]epibatidine binding to mouse brain membrane
preparations. In addition, the same data showed that the density of
-CtxMII-sensitive [3H]epibatidine binding
sites varied widely among regions. Because the proportion of
-CtxMII-sensitive [3H]epibatidine binding
never exceeded that of the cytisine-resistant population (and in many
regions was lower), it seemed possible that -CtxMII was selectively
interacting with a subpopulation of cytisine-resistant
[3H]epibatidine binding nAChRs, as suggested by
the earlier 125I--CtxMII autoradiography
experiments. Crude regional dissection of the mouse brain provided
limited anatomical resolution, so the relationship between
cytisine-resistant and -CtxMII-sensitive [3H]epibatidine binding sites was explored
using an autoradiographic approach. Inhibition binding experiments
conducted using filtration binding indicated that 100 nM cytisine would
essentially eliminate [3H]epibatidine binding
to cytisine-sensitive sites (at a
[3H]epibatidine concentration of 500 pM) but
would leave the cytisine-resistant [3H]epibatidine binding largely unaffected
(Fig. 4, right). For this reason, cytisine-resistant
[3H]epibatidine binding was visualized using
500 pM [3H]epibatidine, in combination with 100 nM cytisine. The same series of experiments also suggested that 50 nM
-CtxMII would be sufficient to displace -CtxMII-sensitive
[3H]epibatidine binding, without affecting
binding to other types of [3H]epibatidine
binding sites.
|
|
-CtxMII (50 nM) resulted in a loss of
[3H]epibatidine binding from the
cytisine-resistant population (defined in the presence of 100 nM
cytisine). This loss was particularly noticeable in the superficial
layers of the superior colliculus, the optic tract, supraoptic
decussation, mediolateral and ventrolateral geniculate nuclei, the
olivary pretectal nucleus, striatum, olfactory tubercles, and
oculomotor nerve, where 
75% of cytisine-resistant [3H]epibatidine binding was
-CtxMII-sensitive. In contrast, in regions such as the inferior
colliculus, interpeduncular nucleus, olfactory bulbs, medial habenula,
and fasciculus retroflexus, much less -CtxMII sensitivity was seen
(Fig. 5; Table 3). Again, the distribution of -CtxMII-sensitive
[3H]epibatidine binding established by
quantitative autoradiography paralleled that measured by inhibition
binding and 125I--CtxMII autoradiography experiments.
Levels of specific 125I--CtxMII binding were
compared with those of -CtxMII-sensitive
[3H]epibatidine binding (Fig.
6). In the majority of regions in which
specific 125I--CtxMII binding was seen, the
two measures were highly correlated (r = 0.98;
slope = 2.6). However, in six regions (medial habenula, lateral
habenula, oculomotor nerve, zonal layer of the superior colliculus,
fasciculus retroflexus, and interpeduncular nucleus), levels of
-CtxMII-sensitive [3H]epibatidine binding
fell above the correlation line. In each of these regions,
autoradiography showed that in addition to detectable levels of
specific 125I--CtxMII binding, substantial
amounts of [3H]epibatidine binding less
sensitive to both cytisine and unlabeled -CtxMII were found.
|
3 Subunit Expression: In Situ Hybridization.
A previous
investigation (Marks et al., 1998) suggested that cytisine-resistant
[3H]epibatidine binding is found in regions
that express 3 nAChR subunit mRNA or in regions innervated by those
that do. To explore this link more thoroughly, the distribution of 3
mRNA was mapped by in situ hybridization, and compared with that of
cytisine-resistant [3H]epibatidine binding.
Hybridization was performed in brain slices prepared from the same
animals used for autoradiographic investigations of ligand binding in
this study, allowing direct comparisons to be made.
3 hybridization is shown in Fig. 5 (right column).
Expression of 3 mRNA was restricted to a number of small, well-defined nuclei distributed throughout the brain. By far the highest level of hybridization was detected in the medial habenula (48 pCi/mm2), the next highest amount (10 pCi/mm2)
being found in the mitral layer of the accessory olfactory bulbs. Where
detected, hybridization in other brain regions was much weaker than in
these two regions (3.2-0.6 pCi/mm2). The distribution of
3 hybridization is summarized in Table 4. The highest densities of
cytisine-resistant [3H]epibatidine binding were
found in the medial habenula and accessory olfactory bulbs, matching
the data for 3 mRNA expression. In addition, patterns of 3
hybridization and cytisine-resistant [3H]epibatidine binding were superimposed in
many brain regions (including the dorsal cortex of the inferior
colliculus, medial habenula, medial geniculate nucleus, superficial
layers of the superior colliculus, and the medial vestibular and
prepositus hypoglossal nuclei). In other cases, cytisine-resistant
[3H]epibatidine binding was found in nuclei
innervated by regions where 3 hybridization can be detected (nAChRs
are known to be transported from the ventral tegmental area and
substantia nigra to the striatum, frontal cortex, olfactory tubercles,
and nucleus accumbens (Schwartz et al., 1984), from the retina to the
superior colliculus and the geniculate nuclei (Swanson et al., 1987),
and from the medial habenula to the interpeduncular nucleus (Clarke et
al., 1986). In addition, cytisine-resistant
[3H]epibatidine binding was detectable on fiber
tracts in the fasciculus retroflexus, oculomotor nerve, optic tract,
brachium of the superior colliculus, and supraoptic decussation. Small
amounts of 3 hybridization were also found in a number of regions
that have no apparent cytisine-resistant [3H]epibatidine binding, most notably the motor
trigeminal nucleus and somatosensory cortex.
|
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
-CtxMII, originally isolated from the venom of the predatory
cone snail, C. magus (Cartier et al., 1996), has been used
to investigate the diversity of nicotinic receptor binding sites in
mouse brain. The data presented here demonstrate that mouse brain
high-affinity epibatidine binding has three components: 1) a component
that corresponds to the ()-[3H]nicotine- and
[3H]cytisine-binding sites
["cytisine-sensitive" sites, probably the 42 subtype
(Whiting and Lindstrom, 1987; Flores et al., 1992)] and two sites with
lower cytisine affinity ("cytisine-resistant" sites); 2) a
cytisine-resistant component that displays high affinity for -CtxMII
and that has been visualized here using
125I--CtxMII; 3) a cytisine-resistant
component that displays lower affinity for -CtxMII. This subdivision
of mouse brain [3H]epibatidine-binding nAChRs
is illustrated in Fig. 7.
|
125I--CtxMII Binds to a Novel nAChR Population.
Competition binding experiments demonstrated that -CtxMII has a low
affinity (Ki >10 µM) at mouse brain
()-[3H]nicotine and
125I--Bgt binding sites (Fig. 4). This shows
that it binds to a novel neuronal nAChR population, distinct from the
well- characterized ()-[3H]nicotine- and
125I--Bgt-binding sites (corresponding to
42 and 7-containing subtypes in mammalian neurons; Whiting and
Lindstrom, 1987; Schoepfer et al., 1990; Flores et al., 1992; Seguela
et al., 1992).
-CtxMII in tissue slices yielded
Ki values versus
125I--CtxMII for unlabeled epibatidine and
cytisine of 80 to 90 pM and 14 to 18 nM, respectively. These values are
similar to those reported by Marks et al. (1998) for cytisine-resistant
[3H]epibatidine-binding sites. As shown in Fig.
3, the cytisine sensitivity of
125I--CtxMII-binding sites was constant across
brain regions, suggesting that they represent a single population (an
alternate but less likely explanation is that multiple sites with a
mean cytisine Ki of 20 nM exist in all
125I--CtxMII-binding regions). Additionally,
125I--CtxMII binding sites have no appreciable
affinity for -Bgt (no displacement by 1 µM -Bgt) and have a
relatively low affinity for ()-nicotine
[Ki = 280-380 nM, compared with 8.9 nM at
the mouse brain 42 high affinity
[3H]nicotine binding subtype (Marks et al.,
1998)].
Quantitative autoradiographic analysis showed that specific
125I--CtxMII binding occurs in discrete nuclei
distributed throughout the mouse brain. The sites' regional
distribution is unlike previously reported nicotinic binding patterns
and seemed to represent a subset of the cytisine-resistant
[3H]epibatidine binding sites reported by Marks
et al. (1998). The unusual distribution and nicotinic pharmacology of
125I--CtxMII-binding sites confirms that they
represent a novel native neuronal nAChR subtype.
-CtxMII-Binding nAChRs are a Subset of Cytisine-Resistant
[3H]Epibatidine Binding Sites.
Confirming the data
provided by the 125I--CtxMII autoradiography
experiments, -CtxMII was a potent inhibitor (mean
Ki = 2.7 nM) of a fraction of
[3H]epibatidine binding sites in mouse brain
regional homogenates (Fig. 4; Table 2). Densities of these
-CtxMII-sensitive [3H]epibatidine-binding
sites varied among brain regions, in many cases having a lower density
than cytisine-resistant [3H]epibatidine binding
sites. Indeed, quantitative autoradiography (Fig. 5) showed that
-CtxMII-sensitive sites are a subset of the cytisine-resistant
[3H]epibatidine-binding sites described by
Marks et al. (1998).
) and corresponded to the distribution of
high affinity nicotine or cytisine binding (Perry and Kellar, 1995;
Marks et al., 1998). The autoradiograms demonstrated that these sites
were confined to a limited set of nuclei, scattered throughout the
brain. Some (but not all) cytisine-resistant
[3H]epibatidine binding was highly
-CtxMII-sensitive (Fig. 5, column 3 versus column 2). The ability of
unlabeled -CtxMII (50 nM) to selectively displace
[3H]epibatidine from some cytisine-resistant
populations, but not others, further confirms that -CtxMII interacts
only with a subset of cytisine-resistant
[3H]epibatidine-binding sites.
The regional densities of 125I--CtxMII-binding
sites and -CtxMII-sensitive
[3H]epibatidine-binding sites were compared
directly (Fig. 6). The distributions of specific
125I--CtxMII and -CtxMII-sensitive
[3H]epibatidine-binding sites largely
coincided, but numbers of 125I--CtxMII-binding
sites were consistently lower that those -CtxMII-sensitive [3H]epibatidine-binding sites. This occurred
because a saturating [3H]epibatidine
concentration was used, whereas [125I]CtxMII
was used at a concentration below its Kd
value. However, in six regions more -CtxMII-sensitive
[3H]epibatidine binding was found than would be
predicted from the density of 125I--CtxMII
binding sites (Fig. 6). This finding needs to be interpreted with some
caution: all of these regions contained high levels of
[3H]epibatidine binding, and the additional
-CtxMII-sensitive [3H]epibatidine binding
sites represent a small portion of total binding. However, if the
discrepancy is real, it may represent evidence for a second
-CtxMII-sensitive [3H]epibatidine-binding
nAChR population in a small number of nuclei. A relatively high
unlabeled -CtxMII concentration (50 nM) was used to displace
[3H]epibatidine binding, so these putative
sites could have a relatively low affinity for -CtxMII (making it
undetectable by direct binding of 0.5 nM
125I--CtxMII). Thus, although -CtxMII
displays good selectivity for its primary site of action versus
[3H]nicotine and
125I--Bgt binding sites, high concentrations
may distinguish less well between subtypes of cytisine-resistant
[3H]epibatidine binding. As a result, it is
uncertain whether 125I--CtxMII binding to the
medial habenula/interpeduncular nucleus tract reflects faint
cross-labeling to the high density of other cytisine-resistant
[3H]epibatidine binding sites, or the
expression of a minor 125I--CtxMII binding population.
Physiological Relevance of -CtxMII-Binding nAChRs.
Although
-CtxMII binding nAChRs are relatively rare, they probably exert
important physiological effects. -CtxMII inhibits a component of
nicotine-evoked mouse striatal synaptosomal
[3H]dopamine release with an
IC50 value of 2 nM (Grady et al., 1997), similar
to the binding affinity reported here (Ki
versus [3H]epibatidine = 2.7 nM). Similar
IC50 values have also been reported for
-CtxMII inhibition of functional responses in rat and avian preparations (Kulak et al., 1997; Ullian et al., 1997; Kaiser et al.,
1998). The similar affinities for binding and functional measures
strongly imply a competitive mode of antagonism for -CtxMII in these
preparations. Although less than 15% of
[3H]epibatidine binding in mouse striatum is
-CtxMII-sensitive, about 50% of nicotine-evoked
[3H]dopamine release is inhibited by -CtxMII
(Grady et al., 1997). This disproportionate -CtxMII sensitivity may
arise because of preferential -CtxMII-sensitive nAChR location on
dopaminergic termini.
Identity of -CtxMII-Binding nAChRs.
Marks et al. (1998)
noted a resemblance between the patterns of 3 nAChR subunit mRNA
expression and cytisine-resistant
[3H]epibatidine binding in mouse brain. Direct
comparison of the two measures in brain slices prepared from the same
animals reinforced this initial impression (Fig. 5). The highest
densities of both 3 hybridization and cytisine-resistant
[3H]epibatidine binding are found in the medial
habenula and accessory olfactory bulbs, and in many nuclei 3
hybridization and cytisine-resistant binding are completely
superimposed. The majority of the remaining cytisine-resistant binding
sites are found in regions known to be innervated by 3 mRNA
expressing regions. Cytisine-resistant nAChRs may also contain other
subunits: for instance, the 6 subunit is extensively coexpressed
with 3 in rat brain (LeNovere et al., 1996), and Vailati et al.
(1999) have shown that 6-containing nAChRs also represent a class of
cytisine-resistant nAChRs (Ki values versus
[3H]epibatidine binding for cytisine and
epibatidine = 11 nM and 20 pM, respectively). nAChRs containing
6 also bind -CtxMII with moderate affinity
(Ki versus
[3H]epibatidine = 66 nM), making them
possible candidates for the putative second, lower affinity
-CtxMII-binding site discussed previously.
3 subunits in
-CtxMII-binding sites. -CtxMII is a selective antagonist of heterologously expressed rat 32 nAChRs in X. laevis
oocytes (Cartier et al., 1996) and human 32 in human embryonic
kidney 293 cells (Crona et al., 1997). Parker et al. (1998) showed that X. laevis oocyte-expressed rat 32 nAChRs bind
[3H]epibatidine with high affinity and have a
low cytisine affinity. Furthermore, the pattern of -CtxMII-sensitive
[3H]epibatidine binding was similar to that
described by Schultz et al. (1991) for -Bgt-insensitive
[125I]neuronal Bgt binding. Although neuronal
Bgt exhibits complex kinetics of interaction at a variety of nAChR
subtypes (Papke et al., 1993), it is a comparatively selective
antagonist of heterologously expressed 32 nAChRs (Luetje et al.,
1990). Together, these data suggest that mouse brain
-CtxMII-sensitive [3H]epibatidine binding
occurs at receptors containing 3 and 2 subunits.
The remaining cytisine-resistant
[3H]epibatidine binding was found in regions
expressing high levels of both the 3 and 4 nAChR subunits
(Dinelly-Miller and Patrick, 1992). Again, Parker et al. (1998) report
that 34-subtype nAChRs bind
[3H]epibatidine with detectable affinity and
exhibit low cytisine affinity. Thus, it is possible that in mouse
brain, the sites less sensitive to -CtxMII cytisine-resistant
binding are a combination of (minimally) 3 and 4 subunits.
In conclusion, this study shows that -CtxMII is a potent, selective,
competitive antagonist at a novel population of mouse brain nAChRs.
125I--CtxMII was used in this study to
quantify the high -CtxMII affinity population and map its
distribution. Selective inhibition with cytisine and -CtxMII
revealed high affinity [3H]epibatidine binding
at three nAChR pharmacological subtypes. The largest
[3H]epibatidine binding population was highly
cytisine-sensitive and corresponds to the high affinity
()-[3H]nicotine binding, 42 nAChR
subtype. Cytisine-resistant sites are likely to be 3
subunit-containing and exhibited differential -CtxMII sensitivity
that may be caused by differing subunit composition.
| |
Footnotes |
|---|
Received October 18, 1999; Accepted February 3, 2000
This work was supported by National Institute on Drug Abuse Grants DA12242, DA03194, and DA10156, National Institutes of Mental Health Grant MH53631, and National Institute of General Medical Sciences Grant GM48677. A.C.C. is supported, in part, by Research Scientist Award DA00197 from the National Institute on Drug Abuse.
Send reprint requests to: Dr. A.C. Collins, Institute for Behavioural Genetics, University of Colorado, Campus Box 447, Boulder, CO 80303-0447.
| |
Abbreviations |
|---|
nAChR, nicotinic acetylcholine receptor;
CtxMII, conotoxin MII;
Bgt, bungarotoxin;
PEI, polyethylenimine;
Yo--CtxMII, N-terminal tyrosine-tagged -conotoxin
MII;
TFA, trifluoroacetic acid;
SSC, standard saline citrate.
| |
References |
|---|
|
Top
Abstract
Introduction
Experimental Procedures
Results
Discussion
References
|
|---|
-conotoxin which targets 32 nicotinic acetylcholine receptors.
J Biol Chem
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407-413[Medline].-bungarotoxin and -conotoxin IMI and -conotoxin MII on human nicotinic receptor-mediated calcium responses and ionic currents.
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23:
155.6.4, are distributed in multiple areas of the rat central nervous system.
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16:
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5263-52706 subunit mRNA is selectively concentrated in catecholaminergic nuclei of the rat brain.
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2428-2439[Medline].-bungarotoxin.
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427-436[Abstract]. subunit domain regulates the kinetics of inhibition by neuronal-bungarotoxin.
Proc R Soc Lond B Biol Sci
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141-148[Medline].2 and 4 subunits confer large differences in agonist binding affinity.
Mol Pharmacol
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1132-1139-bungarotoxin binding protein cDNAs and mAbs reveal subtypes of this branch of the ligand-gated ion channel gene superfamily.
Neuron
5:
35-48[Medline].7: a nicotinic cation channel highly permeable to calcium.
J Neurosci
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596-604[Abstract].6-containing nicotinic receptors are present in chick retina.
Mol Pharmacol
56:
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O. Salminen, J. A. Drapeau, J. M. McIntosh, A. C. Collins, M. J. Marks, and S. R. Grady Pharmacology of {alpha}-Conotoxin MII-Sensitive Subtypes of Nicotinic Acetylcholine Receptors Isolated by Breeding of Null Mutant Mice Mol. Pharmacol., June 1, 2007; 71(6): 1563 - 1571. [Abstract] [Full Text] [PDF]
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L. Azam and J. M. McIntosh Characterization of Nicotinic Acetylcholine Receptors That Modulate Nicotine-Evoked [3H]Norepinephrine Release from Mouse Hippocampal Synaptosomes Mol. Pharmacol., September 1, 2006; 70(3): 967 - 976. [Abstract] [Full Text] [PDF]
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M. Quik and J. M. McIntosh Striatal {alpha}6* Nicotinic Acetylcholine Receptors: Potential Targets for Parkinson's Disease Therapy J. Pharmacol. Exp. Ther., February 1, 2006; 316(2): 481 - 489. [Abstract] [Full Text] [PDF]
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T. Endo, Y. Yanagawa, K. Obata, and T. Isa Nicotinic Acetylcholine Receptor Subtypes Involved in Facilitation of GABAergic Inhibition in Mouse Superficial Superior Colliculus J Neurophysiol, December 1, 2005; 94(6): 3893 - 3902. [Abstract] [Full Text] [PDF]
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A. M. Marritt, B. C. Cox, R. P. Yasuda, J. M. McIntosh, Y. Xiao, B. B. Wolfe, and K. J. Kellar Nicotinic Cholinergic Receptors in the Rat Retina: Simple and Mixed Heteromeric Subtypes Mol. Pharmacol., December 1, 2005; 68(6): 1656 - 1668. [Abstract] [Full Text] [PDF]
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S. E. McCallum, N. Parameswaran, T. Bordia, J. M. McIntosh, S. R. Grady, and M. Quik Decrease in {alpha}3*/{alpha}6* Nicotinic Receptors but Not Nicotine-Evoked Dopamine Release in Monkey Brain after Nigrostriatal Damage Mol. Pharmacol., September 1, 2005; 68(3): 737 - 746. [Abstract] [Full Text] [PDF]
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C. Gotti, M. Moretti, F. Clementi, L. Riganti, J. M. McIntosh, A. C. Collins, M. J. Marks, and P. Whiteaker Expression of Nigrostriatal {alpha}6-Containing Nicotinic Acetylcholine Receptors Is Selectively Reduced, but Not Eliminated, by {beta}3 Subunit Gene Deletion Mol. Pharmacol., June 1, 2005; 67(6): 2007 - 2015. [Abstract] [Full Text] [PDF]
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A. Lai, N. Parameswaran, M. Khwaja, P. Whiteaker, J. M. Lindstrom, H. Fan, J. M. McIntosh, S. R. Grady, and M. Quik Long-Term Nicotine Treatment Decreases Striatal {alpha}6* Nicotinic Acetylcholine Receptor Sites and Function in Mice Mol. Pharmacol., May 1, 2005; 67(5): 1639 - 1647. [Abstract] [Full Text] [PDF]
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M. Quik, S. Vailati, T. Bordia, J. M. Kulak, H. Fan, J. M. McIntosh, F. Clementi, and C. Gotti Subunit Composition of Nicotinic Receptors in Monkey Striatum: Effect of Treatments with 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine or L-DOPA Mol. Pharmacol., January 1, 2005; 67(1): 32 - 41. [Abstract] [Full Text] [PDF]
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O. Salminen, K. L. Murphy, J. M. McIntosh, J. Drago, M. J. Marks, A. C. Collins, and S. R. Grady Subunit Composition and Pharmacology of Two Classes of Striatal Presynaptic Nicotinic Acetylcholine Receptors Mediating Dopamine Release in Mice Mol. Pharmacol., June 1, 2004; 65(6): 1526 - 1535. [Abstract] [Full Text] [PDF]
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J. M. McIntosh, L. Azam, S. Staheli, C. Dowell, J. M. Lindstrom, A. Kuryatov, J. E. Garrett, M. J. Marks, and P. Whiteaker Analogs of {alpha}-Conotoxin MII Are Selective for {alpha}6-Containing Nicotinic Acetylcholine Receptors Mol. Pharmacol., April 1, 2004; 65(4): 944 - 952. [Abstract] [Full Text]
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S. L. Parker, Y. Fu, K. McAllen, J. Luo, J. M. McIntosh, J. M. Lindstrom, and B. M. Sharp Up-Regulation of Brain Nicotinic Acetylcholine Receptors in the Rat during Long-Term Self-Administration of Nicotine: Disproportionate Increase of the {alpha}6 Subunit Mol. Pharmacol., March 1, 2004; 65(3): 611 - 622. [Abstract] [Full Text] [PDF]
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C. Cui, T. K. Booker, R. S. Allen, S. R. Grady, P. Whiteaker, M. J. Marks, O. Salminen, T. Tritto, C. M. Butt, W. R. Allen, et al. The {beta}3 Nicotinic Receptor Subunit: A Component of {alpha}-Conotoxin MII-Binding Nicotinic Acetylcholine Receptors that Modulate Dopamine Release and Related Behaviors J. Neurosci., December 3, 2003; 23(35): 11045 - 11053. [Abstract] [Full Text] [PDF]
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H. N. Nguyen, B. A. Rasmussen, and D. C. Perry Subtype-Selective Up-Regulation by Chronic Nicotine of High-Affinity Nicotinic Receptors in Rat Brain Demonstrated by Receptor Autoradiography J. Pharmacol. Exp. Ther., December 1, 2003; 307(3): 1090 - 1097. [Abstract] [Full Text] [PDF]
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A. J. Mogg, P. Whiteaker, J. M. McIntosh, M. Marks, A. C. Collins, and S. Wonnacott Methyllycaconitine Is a Potent Antagonist of alpha -Conotoxin-MII-Sensitive Presynaptic Nicotinic Acetylcholine Receptors in Rat Striatum J. Pharmacol. Exp. Ther., July 1, 2002; 302(1): 197 - 204. [Abstract] [Full Text] [PDF]
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S. R. Grady, K. L. Murphy, J. Cao, M. J. Marks, J. M. McIntosh, and A. C. Collins Characterization of Nicotinic Agonist-Induced [3H]Dopamine Release from Synaptosomes Prepared from Four Mouse Brain Regions J. Pharmacol. Exp. Ther., May 1, 2002; 301(2): 651 - 660. [Abstract] [Full Text] [PDF]
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P. Whiteaker, C. G. Peterson, W. Xu, J. M. McIntosh, R. Paylor, A. L. Beaudet, A. C. Collins, and M. J. Marks Involvement of the alpha 3 Subunit in Central Nicotinic Binding Populations J. Neurosci., April 1, 2002; 22(7): 2522 - 2529. [Abstract] [Full Text] [PDF]
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R. S. Broide, R. Salas, D. Ji, R. Paylor, J. W. Patrick, J. A. Dani, and M. De Biasi Increased Sensitivity to Nicotine-Induced Seizures in Mice Expressing the L250T alpha 7 Nicotinic Acetylcholine Receptor Mutation Mol. Pharmacol., March 1, 2002; 61(3): 695 - 705. [Abstract] [Full Text] [PDF]
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N. Champtiaux, Z.-Y. Han, A. Bessis, F. M. Rossi, M. Zoli, L. Marubio, J. M. McIntosh, and J.-P. Changeux Distribution and Pharmacology of alpha 6-Containing Nicotinic Acetylcholine Receptors Analyzed with Mutant Mice J. Neurosci., February 15, 2002; 22(4): 1208 - 1217. [Abstract] [Full Text] [PDF]
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J. M. Kulak, J. M. McIntosh, and M. Quik Loss of Nicotinic Receptors in Monkey Striatum after 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine Treatment Is Due to a Decline in alpha -Conotoxin MII Sites Mol. Pharmacol., January 1, 2002; 61(1): 230 - 238. [Abstract] [Full Text] [PDF]
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P. H. Lee, M. Schmidt, and W. C. Hall Excitatory and Inhibitory Circuitry in the Superficial Gray Layer of the Superior Colliculus J. Neurosci., October 15, 2001; 21(20): 8145 - 8153. [Abstract] [Full Text] [PDF]
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M. Quik, Y. Polonskaya, J. M. Kulak, and J. M. McIntosh Vulnerability of 125I-{alpha}-Conotoxin MII Binding Sites to Nigrostriatal Damage in Monkey J. Neurosci., August 1, 2001; 21(15): 5494 - 5500. [Abstract] [Full Text] [PDF]
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,
IC50 = 103 ± 41 pM,
nH = 
, IC50 = 18 ± 8 nM,
nH = 
, IC50 = 481 ± 54 nM,
nH = 
