From the Receptor Biology Laboratory, Department of Physiology and Biophysics, Mayo Foundation, Rochester, Minnesota 55905
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ABSTRACT |
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To identify residues in the neuronal
7 acetylcholine subunit that confer high affinity
for the neuronal-specific toxin conotoxin ImI (CTx ImI), we constructed
7-
1 chimeras containing segments of the
muscle
1 subunit inserted into equivalent positions of the neuronal
7 subunit. To achieve high expression in
293 human embryonic kidney cells and formation of homo-oligomers, we
joined the extracellular domains of each chimera to the M1 junction of the 5-hydroxytryptamine-3 (5HT-3) subunit. Measurements of CTx ImI
binding to the chimeric receptors reveal three pairs of residues in
equivalent positions of the primary sequence that confer high affinity
of CTx ImI for
7/5HT-3 over
1/5HT-3
homo-oligomers. Two of these pairs,
7Trp55/
1Arg55 and
7Ser59/
1Gln59,
are within one of the four loops that contribute to the traditional non-
subunit face of the muscle receptor binding site. The third pair,
7Thr77/
1Lys77, is
not within previously described loops of either the
or non-
faces and may represent a new loop or an allosterically coupled loop.
Exchanging these residues between
1 and
7
subunits exchanges the affinities of the binding sites for CTx ImI,
suggesting that the
7 and
1 subunits,
despite sequence identity of only 38%, share similar protein
scaffolds.
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INTRODUCTION |
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The two neurotransmitter binding sites of muscle nicotinic
acetylcholine receptors
(AChR)1 are generated by
apposition of pairs of nonequivalent subunits, 1/
,
1/
, and
1/
. By contrast, the
binding sites of
7 neuronal nicotinic receptors are
generated by apposition of pairs of identical subunits,
7/
7 (1). Because only the
7 subunit contributes to both faces of the ligand
binding site, one can study the traditional
and non-
faces by
mutagenesis of a single
7 cDNA.
Ligand affinities of 7 neuronal and muscle AChRs differ
owing to the different subunits that form their binding site
interfaces. For example, the muscle-specific
-conotoxins MI, GI, and
SI bind with high affinity to muscle receptors, whereas they bind with low affinity to
7 neuronal receptors (2). On the other
hand,
-conotoxin ImI (CTx ImI) binds with high affinity to
7 receptors but binds with low affinity to muscle
receptors (3). As the only known
7-specific
-conotoxin, CTx ImI is a valuable probe of the homo-oligomeric
7 binding site.
Understanding of the 7 binding site has been limited by
low expression of
7 receptors in mammalian cell lines
(4, 5). Part of the problem appears due to cell type, as neuronal cell lines promote expression of
7 receptors more efficiently
than non-neuronal cell lines (6). The sequence of the subunit also affects expression, as chimeras derived from
7 and 5HT-3
subunits express high levels of functional homo-oligomers in
non-neuronal cells (7). Joining the
7 extracellular
domain to the M1 junction of 5HT-3 permits expression in 293 HEK cells
and preserves the pharmacology of the
7 binding site
(7). Thus, inserting portions of 5HT-3 sequence is a powerful tool to
express receptors with an intact
7 binding site.
Studies of the muscle AChR have led to a basic scaffold hypothesis to
account for observations that residues in equivalent positions of the
homologous ,
, and
subunits contribute similarly to ligand
affinity (8-10). The hypothesis postulates that owing to their high
degree of homology, these subunits fold into similar peptide scaffolds
such that residues equivalent in the linear sequence occupy equivalent
positions in three-dimensional space. Thus, ligand affinity for a
particular site is dictated by differences in primary structure rather
than differences in secondary or tertiary structures.
The primary sequence of the 7 subunit reveals conserved
residues that contribute to both the
and non-
faces of the
ligand binding site. Within the
face of the binding site,
7 and
1 share conserved aromatic residues
that stabilize agonists, including
7Tyr92,
7Trp148,
7Tyr187,
and
7Tyr195 (11-13). On the other hand,
7 and non-
muscle subunits (
,
, and
) share
the conserved
7Trp55, which contributes to
binding of agonists and antagonists (14, 15). Thus, despite only
31-38% sequence homology with muscle subunits,
7
subunits maintain conserved residues that contribute to both faces of
the ligand binding site.
The experiments described herein identify residues of the
7 binding site that determine selectivity for the
competitive antagonist CTx ImI and examine the question of whether
7 neuronal and
1 muscle subunits form
similar protein scaffolds. By constructing chimeras composed of
7 and
1 subunits, we identified three
pairs of residues in equivalent positions of the subunits that confer selectivity of CTx ImI for binding sites formed from
7
versus
1 subunits. Moreover,
exchanging these three selectivity determinants between
7 and
1 subunits exchanges the affinity
conferred by the subunit, indicating that
7 and
1 subunits share similar protein scaffolds.
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EXPERIMENTAL PROCEDURES |
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Materials--
125I-Labeled -bungarotoxin
(
-bgt) was purchased from NEN Life Science Products,
d-tubocurarine chloride from ICN Pharmaceuticals, (+)-epibatidine and methyllycaconitine from Research Biochemicals, 293 human embryonic kidney cell line (293 HEK) from the American Type
Culture Collection, and unlabeled
-bgt from Sigma. Human
7 and rat 5HT-3 subunit cDNAs were generously
provided by Drs. John Lindstrom and William Green. Sources of the human
acetylcholine receptor subunit cDNAs were as described previously
(16).
Synthesis and Purification of Conotoxin ImI-- Conotoxin ImI was synthesized by standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry on an Applied Biosystems 431A peptide synthesizer. During synthesis, cysteine (S-triphenylmethyl)-protecting groups were incorporated at cysteines 3 and 12, and acetamidomethyl-protecting groups were incorporated at cysteines 2 and 8. The linear peptide was purified by reversed phase high performance liquid chromatography using a Vydac C18 preparative column with trifluoroacetic acid/acetonitrile buffers. Two intramolecular disulfide bridges were formed as follows: the cysteine S-triphenylmethyl-protecting groups of cysteines 3 and 12 were removed during trifluoroacetic acid cleavage of the linear peptide from the support resin, and the peptide was oxidized by molecular oxygen to form the 3-12 disulfide by stirring in 50 mM ammonium bicarbonate buffer, pH 8.5, at 25 °C for 24 h. The peptide was lyophilized prior to the formation of the second bridge. The acetamidomethyl-protecting groups on cysteine 2 and 8 were removed oxidatively by iodine as described (17) except that the peptide/iodine reaction was allowed to progress 16 h prior to carbon tetrachloride extraction. Residual iodine was separated from the pure product by high performance liquid chromatography, and CTx ImI was characterized by mass spectrometry.
Mutagenesis and Expression in HEK Cells--
Acetylcholine
receptor subunit cDNAs were subcloned into the
cytomegalovirus-based expression vector pRBG4 (18). Mutant cDNAs
were constructed by bridging naturally occurring or mutagenically installed restriction sites with double-stranded oligonucleotides. The
chimeras are named as follows: the first subunit is the amino-terminal sequence of the chimera, the number following gives the position of the
chimeric junction, and the final subunit gives the subunit from which
the carboxyl-terminal sequence of the extracellular domain is taken.
The extracellular domains of all chimeras are joined at M1 to the rat
5HT-3 sequence. Chimera 7/5HT-3
(
7200/5HT-3) was constructed by bridging a 58-bp
synthetic oligonucleotide from a TfiI site in
7 to an EcoRV site in rat 5HT-3. Chimera
1/5HT-3 (
1205/5HT-3) was constructed by
bridging a 69-bp synthetic oligonucleotide from a DraIII
site in
1 to a StuI site in rat 5HT-3. All
constructs were confirmed by dideoxy sequencing. HEK cells were
transfected with wild type or mutant cDNAs using calcium phosphate
precipitation as described (18). Two days after transfection, intact
cells were harvested by gentle agitation in phosphate-buffered saline
with 5 mM EDTA.
Ligand Binding Measurements--
Ligand binding to intact cells
was measured by competition against the initial rate of
125I-labeled -bgt binding (18). The cells were briefly
centrifuged, resuspended in potassium Ringer's solution, and divided
into aliquots for ligand binding. Potassium Ringer's solution contains
140 mM KCl, 5.4 mM NaCl, 1.8 mM
CaCl2, 1.7 mM MgCl2, 25 mM HEPES, and 30 mg/liter bovine serum albumin, adjusted to
a pH of 7.4 with 10 mM NaOH. Specified concentrations of
ligand were added 30 min prior to addition of 3.75 nM
125I-labeled
-bgt, which was allowed to bind for 15 min
to occupy approximately half of the surface receptors. Binding was
terminated by addition of 2 ml of potassium Ringer's solution
containing 600 µM of d-tubocurarine chloride.
All experiments were performed at 24 ± 2 °C. Cells were
harvested by filtration through Whatman GF-B filters using a Brandel
cell harvester and washed three times with 3 ml of potassium Ringer's
solution. Prior to use, filters were soaked in potassium Ringer's
solution containing 4% skim milk. Nonspecific binding was determined
in the presence of 10 nM
-bgt and was typically 1% of
the total number of binding sites. The total number of binding sites
was determined by incubation with toxin for 120 min. The initial rate
of toxin binding was calculated as described previously (19) to yield
the fractional occupancy of ligand. Binding measurements were analyzed
according to the Hill equation: 1
Y = 1/(1 + ([ligand]/Kapp)nH), where Y is
fractional occupancy of ligand, Kapp is the
apparent dissociation constant and nH is the Hill
coefficient. Parameter estimates and standard errors were obtained
using UltraFit (BIOSOFT). For multiple experiments, means of the
individual fitted parameters and standard deviations are presented
(Tables I and II).
Sucrose Gradient Centrifugation--
Two days after
transfection, 293 HEK cells expressing various receptors were harvested
by gentle agitation in phosphate-buffered saline and resuspended in
high potassium Ringer's solution. Following labeling with 3.75 nM 125I-labeled -bgt for 2 h, cells
were washed free of unbound radioactivity. Samples for sucrose gradient
centrifugation were solubilized in 1 ml of Triton X-100 buffer (0.6%
Triton X-100, 150 mM NaCl, 5 mM EDTA, 50 mM Tris, 35 µg/ml phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 1 µg/ml pepstatin A, pH 7.5). Extracts were layered on sucrose gradients (3-30%) and centrifuged for 22 h at
40,000 rpm, and fractions were collected and counted with a
counter. Radioactivity in each fraction was normalized to that of the
fraction containing the maximum radioactivity in each gradient.
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RESULTS |
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Characterization of 7/5HT-3 and
1/5HT-3 Chimeric Receptors--
Previous studies
described construction of a chimera containing the extracellular domain
of chick
7 joined to the M1 junction of the rat 5HT-3
subunit (
7201/5HT-3) (7). The studies further showed
that addition of 5HT-3 sequence maintained ligand recognition properties of the native
7 binding site. We constructed
a similar chimera by joining the extracellular domain of human
7 to the rat 5HT-3 subunit, with the chimera junction
formed at position 200 (
7200/5HT-3) (Fig.
1A). To determine whether our
human
7/5HT-3 receptor has similar ligand recognition
properties to wild type
7, we expressed the constructs
in 293 HEK cells and measured binding of agonists and antagonists by
competition against the initial rate of 125I-labeled
-bgt binding. Although expression of
7/5HT-3
receptors exceeds that of wild type
7 receptors by
1000-fold (see Fig. 1 legend), the competitive antagonists CTx ImI and
methyllycaconitine and the agonist (+)-epibatidine bind with identical
affinities to the two types of receptors (Fig. 1, B-D;
Table I). Thus, the
7
ligand binding domain is preserved in
7/5HT-3 receptors,
and expression is greatly enhanced by addition of 5HT-3 sequence.
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Determinants of CTx ImI Selectivity Identified Using
7-
1 Chimeras--
We constructed a
series of
7-
1 chimeras to identify
residues of the
7 receptor that confer the 50-fold
higher affinity of CTx ImI for
7/5HT-3 over
1/5HT-3 receptors. For all chimeras, we joined the
extracellular domains to the M1 junction of the rat 5HT-3 subunit. Our
first informative chimera contained a small insertion of
1 sequence between residues 55 and 77 of the
7 subunit (Fig.
3A). This short segment of
1 sequence fully decreases CTx ImI affinity to that of
the pure
1/5HT-3 chimera (Fig. 3B), suggesting that the 55-77 segment is the entire source of high affinity in
7.
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Dissection of the 55-77 Segment--
We constructed a series of
stepwise chimeras to further localize selectivity determinants within
the 55-77 segment. Starting with our reference chimera
755
177
7, we maintained the
7/
1 junction at position 55 but shifted
the carboxyl-terminal junction from position 77 to position 57 (Fig.
4). Surpassing only one mismatched
residue, the chimera
755
176
7 increases CTx ImI
affinity toward that of
7, suggesting that the pair
7Thr77/
1Lys77
contributes to selectivity. Shifting the junction from position 76 to
59 produces no further change in affinity, but shifting from position
59 to 57 reveals an additional increase in affinity toward that of
7. The last chimera in this series,
755
157
7, falls 3-fold
short of pure
7-like affinity, indicating that residues 55-57 contribute the remaining increment of selectivity. Thus, the
stepwise chimeras reveal at least three subsets of selectivity determinants within the 55-77 segment; one is between positions 55 and
57, the second is between positions 57 and 59, and the third is the
pair
7Thr77/
1Lys77.
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Point Mutants of Selectivity Determinants--
We further
localized selectivity determinants by constructing point mutations in
the 7/5HT-3 cDNA. Beginning with the 55-57 segment,
the point mutation
7W55R decreased CTx ImI affinity by
the same amount observed in the stepwise chimeras. Similarly, mutating
within the 57-59 segment,
7S59Q decreases affinity by the same amount observed in the stepwise chimeras. Finally, the mutation
7T77K decreases affinity as observed in the
chimeras. Thus, three residues,
7Trp55,
7Ser59, and
7Thr77, contribute to CTx ImI selectivity
for
7 (Fig. 5).
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Exchange of Selectivity Determinants between the 7
and
1 Subunits--
To further confirm that
7W55R,
7S59Q, and
7T77K
confer CTx ImI selectivity, we sought to convert
1 to
7 affinity by constructing the converse mutations in the
1/5HT-3 subunit (Fig. 5). Two of the three point
mutants,
1Q59S and
1K77T, maintain good
levels of expression and increase affinity for CTx ImI, as expected
from the chimeras. Combining these two mutations into a single receptor with
1(Q59S/K77T) increases CTx ImI affinity in an
additive manner, again showing that these determinants contribute
independently. However, the third point mutation,
1R55W,
does not express alone nor when present in the triple mutant
1(R55W/Q59S/K77T). The residual affinity between the
double point mutation
1(Q59S/K77T) and
7/5HT-3 equals that conferred by the point mutant
7W55R, further suggesting that basic scaffold of the
1 subunit is similar to that of the
7
subunit.
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DISCUSSION |
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We probed the binding site of the 7 receptor using
the neuronal-specific toxin CTx ImI together with chimeras containing portions of
1 sequence substituted into the
extracellular domain of
7. The results reveal three
pairs of residues in equivalent positions of the subunits that confer
selectivity of CTx ImI for
7 over muscle-like AChRs.
Because exchange of these residues between
7/5HT-3 and
1/5HT-3 exchanges affinity for CTx ImI, the
extracellular domains of
7 and
1 subunits
appear to fold into similar basic scaffolds. This basic scaffold
hypothesis was originally developed to explain ligand selectivity
conferred by subunits with high homology, such as
and
subunits
(49% identity),
and
subunits (47%), and
and
subunits
(53%) (8-10, 18, 21). We find that the hypothesis extends to the less
homologous
7 and
1 subunits, which are
only 38% identical.
To investigate the basis for neuronal specificity of CTx ImI, we needed
to achieve high levels of expression of receptors with 7
binding sites and a homologous, muscle-like frame of reference. As
described by others (7), we find that substituting 5HT-3 sequence from
the M1 domain to the carboxyl terminus markedly increases expression,
yet preserves the ligand recognition properties of the
7
binding site. The increased expression was initially surprising because
the extracellular domain was widely known for its importance in
receptor assembly (22-24). However, studies of
7-
3 chimeras show that formation of
homo-oligomers requires matching of particular residues in the M1 and
M2 transmembrane domains (25). Thus, our results further confirm that
the region carboxyl-terminal to M1 contributes to assembly of
homo-oligomers.
Another surprise is that our 1/5HT-3 construct forms
homo-oligomers on the cell surface. This observation contrasts with expression of the
1 subunit alone, which remains
monomeric and retained within the cell. Thus, 5HT-3 sequence between M1
and the carboxyl terminus promotes homo-oligomer formation. We found differences, however, between
1/5HT-3 and
7/5HT-3 homo-oligomers in their kinetics of
-bgt
dissociation.
-bgt dissociates from
7/5HT-3
homo-oligomers with a single slow rate constant, whereas the toxin
dissociates from
1/5HT-3 homo-oligomers with one rapid and one slow rate constant. Thus, despite the presence of only one type
of subunit, binding sites in
1/5HT-3 homo-oligomers appear to be nonequivalent. The origin of nonequivalent sites is not
known, but they may arise during the course of subunit folding and
oligomerization that produces a fully assembled pentamer with high
affinity for
-bgt (26). Because acquisition of the toxin binding
site is associated with a protein folding event, and folding requires
interaction with specific subunits, successive addition of
1/5HT-3 subunits may produce unusual interactions, leading to nonequivalence of the binding sites.
In addition, we find that 1/5HT-3 and
2
muscle receptors bind CTx ImI with similar
low affinities. Because the muscle receptor contains the stabilizing
residues
Trp55/
Trp57, and the
1/5HT-3 homo-oligomer contains the destabilizing residue
1Arg55, one might expect higher affinity of
the muscle receptor compared with that of
1/5HT-3.
Resolution of the apparent paradox likely lies in the different
contributions of the (
) face in the two types of receptors, because
they contain identical (+) faces. Potentially, residues flanking the
three determinants identified here, but unique to the
and
subunits, may decrease affinity, despite the presence of
Trp55/
Trp57 in the muscle receptor. The
unique residue differences may cause small changes in the protein
scaffold and prevent interaction between CTx ImI and
Trp55/
Trp57, as well as other
determinants of affinity. In addition, the low affinity may result from
reorientation of CTx ImI at the binding site, and the low affinity may
be due to stabilization by residues common to the
1,
, and
subunits.
We chose CTx ImI to probe the 7 binding site because it
is a constrained two-loop structure owing to its two disulfide bridges, similar to the muscle-specific
-conotoxins (Fig.
6). CTx ImI contains four amino acids in
its first loop and three in its second, whereas muscle-specific
-conotoxins contain three and five amino acids in its first and
second loops, respectively. In addition, CTx ImI contains basic
residues in both loops (Arg7 and Arg11) and an
acidic residue in the first loop (Asp5), unlike
muscle-specific
-conotoxins; these unique residues may further
contribute to neuronal specificity of CTx ImI.
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Photoaffinity labeling and mutagenesis studies establish that the
ligand binding sites of the muscle AChR contain contributions of both
and non-
subunits. Residues of the
or (+) face of the
binding site cluster into three linearly separate regions, leading to a
three-loop model of the
subunit contribution to the binding site.
Similarly, residues of the non-
or (
) face of the binding site
cluster into four separate regions of the linear sequence, leading to a
four-loop model of the non-
contribution to the site (reviewed in
Refs. 27 and 28). Studies of the highly homologous
,
, and
subunits show that residues in equivalent positions of the subunit make
equivalent contributions to ligand affinity (8-10, 18, 21). Thus,
the (
) face contributed by these subunits harbors virtually
superimposable peptide scaffolds. Here, we extend the basic scaffold
hypothesis to the less homologous
7 and
1
subunits by showing that exchanging selectivity determinants between
7 and
1 subunits exchanges affinity for
CTx ImI.
Two of the three pairs of 7 selectivity determinants,
7Trp55/
1Arg55 and
7Ser59/
1Gln59,
are within one of the four loops that contribute to the (
) face
of the binding site. The contribution of
7Trp55 was first demonstrated by
photoaffinity labeling with
d-[3H]tubocurarine, which labeled the
equivalent residues
Trp55/
Trp57 in
Torpedo receptors (29). Subsequent mutagenesis of
7201/5HT-3 homo-oligomers revealed contributions of the
equivalent residue in chick
7Trp54 to
agonist and antagonist affinity (15). The second pair in this segment,
7Ser59/
1Gln59, is
equivalent to the residues in non-
subunits in muscle receptors,
Asp59/
Ala61, which contribute to
dimethyl-d-tubocurarine selectivity of the adult receptor
(9). Thus, approximately half of the neuronal specificity of CTx ImI is
due to stabilization by one of the four loops at the (
) face of the
subunit.
The third pair of selectivity determinants,
7Thr77/
1Lys77, is
not contained within previously described loops of either the
or
non-
faces of the binding site. One member of this pair,
1Lys77, is immediately carboxyl-terminal to
the main immunogenic region, which extends from the tip of the
extracellular lobe of the AChR (30).
1Lys77
may be far enough from the main immunogenic region that it can fold
back to the binding site. We cannot say whether
7Thr77/
1Lys77
contributes directly or allosterically to the binding site. However, the positively charged
1Lys77 may repel one
of the arginine side chains in CTx ImI, whereas
7Thr77 may be neutral or stabilize CTx ImI
through hydrogen bonding. We observed equal and opposite changes in
free energy of binding with
7T77K and
1K77T, suggesting direct contributions to affinity. A
long range interaction would not be expected to show such equal and
opposite free energy changes because it would have to propagate through
intervening residues.
The overall results reveal three pairs of equivalent residues in the
7 and
1 subunits that confer selectivity
of CTx ImI and show that the extracellular domains of
7
and
1 subunits fold into similar basic scaffolds. The
precise contacts between CTx ImI and
7 await experiments
that mutate residues in both the toxin and the receptor.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Receptor Biology
Laboratory, Dept. of Physiology and Biophysics, Mayo Foundation, 200 First St. S.W., Rochester, Minnesota 55905. Tel.: 507-284-5612; Fax:
507-284-9420; E-mail: sine.steven{at}mayo.edu.
1
The abbreviations used are: AChR, acetylcholine
receptor; CTx ImI, -conotoxin ImI; HEK, human embryonic kidney;
-bgt,
-bungarotoxin; 5HT, 5-hydroxytryptamine.
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REFERENCES |
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