Site-specific Charge Interactions of 
-Conotoxin MI with the
Nicotinic Acetylcholine Receptor*
Rao V. L.
Papineni
,
Jovanny Ulloa
Sanchez§,
Krishna
Baksi§,
Irmgard Ursula
Willcockson¶, and
Steen E.
Pedersen
From the Department of Molecular Physiology and
Biophysics, Baylor College of Medicine, Houston, Texas 77030 and the
§ Department of Anatomy and Cell Biology, Universidad
Central del Caribe, Bayamon, Puerto Rico 00960-6032
Received for publication, March 15, 2001, and in revised form, April 20, 2001
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ABSTRACT |
We have tested the importance of charge
interactions for 
-conotoxin MI binding to the nicotinic
acetylcholine receptor (AChR). Ionic residues on -conotoxin MI were
altered by site-directed mutagenesis or by chemical modification. In
physiological buffer, removal of charges at the N terminus,
His-5, and Lys-10 had small (2-4-fold) effects on binding
affinity to the mouse muscle AChR and the Torpedo AChR. It
was also demonstrated that conotoxin had no effect on the
conformational equilibrium of either receptor, as assessed by the
effects of the noncompetitive antagonist proadifen on conotoxin binding
and, conversely, the effect of conotoxin on the affinity of
phencyclidine, proadifen, and ethidium. Conotoxin displayed higher
binding affinity in low ionic strength buffer; neutralization of Lys-10
and the N terminus by acetylation blocked this affinity shift at the
site but not at the 
site. It is concluded that Ctx
residues Lys-10 and the N terminal interact with oppositely charged
receptor residues only at the site, and the two sites have
distinct arrangements of charged residues. Ethidium fluorescence
experiments demonstrated that conotoxin is formally competitive with a
small cholinergic ligand, tetramethylammonium. Thus, -conotoxin MI
appears to interact with the portion of the binding site responsible
for stabilizing agonist cations but does not do so with a cationic
residue and is, consequently, incapable of inducing a conformational change.
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INTRODUCTION |
The muscle type nicotinic acetylcholine receptor
(AChR)1 is a pentameric
ligand-gated cation channel with a subunit stoichiometry of
2
(see Ref. 1 for review). The homologous
subunits surround a central pore that forms the ion conductive
pathway. The AChR comprises two extracellular ligand-binding
sites for ACh that lie at the interfaces between the -- and the
--subunits (2, 3). Competitive antagonists and natural toxins
often have different affinities for the two sites that arise from the
distinct contributions of the - and -subunits to each site (4).
For example, d-tubocurarine binds the sites of the
Torpedo AChR with an affinity more than 100-fold greater
than for the site (5).
A similar characteristic was observed for the marine snail
-conotoxins, some of which bind the mouse muscle AChR sites with affinities that differ by more than 10,000-fold, where the site
is bound with higher affinity (see Ref. 6 for review). The differences
in affinity have been attributed to three specific residues in mouse
muscle AChR -subunit (Lys-34, Tyr-111, and Phe-172) and the
corresponding homologous residues in the -subunit (7). Recent work
(8) has also identified -subunit residues that affect binding of
-conotoxin MI (Ctx) in a site-selective manner, which suggests that
Ctx may bind with distinct configurations at the two sites. Ctx
residues Gly-9 and Pro-6 (see Table
I) and residues on the -subunit and
between Ala-7 and Pro-6 and binding site residues in the -subunit
appear to stabilize binding through hydrophobic interactions (9).
Significant affinity differences have also been observed for binding to
the Torpedo AChR (10-12); however, in this case the
site is bound with higher affinity. Chiara et al. (12)
showed that one AChR residue, Tyr-111, and its -subunit homolog,
Arg-113, accounted for much of the affinity differences in
Torpedo AChR. They further suggested that the affinity
change arose from specific charge repulsion between Arg-113 and Ctx
residue Lys-10. This Ctx residue is highly conserved among
-conotoxins; a cationic amino acid is present in this position (see
Table I), with the exception of -conotoxin SI, where it is in the
adjacent position, or in SII where it is absent (Table I). The
homologous residue in -conotoxin GI (Arg-9) was identified as being
responsible for the high affinity binding of conotoxins to the
site; when it was substituted for a proline, affinity decreased
dramatically for the site (13). This observation was consistent
with the low affinities of -conotoxins SI and SII for the
site, which have a Pro at this position.
Several studies suggest that ionic interactions may not be the critical
aspect of the role of this residue in binding. Analysis of pairwise
interactions in the mouse muscle AChR failed to reveal strong
interactions between Tyr-113 and Ctx Lys-10 (9). However, that
analysis did not examine a charge-change at Tyr-113 to Arg, the
amino acid present in the Torpedo AChR. Studies on analogs of -conotoxin GI that changed the corresponding Arg-9 to alanine displayed affinity changes that appeared site-independent (14), suggesting that the charge per se was not important to binding.
Conotoxin binding appears to block competitively the binding of
-bungarotoxin and to block biological activity of AChRs. Yet,
-conotoxins do not appear to influence the conformation of the mouse
AChR (15). The structural model of Unwin and co-workers (16, 17)
suggests the presence of transverse tunnels leading to a buried site
for ACh binding. But such tunnels appear unlikely to permit access of a
ligand the size of Ctx to the inner recesses of the binding site. An
alternative model is that Ctx binds at the ACh-binding site entrance to
block access of smaller ligands but may not interact closely with
residues critical for stabilizing agonist binding. However, Bren and
Sine (9) measured interactions of Ctx with -subunit residues Tyr-93,
Tyr-190, and Tyr-198, residues thought to be critical in stabilizing
agonist binding. The evidence that Ctx interacts with AChR-binding site
residues appears incompatible with the structural model of the AChR and
with a role for cationic residues in binding.
To examine the role of charged residues in stabilizing
binding of Ctx to the AChR and to examine whether Ctx interacts
intimately with residues critical for agonist binding, we determined
the binding properties of charge variants of Ctx, as well as several other Ctx mutants. We present data that the charge of Ctx Lys-10 and
that of His-5 and the N terminus are relatively unimportant to the net
binding affinity toward either the mouse muscle type AChR or the
Torpedo electric organ AChR. We further demonstrate that
binding of Ctx is conformationally insensitive. Nonetheless, the toxin
is formally competitive with the ammonium moiety of ACh. This cationic
toxin does not appear to take advantage of the innate charge
stabilization the receptor confers on agonists and many competitive
antagonists but nonetheless occupies the same steric space. Studies in
low ionic strength, however, did reveal distinct contributions of
charge interactions from the - and -subunits, but these likely
arise from interactions with negatively charged residues on the
AChR.
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EXPERIMENTAL PROCEDURES |
Materials--
All the enzymes for molecular biology, the pMALp2
plasmid vector, and amylose resin were supplied by New England Biolabs
(Beverly, MA). Oligonucleotides were synthesized by Genosys (The
Woodlands, TX). Culture media, competent cells, and
isopropylthio--galactoside were from Life Technologies, Inc. DNA was
sequenced using a kit, version 2.0, from U. S. Biochemical Corp.
Peptidylglycine -amidating enzyme was from Takara (Beverley, CA),
and synthetic Ctx was purchased from Sigma. AChR-rich membranes were
prepared from fresh or frozen Torpedo californica electric
organ as described previously (18).
Synthesis and Modification of Conotoxins--
-Conotoxin MI
(Ctx) was prepared by peptide synthesis at the Baylor College of
Medicine protein core facility. The reduced peptide was purified by
preparative reversed phase HPLC (Vydac 250 × 22 mm, C18); elution
was by a linear gradient of CH3CN containing 0.09%
trifluoroacetic acid. The peptide was renatured by oxidation of
cysteines to form disulfides by stirring in air according to the
procedures described by Zafaralla et al. (19) and by Myers (20). The renatured peptide was purified by reversed phase HPLC; it
displayed the skewed peak profile characteristic of the conformational transition between two states (21). The elution profile was identical
to that of a commercial preparation of Ctx (Sigma) and gave identical
binding constants as assessed by inhibition of [3H]ACh
binding on AChR-rich membranes from Torpedo (22).
Two variants of Ctx were also prepared by peptide synthesis, Ctx
Ala-7 
Ser and Ctx Tyr-12 Trp. These were purified as the
linear peptide. Oxidation of the sulfhydryls to form the correct disulfide bonding was carried out by oxidation in 1 mM
GSSG. This redox buffer required only 30 min to 1 h for complete
oxidation of the peptide, rather than the several days required for air oxidation. The peptides were then purified by preparative reversed phase HPLC as described above. These variants also displayed the skewed
peak characteristic of the conformational transition observed for
native Ctx. In all cases, the composition was confirmed by amino acid analysis.
Chemical modifications of Ctx at the N terminus and Lys-10 were
carried out as follows. Reductive methylation of Ctx was carried out by
the method of Jentoft and Dearborn (23). Briefly, 2 mg of Ctx was
reacted in 3 ml of 50 mM sodium phosphate, pH 7.0, 8.3 mM NaCNBH4, and 8.3 mM
H2CO overnight at 4 °C. The reaction was terminated by
addition of trifluoroacetic acid, and the product was purified by
preparative reversed phase HPLC. The reaction resulted in addition of
two methyl groups each at the N terminus and at Lys-10
(-N-dimethylglycyl, 
-N-dimethyllysyl-Ctx;
Met-Ctx). Acetylation was carried out by reaction with acetic anhydride according to Means and Feeney (24). 2 mg of Ctx was dissolved in cold,
50% saturated sodium acetate (200 µl) and kept on ice during the
reaction. A total of 10 µl of acetic anhydride was added in small
aliquots at regular intervals over 1 h (acetyl-Ctx). The product
was purified by reversed phase HPLC. For trinitrobenzoylation (TNB), 2 mg of Ctx was dissolved in 1 ml of sodium borate buffer, pH 9.5, with
20 mM trinitrobenzoyl sulfonate and incubated for 2 h
at ambient temperature (~21 °C). The product was purified by
reversed phase HPLC. The reaction yielded two peaks. Amino acid
analysis and mass spectroscopy revealed that the larger second peak
contained the product with reaction of TNB at both the N terminus and
Lys-10.
Amino acid analysis of the products of chemical reactions revealed loss
of detectable Lys and one of the two Gly residues after methylation and
after reaction with trinitrobenzoyl sulfonate. The analysis of the
acetylated Ctx was similar to that of unmodified toxin, as expected for
an acid-labile adduct. Mass spectroscopy revealed parent ion masses of
of 1493, 1549, 1577, and 1915 for Ctx, Met-Ctx, Ac-Ctx, and TNB-Ctx
respectively; these masses corresponded to those expected for each
modification. The loss of primary amines in the modified toxins was
verified by loss of reactivity to fluorescamine. Fluorescence
measurements showed that there were no amines available for reaction
for each of the modified toxins, whereas Ctx showed reactivity that
corresponded to the presence of two reactive sites.
Design and Construction of Synthetic Gene for Conotoxin
M1--
We have constructed a recombinant expression plasmid encoding
a maltose-binding protein-Ctx fusion protein, using pMAL-p2 system
(25). The system was chosen to direct secretion of the MBP fusion
protein into the periplasmic space for proper disulfide bond formation.
Codons for Ctx residues were chosen according to Escherichia
coli codon usage (26). Glycine was appended to the Ctx sequence to
allow cleavage by peptidylglycine -amidation enzyme. Two stop codons
were added at the end of the gene followed by a PstI site
for cloning the synthetic gene into pMALp2 at the XmnI and
PstI site. The XmnI site included sequence
encoding the protease factor Xa cleavage site, located just 5' to the
toxin gene sequence (25, 27) such that cleavage of the fusion protein with factor Xa liberates Ctx with no additional amino acids.
Complementary oligonucleotides were purified by gel electrophoresis in
20% polyacrylamide containing 7 M urea. They were then
5'-phosphorylated using polynucleotide kinase and annealed by combining
equimolar amounts of each oligomer (300 pmol) in 100 µl of 50 mM Tris-HCl, pH 7.5, 5 mM MgCl2,
and 50 mM NaCl, followed by heating to 95 °C for 5 min
and slowly cooling to room temperature. The duplex DNA was
gel-purified, digested with PstI, and inserted into the
expression plasmid pMALp2 at XmnI and PstI sites.
Transformants containing the conotoxin M1 gene
(pMAL-Cono) were verified by DNA sequencing (28).
Expression and Purification of Recombinant Ctx--
Cells
containing pMAL-Cono clone were grown at 37 °C in LB broth
containing 0.2% glucose and 100 µg/ml ampicillin to an
absorbance of 0.5 at 600 nm, and then induced with 0.5 mM isopropylthio--galactoside for 3 h. A
periplasmic fraction was prepared by osmotic shock (29), and the fusion
protein was purified by affinity chromatography over an amylose resin
(30) column (10 ml) that had been equilibrated previously with 10 mM Tris-HCl, pH 7.4, 200 mM NaCl, 1 mM EDTA. The column was washed with 10 volumes of this
buffer, and the bound MBP-conotoxin fusion protein was then eluted with
buffer containing 5 mM maltose. Fractions were analyzed by
SDS-PAGE.
The purified fusion protein (MBP-conotoxin) was cleaved with factor Xa
at a w/w ratio of 1% for 48 h at room temperature followed by
SDS-PAGE analysis. The result showed about 50-70% release of Ctx. The
recombinant Ctx was then purified by HPLC on a semi-preparative Vydac
C8 column. Elution was by a linear gradient of 0-60% acetonitrile containing 0.09% trifluoroacetic acid at a flow rate of 3 ml/min. HPLC
fractions were analyzed by Tricine/SDS-PAGE (31). To amidate Ctx, the
pooled fractions were treated with peptidylglycine -amidating enzyme
according to a standard procedure (32) for 4 h at 37 °C
followed by repurification on HPLC as described above. Ctx mutants
Lys-10 His, Lys-10 Pro, and His-5 Glu were produced as
described above for Ctx with appropriate codon replacements in the
synthetic oligonucleotides. The yields of the Ctx analogs were lower
than that of native Ctx because the extent of factor Xa digestion was
lower. Because of the limited amount of material, duplicate experiments
on Torpedo AChR were carried out just once.
Binding Assays--
Binding of the various conotoxin derivatives
was measured by competitive inhibition of the initial rate of
bungarotoxin binding, as described previously (33). Binding to
Torpedo AChR was measured using the assay described by
Schmidt and Raftery (34) or by filtration over GF/F filters. For
filtration the following procedure was followed. Samples of AChR-rich
membranes and competitive ligand were preincubated in HTPS (250 mM NaCl, 5 mM KCl, 3 mM
CaCl2, 2 mM MgCl2, 0.002%
NaN3, 20 mM Hepes, pH 7.0) containing 0.1% BSA
for 30 min to reach equilibrium. The binding was initiated by addition
of 125I--BgTx (0.5-3 nM), and the
samples were allowed to incubate at ambient temperature for 45 min.
Reactions were quenched by addition of 300 nM
non-radioactive -BgTx in HTPS. The samples were then filtered over
Whatman type GF/F filters that had been presoaked in 1%
polyethyleneimine. The filters were then washed with 10 ml of HTPS and
counted in an -radiation counter (Beckman Instruments). For low
ionic strength experiments, binding was carried out in 10 mM Hepes, pH 7.0, with 0.1% BSA. Binding reactions were
quenched at 30 s to remain in the linear range of the initial association rate.
Binding to mouse muscle type AChR expressed on the surface of
tissue culture cells (BC3H1) was carried out as described earlier (35)
using a 24-well plate assay or, alternatively, by a filtration assay
(4). The latter assay was carried out by suspending the cells in
potassium-Ringer buffer (140 mM KCl, 5.4 mM NaCl, 12.8 mM CaCl2, 1.7 mM MgCl2, 25 mM Hepes, 30 µg/ml
BSA, pH 7.4). Assays were incubated as described above and then
filtered over Whatman type GF/C filters that had been presoaked
overnight in 4% Carnation instant nonfat dry milk. The filters were
then washed with 12 ml of phosphate-buffered saline and counted in an
-radiation counter (Beckman Instruments).
Assessment of competitive binding between TMA and Ctx was carried
out by monitoring their effects on the conformational state of the
AChR. The conformation, in turn, was measured by the binding of
ethidium. Ethidium preferentially binds the desensitized state of the
AChR, and the extent of binding reflects the extent of desensitization
of the AChR when the AChR and ethidium concentrations are low relative
to the KD values for ethidium (~500 nM). Ethidium binding was measured by fluorescence
enhancement, essentially as described by Lurtz and Pedersen (36),
except that fluorescence was monitored through a filter (550 nm cut on, Oriel number 59502) to obtain a higher signal to noise ratio. Binding
to the NCA site was also measured by [3H]phencyclidine
binding using a centrifugation assay as described previously (22).
Binding was carried out in low concentrations of
[3H]phencyclidine (~1 nM) and is reported
as bound over free, which compensates for the change in free
[3H]phencyclidine upon binding of competing ligand.
Binding was analyzed by nonlinear least squares fitting to
Equation 1 for inhibition by competitive binding to two
independent sites present in equal concentrations,
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(Eq. 1)
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where KI1 and
KI2 represent the binding constants for
the inhibitor, Bmax the maximum amount of
binding at each site, and I the concentration of inhibitor. In some
cases, binding was fit to Equation 2 for the binding of two sites
present in differing concentrations.
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(Eq. 2)
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This was necessary because 125I--BgTx binds with
unequal rates to the two sites under some circumstances. Data for
inhibition of Ctx binding to the mouse muscle AChR were generally fit
with Equation 2, whereas data for inhibition of binding to
Torpedo AChR could be fit with Equation 1. For the analysis
of ethidium fluorescence, the enhancement of binding by agonists
was fit to the Hill Equation 3,
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(Eq. 3)
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or to the binding isotherm (the Hill equation with
n = 1). Inhibition of TMA binding by Ctx was fit to
Equation 4 for inhibition at a single binding site.
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(Eq. 4)
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RESULTS |
Like other -toxins that interact with the nicotinic AChR,
-conotoxins are cationic. Ctx carries a net charge of +3.5, counting His-5 as 1/2 charge and including the N-terminal charge. To test whether these charged moieties play a significant role in the interaction of -conotoxins with the AChR, we examined the binding energies of toxins that had been modified or mutated at the charged loci. One receptor residue that had been identified as constituting an
electrostatically repulsive interaction was Arg-113 in the Torpedo AChR. The homologous residue in the mouse muscle
AChR is a Tyr. Therefore, we compared affinities of toxins in both these species as a means of assessing the importance of charge repulsion between this residue and Ctx. We utilized two approaches to
generate Ctx structural analogs, expression of recombinant Ctx in
E. coli, and chemical modification of synthetic Ctx.
Expression and Purification of Recombinant Ctx in E. coli--
Ctx
could be expressed and refolded in E. coli by attaching a
synthetic gene to the secreted maltose-binding protein. The fusion
protein was isolated by amylose affinity chromatography and Ctx cleaved
by factor Xa. Purification by HPLC, amidation, and a final HPLC
purification yielded a product that was indistinguishable from
commercial Ctx and from synthetic Ctx, as judged by migration SDS-polyacrylamide gel electrophoresis and by HPLC (Fig.
1, insets). The net yield of
recombinant toxin was about 50 µg/liter of cell. Recombinant Ctx was
compared with synthetic Ctx and with a commercial product (Sigma) for
binding activity on mouse muscle AChR and on T. californica
AChR by inhibition of 125I--BgTx binding as described
under "Experimental Procedures." The affinities of each material
were indistinguishable (data not shown). Proper refolding and disulfide
bond formation was apparent from comigration of recombinant and
synthetic Ctx on HPLC and because it is required for biological
activity (37, 38).
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Fig. 1.
Purification of recombinant Ctx.
Products resulting from MBP-conotoxin fusion protein digested with
factor Xa were separated on a Vydac C8 column as described under
"Experimental Procedures". Inset A, 16%
Tricine/SDS-PAGE analysis of the recombinant conotoxin. Lane
1, molecular mass markers, 116.3, 66.73, 55.4, 36.5, 31.0, 21.5, and 6.0 kDa; lane 2, purified recombinant conotoxin;
and lane 3, synthetic Ctx. Inset B, analytical
HPLC analysis of 10 µg of recombinant conotoxin mixed with 10 µg of
synthetic Ctx.
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Effects of Ctx Modifications on Binding Affinity--
Ctx was
chemically modified by reductive methylation, by acetylation, or by
trinitrobenzoylation, using standard reaction methods (see
"Experimental Procedures"). Each modified toxin was purified by
reversed phase HPLC and shown to be quantitatively modified at Lys-10
and at the N terminus by mass spectroscopy, by lack of reactivity to
fluorescamine, and by amino acid analysis. Methylation will increase
the bulk of the reacted amines but will not alter the charge; both the
N-terminal amine and Lys-10 were found to be dimethylated. The effect
of amine dimethylation on affinity was negligible
(Fig. 2 and Table II), demonstrating that the simple size of these amines was relatively unimportant for binding.
Acetylation of the amines resulted in formation of a neutral acetamide
with more steric bulk than the methylation. Nonetheless, there was less
than a 2-fold effect on affinity for the mouse AChR and a 3-fold
decrease for the Torpedo AChR site (Fig. 2 and Table
II). The small affinity changes suggest that the charges of Lys-10 and
the N terminus do not contribute substantially to the binding energy.
The modification by addition of a picric acid moiety, (TNB-Ctx) does
cause a significant shift of binding to the site of
Torpedo AChR, and the site of the mouse AChR of
13-15-fold. The effects on mouse and Torpedo
sites were smaller (Table II).
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Fig. 2.
Chemical modification of Ctx lysine 10 affects affinity weakly. Met-Ctx ( ), acetyl-Ctx ( ), and
TNB-Ctx ( ) were compared with Ctx ( ) for binding to the mouse
AChR (A) or the Torpedo AChR (B).
Binding was measured by inhibition of the initial rate of
125I--BgTx binding using the 24-well plate assay
(A) and to the Torpedo AChR using the DE81 filter
binding assay (B) as described under "Experimental
Procedures." Each symbol represents the average of duplicate
determinations that generally varied less than 5%. The data are
normalized to the initial rate of binding observed in the absence of
competitor. The solid lines represent the best fits to a
model for inhibition at two binding sites (Equations 1 and 2).
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These results indicated that the Lys-10 side chain charge was
relatively unimportant for binding, although added steric bulk could
affect binding at some sites. We further tested the ability of
histidine, an aromatic cationic residue, and proline to affect the
overall affinity at this position. Proline occurs as a natural substitution in -conotoxin SI and SII at this position; both these
toxins have substantially lower affinity for the AChR (13).
-Conotoxin Lys-10 Pro and Lys-10 His were made by expression
in E. coli as MBP fusions. Substitution of His for Lys had a
5-fold effect on the affinity for the Torpedo AChR, and
decreased the affinity for the mouse site more than 20-fold
(Fig. 3 and Table II). Pro-substitution
decreased affinity on the Torpedo AChR 7-23-fold, whereas
the change in affinity for the mouse site was more than
7000-fold. These results indicate that the mouse AChR is substantially
more sensitive to changes in this position than the Torpedo
AChR. The results with His indicate that change in side chain structure
at this position can affect affinity substantially. The effects of Pro
substitution must be interpreted more cautiously, as it will also
constrain the backbone conformation, but the results do confirm that a
Pro in this position likely contributes to the weaker affinity of
conotoxins SI and SII. We examined the role of His-5 as a possible
contributor to electrostatic stabilization. This residue was mutated to
Glu, providing 2 units charge change. Remarkably, Ctx His-5 Glu
showed only a 3-6-fold change in affinity, indicating a modest effect of electrostatic attraction (Fig. 4 and
Table II).
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Fig. 3.
Mutagenesis of Ctx lysine 10 decreases
affinity. Ctx Lys-10 Pro () and Ctx Lys-10 His ()
were produced by expression in E. coli, and their affinities
for the mouse AChR (A) and the Torpedo AChR
(B) were compared with Ctx (). Binding was performed as
described in Fig. 2. Each symbol represents the average of two
determinations, and the data are normalized to the initial rate of
binding observed in the absence of competitor. The solid
lines represent the best fits to a model for inhibition at two
binding sites.
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Fig. 4.
Ctx His-5 mutation alters affinity similarly
at all sites. Ctx His-5 Glu () was produced by
expression in E. coli, and its affinities for the mouse AChR
(A) and the Torpedo AChR (B) were
compared with Ctx (). Binding was performed as described in Fig. 2.
Each symbol represents the average of two determinations, and the data
are normalized to the initial rate of binding observed in the absence
of competitor. The solid lines represent the best fits to a
model for inhibition at two binding sites.
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We examined the importance of two additional conserved side chains by
making the toxins using peptide synthesis. Tyr-12 of Ctx represents a
residue conserved as either a Tyr or Phe residue among all
-conotoxins (Table I). We synthesized
the analog Ctx Tyr-12 Trp and examined its binding properties (Fig.
5 and Table II). The binding for the mouse AChR
was unchanged from that of conotoxin MI but had higher affinity for the
Torpedo site. Thus, this position is not acutely
sensitive to a significant change in the size of the side chain.
Alanine 7 of Ctx MI is highly conserved among the -conotoxins. A
change to serine affected affinity to both mouse and Torpedo
AChR but affected Torpedo more strongly. This observation
confirms the importance of this residue but also shows that the
addition of a hydroxyl group can be tolerated without dramatic loss of
affinity.
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Fig. 5.
Modification of Ctx positions 7 and 12 affect
binding to the AChR. Ctx A7S () and Ctx Y12W () were
produced by peptide synthesis, and their affinities as well as that for
Ctx () for the mouse AChR (A) and Torpedo AChR
(B) were determined by the glass fiber filtration assays
described under "Experimental Procedures." Each symbol represents
the average of three determinations, and the data are normalized to the
initial rate of binding observed in the absence of competitor; standard
deviations for each point were generally less than 5%. The solid
lines represent the best fits to a model for inhibition at two
binding sites.
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Table II
Binding of Conotoxin analogs to the high and low affinity sites of
mouse and Torpedo AChR
KI values for inhibition of 125I--BgTx
binding to mouse and Torpedo AChR by Ctx and its analogs
were determined as described under "Experimental Procedures." The
errors listed are the standard errors of the mean.
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Conformational Changes in the AChR Do Not Affect Ctx
Binding--
The cation associated with agonists and many competitive
antagonists affects the conformational equilibrium of the AChR,
generally to increase the proportion of desensitized AChR upon binding. Even the smaller agonists such as TMA can desensitize the AChR. If a
cationic moiety of Ctx bound the ACh-binding site in the same manner as
the ammonium of ACh, it would be expected to affect the conformation as
well. We tested the ability of Ctx to affect the conformation by
measuring the effect of noncompetitive antagonists on the affinity of
conotoxin and, conversely, the effect of conotoxin on the affinities of
proadifen and phencyclidine. As shown in Fig. 6, A and B,
proadifen does not appreciably change the affinity of Ctx for either
the mouse or Torpedo AChR (Table II), nor does tetracaine
affect binding, an NCA that stabilizes the resting state of the
Torpedo AChR (Table II). The presence of Ctx in nearly saturating conditions does not affect PCP binding to the
Torpedo AChR and decreases the affinity of proadifen binding
less than 2-fold, as measured by inhibition of [3H]PCP
binding (Fig. 6C). As a control, the agonist carbamylcholine increased the affinity of proadifen more than 10-fold (Fig.
6C). Ctx also does not affect the apparent affinity of
ethidium, another strongly desensitizing noncompetitive antagonist (see
below).
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Fig. 6.
Ctx binding to the AChR is independent of
conformation. A, BC3H-1 cells were incubated in the
indicated concentrations of Ctx in the absence () or presence ()
of 26 µM proadifen, and the initial rate of binding of
125I--BgTx was measured using the 24-well assay as
described under "Experimental Procedures." The value for the
presence of excess BgTx to define background binding is also shown
( ). The solid curves are the best fits to a model for
inhibition at two binding sites (Equation 2). The
KI1 values for this experiment are 11 and 5 nM in the absence and presence of proadifen,
respectively. B, Torpedo AChR-rich membranes (3.4 nM ACh sites) were incubated with the indicated
concentrations of Ctx in the presence of 26 µM proadifen
() or no added ligand (). The initial rate of
125I--BgTx (0.75 nM) binding was measured
using the glass fiber filtration method described under "Experimental
Procedures." The solid curves represent the best fits to a
model for binding two independent sites. C, Ctx does not
alter the affinity of proadifen. [3H]PCP binding to the
Torpedo AChR (0.1 mg/ml) was measured in the indicated
concentrations of proadifen as described under "Experimental
Procedures." Measurements were carried out in the absence ( ) or
presence () of 2 µM Ctx or the presence of 100 µM carbamylcholine (). The data were normalized to
maximal binding. The maximal values of the bound/free ratios were
similar in the absence or presence of Ctx. The solid lines
show the best fits to models for inhibition at a single binding site;
for this experiment, the Kapp values determined
were 1.1 µM for the absence of Ctx, 2.1 µM
in its presence and 0.09 µM in the presence of
carbamylcholine. In all panels, each symbol represents the average of
duplicate determinations.
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Electrostatic Dependence of Ctx Binding--
The charge changes in
Ctx affected by mutation or modification did not reveal any discrete
charge-charge interactions critical for stabilizing binding; however,
it was possible that such interactions might be revealed at low ionic
strength conditions that would enhance electrostatic attraction or
repulsion. We examined the ability of ionic strength to influence the
affinity of Ctx and acetyl-Ctx for Torpedo AChR by
inhibition of 125I--BgTx binding in 10 mM
Hepes. As can be seen in Fig. 7, lowered ionic strength dramatically increased the affinity of Ctx (compare dotted lines in Fig. 7A). The change was more
pronounced at the lower affinity site, suggesting that there are
stronger charge interactions at this site. In low ionic strength
buffer, inhibition by Ctx was characterized by a single site inhibition
curve rather than a two-site inhibition curve as observed in
physiological ionic strength. To ensure that this reflected similar
binding of Ctx to each of the two sites, rather than an artifact of
125I--BgTx binding kinetics, we examined the ability of
d-tubocurarine to inhibit the rate of
125I--BgTx binding under the same conditions (Fig.
7B). The data clearly reveal two-site inhibition, with
affinities for d-tubocurarine similar to those observed at
physiological ionic strength. Therefore, Ctx binding has similar
affinities to the site and site in low ionic
strength.
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Fig. 7.
Ionic interactions are site-specific.
A, the binding affinities of Ctx () and acetyl-Ctx ()
were measured by inhibition of the initial rate of
125I--BgTx binding to AChR-rich membranes in 10 mM Hepes, 0.1% BSA by filtration as described under
"Experimental Procedures". The dotted and solid
lines represent the best fits to a single site inhibition function
or a two-site inhibition function, respectively. For reference are
shown the fitted curves for Ctx (····) and acetyl-Ctx
( -) inhibition in HTPS from parallel experiments. B,
inhibition of 125I--BgTx binding by
d-tubocurarine (dTC, ) and by acetyl-Ctx ()
in the presence of 1 µM d-tubocurarine. The
best fits to a two-site binding function with variable site
stoichiometry and a single site binding function, respectively, are
shown as solid curves. The data points in both panels
represent the average of two independent determinations that generally
varied by less than 5% and are plotted as fractional binding of the
maximum (f). The K values obtained from
the fits are as follows: Ctx, K = 4.4 nM;
acetyl-Ctx, K1 = 6.3 nM and
K2 = 194 nM; acetyl-Ctx plus
d-tubocurarine, K = 373 nM;
d-tubocurarine, K1 = 48 nM and K2 = 17 µM.
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We also determined the affinity of acetyl-Ctx (Fig. 7A) to
test whether neutralizing the N terminus and Lys-10 was important to
the charge interactions observed in low ionic strength buffer. Acetyl-Ctx displayed clear two-site binding in low ionic strength, whereas binding was well fit by single site function in physiological buffer. Therefore, we could not, a priori, assign sites to
the high and low affinity components. To determine unambiguously which site corresponded to the high affinity component, we carried out inhibition by acetyl-Ctx with the site blocked by including 1 µM d-tubocurarine (Fig. 7B,
). The inhibition was well fit by a single site binding
function with a K value that corresponded to that of the low
affinity component (Table III). This
demonstrated that the high affinity component reflected acetyl-Ctx
binding to the site and that the lower affinity reflected
binding to the site.
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Table III
Charge effects on binding observed in low ionic strength buffer
The K values for binding of conotoxin M1 and
acetyl-conotoxin to the AChR in low ionic strength buffer (10 mM Hepes) were determined as described in Fig. 7 and under
"Experimental Procedures." Errors are the standard deviation of the
number of independent determinations indicated by n.
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In low versus high ionic strength, the shift in
affinity of acetyl-Ctx was similar to the shift observed for Ctx itself
at the high affinity, site, ~10-30-fold. Acetylation,
therefore, does not affect ionic interactions at the site. The
ionic strength-induced change in affinity must arise from charges other
than the N terminus or Lys-10. The ionic strength shift of acetyl-Ctx
affinity at the low affinity site was much smaller (1-3-fold),
particularly when compared with the shift of Ctx itself (~300-fold).
Thus, the ionic strength change affects interactions between negatively charged residues at the site and Ctx residues Lys-10 or the N
terminus or both. The attractive interactions at the site must
be mediated by other residues since the shift by ionic strength is
unaffected by acetylation.
Ctx Competes with a Small Agonist--
The data strongly suggested
that charged moieties on Ctx do not participate in binding and that
there was not a functional group that acted as the equivalent of the
ammonium group present in most agonists and antagonists. In addition,
Ctx did not affect the conformational state of the AChR as would be
expected if it occupied the same space as that occupied by an agonist
ammonium group. Nonetheless, Ctx appears to be competitive with other
toxins and with ACh; we have shown that Ctx blocks
[3H]ACh binding and that it blocks
[3H]d-tubocurarine binding (data not shown).
However, it was possible that Ctx sterically blocked binding by
interaction with the sites that stabilize moieties outside of the
ammonium group, such as the acetate of ACh or the larger bulk of other
toxins. To test whether Ctx directly interacts with the AChR in the
most critical part of the ACh-binding sites, the portion that
stabilizes ammonium binding, we tested whether Ctx was directly
competitive with the small agonist TMA.
To assess whether Ctx and TMA were mutually competitive, we utilized
the observation that Ctx does not affect the conformation of the AChR,
whereas TMA desensitizes the AChR. To assess the conformational state,
we utilized the fluorescent enhancement of ethidium upon binding the
AChR. Ethidium preferentially binds the desensitized conformation (22,
39). Conditions of relatively low ethidium and low AChR concentrations
were chosen such that the fluorescence increase due to ethidium binding
would reflect the increase in affinity due to desensitization. Fig.
8A shows the increases in
fluorescence caused by titration of ethidium/AChR suspensions with
carbamylcholine and with TMA. Both ligands caused concentration-dependent changes in fluorescence that
correspond to the binding of ligand and concomitant desensitization of
the AChR. Carbamylcholine increased fluorescence with a Hill
coefficient of 1.5 and a Kobs of 170 nM, which corresponds closely to the values for equilibrium
binding (data not shown). The Kobs for TMA was 3 µM with a Hill coefficient of 1.
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Fig. 8.
Ctx competes with the small agonist,
TMA. Binding of 200 nM ethidium to 100 nM
AChR was measured by fluorescence enhancement in HTPS buffer as
described under "Experimental Procedures." A, ethidium
binding was enhanced by increasing concentrations of carbamylcholine
() or of TMA (); controls are also shown in the presence of 20 µM proadifen (). The data for carbamylcholine were fit
to the Hill equation (Equation 3) and yielded a
Kapp of 170 nM with a Hill
coefficient of n = 1.5. The data for TMA were fit to a
single-site binding isotherm (Equation 4) and yielded a
Kapp of 3.3 µM. B,
inhibition of ethidium fluorescence by varying concentrations of Ctx
was measured in the presence of no TMA (), 10 µM TMA
(), 20 µM TMA (), and 100 µM TMA
( ). Panel C, the Kobs for
inhibition of TMA binding by Ctx was determined by titration of
increasing concentrations of Ctx into suspensions of AChR, ethidium,
and TMA as shown in B. The Kobs for
inhibition at various TMA concentrations are replotted
versus the TMA concentration. The fit to a line yields a
slope of 0.028 with an intercept of 0.088 µM.
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To determine whether conotoxin competes with TMA binding, Ctx was
titrated into suspensions of ethidium/AChR that contained varying
concentrations of TMA. Example titrations with 0, 10, 20, and 100 µM TMA are shown in Fig. 8B. The curve with no
TMA demonstrates that Ctx by itself has only a small effect on ethidium fluorescence. As titration with Ctx displaced TMA from the ACh-binding sites, the AChR displayed less ethidium binding and fluorescence as the
desensitized conformation was not stabilized by Ctx. In each case the
inhibition was well fit by a single site inhibition function with a
background fluorescence approaching that found in the absence of TMA.
The Kobs was determined for each titration and
replotted against the TMA concentration (Fig. 8C). There is a linear relationship between the TMA concentration and the
Kobs, indicative of direct competition.
The results cannot be accounted for by inhibition through a ternary
complex of AChR, TMA, and Ctx unless one ligand conformationally reduces the binding of the second ligand with a coupling energy of more
than 3.4 kcal/mol. This value was determined from the maximum TMA
concentration used (1 mM) and the
Kobs for TMA binding. The experiment instead
suggests direct, steric competition between TMA and Ctx, where Ctx
occupies the space normally taken by the agonist ammonium moiety.
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DISCUSSION |
We examined various charged Ctx residues to determine whether they
were critical for high affinity binding to the AChR, in a manner
similar to the cationic moiety of agonists. Elimination of charge at
Lys-10 and the N terminus affected binding affinity 2-4-fold. A charge
reversal at His-5 consistently changed affinity 6-fold at all sites.
Such modest changes show that individual charge-charge interactions are
not a highly significant component of stabilizing Ctx binding in
physiological buffer, despite the generally conserved cationic nature
of these toxins. Binding affinity to Torpedo AChR was,
however, significantly modified upon neutralization of Lys-10 when
examined at low ionic strength, and the effect was much stronger at the
site than the site. The results indicate an attractive
ionic interaction of Lys-10, the N terminus, or both with the
site that is not present at the site. The two sites must have
distinct orientations of charged residues or the toxin must bind in
distinctly different orientations at each site.
We conclude that Lys-10 or His-5 do not interact with the AChR in the
same manner as cationic groups of agonists that are required for
channel activation. This is shown by the modest affinity changes upon
modification and by the general failure of Ctx to modulate the
conformational state of the AChR. Ctx does not appear to interact with
other residues that influence the conformational equilibrium between
the resting and desensitized states of the AChR. However, Ctx does
formally compete with the binding of TMA, suggesting that steric
hindrance prevents the simultaneous occupancy of both Ctx and TMA.
Consequently, Ctx does interact with the portion of the binding site
responsible for stabilizing agonist cations but does not do so with a
cationic residue and is therefore incapable of inducing a
conformational change.
Our binding data reveal affinities of Ctx for the Torpedo
site that differ from those observed by others (10, 11). Some of
the larger differences can be accounted for by the distinct buffer
conditions used as follows: Hann et al. (13) and Groebe et al. (10) used low ionic strength buffers to measure
binding in the presence of detergents. However, the data of Chiara
et al. (12) was carried out in physiological buffer that
revealed affinities 5-10-fold higher than we observed. Our binding
data on the mouse muscle AChR is in complete agreement with that of others (7). We also obtained similar binding data for commercial, synthetic, and recombinant Ctx and observed consistent inhibition of
[3H]ACh binding and of
[3H]d-tubocurarine binding (22) (data not
shown). The source of the difference remains unknown.
Ionic Interactions in Binding--
It has long been argued
that localized charges stabilize the binding of agonists and
competitive antagonists. Cholinergic agonists universally carry a
positively charged group (40), and for the AChR, a simple quaternary
ammonium in the form of TMA is a completely competent agonist.
Localization of several anionic residues (Asp-180 and Asp-152) in
the vicinity of the ACh-binding sites has supported the idea of charge
stabilization of ligand binding (41, 42), but it remains unclear
whether these residues act as countercharges to the ligand ammonium.
Experiments that examined the effect of ionic strength on ligand
binding were more consistent with a diffuse charge distribution near
the binding site than a single countercharge in close proximity (43).
-Toxins of snakes and marine snails are generally basic and have
binding that is strongly dependent on ionic strength (34, 44),
suggesting that ionic interactions constitute a significant component
of the net binding energy.
Sine et al. (7) identified several residues critical
for the site selectivity of Ctx binding to mouse muscle receptor, including residues Lys-34, Tyr-111, and His-172. The
homologous residue to Tyr-111 in Torpedo -subunit is
Arg-113. Chiara et al. (12) demonstrated that Arg-113
dramatically weakened binding of Ctx to the site, and they
inferred an electrostatic repulsion with Lys-10. At the
Torpedo AChR -subunit, acetylation of Lys-10 does appear
to cause an increase in affinity of 2-4-fold, consistent with lessened
repulsion. However, this change is much smaller than the 1000-fold
effect observed by Chiara et al. (12). Our data suggest that
the Arg-113 interaction is not through electrostatic repulsion at
the N terminus, His-5, or Lys-10 of Ctx. We cannot rigorously exclude
an interaction through Ctx Arg-2, which we have not modified; however,
it appears unlikely that Arg-2 is responsible because conotoxins GI and
SIA bind with similar selectivity to the Torpedo AChR (13)
and have a Glu and Tyr in the homologous positions, respectively (see
Table I). We conclude that Lys-10 of Ctx and Arg-113 are not in
close apposition because electrostatic repulsion is not alleviated by
charge neutralization. This is in agreement with the conclusions of
Groebe et al. (14) who used a Arg-9 Ala mutation of
-conotoxin GI to analyze binding to the Torpedo and mouse AChRs.
Ionic interactions become more apparent in low ionic strength, where we
observed similar affinities of Ctx for both sites on the
Torpedo AChR, indicating a larger change in affinity for the
site. This shift is consistent with stronger attraction among
opposite charges. If charge repulsion among cationic residues had been
significant, lower ionic strength should have exacerbated the
repulsion, resulting in relatively lower affinity. The observation of
higher affinity, rather than lower, at the site, reinforces the
conclusion that charge repulsion between Lys-10 and the Arg-113 of
the Torpedo AChR is not a factor that causes site
selectivity in binding.
In low ionic strength, reducing the charge on Ctx by acetylation of Ctx
and the N terminus results in a larger shift on the Torpedo
-subunit than the -subunit. This suggests that attractive forces are lost upon neutralization and that they are more significant for the -subunit than the -subunit. The results further
suggest the presence of distinct charge distributions at the and
sites. For the site, binding of Ctx is enhanced
300-600-fold upon lowering ionic strength, and this is reduced to a
1-3-fold effect for diacetyl-Ctx. This suggests that the primary
stabilizing ionic interactions occur from the site to
Lys-10 or the N terminus. In contrast for the site, the binding
affinity of Ctx is enhanced about 30-fold upon lowering ionic strength,
and this is similar for diacetyl-Ctx. Thus, ionic strength effects are
similar even when charge is neutralized at Ctx Lys-10 and the N
terminus. This suggests that the weaker stabilizing ionic interactions
at the site occur through one or more of the remaining ionic
residues on Ctx, Arg-2 or His-5. The His-5 Glu mutation shows
similar effects at both sites of Torpedo AChR, suggesting a
role for Arg-2; however, those experiments will need to be conducted in
low ionic strength.
Examination of the - and -subunit N-terminal domain
sequences does not lead to immediate insights into the difference in amino acids that could account for the binding in low ionic strength. Since the ionic changes reflect attractive interactions, the difference in binding should reflect differences in the distribution of anionic residues near the Ctx-binding sites. There are nearly two dozen such
possible sites, some near the binding site loops and many not.
Unfortunately, it is also unclear whether such differences in charge
distribution will be important for binding since charge changes have
slight effects on binding in physiological ionic strength.
Conotoxin Binds Independently of Conformation--
Our data shows
that Ctx binding is affected less than 2-fold by noncompetitive
antagonists on both Torpedo and mouse AChR and that Ctx
affects the binding of NCAs on the Torpedo AChR less than
2-fold. Ctx, therefore, binds equivalently to the desensitized and
resting forms of the AChR. The results are consistent with a similar
finding of Prince and Sine (15) that Ctx binding to mouse muscle AChR
was insensitive to the presence of proadifen. It can be inferred that
Ctx does not interact strongly with residues that mediate
conformational changes even though Ctx competes for ACh binding.
An alternative interpretation is that Ctx acts as a cap to block
an entrance to the binding site but does not intimately contact the
residues that normally stabilize binding. A capping mechanism should
have permitted us to observe simultaneous binding of Ctx and a small
ligand, such as TMA. We tested this notion explicitly by examining
competition of Ctx with TMA. The mutual inhibition was linear over a
300-fold range of TMA concentrations, which represents an interaction
energy of >3.4 kcal/mol. Allosteric effects were unlikely to account
for this interaction since we had shown Ctx binding to be insensitive
to the fundamental conformational change of desensitization. The
interaction most likely represents steric repulsion. Such a conclusion
suggests that Ctx inserts a moiety into the most critical part of the
ACh-binding site and does not merely cap access to the site. The
moiety, however, is clearly not one of the Ctx cationic residues
because they affect binding only weakly.
Our results show that charge-charge interactions between Ctx and the
ACh-binding sites are weak, at best, under physiological conditions but
reveal distinct arrangements of charged residues at each site or, as
suggested by Sugiyama et al. (8), that Ctx binds in distinct
orientations at the two sites. They furthermore show intimate binding
of Ctx with the ACh-binding site that is independent of cationic
residues on Ctx and that do not affect the conformation of the AChR.
The lack of effect on conformation may be a consequence of not placing
a cation in the critical part of the binding site but seems remarkable nonetheless.
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ACKNOWLEDGEMENTS |
We thank Cynthia Edwards for maintaining
tissue culture cell lines used for these experiments. We thank Haijun
Wang, Hyunah Choi, and Arlene Samano for excellent technical assistance
in carrying out many of the ligand binding experiments. We thank R. Hann and O. Pagan, Universidad Central Del Caribe, Bayamon, Puerto
Rico, for performing binding studies on recombinant toxins at the
initial stages of the project. All experiments involving expressed and
synthetic conotoxins and its derivatives were reviewed by Biohazards
Safely Committees of UCC and at Baylor College of Medicine according to
the National Institutes of Health Recombinant DNA Guidelines.
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FOOTNOTES |
*
This work was supported in part by United States Public
Health Service Grants NS35212 (to S. E. P.) and 2G12RR3035 (to
K. B.) and Robert A. Welch Foundation Grant Q-1406 (to S. E. P.).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.
¶
Supported by USPHS Grant T32-HL07676. Current address:
Dept. of Health Informatics, School of Allied Health Sciences,
University of Texas Health Science Center at Houston, 7000 Fannin,
Suite 600, Houston, TX 77030.
To whom correspondence should be addressed. Tel.:
713-798-3888; Fax: 713-798-3475; E-mail: pedersen@bcm.tmc.edu.
Published, JBC Papers in Press, April 25, 2001, DOI 10.1074/jbc.M102350200
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ABBREVIATIONS |
The abbreviations used are:
AChR, nicotinic
acetylcholine receptor;
acetyl-Ctx, -N-acetyl-glycyl,
-N-acetyl-lysyl--conotoxin MI;
ACh, acetylcholine;
Ctx, -conotoxin MI;
HPLC, high performance liquid chromatography;
, Met-Ctx, -N-dimethylglycyl,
-N-dimethyllysyl--conotoxin MI, MBP, maltose-binding
protein;
NCA, noncompetitive antagonist of the nicotinic acetylcholine
receptor;
PAGE, polyacrylamide gel electrophoresis;
PCP, phencyclidine;
TMA, tetramethyl ammonium;
TNB-Ctx, -N-trinitrobenzoylglycyl,
-N-trinitrobenzoyllysyl--conotoxin MI;
BSA, bovine serum albumin;
Tricine, N-[2-
hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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ABSTRACT
INTRODUCTION
