From the Departments of Biology and
¶ Psychiatry, University of Utah,
Salt Lake City, Utah 84112
Received for publication, May 9, 2002, and in revised form, October 11, 2002
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ABSTRACT |
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A novel conotoxin, Marine snails in the genus Conus have venoms that
contain a remarkable number of small peptide neurotoxins. Many of these peptides, the conotoxins, are rich in cysteine residues and are highly
disulfide-bonded. Known conotoxins may be divided into families based
on shared features (reviewed in Refs. 1 and 2). Members of a given
conotoxin family have a characteristic number and spacing of cysteines,
a conserved disulfide connectivity, and similar receptor targets.
However, the toxins in a given family show great variability in their
intercysteine sequence, and this accounts for the high degree of
receptor subtype specificity within a toxin family. For example, the
In this report, we describe the discovery of a novel Materials--
Puregene reagents were purchased from Gentra
(Minneapolis, MN); PCR and molecular biology reagents were from
Invitrogen (Carlsbad, CA); salts, acetylcholine, and Discovery of Peptide Synthesis and Folding--
Linear Bioassays--
Biological activity of synthetic Electrophysiology--
Complementary RNA encoding rat
Voltage clamping was done essentially as has been described elsewhere
(11). Briefly, oocytes were clamped at a holding potential of
To show that the 5-min exposure to toxin was sufficient for
toxin/receptor binding to reach equilibrium, 5- and 10-min exposures of
1, 0.33, and 0.033 µM
To investigate the voltage dependence of Preparation of Cells Expressing Rat Preparation of Crude Rat Brain Membranes--
Crude rat brain
membranes were prepared as described previously (3) except that
membranes were frozen and stored in PR.
Competition Binding Assays--
Each assay (300 µl total
volume) consisted of the following in PR: 200 µl of thawed cells or
crude rat brain membranes, a final concentration of 4 nM
3-125I- Data Analysis--
Data were analyzed and plotted using PRISM
software (Graphpad). Competition binding dose response curves were fit
to the equation: % binding = 100/(1 + ([toxin]/EC50)nH), and
electrophysiological dose response curves were fit to: % response = 100/(1 + ([toxin]/IC50)nH). For
PCR-based Discovery of The Biological Activity of Block by
The ability of
The 5-HT3 receptor is highly homologous to the Competition for Functional
However, when oocytes were pretreated for 5 min with 100 µM Preincubation with a High Concentration of
When 100 µM
This suggests that 100 µM Analogs of
Both analogs are significantly less functionally potent than the
corresponding native peptides as determined by electrophysiological characterization of We report the discovery and characterization of The first surprising result was obtained when The binding site for competitive antagonists of nAChRs is located at
the interfaces between subunits that make up the receptor (reviewed in
Refs. 19 and 20). The site includes contacts in three conserved loops
from one subunit (loops A, B, and C) that make up the "+" face, and
four loops from an adjacent subunit (loops I to IV) that make up the
" The data in Fig. 5 suggest that Because the primary structures of One potential explanation for the results is that An alternative explanation is based on the work of Green and co-workers
(23), who have shown that despite amino acid sequence identity, the
subunits of a functional Although The experiments with analogues suggest that although Additional information about interactions of The discovery of Previously, it has been shown that very minor changes in the
intercysteine amino acid sequences of conotoxins can drastically affect
their specificity. The toxins -conotoxin ImII (
-CTx
ImII), identified from Conus imperialis venom ducts, was
chemically synthesized. A previously characterized C. imperialis conotoxin,
-conotoxin ImI (
-CTx ImI), is
closely related; 9 of 12 amino acids are identical. Both
-CTx ImII
and
-CTx ImI functionally inhibit heterologously expressed rat
7
nAChRs with similar IC50 values. Furthermore, the
biological activities of intracranially applied
-CTx ImI and
-CTx
ImII are similar over the same dosage range, and are consistent with
7 nAChR inhibition. However, unlike
-CTx ImI,
-CTx ImII was
not able to block the binding of
-bungarotoxin to
7
nAChRs.
-Conotoxin ImI and
-bungarotoxin-binding sites have been
well characterized as overlapping and located at the cleft between
adjacent nAChR subunits. Because
-CTx ImI and
-CTx ImII share
extensive sequence homology, the inability of
-CTx ImII to compete
with
-BgTx is surprising. Furthermore, functional studies in oocytes
indicate that there is no overlap between functional binding sites of
-CTx ImI and
-CTx ImII. Like
-CTx ImI, the block by
-CTx
ImII is voltage-independent. Thus,
-CTx ImII represents a probe for
a novel antagonist binding site, or microsite, on the
7 nAChR.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-conotoxins are inhibitors of nicotinic acetylcholine receptors
(nAChRs),1 but individual
-conotoxins show a high degree of selectivity for different nAChR
subtypes including the neuromuscular subtype and various neuronal
subtypes (1). Minor changes in the sequence of the non-Cys residues of
conotoxins can profoundly change their receptor subtype specificity.
For example, the conotoxin
-CTx PnIA preferentially targets the
7
nAChR and
-CTx PnIB preferentially targets the
3
2 nAChR
despite the fact that the toxins only vary in two of 16 amino acids
(3).
-conotoxin,
-conotoxin ImII (
-CTx ImII) from the worm-hunting snail, Conus imperialis. This molecule is very similar to the
previously characterized C. imperialis toxin
-conotoxin
ImI (
-CTx ImI) (it is identical in 9 of 12 amino acids). Like
-CTx ImI,
-CTx ImII inhibits the
7 nAChR, and both toxins
display very similar potencies against this receptor. Unlike
-CTx
ImI, however,
-CTx ImII does not compete with
-bungarotoxin
(
-BgTx), a classical competitive inhibitor of the
7 nAChR.
Additionally, we show that
-CTx ImI and
-CTx ImII share little,
if any, overlap in their functional binding sites on the receptor. The
discovery of
-CTx ImII thus illustrates that not only can small
changes in intercysteine amino acids alter subtype specificity, but
they can also result in toxins that target the same receptor subtype at
different sites.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-BgTx were from
Sigma; 3-125I-
-BgTx (>200 Ci/mmol) was from
Amersham Biosciences; rat brains minus cerebellum were from
Zivic Miller (Zelionople, PA); HEK293 cells, Dulbecco's modified
Eagle's media, and fetal bovine serum were from ATCC (Manasses, VA);
and all other cell culture reagents were from Sigma. The plasmid
pZeoSV2-
7(V201)/5-HT3 was a gift from Dr.
N. S. Millar (4). The plasmid for generation of rat
7 nAChR
RNA was a gift from Dr. J. Boulter.
-CTx ImII--
The sequence of
-CTx ImII was
obtained as part of a systematic analysis of
-conotoxin sequences,
using PCR amplification of both cDNA and genomic DNA (5-7). The
specimen of C. imperialis analyzed was collected
in the Philippines, and hepatopancreas and venom duct tissue was
isolated and stored at
70 °C. The cDNA was prepared from venom
duct as described previously (8), and genomic DNA was extracted from
hepatopancreas using Puregene reagents and the marine invertebrates
protocol provided by the manufacturer (Gentra).
-CTx ImI was
synthesized and oxidized to form disulfide bridges (folded) as
described previously (9). Linear
-CTx ImII was synthesized by
standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry,
using an ABI model 430A peptide synthesizer at the University of Utah
core facility. The peptide was folded to give the correct disulfide
connectivity (first Cys to third Cys and second Cys to fourth Cys)
using orthogonal Cys protection. The first and third Cys residues had
stable Cys(S-acetomidomethyl) protection, whereas the second
and fourth Cys residues had acid-labile Cys(S-trityl)
protection. A previously described folding scheme (3) that sequentially
closed the second Cys to fourth Cys bridge and then the first Cys to
third Cys bridge was used to generate toxin. The analogs [P6R]
-CTx
ImI and [R6P]
-CTx ImII were generated in the same way as
-CTx ImII.
-conotoxins
was tested by intracranial injection into young mice as described
previously (10).
7 nAChR
was prepared and injected into Xenopus laevis
oocytes as described previously (11). The RNA was generated by in
vitro transcription using a plasmid that was a gift from Dr. J. Boulter. The plasmid carries a rat
7 nAChR cDNA clone (accession
number M85273) inserted into the EcoRI site of pBS SK(
).
RNA was transcribed from the T7 promoter of SmaI linearized
plasmid. Oocytes were injected 1-2 days after harvesting and used for
voltage clamping 1-7 days after injection.
70 mV
with a two-electrode system and were perfused in a 30-µl bath with
ND96 (96 mM NaCl, 2.0 mM KCl, 1.8 mM CaCl2, 1.0 mM MgCl2, 5 mM HEPES, pH 7.1-7.5). Currents were elicited with 1-s
pulses of 200 µM ACh in ND96 every 1 min. Only oocytes
that yielded stable responses to successive ACh pulses were used. To
determine the concentration dependence of inhibition of rat
7
nAChRs, toxin was applied using a static bath method. That is, the ACh
pulses and ND96 flow were halted, and conotoxin was applied to the
bath. The bath was allowed to equilibrate for 5 min before the ND96 flow was resumed at the same time an the ACh pulse was applied. ACh
pulses and ND96 flow continued until stable ACh-evoked currents were
re-established. To determine the inhibition at different conotoxin
concentrations, the peak current elicited by the first ACh pulse
following toxin exposure was normalized to the peak current elicited
following controls where ND96 alone, instead of toxin, were applied.
-CTx ImI or 1, 0.33, and 0.1 µM
-CTx ImII were carried out, and no difference in
percentage inhibition was seen at the two times.
-CTx ImII inhibition, the
block caused by 1 µM
-CTx ImII was measured as
described above, but at a range of holding potentials randomly altered
between
110,
90,
70,
50,
30, and
10 mV. To determine the
current-voltage relationship in the absence of toxin, the ratio of
current amplitudes at two successive potentials was determined from the
average of at least five currents before and after the voltage change.
7-5-HT3
Chimera--
The plasmid
pZeoSV2-
7(V201)/5-HT3 was a gift from Dr.
N. S. Millar (4). It encodes a chimeric receptor that has the
N-terminal ACh-binding domain of the rat
7 nAChR and the C-terminal
of the homologous 5-HT3 receptor. The chimera was expressed
in HEK293 cells, which are null for endogenous
-BgTx binding. HEK293
cells were grown in Dulbecco's modified Eagle's medium containing
10% heat-inactivated fetal calf serum, 100 µg/ml streptomycin, and 100 units/ml penicillin. Cells were transfected with
pZeoSV2-
7(V201)/5-HT3 using LipofectAMINE
(Invitrogen) according to the manufacturer's instructions for HEK293
cells. After 48 h the cells were washed with ice-cold potassium
Ringer's (PR) solution (140 mM KCl, 5.4 mM
NaCl, 1.7 mM MgCl2, 25 mM HEPES, and 30 µg/ml bovine serum albumin, adjusted to
a pH of 7.4 with 10 mM NaOH). The cells were detached into
fresh, ice-cold PR (3 ml/10-cm cell culture plate) with a cell scraper
and were spun down (10,000 × g). The cells were washed
twice by resuspension in ice-cold PR followed by recentrifugation. The
suspension was passed 10 times through an 18-gauge needle, divided into
1-ml aliquots, snap-frozen in liquid nitrogen, and stored at
70 °C
until use.
-BgTx (Amersham Biosciences) and various
concentrations of
-CTx ImI,
-CTx ImII, or 100 µM
MLA (to determine nonsaturatable binding).
-CTx ImI,
-CTx ImII,
or MLA were preincubated with cells for 30 min prior to the addition of
3-125I-
-BgTx (applied in a volume of 4 µl). The
radioligand was allowed to bind for 15 min during which its association
with receptor was linear with time (data not shown). The assays were
quenched with 500 µl of ice-cold d-tubocurarine (2400 µM). The cells were harvested (using a Brandell cell
harvester) through Whatman GF-B filters pretreated with 4% nonfat dry
milk. The filters were washed three times with about 800 µl of PR and
were counted using a COBRAII
-counter (Packard). Nonsaturatable
binding determined in assays containing 100 µM MLA was
subtracted from all readings and the resulting specific
3-125I-
-BgTx binding was normalized as a percentage of
specific binding in the absence of toxin. Assays were done at 25 ± 2 °C.
-CTx ImII and [P6R]
-CTx ImI, competition binding assay data
were fit to a straight line by linear regression.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-CTx ImII--
Members of a conotoxin
family, both from a given Conus species as well as from
different species, share conserved sequence elements in their gene
structure (12, 13). Thus, PCR strategies can amplify fragments of
conotoxin genes that include sequence encoding the mature toxin. PCR
was used to amplify
-conotoxin gene fragments from C. imperialis genomic DNA and cDNA. The heterogeneous pools of
PCR product were cloned and independent clones were sequenced; sequences encoding two closely related peptides,
-CTx ImI and
-CTx ImII, were found (Fig. 1). The
-CTx ImI peptide had previously been purified from C. imperialis venom (9) and is a potent and specific competitive
inhibitor of rat
7 nAChRs (14, 15). Based on the predicted
sequence from the clone,
-CTx ImII was chemically synthesized and
folded to form disulfide bonds (see "Experimental Procedures"),
and the synthetic peptide was then used to evaluate potential
interactions with
7 nAChRs.
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Fig. 1.
A, fragments of the -CTx ImII
gene were PCR amplified from genomic DNA and cDNA prepared from
C. imperialis tissue collected in the Philippines. The
nucleotide sequence in the vicinity of the region encoding mature toxin
is shown with the predicted translation product. The putative mature
peptide sequence is in bold letters. The N-terminal of the
mature toxin is deduced by the presence of an Arg (R) in the
larger precursor molecule that can act as a cleavage site for the
release of the mature toxin. The C terminus is deduced from the
presence of a stop codon; for the mature peptide we assume that the
C-terminal Gly (G) is post-translationally removed, leaving
Cys-12 amidated (amidation is represented by #). Fragments of the
-CTx ImI gene were also found in the pools of PCR products. A
fragment of the
-CTx ImI gene in the vicinity of the region encoding
mature toxin is shown, as is the known amino acid sequence of mature
-CTx ImI (9). B, the mature toxin sequences of
-CTx ImI and
-CTx ImII. The residues of
-CTx ImII that differ
from
-CTx ImI are underlined.
-CTx ImII Is Similar to That Seen for
the
7 nAChR-targeting Toxins
-CTx ImI and
-BgTx--
-CTx ImI and
-BgTx have been shown to cause
complex seizures when introduced intracranially into rats (14). This
behavior is believed to be because of inhibition of
7 nAChRs. To see
if
-CTx ImII caused similar effects, intracranial injections of
-CTx ImI and
-CTx ImII were made in young mice. As can be seen in
Table I, the effects of both toxins were
generally similar and are consistent with both toxins acting on the
neuronal
7 subtype of the nAChR.
Dose dependence of effects of -CTx ImI and
-CTx ImII on mice
-CTx ImII, Like
-CTx ImI, Inhibits ACh-gated Currents in Rat
7 nAChRs--
The ability of
-CTx ImII to inhibit ACh-gated
currents through rat
7 nAChRs heterologously expressed in X. laevis oocytes was determined as described under "Experimental
Procedures."
-CTx ImII inhibits these currents; the concentration
dependence of inhibition is shown in Fig.
2. As previously reported (14),
-CTx
ImI was also found to be an inhibitor of oocyte-expressed rat
7
nAChR. Using the protocol described under "Experimental Procedures,"
-CTx ImI and
-CTx ImII were found to have similar IC50 values (191 nM for
-CTx ImI and 441 nM for
-CTx ImII).
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Fig. 2.
A, concentration dependence of
conotoxin inhibition of ACh-gated currents through Xenopus
oocyte-expressed rat 7 nAChRs. Curves were determined as described
under "Experimental Procedures." The size of ACh-gated currents
at a given conotoxin concentration are plotted as the percent of
no-toxin controls.
-CTx ImI (squares) and
-CTx ImII
(triangles) have very similar curves (
-CTx ImI,
IC50 = 191 nM, nH = 0.879;
-CTx ImII, IC50 = 441 nM,
nH = 1.195). B, representative
traces showing block of the ACh-gated currents by
-CTx ImII. Pulses
of ACh (1 s at 1-min intervals) gate currents in oocytes expressing rat
7 nAChRs.
-CTx ImII applied in a static bath (see "Experimental
Procedures") results in a concentration-dependent
reduction of the peak height of ACh-gated currents obtained
simultaneously with the resumption of buffer flow. C,
filled circles, the amplitudes of ACh-gated currents
through oocyte-expressed rat
7 nAChR at different holding potentials
are normalized such that the average amplitude of responses gated at
70 mV is
1. Open circles, the amplitudes of ACh-gated
currents at different holding potentials following 5-min applications
of 1 µM
-CTx ImII are normalized such that the
70 mV
response after toxin application is equal to the
70 mV response in
the absence of toxin (i.e.
1). Data points and
error bars, mean ± S.E. for 3 to 6 measurements.
-CTx ImII, Like That by
-CTx ImI, Is
Voltage-independent--
The functional inhibition of oocyte-expressed
rat
7 nAChRs by 1 µM
-CTx ImII was measured at
different holding potentials. As can be seen from Fig. 2C,
the percent block was independent of holding potential indicating that
the activity of
-CTx ImII is not voltage-dependent.
Block by
-CTx ImI is also voltage-independent (15).
-CTx ImII, Unlike
-CTx ImI, Does Not Inhibit
-BgTx Binding
to Rat
7 nAChRs--
-BgTx is a classical competitive inhibitor
of some nAChR subtypes, including the
7 subtype. Therefore, the
abilities of
-CTx ImII and
-CTx ImI to inhibit
3-125I-
-bungarotoxin binding to two different rat
7
nAChR preparations were assessed.
-CTx ImII and
-CTx ImI to compete with
3-125I-
-BgTx for binding to crude rat brain membranes
was determined as described under "Experimental Procedures." As
shown in Fig. 3A,
-CTx ImII
is unable to significantly inhibit 3-125I-
-BgTx binding,
whereas
-CTx ImI inhibits all specific 3-125I-
-BgTx
binding. This contrasts with the functional inhibition of receptors
expressed in oocytes, where both conotoxins exhibited roughly equal
IC50 values (compare Fig. 3A with Fig.
2A).
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Fig. 3.
Inhibition of
3-125I- -BgTx binding to crude rat
brain membranes (A) and
7-5-HT3 chimera
(B).
-CTx ImI (squares) and
-CTx
ImII (circles) were added to compete with radiolabeled
-bungarotoxin as described under "Experimental Procedures."
The specific binding of 3-125I-
-BgTx at each conotoxin
concentration is normalized to that obtained in the absence of
conotoxins. For
-CTx ImI, the EC50 is 1,560 nM (nH = 0.59) on crude membranes
and 407 nM (nH = 0.71) on
7-5-HT3 chimera. The
-CTx ImI data were fit to a
curve and the
-CTx ImII data were fit to a straight
line as described under "Experimental Procedures." Data
points and error bars, mean ± S.E. for 3 to 6 measurements.
7 nAChR,
and the N-terminal ACh-binding domain of the
7 nAChR has been used to replace the N-terminal 5-HT-binding domain from the
5-HT3 receptor (4, 16, 17). The resulting chimera can be
expressed in HEK293 cells such that
-BgTx-binding sites are produced
at a level ~1000-fold higher than when the native
7 receptor is
used (17). In addition, the chimera retains the pharmacology of the wild-type receptor with respect to many cholinergic agonists and antagonists (16, 17). The ability of
-CTx ImII and
-CTx ImI to
inhibit 3-125I-
-BgTx binding to rat
7-5-HT3 chimera was tested as described under
"Experimental Procedures." As shown in Fig. 3B, the same pattern of inhibition was seen with the chimera as with the native
7
receptor. Again,
-CTx ImII was unable to significantly block 3-125I-
-BgTx binding, but
-CTx ImI inhibited all
specific binding of the radiolabel.
-BgTx-binding
Sites--
It was previously shown using rat hippocampal neurons (15)
that preincubation of
7 nAChRs with
-CTx ImI prevents the very slowly reversible functional block by
-BgTx. We have used a similar approach to investigate the functional binding sites of
-CTx ImII
and
-CTx ImI on oocyte-expressed rat
7 nAChRs. It was found that a 5-min bath application of 100 nM
-BgTx is
sufficient to block about 95% of ACh-gated current in oocytes
expressing rat
7 nAChRs. Because of the very slow off-rate of
-BgTx, no significant recovery was observed after washing toxin from
the oocyte bath (Fig. 4A).
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Fig. 4.
Functional competition between
-BgTx and
-conotoxins. Xenopus oocytes
expressing rat
7 nAChRs were voltage-clamped as described under
"Experimental Procedures" and their responses to 1-s ACh pulses
at 1-min intervals were recorded. The peak heights are all normalized
to the average of 5 peaks recorded prior to toxin application.
A, bath perfusion was paused for 11 min. After 1 min, 1 µl of ND96 was added; after 6 min,
-BgTx was added (black
bar) in 1 µl of ND96 to a final concentration of 100 nM; after 11 min, ND96 flow and ACh pulses were resumed.
B, bath perfusion was paused for 11 min. After 1 min,
-CTx ImI in 1 µl of ND96 was added (striped bar) to a
final concentration of 100 µM; after 6 min,
-BgTx was
added (black bar) in 1 µl of ND96 to a final concentration
of 100 nM; after 11 min, ND96 flow and ACh pulses were
resumed. C, the same protocol as in B was
used except that
-CTx ImII was added (white bar) instead
of
-CTx ImI. Data points and error bars,
mean ± S.D. for four repetitions.
-CTx ImI and then subjected to a 5-min
co-application of
-BgTx and
-CTx ImI, very rapid and essentially
full recovery was observed after washing out the toxins. This result is
consistent with
-CTx ImI binding preventing the slowly reversible
block by
-BgTx, i.e. that the two toxins compete for the
same functional site. However, a much more limited ability to protect
against block by
-BgTx (Fig. 4C) is achieved by a similar
preincubation with
-CTx ImII. Note that 5 min of bath application of
-CTx ImI and
-CTx ImII is sufficient for both to reach
equilibrium with receptor (see "Experimental Procedures").
-CTx ImI Does Not
Inhibit Binding of
-CTx ImII to Oocyte-expressed Rat
7
nAChRs--
The ability of
-CTx ImII to bind to oocyte-expressed
receptor was tested with and without pre-equilibration of oocytes with a high concentration of
-CTx ImI. As can be seen in Fig.
5A, a 5-min bath application
of
-CTx ImI (100 µM) or
-CTx ImII (10 µM) is sufficient to completely inhibit ACh-gated ion
currents in oocyte-expressed rat
7 nAChRs. Subsequent washout
results in full recovery for both toxins; however,
-CTx ImII has a
noticeably slower off-rate than
-CTx ImI. Although the differences
are subtle, they are highly reproducible and a diagnostic functional
difference between the toxins.
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Fig. 5.
Functional competition between
-CTx ImI and
-CTx
ImII. Xenopus oocytes expressing rat
7 nAChRs were
voltage-clamped as described under "Experimental Procedures" and
their response to brief ACh pulses at 1-min intervals were recorded.
The peak heights are all normalized to the average of 5 peaks recorded
prior to toxin application. A1, bath perfusion was
paused for 6 min. After 1 min,
-CTx ImI in 1 µl of ND96 was added
to a final concentration of 100 µM (striped
bar); after 6 min, ND96 flow and ACh pulses were resumed.
A2, the same protocol as in A1 was applied
except that
-CTx ImII was added to a final concentration of 10 µM (white bar). B, bath
perfusion was paused for 11 min. After 1 min, 1 µl of ND96 was added;
after 6 min,
-CTx ImII was added in 1 µl of ND96 to a final
concentration of 10 µM (white bar); after 11 min, ND96 flow and ACh pulses were resumed. C, bath
perfusion was paused for 11 min. After 1 min,
-CTx ImI was added in
1 µl of ND96 to a final concentration of 100 µM
(striped bar); after 6 min, 1 µl of ND96 was added; after
11 min, ND96 flow and ACh pulses were resumed. D, bath
perfusion was paused for 11 min. After 1 min,
-CTx ImI was added in
1 µl of ND96 to a final concentration of 100 µM; after
6 min,
-CTx ImII was added in 1 µl of ND96 to a final
concentration of 10 µM; after 11 min, ND96 flow and ACh
pulses were resumed. Data points and error
bars, mean ± S.D. for four repetitions for
B, C, and D. A1 and
A2 are representative traces.
-CTx ImI was bath-applied to oocytes
expressing rat
7 nAChRs for 10 min, the characteristic fast off-rate
of
-CTx ImI was observed (Fig. 5C). However, when 100 µM
-CTx ImI was bath-applied for 5 min and 10 µM
-CTx ImII was then added, giving 5 min of
co-application of
-CTx ImI and
-CTx ImII, the characteristic slow
off-rate for
-CTx ImII was observed, and the result was not
detectably different from that of the control experiment in Fig.
5B (no toxin was applied for 5 min, 10 µM
-CTx ImII was then added for 5 min).
-CTx ImI does not inhibit
-CTx ImII binding to rat
7 nAChRs despite this concentration
being about 520 times greater than the functional IC50.
These, as well as the previous data, are consistent with the conclusion
that
-CTx ImI and
-CTx ImII have little if any overlap in their
high affinity binding sites on the
7 receptor. Nevertheless,
occupancy of the two different sites by each
-conotoxin leads to
functional block of the receptor.
-Conotoxins ImI and ImII--
The peptides
-CTx
ImI and
-CTx ImII are identical in 9 of 12 amino acids. Because they
appear to target different sites on the
7 nAChR, we performed a
structure/function study to identify which amino acids were critical
for the difference in targeting. Of the three differences, those at
positions 6 (Pro versus Arg) and 9 (Ala versus
Arg) seem the most striking. At position 1 (Gly versus Ala),
the two residues differ only by a methyl group. Additionally, the
absence of a first loop Pro is very unusual in
-conotoxins (see
Table II). The two analogs shown in Table
II were thus synthesized (see "Experimental Procedures") and
characterized.
-Conotoxin and analog sequences
-CTx ImII are from a recent review
(1). O, hydroxy proline; #, C-terminal amidation. Y15
is sulfated in EpI.
-Conotoxins have the disulfide connectivity:
first Cys to third Cys and second Cys to fourth Cys. Proline residues
in the first loop are underlined. P6R
-CTx ImI and R6P
-CTx ImII
are the analogs described in this study.
7 nAChR inhibition (data not shown).
Nevertheless, what is clearly indicated by the data is that the
presence of a proline residue at position 6 is the major determinant of
whether a peptide will compete with radiolabeled
-bungarotoxin for
binding to the
7 receptor (Fig. 6).
Thus, R6P
-CTx ImII is better at displacing
-bungarotoxin than
native
-CTx ImII. In contrast, replacement of the Pro-6 residue in
-CTx ImI with Arg results in failure to displace
-bungarotoxin
even at a concentration of 100 µM (compared with an
EC50 for native
-CTx ImI of 407 nM). Thus,
the presence or absence of proline at position 6 determines whether or
not these peptides preferentially bind to a site that overlaps with the
-bungarotoxin-binding site or another site.
View larger version (14K):
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Fig. 6.
Competition binding of
-conotoxin analogs. A,
inhibition of 3-125I-
-BgTx binding to
7-5-HT3 chimera by
-CTx ImI (squares) and
P6R
-ImI (circles). The specific binding of
3-125I-
-BgTx at each conotoxin or mutant toxin
concentration is normalized to specific 3-125I-
-BgTx
binding in the absence of peptide. The
-CTx ImI data was fit to a
curve and the P6R
-CTx ImI data was fit to a straight line as
described under "Experimental Procedures." For
-CTx ImI, the
EC50 is 407 nM and the
nH is 0.71. B, inhibition of
3-125I-
-BgTx binding to rat
7-5-HT3
chimeras by
-CTx ImII (circles) and R6P
-CTx ImII
(squares) was determined as described under "Experimental
Procedures." The specific binding of 3-125I
-BgTx at
each conotoxin or mutant toxin concentration is normalized to specific
3-125I-
-BgTx binding in the absence of peptide. The R6P
-CTx ImII data was fit to a curve and the
-CTx ImII data was fit
to a straight line as described under "Experimental Procedures."
For R6P
-CTx ImII, the EC50 is 19.3 µM and
the nH is 0.78. Data points and
error bars, mean ± S.E. for 3 to 6 measurements.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-CTx ImII that
has high sequence identity (9 of 12 amino acids) to
-CTx ImI (Fig.
1); both peptides are from the venom ducts of C. imperialis (9).
-CTx ImI is a specific competitive
inhibitor of the
7 nAChR subtype (14, 15). Given the close sequence
similarity of
-CTx ImI and
-CTx ImII, it was not surprising that
-CTx ImII was also found to inhibit the
7 nAChR. However, most
unexpectedly, the two closely related peptides appear to cause their
similar functional effects by binding to different sites on the
7 nAChR.
-CTx ImII was found to be similar to
-CTx ImI in the behavioral
effects observed when injected intracranially into mice; both peptides
elicited complex seizures, weakness, tremors and, at higher doses,
death. Similar behavior was also observed following intracerebral-ventricular injection of
-BgTx, another
7 nAChR inhibitor, into rats (14). In view of its homology to
-CTx ImI and
the characteristic symptoms observed when it was injected into the
central nervous system,
-CTx ImII was tested for its ability
to inhibit ACh-gated currents in Xenopus oocytes
expressing rat
7 nAChRs.
-CTx ImII was found to inhibit the
receptor with an IC50 similar to that of
-CTx ImI (Fig.
2) when the toxins were tested using identical protocols.
-CTx ImII was tested
in a competition assay with 3-125I-
-BgTx. As had been
previously demonstrated by others (18), we found that
-CTx ImI
competed with
-BgTx for binding to the receptor. In contrast,
-CTx ImII did not appreciably displace
-BgTx binding in the
concentration range tested. These results for
-CTx ImI and
-CTx
ImII were obtained with both rat brain
7 nAChRs (Fig. 3A)
as well as rat
7-5-HT3 chimeras (Fig. 3B). Furthermore, experiments using rat
7 nAChRs expressed in oocytes (Fig. 4) demonstrated that preincubation with
-CTx ImI prevents
-BgTx from binding to its functionally relevant site, a result consistent with competitive antagonism, and previously shown by others
(15). On the other hand,
-CTx ImII had only a very weak effect on
-BgTx inhibition of oocyte-expressed receptor, consistent with a
different binding site.
" face. The binding of
-CTx ImI to the
7 nAChR is affected
by mutations in or near loops A, B, and C, and II and III (17, 18). The
-BgTx site on the
7 nAChR has also been mapped to the A, B, and C
loops (21), and a loop II mutation causes a minor reduction in
-BgTx
affinity (22). This and other evidence are consistent with
-CTx
ImI-binding sites in
7 nAChRs overlapping with ACh and
-BgTx-binding sites, and being at subunit interfaces.
-CTx ImI and
-CTx ImII do not
bind to the same site at a subunit interface. Assuming a potential five
identical subunit interfaces in the
7 nAChR pentamer, and that
occupation of even one site by
-CTx ImI results in inhibition of the
receptor, then the concentration of
-CTx ImI that occupies half the
potential sites, Kd, is related to the functional IC50 by IC50/Kd = 0.15 (3),
i.e. Kd = IC50 × 6.67. The
IC50 of
-CTx ImI on the
7 nAChR is 191 nM
(Fig. 2). Therefore, 100 µM
-CTx ImI (see Fig. 5)
would clearly occupy most subunit interface-binding sites on the
7
nAChR (assuming these are identical) and should significantly reduce
binding of
-CTx ImII if
-CTx ImI and
-CTx ImII share a binding
site. The
-CTx ImII-binding site awaits definitive characterization;
however, several possibilities are outlined below.
-CTx ImI and
-CTx ImII are so
similar, and because they share the characteristic
-conotoxin disulfide framework, it seems possible that
-CTx ImII also binds to
the interface between
7 subunits. In this case, the inability of
-CTx ImII to compete with
-BgTx or
-CTx ImI might be explained by the following models.
-CTx ImI and
-CTx ImII can simultaneously bind at a single subunit interface by
positioning differently within the cleft at different microsites. In
fact,
-BgTx appears to make more contacts with the + face than with
the
face at
7 nAChR subunit interfaces (21, 22). It is
possible, for example, that
-CTx ImII binds predominantly to
the
face and is thus unable to displace
-BgTx, whereas
-CTx ImI, because of many contacts in the + face, disrupts many
-BgTx-receptor interactions, and is thus able to compete with this toxin.
7 nAChR receptor are not identical.
Evidence was presented that the functional
7 nAChR complex requires
a mixture of
7 subunits that are in at least two states that differ
in their N-terminal domain conformation and the oxidation state of Cys
residues (23). A direct consequence of this nonidentity is that
putative ligand-binding sites located between subunits become
distinguishable. One possibility is that one type of interface between
7 subunits is the
-CTx ImI and
-BgTx-binding site, whereas
another type of subunit interface does not bind
-BgTx, but is the
-CTx ImII target site. Bertrand and co-workers (24) have shown that
for the competitive
7 nAChR antagonist MLA there are five identical
binding sites. This is not necessarily incompatible with a
heterogeneous interface model. MLA may recognize structural elements at
interfaces that are unaffected by the state of flanking subunits.
However, other ligands might be sensitive to the state of flanking
subunits and thus have distinguishable interface-binding sites. In
fact, there is evidence to support the notion of nonhomogeneous
-BgTx-binding sites on
7 nAChRs (25). Additionally, in mouse
brain, some [3H]MLA-binding sites are resistant to
competition by
-BgTx (26); because the resistant fraction does not
appear to be because of a distinct MLA receptor, a simple explanation
could be that MLA binds to all five subunit interfaces but
-BgTx,
even at high concentrations, cannot.
-CTx ImI and
-CTx ImII show extensive sequence homology,
it is possible that
-CTx ImII binds to a nonsubunit-interface site
on the receptor. For example, it might bind extracellular regions of
the receptor that are not in the N-terminal ACh-binding domain,
i.e. the extracellular loop that occurs between two
transmembrane helices of the
7 nAChR or the C-terminal extracellular
region.
-CTx ImII could also potentially bind to nonsubunit
interface regions on the N-terminal ACh-binding domain or the channel
pore; however, because
-CTx ImII block is not voltage dependent,
this supports the model that it is not an open channel blocker.
-CTx ImI and
-CTx ImII have very similar sequences, the amino acid residue at
position 6 (Pro in
-CTx ImI, Arg in
-CTx ImII) is critical in
determining where they bind on the
7 nAChR. Relative to wild-type
-CTx ImI, P6R
-CTx ImI is a very poor competitor of
-BgTx
binding to
7-5-HT3 chimera. In contrast, R6P
-CTx ImII has an enhanced ability to compete with
-BgTx compared with wild-type
-CTx ImII. Because the two native toxins apparently target
different sites, a key determinant for selectivity is which amino acid
is present at position 6.
-CTx ImI and
-CTx
ImII with their distinct binding sites can be derived from the analog
toxin data if one assumes the initial Gly and Ala residues in the two
toxins are functionally equivalent. In this case, the P6R
-CTx ImI
analog is equivalent to R9A
-CTx ImII and the R6P
-CTx-ImII analog
is equivalent to A9R
-CTx-ImI. Because P6R
-CTx ImI does not
compete with
-BgTx for binding to the
7 nAChR, this strongly suggests that R9A
-CTx ImII would be like
-CTx ImII and also not
compete with
-BgTx. Because R6P
-CTx ImII has some ability to
compete
-BgTx but is not as potent as
-CTx ImI, this strongly suggests that A9R
-CTx ImI would compete with
-BgTx for binding to the
7 nAChR but would be a less potent competitor than
-CTx ImI. Taken together, these observations imply that the residues at
position 9 in
-CTx ImI and
-CTx ImII are not critical in determining whether the
-CTx ImI or
-CTx ImII site is targeted, but are important for ensuring optimal affinity of
-CTx ImI and
-CTx ImII for their respective sites.
-CTx ImII reveals that C. imperialis has
two toxins that inhibit the rat
7 nAChR, and that these act at different sites. Although caution must be applied when extrapolating this observation to the native prey, it suggests that C. imperialis may target marine worms with both
-CTx ImI and
-CTx ImII, which may bind to different sites on an
"
7-like" receptor in native prey. This would
represent a second example of cone snail venom containing two distinct
antagonists of the same nAChR. It was previously demonstrated that
Conus purpurascens produces two structurally unrelated nAChR
antagonists, a competitive
A-conotoxin and a noncompetitive
-conotoxin (reviewed in Ref. 1). The present case is different,
however, in that the toxins are both
-conotoxins that are very
closely related to each other in sequence. A caveat that must be
applied to this model is that natural, venom-derived
-CTx ImII may
possess post-translational modifications that were not incorporated in
the synthetic peptide used in this study. The native toxin may thus
differ from the synthetic molecule in its functional properties,
i.e. it may not target an
7-like receptor at all.
On the other hand, post-translational modification in
-conotoxins
isolated from venom have so far been limited to C-terminal amidation
and tyrosine sulfation (
-CTx ImI and
-CTx ImII lack tyrosine residues).
-CTx PnIA and
-CTx PnIB from
Conus pennaceus are different in only 2 of 16 amino acids, but preferentially block
3
2 and
7 nAChRs, respectively (3). The discovery of
-CTx ImII illustrates that in C. imperialis, minor differences between two toxins result in
molecules that target, not distinct receptor subtypes, but distinct
sites on a single nAChR subtype.
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ACKNOWLEDGEMENT |
---|
We thank Doju Yoshikami for helpful discussions and critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants GM48677 and MH53631.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.
The nucleotide sequence(s) reported in this paper for portions of
the -CTx ImI and
-CTx ImII genes that encode the mature toxins
has been submitted to the GenBankTM/EBI Data
Bank under accession numbers AY159317 and AY159318, respectively.
§ Supported in part by National Institutes of Health Grant GM08537 (to the University of Utah). To whom correspondence should be addressed: Dept. of Biology, University of Utah, 257 South 1400 East, Salt Lake City, UT 84112. Tel.: 801-581-8370; Fax: 801-585-5010; E-mail: michael.ellison@m.cc.utah.edu.
Published, JBC Papers in Press, October 15, 2002, DOI 10.1074/jbc.M204565200
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ABBREVIATIONS |
---|
The abbreviations used are:
ACh, acetylcholine;
-BgTx,
-bungarotoxin;
-CTx,
-conotoxin;
MLA, methyllycaconitine;
PR, potassium Ringer's solution;
5-HT, 5-hydroxytryptamine (serotonin);
5-HT3 receptor, type 3 serotonin receptor.
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