alpha -Conotoxins ImI and ImII

SIMILAR alpha 7 NICOTINIC RECEPTOR ANTAGONISTS ACT AT DIFFERENT SITES*

Michael EllisonDagger §, J. Michael McIntoshDagger , and Baldomero M. OliveraDagger

From the Departments of Dagger  Biology and  Psychiatry, University of Utah, Salt Lake City, Utah 84112

Received for publication, May 9, 2002, and in revised form, October 11, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A novel conotoxin, alpha -conotoxin ImII (alpha -CTx ImII), identified from Conus imperialis venom ducts, was chemically synthesized. A previously characterized C. imperialis conotoxin, alpha -conotoxin ImI (alpha -CTx ImI), is closely related; 9 of 12 amino acids are identical. Both alpha -CTx ImII and alpha -CTx ImI functionally inhibit heterologously expressed rat alpha 7 nAChRs with similar IC50 values. Furthermore, the biological activities of intracranially applied alpha -CTx ImI and alpha -CTx ImII are similar over the same dosage range, and are consistent with alpha 7 nAChR inhibition. However, unlike alpha -CTx ImI, alpha -CTx ImII was not able to block the binding of alpha -bungarotoxin to alpha 7 nAChRs. alpha -Conotoxin ImI and alpha -bungarotoxin-binding sites have been well characterized as overlapping and located at the cleft between adjacent nAChR subunits. Because alpha -CTx ImI and alpha -CTx ImII share extensive sequence homology, the inability of alpha -CTx ImII to compete with alpha -BgTx is surprising. Furthermore, functional studies in oocytes indicate that there is no overlap between functional binding sites of alpha -CTx ImI and alpha -CTx ImII. Like alpha -CTx ImI, the block by alpha -CTx ImII is voltage-independent. Thus, alpha -CTx ImII represents a probe for a novel antagonist binding site, or microsite, on the alpha 7 nAChR.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -conotoxins are inhibitors of nicotinic acetylcholine receptors (nAChRs),1 but individual alpha -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 alpha -CTx PnIA preferentially targets the alpha 7 nAChR and alpha -CTx PnIB preferentially targets the alpha 3beta 2 nAChR despite the fact that the toxins only vary in two of 16 amino acids (3).

In this report, we describe the discovery of a novel alpha -conotoxin, alpha -conotoxin ImII (alpha -CTx ImII) from the worm-hunting snail, Conus imperialis. This molecule is very similar to the previously characterized C. imperialis toxin alpha -conotoxin ImI (alpha -CTx ImI) (it is identical in 9 of 12 amino acids). Like alpha -CTx ImI, alpha -CTx ImII inhibits the alpha 7 nAChR, and both toxins display very similar potencies against this receptor. Unlike alpha -CTx ImI, however, alpha -CTx ImII does not compete with alpha -bungarotoxin (alpha -BgTx), a classical competitive inhibitor of the alpha 7 nAChR. Additionally, we show that alpha -CTx ImI and alpha -CTx ImII share little, if any, overlap in their functional binding sites on the receptor. The discovery of alpha -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Puregene reagents were purchased from Gentra (Minneapolis, MN); PCR and molecular biology reagents were from Invitrogen (Carlsbad, CA); salts, acetylcholine, and alpha -BgTx were from Sigma; 3-125I-alpha -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-alpha 7(V201)/5-HT3 was a gift from Dr. N. S. Millar (4). The plasmid for generation of rat alpha 7 nAChR RNA was a gift from Dr. J. Boulter.

Discovery of alpha -CTx ImII-- The sequence of alpha -CTx ImII was obtained as part of a systematic analysis of alpha -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).

Peptide Synthesis and Folding-- Linear alpha -CTx ImI was synthesized and oxidized to form disulfide bridges (folded) as described previously (9). Linear alpha -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]alpha -CTx ImI and [R6P]alpha -CTx ImII were generated in the same way as alpha -CTx ImII.

Bioassays-- Biological activity of synthetic alpha -conotoxins was tested by intracranial injection into young mice as described previously (10).

Electrophysiology-- Complementary RNA encoding rat alpha 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 alpha 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.

Voltage clamping was done essentially as has been described elsewhere (11). Briefly, oocytes were clamped at a holding potential of -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 alpha 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.

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 alpha -CTx ImI or 1, 0.33, and 0.1 µM alpha -CTx ImII were carried out, and no difference in percentage inhibition was seen at the two times.

To investigate the voltage dependence of alpha -CTx ImII inhibition, the block caused by 1 µM alpha -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.

Preparation of Cells Expressing Rat alpha 7-5-HT3 Chimera-- The plasmid pZeoSV2-alpha 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 alpha 7 nAChR and the C-terminal of the homologous 5-HT3 receptor. The chimera was expressed in HEK293 cells, which are null for endogenous alpha -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-alpha 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.

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-alpha -BgTx (Amersham Biosciences) and various concentrations of alpha -CTx ImI, alpha -CTx ImII, or 100 µM MLA (to determine nonsaturatable binding). alpha -CTx ImI, alpha -CTx ImII, or MLA were preincubated with cells for 30 min prior to the addition of 3-125I-alpha -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 gamma -counter (Packard). Nonsaturatable binding determined in assays containing 100 µM MLA was subtracted from all readings and the resulting specific 3-125I-alpha -BgTx binding was normalized as a percentage of specific binding in the absence of toxin. Assays were done at 25 ± 2 °C.

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 alpha -CTx ImII and [P6R]alpha -CTx ImI, competition binding assay data were fit to a straight line by linear regression.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

PCR-based Discovery of alpha -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 alpha -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, alpha -CTx ImI and alpha -CTx ImII, were found (Fig. 1). The alpha -CTx ImI peptide had previously been purified from C. imperialis venom (9) and is a potent and specific competitive inhibitor of rat alpha 7 nAChRs (14, 15). Based on the predicted sequence from the clone, alpha -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 alpha 7 nAChRs.


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Fig. 1.   A, fragments of the alpha -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 alpha -CTx ImI gene were also found in the pools of PCR products. A fragment of the alpha -CTx ImI gene in the vicinity of the region encoding mature toxin is shown, as is the known amino acid sequence of mature alpha -CTx ImI (9). B, the mature toxin sequences of alpha -CTx ImI and alpha -CTx ImII. The residues of alpha -CTx ImII that differ from alpha -CTx ImI are underlined.

The Biological Activity of alpha -CTx ImII Is Similar to That Seen for the alpha 7 nAChR-targeting Toxins alpha -CTx ImI and alpha -BgTx-- alpha -CTx ImI and alpha -BgTx have been shown to cause complex seizures when introduced intracranially into rats (14). This behavior is believed to be because of inhibition of alpha 7 nAChRs. To see if alpha -CTx ImII caused similar effects, intracranial injections of alpha -CTx ImI and alpha -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 alpha 7 subtype of the nAChR.

                              
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Table I
Dose dependence of effects of alpha -CTx ImI and alpha -CTx ImII on mice
Intracranial injections were made in young mice as described under "Experimental Procedures." The behavior of the mice was observed for at least 1 h. The numbers of animals exhibiting clearcut effects divided by the total number injected are shown for each dose. The effects seen were fine to coarse tremors, circling, weak gait and at the higher doses, rolling and death.

alpha -CTx ImII, Like alpha -CTx ImI, Inhibits ACh-gated Currents in Rat alpha 7 nAChRs-- The ability of alpha -CTx ImII to inhibit ACh-gated currents through rat alpha 7 nAChRs heterologously expressed in X. laevis oocytes was determined as described under "Experimental Procedures." alpha -CTx ImII inhibits these currents; the concentration dependence of inhibition is shown in Fig. 2. As previously reported (14), alpha -CTx ImI was also found to be an inhibitor of oocyte-expressed rat alpha 7 nAChR. Using the protocol described under "Experimental Procedures," alpha -CTx ImI and alpha -CTx ImII were found to have similar IC50 values (191 nM for alpha -CTx ImI and 441 nM for alpha -CTx ImII).


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Fig. 2.   A, concentration dependence of conotoxin inhibition of ACh-gated currents through Xenopus oocyte-expressed rat alpha 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. alpha -CTx ImI (squares) and alpha -CTx ImII (triangles) have very similar curves (alpha -CTx ImI, IC50 = 191 nM, nH = 0.879; alpha -CTx ImII, IC50 = 441 nM, nH = 1.195). B, representative traces showing block of the ACh-gated currents by alpha -CTx ImII. Pulses of ACh (1 s at 1-min intervals) gate currents in oocytes expressing rat alpha 7 nAChRs. alpha -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 alpha 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 alpha -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.

Block by alpha -CTx ImII, Like That by alpha -CTx ImI, Is Voltage-independent-- The functional inhibition of oocyte-expressed rat alpha 7 nAChRs by 1 µM alpha -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 alpha -CTx ImII is not voltage-dependent. Block by alpha -CTx ImI is also voltage-independent (15).

alpha -CTx ImII, Unlike alpha -CTx ImI, Does Not Inhibit alpha -BgTx Binding to Rat alpha 7 nAChRs-- alpha -BgTx is a classical competitive inhibitor of some nAChR subtypes, including the alpha 7 subtype. Therefore, the abilities of alpha -CTx ImII and alpha -CTx ImI to inhibit 3-125I-alpha -bungarotoxin binding to two different rat alpha 7 nAChR preparations were assessed.

The ability of alpha -CTx ImII and alpha -CTx ImI to compete with 3-125I-alpha -BgTx for binding to crude rat brain membranes was determined as described under "Experimental Procedures." As shown in Fig. 3A, alpha -CTx ImII is unable to significantly inhibit 3-125I-alpha -BgTx binding, whereas alpha -CTx ImI inhibits all specific 3-125I-alpha -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-alpha -BgTx binding to crude rat brain membranes (A) and alpha 7-5-HT3 chimera (B). alpha -CTx ImI (squares) and alpha -CTx ImII (circles) were added to compete with radiolabeled alpha -bungarotoxin as described under "Experimental Procedures." The specific binding of 3-125I-alpha -BgTx at each conotoxin concentration is normalized to that obtained in the absence of conotoxins. For alpha -CTx ImI, the EC50 is 1,560 nM (nH = 0.59) on crude membranes and 407 nM (nH = 0.71) on alpha 7-5-HT3 chimera. The alpha -CTx ImI data were fit to a curve and the alpha -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.

The 5-HT3 receptor is highly homologous to the alpha 7 nAChR, and the N-terminal ACh-binding domain of the alpha 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 alpha -BgTx-binding sites are produced at a level ~1000-fold higher than when the native alpha 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 alpha -CTx ImII and alpha -CTx ImI to inhibit 3-125I-alpha -BgTx binding to rat alpha 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 alpha 7 receptor. Again, alpha -CTx ImII was unable to significantly block 3-125I-alpha -BgTx binding, but alpha -CTx ImI inhibited all specific binding of the radiolabel.

Competition for Functional alpha -BgTx-binding Sites-- It was previously shown using rat hippocampal neurons (15) that preincubation of alpha 7 nAChRs with alpha -CTx ImI prevents the very slowly reversible functional block by alpha -BgTx. We have used a similar approach to investigate the functional binding sites of alpha -CTx ImII and alpha -CTx ImI on oocyte-expressed rat alpha 7 nAChRs. It was found that a 5-min bath application of 100 nM alpha -BgTx is sufficient to block about 95% of ACh-gated current in oocytes expressing rat alpha 7 nAChRs. Because of the very slow off-rate of alpha -BgTx, no significant recovery was observed after washing toxin from the oocyte bath (Fig. 4A).


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Fig. 4.   Functional competition between alpha -BgTx and alpha -conotoxins. Xenopus oocytes expressing rat alpha 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, alpha -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, alpha -CTx ImI in 1 µl of ND96 was added (striped bar) to a final concentration of 100 µM; after 6 min, alpha -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 alpha -CTx ImII was added (white bar) instead of alpha -CTx ImI. Data points and error bars, mean ± S.D. for four repetitions.

However, when oocytes were pretreated for 5 min with 100 µM alpha -CTx ImI and then subjected to a 5-min co-application of alpha -BgTx and alpha -CTx ImI, very rapid and essentially full recovery was observed after washing out the toxins. This result is consistent with alpha -CTx ImI binding preventing the slowly reversible block by alpha -BgTx, i.e. that the two toxins compete for the same functional site. However, a much more limited ability to protect against block by alpha -BgTx (Fig. 4C) is achieved by a similar preincubation with alpha -CTx ImII. Note that 5 min of bath application of alpha -CTx ImI and alpha -CTx ImII is sufficient for both to reach equilibrium with receptor (see "Experimental Procedures").

Preincubation with a High Concentration of alpha -CTx ImI Does Not Inhibit Binding of alpha -CTx ImII to Oocyte-expressed Rat alpha 7 nAChRs-- The ability of alpha -CTx ImII to bind to oocyte-expressed receptor was tested with and without pre-equilibration of oocytes with a high concentration of alpha -CTx ImI. As can be seen in Fig. 5A, a 5-min bath application of alpha -CTx ImI (100 µM) or alpha -CTx ImII (10 µM) is sufficient to completely inhibit ACh-gated ion currents in oocyte-expressed rat alpha 7 nAChRs. Subsequent washout results in full recovery for both toxins; however, alpha -CTx ImII has a noticeably slower off-rate than alpha -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 alpha -CTx ImI and alpha -CTx ImII. Xenopus oocytes expressing rat alpha 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, alpha -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 alpha -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, alpha -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, alpha -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, alpha -CTx ImI was added in 1 µl of ND96 to a final concentration of 100 µM; after 6 min, alpha -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.

When 100 µM alpha -CTx ImI was bath-applied to oocytes expressing rat alpha 7 nAChRs for 10 min, the characteristic fast off-rate of alpha -CTx ImI was observed (Fig. 5C). However, when 100 µM alpha -CTx ImI was bath-applied for 5 min and 10 µM alpha -CTx ImII was then added, giving 5 min of co-application of alpha -CTx ImI and alpha -CTx ImII, the characteristic slow off-rate for alpha -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 alpha -CTx ImII was then added for 5 min).

This suggests that 100 µM alpha -CTx ImI does not inhibit alpha -CTx ImII binding to rat alpha 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 alpha -CTx ImI and alpha -CTx ImII have little if any overlap in their high affinity binding sites on the alpha 7 receptor. Nevertheless, occupancy of the two different sites by each alpha -conotoxin leads to functional block of the receptor.

Analogs of alpha -Conotoxins ImI and ImII-- The peptides alpha -CTx ImI and alpha -CTx ImII are identical in 9 of 12 amino acids. Because they appear to target different sites on the alpha 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 alpha -conotoxins (see Table II). The two analogs shown in Table II were thus synthesized (see "Experimental Procedures") and characterized.

                              
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Table II
alpha -Conotoxin and analog sequences
All the native sequences except alpha -CTx ImII are from a recent review (1). O, hydroxy proline; #, C-terminal amidation. Y15 is sulfated in EpI. alpha -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 alpha -CTx ImI and R6P alpha -CTx ImII are the analogs described in this study.

Both analogs are significantly less functionally potent than the corresponding native peptides as determined by electrophysiological characterization of alpha 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 alpha -bungarotoxin for binding to the alpha 7 receptor (Fig. 6). Thus, R6P alpha -CTx ImII is better at displacing alpha -bungarotoxin than native alpha -CTx ImII. In contrast, replacement of the Pro-6 residue in alpha -CTx ImI with Arg results in failure to displace alpha -bungarotoxin even at a concentration of 100 µM (compared with an EC50 for native alpha -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 alpha -bungarotoxin-binding site or another site.


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Fig. 6.   Competition binding of alpha -conotoxin analogs. A, inhibition of 3-125I-alpha -BgTx binding to alpha 7-5-HT3 chimera by alpha -CTx ImI (squares) and P6R alpha -ImI (circles). The specific binding of 3-125I-alpha -BgTx at each conotoxin or mutant toxin concentration is normalized to specific 3-125I-alpha -BgTx binding in the absence of peptide. The alpha -CTx ImI data was fit to a curve and the P6R alpha -CTx ImI data was fit to a straight line as described under "Experimental Procedures." For alpha -CTx ImI, the EC50 is 407 nM and the nH is 0.71. B, inhibition of 3-125I-alpha -BgTx binding to rat alpha 7-5-HT3 chimeras by alpha -CTx ImII (circles) and R6P alpha -CTx ImII (squares) was determined as described under "Experimental Procedures." The specific binding of 3-125Ialpha -BgTx at each conotoxin or mutant toxin concentration is normalized to specific 3-125I-alpha -BgTx binding in the absence of peptide. The R6P alpha -CTx ImII data was fit to a curve and the alpha -CTx ImII data was fit to a straight line as described under "Experimental Procedures." For R6P alpha -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

We report the discovery and characterization of alpha -CTx ImII that has high sequence identity (9 of 12 amino acids) to alpha -CTx ImI (Fig. 1); both peptides are from the venom ducts of C. imperialis (9). alpha -CTx ImI is a specific competitive inhibitor of the alpha 7 nAChR subtype (14, 15). Given the close sequence similarity of alpha -CTx ImI and alpha -CTx ImII, it was not surprising that alpha -CTx ImII was also found to inhibit the alpha 7 nAChR. However, most unexpectedly, the two closely related peptides appear to cause their similar functional effects by binding to different sites on the alpha 7 nAChR.

alpha -CTx ImII was found to be similar to alpha -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 alpha -BgTx, another alpha 7 nAChR inhibitor, into rats (14). In view of its homology to alpha -CTx ImI and the characteristic symptoms observed when it was injected into the central nervous system, alpha -CTx ImII was tested for its ability to inhibit ACh-gated currents in Xenopus oocytes expressing rat alpha 7 nAChRs. alpha -CTx ImII was found to inhibit the receptor with an IC50 similar to that of alpha -CTx ImI (Fig. 2) when the toxins were tested using identical protocols.

The first surprising result was obtained when alpha -CTx ImII was tested in a competition assay with 3-125I-alpha -BgTx. As had been previously demonstrated by others (18), we found that alpha -CTx ImI competed with alpha -BgTx for binding to the receptor. In contrast, alpha -CTx ImII did not appreciably displace alpha -BgTx binding in the concentration range tested. These results for alpha -CTx ImI and alpha -CTx ImII were obtained with both rat brain alpha 7 nAChRs (Fig. 3A) as well as rat alpha 7-5-HT3 chimeras (Fig. 3B). Furthermore, experiments using rat alpha 7 nAChRs expressed in oocytes (Fig. 4) demonstrated that preincubation with alpha -CTx ImI prevents alpha -BgTx from binding to its functionally relevant site, a result consistent with competitive antagonism, and previously shown by others (15). On the other hand, alpha -CTx ImII had only a very weak effect on alpha -BgTx inhibition of oocyte-expressed receptor, consistent with a different binding site.

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 "-" face. The binding of alpha -CTx ImI to the alpha 7 nAChR is affected by mutations in or near loops A, B, and C, and II and III (17, 18). The alpha -BgTx site on the alpha 7 nAChR has also been mapped to the A, B, and C loops (21), and a loop II mutation causes a minor reduction in alpha -BgTx affinity (22). This and other evidence are consistent with alpha -CTx ImI-binding sites in alpha 7 nAChRs overlapping with ACh and alpha -BgTx-binding sites, and being at subunit interfaces.

The data in Fig. 5 suggest that alpha -CTx ImI and alpha -CTx ImII do not bind to the same site at a subunit interface. Assuming a potential five identical subunit interfaces in the alpha 7 nAChR pentamer, and that occupation of even one site by alpha -CTx ImI results in inhibition of the receptor, then the concentration of alpha -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 alpha -CTx ImI on the alpha 7 nAChR is 191 nM (Fig. 2). Therefore, 100 µM alpha -CTx ImI (see Fig. 5) would clearly occupy most subunit interface-binding sites on the alpha 7 nAChR (assuming these are identical) and should significantly reduce binding of alpha -CTx ImII if alpha -CTx ImI and alpha -CTx ImII share a binding site. The alpha -CTx ImII-binding site awaits definitive characterization; however, several possibilities are outlined below.

Because the primary structures of alpha -CTx ImI and alpha -CTx ImII are so similar, and because they share the characteristic alpha -conotoxin disulfide framework, it seems possible that alpha -CTx ImII also binds to the interface between alpha 7 subunits. In this case, the inability of alpha -CTx ImII to compete with alpha -BgTx or alpha -CTx ImI might be explained by the following models.

One potential explanation for the results is that alpha -CTx ImI and alpha -CTx ImII can simultaneously bind at a single subunit interface by positioning differently within the cleft at different microsites. In fact, alpha -BgTx appears to make more contacts with the + face than with the - face at alpha 7 nAChR subunit interfaces (21, 22). It is possible, for example, that alpha -CTx ImII binds predominantly to the - face and is thus unable to displace alpha -BgTx, whereas alpha -CTx ImI, because of many contacts in the + face, disrupts many alpha -BgTx-receptor interactions, and is thus able to compete with this toxin.

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 alpha 7 nAChR receptor are not identical. Evidence was presented that the functional alpha 7 nAChR complex requires a mixture of alpha 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 alpha 7 subunits is the alpha -CTx ImI and alpha -BgTx-binding site, whereas another type of subunit interface does not bind alpha -BgTx, but is the alpha -CTx ImII target site. Bertrand and co-workers (24) have shown that for the competitive alpha 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 alpha -BgTx-binding sites on alpha 7 nAChRs (25). Additionally, in mouse brain, some [3H]MLA-binding sites are resistant to competition by alpha -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 alpha -BgTx, even at high concentrations, cannot.

Although alpha -CTx ImI and alpha -CTx ImII show extensive sequence homology, it is possible that alpha -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 alpha 7 nAChR or the C-terminal extracellular region. alpha -CTx ImII could also potentially bind to nonsubunit interface regions on the N-terminal ACh-binding domain or the channel pore; however, because alpha -CTx ImII block is not voltage dependent, this supports the model that it is not an open channel blocker.

The experiments with analogues suggest that although alpha -CTx ImI and alpha -CTx ImII have very similar sequences, the amino acid residue at position 6 (Pro in alpha -CTx ImI, Arg in alpha -CTx ImII) is critical in determining where they bind on the alpha 7 nAChR. Relative to wild-type alpha -CTx ImI, P6R alpha -CTx ImI is a very poor competitor of alpha -BgTx binding to alpha 7-5-HT3 chimera. In contrast, R6P alpha -CTx ImII has an enhanced ability to compete with alpha -BgTx compared with wild-type alpha -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.

Additional information about interactions of alpha -CTx ImI and alpha -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 alpha -CTx ImI analog is equivalent to R9A alpha -CTx ImII and the R6Palpha -CTx-ImII analog is equivalent to A9R alpha -CTx-ImI. Because P6R alpha -CTx ImI does not compete with alpha -BgTx for binding to the alpha 7 nAChR, this strongly suggests that R9A alpha -CTx ImII would be like alpha -CTx ImII and also not compete with alpha -BgTx. Because R6P alpha -CTx ImII has some ability to compete alpha -BgTx but is not as potent as alpha -CTx ImI, this strongly suggests that A9R alpha -CTx ImI would compete with alpha -BgTx for binding to the alpha 7 nAChR but would be a less potent competitor than alpha -CTx ImI. Taken together, these observations imply that the residues at position 9 in alpha -CTx ImI and alpha -CTx ImII are not critical in determining whether the alpha -CTx ImI or alpha -CTx ImII site is targeted, but are important for ensuring optimal affinity of alpha -CTx ImI and alpha -CTx ImII for their respective sites.

The discovery of alpha -CTx ImII reveals that C. imperialis has two toxins that inhibit the rat alpha 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 alpha -CTx ImI and alpha -CTx ImII, which may bind to different sites on an "alpha 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 alpha A-conotoxin and a noncompetitive psi -conotoxin (reviewed in Ref. 1). The present case is different, however, in that the toxins are both alpha -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 alpha -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 alpha 7-like receptor at all. On the other hand, post-translational modification in alpha -conotoxins isolated from venom have so far been limited to C-terminal amidation and tyrosine sulfation (alpha -CTx ImI and alpha -CTx ImII lack tyrosine residues).

Previously, it has been shown that very minor changes in the intercysteine amino acid sequences of conotoxins can drastically affect their specificity. The toxins alpha -CTx PnIA and alpha -CTx PnIB from Conus pennaceus are different in only 2 of 16 amino acids, but preferentially block alpha 3beta 2 and alpha 7 nAChRs, respectively (3). The discovery of alpha -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.

    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 alpha -CTx ImI and alpha -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

    ABBREVIATIONS

The abbreviations used are: ACh, acetylcholine; alpha -BgTx, alpha -bungarotoxin; alpha -CTx, alpha -conotoxin; MLA, methyllycaconitine; PR, potassium Ringer's solution; 5-HT, 5-hydroxytryptamine (serotonin); 5-HT3 receptor, type 3 serotonin receptor.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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