Báo cáo khoa học: Novel a-conotoxins from Conus spurius and the a-conotoxin EI share high-affinity potentiation and low-affinity inhibition of nicotinic acetylcholine receptors doc

Novel a-conotoxins from Conus spurius and the a-conotoxin EI share high-affinity potentiation and low-affinity inhibition of nicotinic acetylcholine receptors Estuardo Lo ´ pez-Vera 1, * , †, Manuel B. Aguilar 1, *, Emanuele Schiavon 2 , Chiara Marinzi 2 , Ernesto Ortiz 3 , Rita Restano Cassulini 2 , Cesar V. F. Batista 3 , Lourival D. Possani 3 , Edgar P. Heimer de la Cotera 1 , Francesco Peri 2 , Baltazar Becerril 3 and Enzo Wanke 2 1 Laboratorio de Neurofarmacologı ´ a Marina, Departamento de Neurobiologı ´ a Celular y Molecular, Instituto de Neurobiologı ´ a, Universidad Nacional Auto ´ noma de Me ´ xico, Campus Juriquilla, Queretaro, Me ´ xico 2 Dipartimento di Biotecnologie e Bioscienze, Universita ` di Milano-Bicocca, Milan, Italy 3 Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnologı ´ a, Universidad Nacional Auto ´ noma de Me ´ xico, Cuernavaca, Me ´ xico Conotoxins are small, disulfide-rich peptides that have been isolated from Conus, a large genus of predatory marine snails. The primary structures of more than 100 conotoxins have been determined and classified into gene superfamilies on the basis of the amino acid sequences of the signal peptides of their Keywords a-conotoxin; conotoxins; Conus spurius; nicotinic receptor; potentiation Correspondence E. Wanke, Dipartimento di Biotecnologie e Bioscienze, Universita ` di Milano-Bicocca, Piazza della Scienza, 2U3, 20126 Milan, Italy Fax: +39 02 64483314 Tel: +39 02 64483303 E-mail: enzo.wanke@unimib.it *These authors contributed equally to this work  Present address Instituto de Ciencias del Mar y Limnologı ´ a, Universidad Nacional Auto ´ noma de Mexico, Mexico (Received 4 April 2007, revised 3 June 2007, accepted 11 June 2007) doi:10.1111/j.1742-4658.2007.05931.x a-Conotoxins from marine snails are known to be selective and potent competitive antagonists of nicotinic acetylcholine receptors. Here we des- cribe the purification, structural features and activity of two novel toxins, SrIA and SrIB, isolated from Conus spurius collected in the Yucatan Chan- nel, Mexico. As determined by direct amino acid and cDNA nucleotide sequencing, the toxins are peptides containing 18 amino acid residues with the typical 4 ⁄ 7-type framework but with completely novel sequences. Therefore, their actions (and that of a synthetic analog, [c15E]SrIB) were compared to those exerted by the a4 ⁄ 7-conotoxin EI from Conus ermineus, used as a control. Their target specificity was evaluated by the patch-clamp technique in mammalian cells expressing a 1 b 1 cd, a 4 b 2 and a 3 b 4 nicotinic acetylcholine receptors. At high concentrations (10 lm), the peptides SrIA, SrIB and [c15E]SrIB showed weak blocking effects only on a 4 b 2 and a 1 b 1 cd subtypes, but EI also strongly blocked a 3 b 4 receptors. In contrast to this blocking effect, the new peptides and EI showed a remarkable potentiation of a 1 b 1 cd and a 4 b 2 nicotinic acetylcholine receptors if briefly (2–15 s) applied at concentrations several orders of magnitude lower (EC 50 , 1.78 and 0.37 nm, respectively). These results suggest not only that the novel a-conotoxins and EI can operate as nicotinic acetylcholine receptor inhibitors, but also that they bind both a 1 b 1 cd and a 4 b 2 nicotinic acetyl- choline receptors with very high affinity and increase their intrinsic cho- linergic response. Their unique properties make them excellent tools for studying the toxin–receptor interaction, as well as models with which to design highly specific therapeutic drugs. Abbreviations a 1 b 1 cd, muscular nicotinic acetylcholine receptor; a 3 b 4 , peripheral nervous system nicotinic acetylcholine receptor; a 4 b 2 , central nervous system nicotinic acetylcholine receptor; Acm, S-acetamidomethyl; ACN, acetonitrile; [c15E]SrIB, synthetic a-conotoxin from Conus spurius; nAChR, nicotinic acetylcholine receptor; PTH, phenylthiohydantoin; SrIA, a-conotoxin IA from Conus spurius; SrIB, a-conotoxin IB from Conus spurius. 3972 FEBS Journal 274 (2007) 3972–3985 ª 2007 The Authors Journal compilation ª 2007 FEBS precursors. In general, the members of each super- family have a characteristic arrangement of their cys- teine residues and a particular connectivity of their disulfide bridges. Each gene superfamily comprises one or more pharmacologic families: the O super- family, containing x-conotoxins, j-conotoxins, d-conotoxins, and lO-conotoxins; the M superfamily, containing l-conotoxins, w-conotoxins, and jM-cono- toxins; the S superfamily, containing r-conotoxins and aS-conotoxins; the T superfamily, containing e-conotoxins and v-conotoxins; the P superfamily, containing the spasmodic peptides; the I superfamily, containing several jI-conotoxins, and the A super- family, containing a-conotoxins, aA-conotoxins and jA-conotoxins [1]. Competitive antagonists of the nicotinic acetylcholine receptors (nAChRs) belong to the a and aA families. On the basis of the number of residues between the sec- ond and third cysteines and on the spacing between the third and fourth cysteines in the mature a-conotoxins, these peptides have been divided into three groups: the a4 ⁄ 7 subfamily, the a3 ⁄ 5 subfamily, and a heterogene- ous group including peptides that do not belong to the two previous groups. These groups have different degrees of antagonistic effect on distinct nAChRs: a3 ⁄ 5 toxins block mostly muscular nicotinic acetylcholine receptors a 1 b 1 cd subtypes, whereas a4 ⁄ 7 peptides, with one exception, block neuronal subtypes [2]. In this article, we describe the purification, amino acid sequence determination and cloning of the cDNA encoding two novel peptides, SrIA and SrIB, found in the venom of Conus spurius. The pattern and the spa- cing of their cysteines indicate that they belong to the a4 ⁄ 7 subfamily of conotoxins [3]. We also describe a third peptide, [c15E]SrIB, synthesized by substituting glutamate for the c-carboxyglutamate residue and used for comparison together with the a-EI conotoxin from Conus ermineus. We showed that results with [c15E]SrIB were not significantly different from those seen with the natural compounds, and then, owing to the limited amounts of the natural toxins SrIA and SrIB, used mainly this synthetic peptide for long-dur- ation electrophysiologic tests. The discovery of new agonists or antagonists is of the utmost importance to widen the understanding of alternative functions of nAChRs, which play a crucial role in cellular and molecular mechanisms underlying brain function. Results Purification of SrIA and SrIB Fractionation of C. spurius venom by HPLC, as des- cribed in Experimental procedures, gave the profile shown in Fig. 1A. The fractions indicated as SrIA and SrIB were repurified by RP-HPLC, yielding the two pure peptides SrIA and SrIB (Fig. 1B,C), named follow- ing the nomenclature proposed by Olivera & Cruz [1]. Amino acid sequences and cDNA cloning Automated Edman sequencing of the native peptides SrIA and SrIB unambiguously defined 12 and 13 resi- dues, respectively. Low glutamine signals at positions 12 and 15 of SrIA and at position 15 of SrIB Fig. 1. Purification of SrIA and SrIB. (A) Fractionation of the crude venom by means of an analytical RP C18 HPLC column. Pep- tides were eluted using a linear gradient of 5–95% solution B (dashed line) at a flow rate of 1 mLÆmin )1 for 90 min. Eluents were: 0.1% v ⁄ v trifluoroacetic acid in water (solution A), and 0.09% v ⁄ v trifluoroacetic acid in 90% v ⁄ v ACN (solution B). (B, C) Fractions indicated in (A) as SrIA and SrIB were repurified using a gradient of 15–30% buffer B (dashed line), at a flow rate of 1mLÆmin )1 for 45 min. E. Lo ´ pez-Vera et al. a-Conotoxins with potentiating effects on nAChRs FEBS Journal 274 (2007) 3972–3985 ª 2007 The Authors Journal compilation ª 2007 FEBS 3973 suggested the presence of c-carboxyglutamate residues at these positions. Residues 3, 4, 9 and 17 of both pep- tides were tentatively assigned as cysteine (Table 1), on the basis of the absence of any amino acid signal at these positions. This assumption was confirmed directly by the experiments used to determine disulfide bridges (see below). We obtained positive results with PCR amplification of a-conotoxin-type cDNA, reverse transcribed from C. spurius venom duct total mRNA. Two primers known to match the con- served signal peptide-coding region and the 3¢-UTR of the a-conotoxin family, respectively [4], were success- fully employed. Exactly the same sequence was obtained from several colonies, which, together with the demonstrated conservation of the signal and pro- peptide regions, indicated that the amplification proto- col was reliable. The deduced SrIA ⁄ SrIB precursor sequence agreed with the results of direct peptide sequencing and MS data (see below), and allowed us to define the final unambiguous primary structure for the mature toxins (Fig. 2). From the precursor sequence, and on the basis of earlier observations by our group with toxic peptides [5], we were also able to predict the amidation of the C-terminal end of the mature toxins. The primary structures of SrIA and SrIB resemble those of previously isolated a-conoto- xins with the cysteine framework 4 ⁄ 7 (Table 2). MS The chemical monoisotopic molecular masses of pep- tides SrIA and SrIB determined by ESI MS are 2202.9 Da and 2158.8 Da, respectively (Table 1). The agreement with the calculated masses (assuming two disulfide bridges and an amidated C-terminus for each peptide, plus one and two c-carboxyglutamate residues for SrIB and SrIA, respectively) supports the Edman sequence assignment for each peptide. The tentative assignments of amidated C-termini, based on the struc- ture of the precursor (see ‘cDNA cloning’), were con- firmed by the ESI MS data. Determination of disulfide bridges Two major and more than 20 minor absorbing peaks were observed during the chromatography of peptide SrIA after partial reduction with Tris(2-carboxyethyl) phosphine hydrochloride and alkylation with N-ethyl- maleimide (Fig. 3). This high number of derivatives of peptides alkylated with N-ethylmaleimide has been observed in several studies [6], and it is thought to reflect diastereoisomers resulting from the introduction of a new chiral center in the maleimide ring after for- mation of the S–C bond during alkylation. Another factor that could generate additional derivatives is the opening of the ring of the N-ethylsuccinimidocysteines by hydrolysis [7]. Selected peptides were sequenced to reveal the positions of the alkylated cysteines. The phe- nylthiohydantoin (PTH) derivative of N-ethylsuccini- midocysteine elutes between PTH-Pro and PTH-Met in the HPLC system of the sequencer employed. The presence of alkylated cysteines at positions 4 and 17 in some peptides, and at positions 3 and 9 in other pep- tides, clearly indicated that the connectivity of the two disulfide bridges in peptide SrIA is of the type I–III, II–IV. The absence of peptides with labeled cysteines at positions 3 and 17 or 4 and 9 gives additional sup- port to the proposed disulfide connectivity. The synthetic peptide [c15E]SrIB It has been reported recently that the c-carboxygluta- mic residues present in toxin peptides may be involved in the folding process but are not relevant for their biological activity [8]. Starting from this hypothesis, a peptide sequence was designed that was analogous to those found for SrIA and SrIB, but bearing glutamic acid residues in place of the c-carbo- Table 1. Amino acid sequences and monoisotopic molecular mas- ses of the peptides from C. spurius and of synthetic peptides [c15E]SrIB and EI. Peptide Sequence Experimental mass (Da) Calculated mass (Da) SrIA RTCCSROTCRMcYPcLCG a 2202.9 2202.8 SrIB RTCCSROTCRMEYPcLCG a 2158.8 2158.8 [c15E]SrIB RTCCSROTCRMEYPELCG a 2114.8 2115.0 EI RDOCCYHPTCNMSNPQIC a 2075.4 2075.8 a Amidated C-terminus; O, hydroxyproline; c, c-carboxyglutamate. Fig. 2. The cloned cDNA sequence and the deduced amino acid sequence of the SrIA ⁄ SrIB conotoxin precursor. The residues present in the mature toxins are underlined. a-Conotoxins with potentiating effects on nAChRs E. Lo ´ pez-Vera et al. 3974 FEBS Journal 274 (2007) 3972–3985 ª 2007 The Authors Journal compilation ª 2007 FEBS xyglutamic residues at positions 12 and 15 (Table 1). Testing the biological properties of such a peptide, prepared by chemical synthesis and thus with a fully defined chemical structure (including disulfide pat- tern), would support the amino acid sequence and folding of the native peptides proposed above, and additional tests would not be limited by the availabil- ity of the peptide, as might occur with the natural toxins SrIA and SrIB. To obtain the desired folding pattern (see Experimental procedures), we protected the cysteine side chains with two orthogonal protect- ing groups that can be removed selectively under different conditions, allowing the formation of one disulfide bridge at a time. For this purpose, Cys3 and Cys9 were introduced as S-trityl-protected amino acids, whereas S-(acetamidomethyl)cysteine was used for positions 4 and 17. At the end of chain assembly on the solid support, achieved using standard 2-(1-H- benzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexa- fluorophosphate activation protocols for Fmoc chemistry as previously described [9,10], the peptide resin was treated with trifluoroacetic acid for cleavage from the solid support and side chain deprotection, with simultaneous liberation of the two thiol groups in positions 3 and 9. The first disulfide bond was then formed by air oxidation. Finally, the bis-aceta- midomethyl-peptide generated was treated with iodine, which caused removal of the protecting group and simultaneous oxidation to disulfide, yielding the fully folded sequence. Physiologic effects of natural conotoxins and their synthetic analogs In order to explore the physiologic role of the novel SrIA and SrIB conotoxins, we performed a series of patch-clamp experiments on single cells from the line TE671, which expresses the human muscle receptor [11], and HEK293 lines stably transfected with the human central nervous system nicotinic acetylcholine a 4 b 2 and peripheral nervous system nicotinic acetyl- choline a 3 b 4 receptor subtypes. As our present perfu- sion system is not sufficiently fast to resolve fast desensitizing currents such as those produced by a 7 re- ceptors, we decided not to test our peptides on these receptors, to avoid reporting putatively invalid data. The experiments were done by voltage-clamping the cells at ) 60 mV and comparing the responses to brief Table 2. Amino acid sequence of SrIA, SrIB and [c15E]SrIB, com- pared with some members of the a3 ⁄ 5, a4 ⁄ 3 and a4 ⁄ 7 subfamilies [16,24,48]. Peptide Amino acid sequence Target SI ICCNPACGPKYSC a a 1 b 1 cd SIA YCCHPACGKNFDC a a 1 b 1 cd GI ECCNPACGRHYSC a a 1 b 1 cd GIA ECCNPACGRHYSCGK a a 1 b 1 cd GII ECCHPACGKHFSC a a 1 b 1 cd MI GRCCHPACGKNYSC a a 1 b 1 cd CnIA GRCCHPACGKYYSC a a 1 b 1 cd >> a 7 ImII ACCSDRRCR-WRC a a 7 , a 1 b 1 > a 3 b 2 AnIB GGCCSHPACAANNQDYC a a 3 b 2 >> a 7 PnIA GCCSLPPCAANNPDYC a a 3 b 2 >> a 7 PnIB GCCSLPPCALSNPDYC a a 7 > a 3 b 2 EpI GCCSDPRCNMNNPDYC a a 3 b 4 , a 3 b 2 ; a 7 AuIA GCCSYPPCFATNSDYC a a 3 b 4 Vc1.1 GCCSDPRCNYDHPEIC a a 3 a 7 b 4 , a 3 a 5 b 4 Vc1a GCCSDORCNYDHPc IC a PeIA GCCSHPACSVNHPELC a a 9 a 10 , a 3 b 2 > a 3 b 4 > a 7 PIA RDPCCSNPVCTVHNPQIC a a 6 ⁄ a 3 b 2 b 3 > a 6 ⁄ a 3 b 4 > a 6 b 4 , a 3 b 2 GIC GCCSHPACAGNNQHIC a a 3 b 2 >> a 4 b 2 , a 3 b 4 MII GCCSNPVCHLEHSNLC a a 3 b 2 >> a 7 > a 4 b 2 , a 3 b 4 GID IR GcCCSNPACRVNNOHVC a 3 ⁄ b 2 , a 7 > a 4 ⁄ b 2 EI RDOCCYHPTCNMSNPQIC a a 1 b 1 cd, a 3 b 4 , a 4 b 2 SrIA RTCCSROTCRMc YPcLCG a a 4 b 2 , a 1 b 1 cd SrIB RTCCSROTCRMEYPcLCG a a 4 b 2 , a 1 b 1 cd [c15E]SrIB RTCCSROTCRMEYPELCG a a 4 b 2 , a 1 b 1 cd a Amidated C-terminus; O, hydroxyproline; c,c-carboxyglutamate; Y, sulfated tyrosine. Fig. 3. Determination of the disulfide bridges of peptide SrIA. Deriv- atives of peptide SrIA formed by partial reduction and alkylation under acidic conditions were separated using two analytical RP C18 HPLC columns. Peptides were eluted using a linear gradient (dashed line) of 10–30% solution B at a flow rate of 1 mLÆmin )1 for 120 min. Eluents were: 0.1% v ⁄ v trifluoroacetic acid in water (solu- tion A), and 0.09% v ⁄ v trifluoroacetic acid in 90% v ⁄ v ACN (solu- tion B). Selected peptides were sequenced, and the positions at which cysteines labeled with N-ethylmaleimide were observed are displayed in the corresponding diagrams. The deduced connectivity of the two disulfide bonds is indicated in the upper right inset. E. Lo ´ pez-Vera et al. a-Conotoxins with potentiating effects on nAChRs FEBS Journal 274 (2007) 3972–3985 ª 2007 The Authors Journal compilation ª 2007 FEBS 3975 applications of 50 lm nicotine with those obtained immediately after pretreatment with the different tox- ins. The concentration of nicotine used during the experiments was fixed at 50 lm, because this value is well below the saturating region of the dose–response curve for the a 1 b 1 cd receptor, as shown in Fig. 4A, and also for the a 4 b 2 and a 3 b 4 receptors [12–14]. The pretreatment time and the concentration of each toxin were varied in the range 3–150 s and 0.2 nm to 10 lm, respectively. A typical experiment performed on a TE671 cell with a-conotoxin [c15E]SrIB at 1 lm is shown in Fig. 4B. As indicated, the first 50 lm nicotine control pulse produced a response that was strongly reduced after 180 s of toxin perfusion. After 4 min of washout, the application of an additional nicotine pulse produced a recovery that was complete. As the amount of purified toxins was limited, we did the majority of the experiments with the synthetic toxin [c15E]SrIB and used a known conotoxin [15], such as EI a-conotoxin, as a control. In the case of the inhibitory effects des- cribed in Fig. 4, the results obtained using natural or synthetic peptides, at the same concentration, were not A BCE D Fig. 4. Blocking properties of a-conotoxins on different types of receptors. (A) Dose–response curve obtained with nicotine in TE671 cells. The continuous line is the Boltzmann curve that best fits the data with the following parameters: an IC 50 of 99 ± 12 lM, and a Hill coefficient of 1.98 ± 0.14 (n ¼ 12). The inset shows a representative example of the recorded currents in a single cell. (B) Inward currents recorded in a TE671 cell during successive 50 l M nicotine (nic) test pulses. The first and the last pulse are control and washout, respectively; the second pulse was preceded by an 180 s pretreatment with a-conotoxin [c15E]SrIB (1 l M). (C) Fractional blockade, at fixed toxin concentration (10 l M for 180 s), on the different subtypes of nAChR. *Statistically different at P < 0.05 as compared to a 4 b 2 ; the numbers of experiments are given in parentheses. (D) Normalized time course of the blockade, at 10 l M EI, of the nicotinic response as a function of the toxin pre- treatment time. Continuous curves are exponentials that best fit the data points with the following time constants: a 1 b 1 cd (open squares), 4.9 ± 0.25 s (n ¼ 5); a 3 b 4 (gray squares), 11 ± 1.9 s (n ¼ 5). Insets: superimposed traces of the nicotine responses obtained in a typical TE671 cell and in an a 3 b 4 -expressing cell during control and toxin perfusion. Left inset: the traces show the block at 30 s and the recovery after 40 s. Right inset: the traces show the block at 5 s and the recovery after 20 s. Scale bars: 2 s, 200 pA. (E) Fractional response data obtained with a-conotoxin [c15E]SrIB and EI. The curves are best fitted with the following IC 50 and Hill coefficient: for [c15E]SrIB, 46 ± 10 n M, 1 ± 0.1, and for EI, 187 ± 43 nM, 0.48 ± 0.06, respectively. The number of experiments for each point ranged from three to 12. a-Conotoxins with potentiating effects on nAChRs E. Lo ´ pez-Vera et al. 3976 FEBS Journal 274 (2007) 3972–3985 ª 2007 The Authors Journal compilation ª 2007 FEBS significantly different, and only the data obtained with the synthetic toxin are displayed. Inhibitory actions Figure 4C summarizes the data obtained at high toxin concentrations (10 lm). It can be seen that the frac- tional blockade obtained is both receptor-dependent and toxin-dependent. [c15E]SrIB was ineffective on a 3 b 4 receptors (n ¼ 4), and was a slightly better blocker of the a 4 b 2 receptors (0.56 ± 0.04, n ¼ 7) than of the a 1 b 1 cd receptors (0.39 ± 0.06, n ¼ 5, not statisti- cally significant). On the other hand, EI toxin was able to potently block muscle (0.95 ± 0.01, n ¼ 5) and ganglionic (0.91 ± 0.03, n ¼ 4) receptors, but was less potent for the central nevous system receptor (0.61 ± 0.02, n ¼ 4). These data not only confirm that EI is an inhibitor of muscle receptors [15], but they also show that it is a strong inhibitor of the a 3 b 4 receptors and a relatively weak antagonist of the a 4 b 2 central nervous system receptors, on which it had never been tested before. To further investigate these new EI data, we also performed a series of kinetic experiments at a concen- tration of 10 lm (see Fig. 4D). EI toxin blocked the a 1 b 1 cd receptors with a s on of 4.9 ± 0.25 s (n ¼ 3), and the a 3 b 4 receptor was blocked with a s on of 11 ± 1.9 s (n ¼ 3). Moreover, the s off values that we observed for these receptors were 150 ± 13 s (n ¼ 4) and 122 ± 6.5 s, respectively. Figure 4E shows the dose–response curve for the [c15E]SrIB and EI a-cono- toxins on the a 1 b 1 cd receptors. The estimated IC 50 and Hill coefficient obtained from these data are: 46 ± 10 nm and 1 ± 0.1 for [c15E]SrIB, and 187 ± 43 nm and 0.48 ± 0.06 for EI, respectively. Because, at a toxin concentration [T], a simple Clark’s model receptor theory predicts s on ¼ s off ⁄ (1 + [T] ⁄ K D ), this relationship can be used to confirm the previous IC 50 of Martinez et al. [15] on a 1 b 1 cd receptors, which was 280 nm (low-affinity site) for the mouse receptors, and to predict the unknown and novel value of K D for the a 3 b 4 receptors. Indeed, we found an IC 50 value of 187 nm for a 1 b 1 cd recep- tors (Fig. 4E), which also agrees with the fractional response of 0.04 at 10 lm EI and a s off of 150 s. For the a 3 b 4 receptors, the above relationship results in a K D value of about 1 lm. On the whole, these experiments, designed to study the antagonistic properties of the toxin [c15E]SrIB, showed a narrower spectrum of specificity for nAChRs than that of the EI a-conotoxin, owing to the null effect of [c15E]SrIB on the a 3 b 4 subtype. In contrast, EI was found to be a broad-spectrum a-conotoxin. Potentiating effects During the experiments designed to study inhibitory actions of the two new peptides SrIA and SrIB, we dis- covered that brief applications, at low toxin concentra- tions, resulted in increased responses that were immediately reversed after washout of the toxin. A typical experiment performed on an a 1 b 1 cd-expressing cell with different concentrations of a-conotoxin [c15E]SrIB is shown in Fig. 5A. It can be seen that the first and the last brief control pulses of 50 lm nicotine produced very similar inward currents. However, if 15 s pretreatments with toxin were immediately fol- lowed by the same brief nicotine pulses, currents increased, and then decreased as a function of the drug concentration. In order to shed light on this novel action of the a-conotoxins, we started to investigate whether the various peptides exerted different levels of potentiation on the same a 1 b 1 cd receptor. To clarify whether this novel mechanism was peculiar to the new conotoxins or common also to other, already known, conotoxins, we chose the EI a-conotoxin, which is considered to be an inhibitory conotoxin [15]. At a fixed toxin concentration of 10 nm, the relative potentiation, (I toxin ) I control ) ⁄ I control , of the synthetic [c15E]SrIB, the natural SrIB and SrIA peptides, and the EI a-conotoxin, were as follows: 0.46 ± 0.09 (n ¼ 10), 0.47 ± 0.08 (n ¼ 10), 0.44 ± 0.15 (n ¼ 9), and 0.54 ± 0.13 (n ¼ 9), respectively. These results suggest that, at least for the a 1 b 1 cd receptor type and during brief periods of time (15 s), pretreatment with a con- centration of 10 nm toxin shows no clear differences among these peptides. As the amount of natural toxin available for experimentation is limited, and as no significant differences were found when using the synthetic peptide as compared to the native peptides, we continued our assays using the two synthetically prepared products, namely [c15E]SrIB and EI. The results of these preliminary experiments, obtained only with very low toxin concentrations (0.2 nm to 1 lm) and brief time intervals, do not conflict with those mentioned in Fig. 4, which were obtained with very long pretreatments. To investigate these mechanisms, the toxins were studied in cells expressing various receptor types. Unexpectedly, we discovered that their effects were also receptor-dependent. To clarify the receptor specif- icity, we used the two toxins ([c15E]SrIB and EI) on three different receptors, namely a 1 b 1 cd, a 4 b 2 , and a 3 b 4 , and the maximally observed relative potentiation values are shown in Fig. 5B. Interestingly, whereas the toxins were unable to produce potentiation in the E. Lo ´ pez-Vera et al. a-Conotoxins with potentiating effects on nAChRs FEBS Journal 274 (2007) 3972–3985 ª 2007 The Authors Journal compilation ª 2007 FEBS 3977 ganglionic a 3 b 4 receptor (n ¼ 17), the mean fractional potentiation in a 1 b 1 cd receptors for [c15E]SrIB (0.75 ± 0.22, n ¼ 7) was higher than that obtained for EI (0.35 ± 0.07, n ¼ 22, statistically different). The effects of both toxins were found to be similar on the a 4 b 2 receptor subtype. Furthermore, we investigated the dose–response curves of the maximal fractional potentiation produced by the [c15E]SrIB and EI conotoxins on the a 1 b 1 cd receptors. These data are shown in Fig. 5C, and were fitted to dose–response curves with EC 50 values of 1.78 ± 1.9 and 0.37 ± 0.23 nm, for [c15E]SrIB and EI, respectively. An example of this type of action (15 s toxin pretreatment) is shown in Fig. 5D, in one example of an a 1 b 1 cd-expressing cell, with both toxins at two different concentrations (10 and 100 nm). In this experiment, the two toxins were delivered alter- nately to gain insight into the differences between their sensitivities. The kinetics of the development of the potentiated response were very fast at concentrations higher than 2–5 nm, and it was almost impossible to determine its time course, given that the rate of bath exchange was < 1 s. However, by reducing the toxin concentra- tion to 0.2 nm, we were able to follow, as a function of the duration of the toxin perfusion, not only the expo- nential increase in potentiation, but also the decay of the potentiation response, up to the appearance of the blockade. Indeed, if the pretreatment of the toxin las- ted for more than 10–15 s, it was possible to observe ABC DEF Fig. 5. Potentiation effects of a-conotoxins on different types of receptor. (A) Inward currents recorded in a TE671 cell during successive 50 l M nicotine test pulses. The first and the last pulse are controls; the second, third, fourth and fifth pulses were each preceded by a 15 s pretreatment with different concentrations of the a-conotoxin [c15E]SrIB. (B) Maximal relative potentiation [(I tox ) I control ) ⁄ I control ] for different receptor types ([c15E]SrIB, line pattern; EI, gray pattern). The maximal concentration used was 100 n M, and pretreatment lasted for 15 s. The number of experiments is shown in parentheses on the bars. *Statistically different at P < 0.05 as compared to the EI effect. (C) Dose– response relationships for potentiation, observed in a 1 b 1 cd receptors, for a-conotoxins [c15E]SrIB (open squares), and EI (gray squares). Con- tinuous lines are dose–response curves fitting the experimental data with the following values of IC 50 (nM) (maximal): for [c15E]SrIB, 1.78 ± 1.9, 0.93 ± 0.11; for EI, 0.37 ± 0.23 n M, 0.46 ± 0.1. Each point represents a variable number of experiments from three to 11. (D) In the same cell, the two toxins were applied alternately, each for 15 s pretreatment intervals at different concentrations as indicated. (E, F) The potentiation ⁄ blockade (open squares) kinetics on a 1 b 1 cd receptors, for [c15E]SrIB (E) at 0.2 nM and EI (F) at 0.2 and 1 nM. Continuous curves are exponentials with the following time constants: [c15E]SrIB, s on 7.07 ± 0.1.1, s off 31 ± 2.3 s; EI, s on (0.2 nM) 6.03 ± 0.32, s off (0.2 nM) 16.4 ± 1.3 s; s off (1 nM) 9.4 ± 1.5 s. See text. a-Conotoxins with potentiating effects on nAChRs E. Lo ´ pez-Vera et al. 3978 FEBS Journal 274 (2007) 3972–3985 ª 2007 The Authors Journal compilation ª 2007 FEBS an exponentially decaying depotentiation process. We show two examples obtained by using the two different toxins on the a 1 b 1 cd receptor. Figure 5E,F shows the potentiation ⁄ blockade (I toxin ⁄ I control ) data versus dur- ation of toxin pretreatment obtained from experiments done at 0.2 nm [c15E]SrIB or 0.2 and 1 nm EI, respect- ively (n ¼ 3). Note the different time scales in Fig. 5E and Fig. 5F. Potentiation data at 1 nm are not shown for [c15E]SrIB, because they were too fast to be resolved. On the contrary, data at 1 nm for EI, although fast (but not fitted to exponentials), are shown because they illustrate the interesting depotenti- ation with a time constant different from that observed at 0.2 nm. From these experiments, it can be seen that both the development of potentiation and the depoten- tiation or block are dependent on the toxin type and concentration. These data suggest a very complex mechanism of toxin–receptor interaction that warrants additional study. Unfortunately, this was beyond the scope of this study. On the whole, these results suggest that the potentia- tion described here could be a property of different clas- ses of a-conotoxins. On the other hand, we do not exclude the possibility that this effect could be confined to the conotoxins that act on both neuronal and muscu- lar receptor subtypes, as those used in this work are the only ones reported to be active on both targets. On the a 1 b 1 cd receptor, the synthetic toxin [c15E]SrIB was less potent than EI, but the latter was less efficient. Discussion Biochemical characterization of SrIA and SrIB The primary structures of peptides SrIA and SrIB iso- lated from the worm-hunting snail C. spurius reflect post-translational modifications of proline and gluta- mine residues, together with the amidation of the C-terminus of a shared toxin precursor. From analysis of the cDNA sequence, the C-terminus, including the last cysteine, is: CGGRR. This sequence is typically present in peptides processed post-translationally. Several rules have emerged from matching the sequences of the mature peptides with the nucleotide sequences of the cDNAs encoding scorpion toxins. If one or two basic residues are present at the C-termi- nus, they are removed post-translationally. If a glycine precedes the basic residue(s), it is used to amidate the residue preceding the glycine [5]. The MS analyses of toxins SrIA and SrIB showed that these peptides are in fact amidated. The amino acid sequences indicate that the peptides share structural features typical of the a-conotoxin family. The two peptides contain four and seven resi- dues between the second and the third cysteines, and between the third and the fourth cysteines, respectively (CCX 4 CX 7 C). This spacing defines the subfamily of the a4 ⁄ 7-conotoxins (Table 2), the most widespread category of nicotinic antagonists present in cone snail venoms [2]. The a4 ⁄ 7-conotoxins have a conserved proline in loop I, which comprises residues between the second and the third cysteines. Together with Vc1a [16], peptides SrIA and SrIB are the only known a4 ⁄ 7- conotoxins in which this constant proline is post-trans- lationally modified to hydroxyproline (Table 2). This derivative has been found in l-conotoxins, x-conot- oxins, j-conotoxins, jA-conotoxins, aA-conotoxins, w-conotoxins, e-conotoxins, v-conotoxins, r-conotox- ins, jM-conotoxins, d-conotoxins, and I-conotoxins [17]. It was also discovered in the a4 ⁄ 7-conotoxin GID [18], although not at the conserved proline of loop I. Another unusual characteristic of SrIA and SrIB is the presence of c-carboxyglutamate residues. This post- translational modification has been described in Conus peptides such as the conantokins, the c-conotoxins, the I-conotoxins, and the e-conotoxins [17], and in the N-terminal region of the a4 ⁄ 7-conotoxin GID [18]. However, Vc1a and peptides SrIA and SrIB are the only a-conotoxins in which c-carboxyglutamate residues occur in loop II, which comprises residues between the third and the fourth cysteines. Peptides SrIA and SrIB have 18 amino acids and an amidated C-terminus. They are predicted to have charges of 0 and + 1, respectively, at physiologic pH. It has been pointed out that a-conotoxins specific for neuronal subtypes of nAChR are neutral or negatively charged [19], whereas a-conotoxins that target muscle receptors have a net positive charge [20]. Because, according to these authors, peptide SrIA could be considered a potential antagonist of neuronal nAChR, and toxin SrIB a probable antagonist of muscle nAChR, we decided to test peptides SrIA and SrIB in biological preparations separately expressing neuronal (central, a 4 b 2 , and ganglionic, a 3 b 4 ) and muscle (a 1 b 1 cd) subtypes of nAChR. Unexpectedly, peptides SrIA and SrIB were active on both central and muscle types of the nAChR, which constitutes a novel activity profile of the conserved a4 ⁄ 7-conotoxin-type scaffold. Even more surprising was the finding that peptides SrIA and SrIB have nAChR-potentiating activity, in contrast to all previously studied a4 ⁄ 7-conotoxins. It has been postulated that divergence within a sin- gle superfamily to produce functionally different famil- ies is one of the neuropharmacologic strategies employed by the Conus genus, and may account in part for its success in nature [21]. E. Lo ´ pez-Vera et al. a-Conotoxins with potentiating effects on nAChRs FEBS Journal 274 (2007) 3972–3985 ª 2007 The Authors Journal compilation ª 2007 FEBS 3979 Structure–function relationship for SrIA, SrIB, and EI Peptides EI, SrIA and SrIB contain structural elements of the two types of conotoxins that act differentially on neuronal and muscle nAChR. Toxin EI [15] (present study) and peptides SrIA, SrIB and [c15E]SrIB are the only conotoxins with a type I cysteine scaffold known to act on muscle nAChR. Except for SrIA, they have positive net charges that might contribute to their activity on muscle receptors [20], and they (except EI) share with most of the a3 ⁄ 5-conotoxins (blockers of a 1 b 1 cd nAChR) a tyro- sine at position 4 of loop II that is not present in any of the a4 ⁄ 7 conotoxins known previously (Table 2). This tyrosine has been found to make an important contribution to the affinity of toxin MI for the a ⁄ d subunit interface of the muscle nAChR [22]. The three peptides have threonines and methio- nines at position 4 of loop I and position 2 of loop II, respectively. These residues are not present at these positions in any of the other a4 ⁄ 7 toxins studied to date, with the exception of Met10 in toxin EpI (Table 2). It seems probable that these threo- nines and methionines are somewhat involved in the binding and ⁄ or activity with muscle nAChR. Alter- natively, the nonpolar methionine residue at position 2 of loop II might be involved in binding to neuron- al nAChR subtypes, because all known a4 ⁄ 7-cono- toxins have a nonpolar residue at this position (Table 2). Peptides EI, SrIA, SrIB and [c15E]SrIB have very similar hydrophobic aliphatic residues occupying position 7 of loop II (isoleucine in toxin EI; leucine in peptides SrIA, SrIB, and [c15E]SrIB); aliphatic residues (leucine, isoleucine, or valine) also occur at this position in toxins MII, PeIA, GIC, Vc1.1, PIA, and GID, which target diverse neuronal subtypes (including a 3 b 4 and a 4 b 2 ) with variable affinities (Table 2). Thus, it is probable that hydro- phobic aliphatic residues at position 7 of loop II contribute to the binding and ⁄ or activity of peptides EI, SrIA, SrIB and [c15E]SrIB with a 3 b 4 and ⁄ or a 4 b 2 nAChRs. Finally, except for toxin GID, peptides SrIA, SrIB, and [c15E]SrIB are the only a4 ⁄ 7-conotoxins known to have an arginine at posi- tion 1 of loop II (Table 2). In GID, this residue has been demonstrated to contribute to the block of the a 4 b 2 subtype [18], which is consistent with the biolo- gical activity of peptides SrIA, SrIB and [c15E]SrIB on a 4 b 2 nAChRs. So far, the toxin with the highest affinity (IC 50 ¼ 152 nm) for the a 4 b 2 subtype is GID, and it blocks the a 3 b 2 and a 7 subtypes with  40- fold higher affinities [18]. The physiologic role of the SrI and EI a-conotoxins In the present article, we have defined the weak antag- onist properties of the novel C. spurius a-conotoxins, and of a synthetic analog of one of them, on three of the more important types of acetylcholine receptor. Moreover, while comparing these properties with those of the well-known a-conotoxin EI, we discovered that it has a selectivity spectrum somewhat different from that known previously. a-Conotoxin EI had been considered a specific blocker of a 1 b 1 cd nAChRs [15,17,23,24], but our results show that it also may block the a 3 b 4 and a 4 b 2 neuronal subtypes. This part of our results emphasizes the importance of testing conotoxins not only on the expected subtypes of the known molecular target (based on the toxin sequence and on the current pharmacologic knowledge in the field), but also on other target subtypes and even on nonrelated targets. Recently, toxin ImII has been found to inhibit both a 7 and a 1 b 1 cd nAChRs to similar extents [25], whereas the a3 ⁄ 5-conotoxin CnIA not only inhibits fetal muscle nAChRs, but also blocks the neur- onal a 7 subtype, although with an  80-fold lower affin- ity [26]. One surprising and distinct activity associated with the same protein scaffold of the a4 ⁄ 7-conotoxins has been reported for toxin q-TIA from C. tulipa;it inhibits the a 1 -adrenoreceptor, and has the same disul- fide connectivity as ‘classic’ a-conotoxins [27]. Like toxin q-TIA, which has an extended N-terminal sequence, peptides SrIA and SrIB have sequence fea- tures (hydroxylated proline in loop I and c-carboxyglut- amate residues in loop II) that differ considerably from those of other a4 ⁄ 7-conotoxins. The second part of our results reveals a novel cono- toxin-induced functional nAChR state consisting of a potentiation of the response; the potentiation can be detected both with the new toxins and with EI. It can be observed and quantitatively characterized at extre- mely low concentrations and with brief applications. Interestingly, longer applications produced either a null effect or an inhibitory effect, as expected from the kinetic data shown in Fig. 5E,F and the affinity of the inhibitory process, which were evaluated with pro- longed pretreatments. The a-conotoxins described in this communication showed that they can regulate the nAChR response. It is known [28] that nAChRs are subjected to a variety of actions, including the increase or decrease of the affinity of the receptor for nicotinic ligands, a phenom- enon that may occur in the absence of agonist, and possibly results from stabilization of the desensitized state [29]. Numerous examples of positive and negative a-Conotoxins with potentiating effects on nAChRs E. Lo ´ pez-Vera et al. 3980 FEBS Journal 274 (2007) 3972–3985 ª 2007 The Authors Journal compilation ª 2007 FEBS allosteric effectors acting at neuronal nAChRs have been reported, illustrating the importance of the allos- teric nature of this protein. For example, it was shown that progesterone and 17-b-estradiol act as negative and positive effectors, respectively, of the a 4 b 2 receptor subtype [30,31]. Atropine and zinc are reported to have similar effects on some nAChRs [32,33], although the required concentrations of these drugs were higher by more than two orders of magnitude than those of our peptides. Interestingly, the same mixed partial agonist and antagonist behavior was observed for the well- known blocker d-tubocurarine [34]. It has been repor- ted recently [35] that a-conotoxin PnIA and a synthetic derivative of it ([A10L]PnIA) weakly potentiate acetyl- choline-activated currents in the wild-type a 7 nAChR; these authors also reported that on mutant (a 7 -L247T) receptors, [A10L]PnIA potentiated the acetylcholine- evoked current and acted as an agonist by itself. The mechanisms involved in these processes may be related to previous findings that a-conotoxin MI binds to two distinct sites on the a 1 b 1 cd nAChR, one at the ad interface, and another at the ac (or ae) interface [36]. Concluding remarks As it is unknown how and where the peptides studied in this work bind the different receptor assemblies, it is premature to suggest any hypothesis regarding the structure–function mechanisms underlying the peptide binding. Single-channel studies are in progress using mutagenized peptides and cells expressing specific nAChRs. These peptides are promising tools for studies at a detailed molecular level of the structure–activity rela- tionship that underlies the action of the nAChR-target- ing conotoxins. Considering that nAChRs are implicated in brain diseases such as schizophrenia, noc- turnal frontal lobe epilepsy [37], and Alzheimer’s dis- ease, these new peptides are also candidate models to develop potentially therapeutic drugs of major import- ance [38]; for example, peptides SrIA, SrIB and [c15E]SrIB might lead to the development of a 4 b 2 -select- ive enhancers, which are beginning to be discovered [39]. Experimental procedures Specimen collection and venom extraction Specimens of C. spurius were collected in the Yucatan Channel, Mexico. The venom was obtained by dissection of the venom ducts. The ducts were homogenized in 10 mL of 0.1% v ⁄ v trifluoroacetic acid and 40% v ⁄ v acetonitrile (ACN). The homogenate was centrifuged at 17 000 g for 30 min at 4 °C using a Beckman Coulter Avanti J20 centri- fuge with JA-20 rotor. The supernatant, containing the pep- tides, was subsequently processed. Peptide purification by RP-HPLC HPLC was performed on a n Agilent 1100 Series LC System (G1322A Degasse r, G1311A Quaternary Pump, G1315B Diode Array Detector, G1328A Man ual Injector; Hewlett-Packard, Waldbronn, German y). The venom e xtract was fractionated with a Vydac (Toluca, Mexico) C18 analytical reverse-phase column (2 18TP54, 5 lm, 4.6 · 250 mm) equipped with a Vydac C18 guard column (218GK54, 5 lm, 4.6 · 10 mm). Peptides were eluted with a linear g radient of 5–95% solution B at a fl ow rate of 1 m LÆmin )1 over 90 min, where solution A is 0.1% v ⁄ v aqueous trifluoroacetic acid and solution B is 0 .09% v ⁄ v trifluoroacetic acid in 90% v ⁄ v aqueous ACN. The same column was also employed to repurify the components of the venom, using a linear gradient of 15–30% of solution B at a flow rate of 1 m LÆmin )1 for 4 5 min. Amino acid sequence Peptides were adsorbed onto polybrene-treated (Biobrene Plus; Applied Biosystems, Foster City, CA) glass fiber fil- ters, and the amino acid sequence was determined by auto- mated Edman degradation using an automatic instrument (Procise 491 Protein Sequencing System; Applied Biosys- tems) by the pulsed-liquid method. MS analysis Native peptides were applied directly into a Finnigan LCQ DUO ion trap mass spectrometer (Finnigan, San Jose, CA). The LCQ mass spectrometer is coupled to a Surveyor syringe pump delivery system. The eluate at 20 lLÆmin )1 was split to allow only 5% of the sample to enter the nano- spray source (1.0 lLÆmin )1 ). The spray voltage was set to 1.6 kV, and the capillary temperature was set to 130 °C. All spectra were obtained in the positive-ion mode. The acquisition and deconvolution of data were performed with xcalibur software (Thermo Electron Corp., Nashville, TN) on a Windows NT PC system. Determination of disulfide bridges The connectivity of the cysteines of toxin SrIA was deter- mined by partial reduction with Tris(2-carboxyethyl) phos- phine hydrochloride and alkylation with N-ethylmaleimide. The peptide (11.8 nmol) was dissolved in 10 lL of denatur- ing buffer (0.1 m sodium citrate containing 6 m guanidine hydrochloride, pH 3.0), and 27 l L of 0.1 m Tris(2-carboxy- ethyl) phosphine hydrochloride in the same buffer was added. The mixture was incubated for 15 min at room E. Lo ´ pez-Vera et al. a-Conotoxins with potentiating effects on nAChRs FEBS Journal 274 (2007) 3972–3985 ª 2007 The Authors Journal compilation ª 2007 FEBS 3981 [...]... gradient from 10% to 30% solution B was developed at 1 mLÆmin)1 for 120 min at room temperature; the absorbance of the eluate was monitored at 220 nm The absorbing peaks not present in the corresponding reagent blank were collected and taken to dryness for automatic sequence analysis cDNA cloning Synthesis of peptides [c15E]SrIB and EI The venom duct and gland from one C spurius specimen was dissected, and. .. isolated using the RNAgents Total RNA Isolation System (Promega, Madison, WI), according to the supplier’s instructions Reverse transcription of mRNA was primed by oligo-p(dT)22NN, where p(dT)22 annealed with the polyA tail of mRNA, and NN localized the primer to the border between the polyA tail and the 3¢-UTR of the mRNA Reverse transcription was performed using the 1st Strand cDNA Synthesis Kit for... Progesterone modulates a neuronal nicotinic acetylcholine receptor Proc Natl Acad Sci USA 89, 9949–9953 Curtis L, Buisson B, Bertrand S & Bertrand D (2002) Potentiation of human a4b2 neuronal nicotinic acetylcholine receptor by estradiol Mol Pharmacol 61, 127–135 Zwart R & Vijverberg HP (1997) Potentiation and inhibition of neuronal nicotinic receptors by atropine: competitive and noncompetitive effects Mol... Subunit-dependent modulation of neuronal nicotinic receptors by zinc J Neurosci 21, 1848–1856 Steibach JH & Chen Q (1995) Antagonist and partial agonist actions of d-tubocurarine at mammalian muscle acetylcholine receptors J Neurosci 15, 230–240 Hogg RC, Hopping G, Alewood PF, Adams DJ & Bertrand D (2003) a-Conotoxin PnIA and [A10L]PnIA stabilize different states of the a7–L247T nicotinic acetylcholine receptor... pharmacological tools and potential drug leads Curr Med Chem 8, 327– 344 24 Janes RW (2005) a-Conotoxins as selective probes for nicotinic acetylcholine receptor subclasses Curr Opin Pharmacol 5, 280–292 25 Ellison M, Gao F, Wang H-L, Sine SM, McIntosh JM & Olivera BM (2004) a-Conotoxins ImI and ImII target distinct regions of the human alpha7 nicotinic acetylcholine receptor and distinguish human nicotinic receptor... 1 · ThermoPol Reaction Buffer [20 mm Tris ⁄ HCl (pH 8.8), 10 mm KCl, 10 mm (NH4)2SO4, 2 mm MgSO4, 0.1% Triton X-100] and 0.2 mm dNTP mix was added 5 lL of reverse transcription product and 20 pmol of each primer The mixture was incubated in the thermal cycler at 95° for 5 min, and then 2 units of Vent DNA Polymerase were added (Hot Start) The parameters for thermal cycling were: 95 °C for 5 min; then... to a final peptide concentration of 5 mm The pH was adjusted to 8 by adding 1.5 m Tris ⁄ HCl buffer (pH 8.8) The resulting solution was stirred for 48 h and then concentrated The bis-Acm-peptides containing disulfide bridges between Cys3 and Cys9 in the case of [c15E]SrIB or between Cys4 and Cys10 in the case of EI were purified by semipreparative HPLC Mass analysis gave the following results: Bis-Acm[c15E]SrIB... a-Conotoxin GIC from Conus geographus, a novel peptide antagonist of nicotinic acetylcholine receptors J Biol Chem 277, 33610–33615 4 Bai-Song L, Fang Y, Dong Z, Pei-Tang H & Cui-Fen H (1999) Conopeptides from Conus striatus and Conos textile by cDNA cloning Peptides 20, 1139–1144 5 Becerril B, Marangoni S & Possani LD (1997) Toxins and genes isolated from scorpions of the genus Tityus Toxicon 35, 821–835... five-fold excess of activated amino acid for a minimum of 30 min, and monitored by the 2,4,6-trinitrobenzenesulfonic acid test [42] At the end of chain assembly, the resin was washed with dimethylformamide ⁄ dichloromethane, dried under nitrogen, and treated with a mixture of cation scavengers in trifluoroacetic acid with magnet stirring for 2 h to detach the peptides from the resin and simultaneously... 275–276 a-Conotoxins with potentiating effects on nAChRs 38 Hogg RC & Bertrand D (2007) Partial agonists and therapeutic agents at neuronal nicotinic acetylcholine receptors Biochem Pharmacol 73, 459–468 39 Broad LM, Swart R, Pearson HK, Lee M, Wallace L, McPhie G, Emkey R, Hollinshead SP, Dell CP, Baker SR et al (2006) Identification and pharmacological profile of a new class of selective nicotinic acetylcholine . Novel a-conotoxins from Conus spurius and the a-conotoxin EI share high-affinity potentiation and low-affinity inhibition of nicotinic acetylcholine receptors Estuardo. [18]. The physiologic role of the SrI and EI a-conotoxins In the present article, we have defined the weak antag- onist properties of the novel C. spurius a-conotoxins, and