a-Conotoxin analogs with additional positive charge show increased selectivity towards Torpedo californica and some neuronal subtypes of nicotinic acetylcholine receptors Igor E. Kasheverov 1 , Maxim N. Zhmak 1 , Catherine A. Vulfius 2 , Elena V. Gorbacheva 2 , Dmitry Y. Mordvintsev 1 , Yuri N. Utkin 1 , Rene ´ van Elk 3 , August B. Smit 3 and Victor I. Tsetlin 1 1 Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia 2 Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Russia 3 Department of Molecular and Cellular Neurobiology, Center for Neurogenomics and Cognitive Research, Vrije Universiteit, Amsterdam, the Netherlands a-Conotoxins are a group of relatively short peptides (12–19 amino acid residues, two disulfide bridges) from the venom of poisonous marine snails of the Conus genus [1]. In addition to peptides isolated from venom new a-conotoxins have recently been identified by cDNA cloning from venomous glands and have been Keywords acetylcholine-binding protein; acetylcholine- elicited Cl – current; a-conotoxin analogs; identified Lymnaea neurons; nicotinic acetylcholine receptor Correspondence V. I. Tsetlin, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya str. 16 ⁄ 10 Moscow, Russia Tel ⁄ Fax: +7 495 335 57 33 E-mail: vits@ibch.ru (Received 28 March 2006, revised 16 June 2006, accepted 4 August 2006) doi:10.1111/j.1742-4658.2006.05453.x a-Conotoxins from Conus snails are indispensable tools for distinguishing various subtypes of nicotinic acetylcholine receptors (nAChRs), and synthe- sis of a-conotoxin analogs may yield novel antagonists of higher potency and selectivity. We incorporated additional positive charges into a-conotox- ins and analyzed their binding to nAChRs. Introduction of Arg or Lys res- idues instead of Ser12 in a-conotoxins GI and SI, or D12K substitution in a-conotoxin SIA increased the affinity for both the high- and low-affinity sites in membrane-bound Torpedo californica nAChR. The effect was most pronounced for [D12K]SIA with 30- and 200-fold enhancement for the respective sites, resulting in the most potent a-conotoxin blocker of the Torpedo nAChR among those tested. Similarly, D14K substitution in a-conotoxin [A10L]PnIA, a blocker of neuronal a7 nAChR, was previously shown to increase the affinity for this receptor and endowed [A10L,D14K]PnIA with the capacity to distinguish between acetylcholine- binding proteins from the mollusks Lymnaea stagnalis and Aplysia califor- nica. We found that [A10L,D14K]PnIA also distinguishes two a7-like anion-selective nAChR subtypes present on identified neurons of L. stag- nalis: [D14K] mutation affected only slightly the potency of [A10L]PnIA to block nAChRs on neurons with low sensitivity to a-conotoxin ImI, but gave a 50-fold enhancement of blocking activity in cells with high sensitiv- ity to ImI. Therefore, the introduction of an additional positive charge in the C-terminus of a-conotoxins targeting some muscle or neuronal nAChRs made them more discriminative towards the respective nAChR subtypes. In the case of muscle-type a-conotoxin [D12K]SIA, the contribu- tion of the Lys12 positive charge to enhanced affinity towards Torpedo nAChR was rationalized with the aid of computer modeling. Abbreviations ACh, acetylcholine; AChBP, acetylcholine-binding protein; IC 50 , ligand concentration at which 50% inhibition is achieved; nAChR, nicotinic acetylcholine receptor; n H , Hill coefficient. 4470 FEBS Journal 273 (2006) 4470–4481 ª 2006 The Authors Journal compilation ª 2006 FEBS synthesized chemically [2–5]. a-Conotoxins have become widely used tools in studies on nicotinic acetylcholine re- ceptors (nAChRs) [6,7] because they can distinguish between different nicotinic acetylcholine receptor (nAChR) subtypes. For example, a-conotoxins GI, MI and SIA selectively block muscle-type nAChRs, whereas some others block distinct neuronal nAChRs, e.g. a-conotoxins ImI and ImII target homo-oligomeric a7 nAChR [8], whereas a-conotoxins MII, PnIA, GIC block heteromeric nAChR containing a3, a6 and b2 subunits [6]. A change in one or several residues of the naturally occurring a-conotoxin might result in a change in its nAChR subtype selectivity [9]. For example, the A10L substitution in a-conotoxin PnIA switched its selectivity from the a3b2 to the a7 nAChR [10,11]. Synthesis of diverse a-conotoxin analogs, mutations in nAChRs and pair-wise mutation analysis have enabled the identification of specific a-conotoxin and ⁄ or nAChR residues taking part in ligand–receptor interactions [12–15]. The crystal structure of the acet- ylcholine-binding protein (AChBP) from the mollusk Lymnaea stagnalis, which provides a high-resolution structure for the extracellular domains of nAChRs [16,17], has been used to build models for a-conotoxin binding to distinct nAChRs [18]. Recently, crystal structures have been solved for AChBP complexes with two a-conotoxins: [A10L, D14K]PnIA, a double mutant of a-conotoxin PnIA [19], and for a-conotoxin ImI [20,21]. These structures provide a solid basis for modeling the spatial structures of a-conotoxins with the cognate nAChRs. Modeling may also be a start- ing point for the rational design of new a-conotoxins with higher affinity and better selectivity towards nAChRs. D14K substitution increased the affinity of the starting [A10L]PnIA for chicken a7 nAChR and L. stagnalis AChBP [19]. X-Ray data on the AChBP)a-conotoxin complex were the basis for con- structing a model for a7 nAChR complexes with [A10L]PnIA and [A10L, D14K]PnIA [19]. We used the X-ray data and cryoelectron microscopy structure of Torpedo nAChR [22] to build a respective model for a-conotoxin [D12K]SIA, wherein the Lys12 positive charge gave the most dramatic increase in the affinity for T. californica nAChR. Anion-selective nAChRs in some identified neurons of the fresh-water snail L. stagnalis and marine mol- lusk Aplysia californica were found to resemble the a7 nAChRs of vertebrates in terms of their pharmacologi- cal profile and the response kinetics to acetylcholine (ACh) [23,24]. To further elucidate the significance of a positive charge in the C-terminus of a-conotoxins we compared the action of [A10L]PnIA and [A10L,D14K]PnIA on a 7-like nAChRs in identified Lymnaea neurons. This is of interest in light of the recent cloning of a set of nAChR subunits from this species and electrophysiological analysis of several of them expressed in Xenopus oocytes [25,26]. Results and Discussion Synthesis of a-conotoxins New analogs of a-conotoxins GI, SI and SIA with arginine, lysine and ⁄ or aspartate introduced at position 12 (Table 1) were synthesized using a solid-phase method with the simultaneous formation of the two disulfides. For a-conotoxin SIA, which has Asp12 in this position, an additional D12S analog was also synthesized. A series of a-conotoxin MI analogs was similarly synthesized. In this case, we employed Lys- scanning mutagenesis for the possibly complete set of MI variants, excluding the substitutions of structurally important amino acid residues (Cys, Pro). As a result, three novel analogs of a-conotoxin MI with a lysine residue introduced at position 5, 7 or 11 were obtained. Simultaneous formation of the disulfides decreases the number of stages and usually gives higher peptide yields, although this is sometimes accompanied by the production of incorrectly bridged isomers [27]. When several isomers were formed, the peptide with correctly formed disulfide bridges was assumed to have a higher potency to bind to the membrane-bound T. californica nAChR in the radioligand-binding assay (see below). It is known that incorrect disulfide formation in a-conotoxins that target the muscle-type nAChRs entails a decrease in the affinity [28]. However, the enhanced affinity of the incorrectly formed isomer of a-conotoxin AuIB, targeting one neuronal-type nAChR, was revealed previously [29] and makes the method less predictive. Therefore, all new synthesized analogs were also characterized using CD spectroscopy to detect secondary structure changes in the ‘incorrect’ isomers (see below). In 13 syntheses of muscle-type conotoxins we found the generation of isomers only in two cases – one additional minor peak for [S12D]GI and two for SIA (all peaks had correct molecular masses). a-Conotoxin [A10L]PnIA, known to act on a7 nAChR [10,11] was obtained by solid-phase peptide synthesis using the simultaneous formation of two disulfides as described previously [30]. In the case of [A10L,D14K]PnIA, orthogonal protecting groups were used for correct pair-wise closing of disulfides to exclude the formation of other isomers (see Experi- mental procedures). The structures of all synthesized I. E. Kasheverov et al. Novel a-conotoxin analogs FEBS Journal 273 (2006) 4470–4481 ª 2006 The Authors Journal compilation ª 2006 FEBS 4471 peptides were verified by MALDI analysis (Table 1) and purity by RP-HPLC (data not shown). CD spectroscopy CD spectra were obtained for aqueous solutions of native a-conotoxins GI, MI, SI, SIA and their analogs [S12R ⁄ K ⁄ D]GI, [H5K]MI, [S12R]SI, [D12S⁄ K]SIA, as well as for one isomer of [S12D]GI and two isomers of SIA, which were produced in noticeable quantities during peptide syntheses. As an example the spectra of a-conotoxin GI analogs are presented in Fig. 1. Amino acid substitutions at position 12 did not result in any noticeable alterations in peptide secondary structure. However, the second (minor) isomer of [S12D]GI dis- played a remarkable change in spectral characteristics (inset in Fig. 1). Similarly, the spectra of the SI and SIA analogs with substitution at position 12 (as well as [H5K]MI) were identical to that of the respective naturally occurring a-conotoxins. However, both minor isomers of SIA had spectra resembling that of minor [S12D]GI isomer (data not shown). The available literature data indicate that single amino acid substitutions do not markedly change the CD curves of a-conotoxins. However, breaking the Cys–Cys disulfide bonds in a-conotoxin ImI [31] or x-conotoxin MVIIA [32], or changing the size of the disulfide-confined peptide loops by introduction of an additional amino acid residue in a-conotoxin ImI [33], resulted in a remarkable change in CD spectra, with shifting of the ellipticity minimum into the 195–200 nm region. This shift resembles that seen for minor isomers of both [S12D]GI and SIA (see curve 4a in the inset of Fig. 1). Taken together, these results indicate that analysis of biological activities (Fig. 2) has been carried out on a series of a-conotoxins with correctly closed disulfides. Binding of synthesized a-conotoxin analogs to membrane-bound Torpedo nAChR The activity of analogs was evaluated in competition with radioiodinated a-conotoxins GI or MI for binding Fig. 1. CD spectra of a-conotoxins GI (1, solid line), [S12K]GI (2, dotted line), [S12R]GI (3, dash-dot line) and main isomer of [S12D]GI (4, dash line) in water. Inset: CD spectra of two [S12D]GI isomers – the main (4, solid line) and minor (4a, dash line) ones. Table 1. The structures of synthesized naturally occurring a-conotoxins and their analogs. All a-conotoxins have amidated C-termini as well as disulfide bridges Cys1–Cys3 and Cys2–Cys4. The substituted residues in the analogs are indicated in bold type. a-Conotoxin Sequence Mol. mass, MH + Calculated MALDI-measured GI ECCNPACGRHYSC 1438.6 1438.4 [S12R]GI ECCNPACGRHYRC 1507.7 1506.6 [S12K]GI ECCNPACGRHYKC 1479.7 1480.9 [S12D]GI ECCNPACGRHYDC 1466.6 1465.5 SI ICCNPACGPKYSC 1354.6 1353.5 [S12R]SI ICCNPACGPKYRC 1423.7 1422.4 SIA YCCHPACGKNFDC 1456.7 1455.6 [D12S]SIA YCCHPACGKNFSC 1428.7 1427.5 [D12K]SIA YCCHPACGKNFKC 1469.9 1469.3 MI GRCCHPACGKNYSC 1494.7 1494.4 [H5K]MI GRCCKPACGKNYSC 1485.7 1484.6 [A7K]MI GRCCHPKCGKNYSC 1553.2 1554.1 [N11K]MI GRCCHPACGKKYSC 1508.8 1507.7 [A10L]PnIA a GCCSLPPCALNNPDYC 1664.7 1664.7 [A10L,D14K]PnIA a GCCSLPPCALNNPKYC 1677.8 1677.6 a Described in Celie et al. [19]. Novel a-conotoxin analogs I. E. Kasheverov et al. 4472 FEBS Journal 273 (2006) 4470–4481 ª 2006 The Authors Journal compilation ª 2006 FEBS to membrane-bound T. californica nAChR (Fig. 2). Both tracers bound specifically to the Torpedo receptor with equal high affinity: K d values for 125 I-labeled GI and MI were 24 ± 3 and 28 ± 6 nm, respectively. By contrast to a-conotoxin GI and M1, a-conotoxin SI has an equal potency to both sites in the Torpedo nAChR [34], whereas a-conotoxin SIA binds to only one site [35] as revealed by competition with 125 I-labe- led a-bungarotoxin. That is why we did not prepare the radioactive forms of these peptides, and 125 I-labeled GI was used as a tracer to test the SI and SIA analogs. In these experiments the synthetic a-conotoxins GI, SI, SIA and MI were used as controls. The respective lig- and concentrations at which 50% inhibition is achieved (IC 50 values) are presented in Table 2. The introduction of a positively charged amino acid residue instead of a neutral one in position 12 of a-conotoxins GI and SI resulted in a three- to seven- fold increase in the affinity to both binding sites of the Torpedo nAChR (Fig. 2A,B; Table 2). The most remarkable was the D12K mutation in a-conotoxin SIA: the binding efficiencies to the high- and low-affin- ity sites increased for the [D12K]SIA analog by 35 and 260 times, respectively (Fig. 2C; Table 2). This increase was due mainly to removal of the negatively charged amino acid residue in this position, because substitu- tion with neutral Ser also resulted in affinity enhance- ment to both sites (25 and 65 times, respectively). Conversely, the introduction of a negative charge in position 12 of a-conotoxin GI caused a considerable decrease in the affinity for the receptor (Fig. 2A; Table 2). However, the introduction of an additional positive charge (Lys) at position 11 of a-conotoxin MI (which corresponds spatially to residue 10 of a-cono- toxins GI, SI and SIA) affected the peptide activity only slightly, whereas H5K or A7K mutations wor- sened the binding characteristics of these analogs (Fig. 2D; Table 2). It should be noted that all three minor isomers (of [S12D]GI and SIA) showed more than tenfold Fig. 2. Inhibition of 125 I-labeled a-conotoxins GI (A–C) and MI (D) binding to membrane-bound Torpedo nAChR with indicated a-conotoxins and their analogs. Final concentrations of the radioligand and toxin-binding sites of receptor were 280 and 230 n M, respectively. The data shown are the averages of two independent experiments. The inhibition curves were fitted using ORIGIN 6.1 (MicroCal Software Inc.) in the frames of a two-site competition model for all peptides (with one exception for [A7K]MI). The respective IC 50 values are presented in Table 2. I. E. Kasheverov et al. Novel a-conotoxin analogs FEBS Journal 273 (2006) 4470–4481 ª 2006 The Authors Journal compilation ª 2006 FEBS 4473 decreased efficacies, compared with the major com- pounds, in competition with radiolabeled a-conotoxin GI for the T. californica nAChR binding (data not shown). Both PnIA variants at concentrations of up to 100 lm were inactive in competition with 125 I-labeled GI for binding to the membrane-bound Torpedo nAChR (data not shown). We synthesized mainly the modified a-conotoxins targeting the muscle-type nAChR. Literature data on the role of charged residues in this group of a-cono- toxins are in part contradictory. Several researchers have shown that charged groups at the N-termini of a-conotoxins GI, MI and SI exert only a weak influence on the activity [33,35–39]. The important role of Arg9 in the interaction with a high-affinity a-conotoxin- binding site on the Torpedo nAChR has been convin- cingly demonstrated: R9P and R9A substitutions in a-conotoxin GI resulted in a two to three order of magnitude loss in the affinity for the a ⁄ c site, whereas the reverse substitutions P9R and P9K in a-conotoxin SI enhanced the affinity for this site [34,35,40]. How- ever, when Ala or Pro residues in a-conotoxin MI were substituted for the Lys10, whose spatial disposition is close to that of Arg9 in a-conotoxin GI, the interac- tion with the high-affinity a ⁄ c-binding site was affected to a much less degree [38–40]. In addition, acylation of Lys10 with azidobenzoyl or benzoylbenzoyl groups practically did not change the capacity of the respect- ive derivatives to interact with the membrane-bound Torpedo nAChR [41]. Of all known muscle-type a-conotoxins, only a-conotoxin SIA interacts exclusively with one a ⁄ c site on the Torpedo nAChR [35]. Interestingly, this peptide contains a negatively charged residue (Asp12) in the C-terminal part of the molecule (Table 1) whose role has not been examined previously. D12S substitution resulted in a 25- and 65-fold increase in the affinity for the high- and low-affinity binding sites, respectively (Fig. 2C; Table 2). Introduction of a positive charge (Lys) at this position resulted in an additional fourfold increase in affinity for the low-affinity site (Fig. 2C; Table 2). Substitution of Lys or Arg for Ser12 in a-conotoxins GI and SI gave a reliable enhancement (three- to sevenfold) of the affinity for both binding sites (Fig. 2A,B; Table 2). By contrast, introduction of a negative charge at this position (S12D) in a-conotox- in GI brought about a marked decrease in the affinity (Fig. 2A; Table 2). It is noteworthy that use of 125 I- labeled a-conotoxin GI in these experiments, instead of the usual 125 I-labeled a-bungarotoxin [34,35,39,40], revealed the differences in potency to two sites for a-conotoxin SI and made possible the detection of a low-affinity binding site for a-conotoxin SIA in the Torpedo nAChR. From literature data it is known that the affinities of muscle-type a-conotoxins to the Tor- pedo nAChRs binding sites (tested in competition with 125 I-labeled a-bungarotoxin) vary from one to three orders of magnitude [12,34,35,39]. The given explan- ation for this scatter is the influence of the receptor state, test conditions, etc. It is therefore not surprising that using a different tracer in the radioligand assay may result in different binding parameters for a-cono- toxins. This was shown previously for one a-conotoxin GI analog on the Torpedo receptor [41]. There is convincing evidence (the crystal structures of the a-cobratoxin and a-conotoxin complexes with acetyl- choline-binding proteins) [19–21,42] that the binding sites for these two groups of competitive antagonists overlap, but are not identical. Grafting positive charges on to other positions of a-conotoxin amino acid sequence resulted in Table 2. Activity of a-conotoxins and their analogs tested in compe- tition binding assays. Using the membrane-bound Torpedo nAChR, the inhibitory activities of a-conotoxins GI, SI, SIA or MI and their analogs were evaluated in competition with 125 I-labeled a-conotox- ins GI (GI, SI, SIA and their analogs) or MI (MI and its analogs): see respective inhibition curves presented in Fig. 2. IC 50 values were calculated using ORIGIN 6.1 in the frames of both one- and two-site models using the joint data from two or three independent experi- ments for each a-conotoxin. The choice was made in favor of the model giving the minimal ‘reduced chi-squared’ parameter comple- mented with reasonable SE values and taking into consideration the Hill coefficients (n H ). For all muscle-type conotoxins (with one exception), a two-site model was found the best. In the case of [A7K]MI analog the program failed to fit the data to a two-site model, so the respective IC 50 value was generated in the frames of one-site model and ascribed to both sites. Both PnIA variants were inactive in competition with 125 I-labeled a-conotoxins GI at 100 lM (4 ± 2% of inhibition). a-Conotoxin n H IC 50 ,lM high affinity site low affinity site GI 0.64 ± 0.04 1.6 ± 0.7 9.3 ± 3.7 [S12R]GI 0.68 ± 0.04 0.49 ± 0.25 1.7 ± 1.0 [S12K]GI 0.56 ± 0.06 0.29 ± 0.13 3.2 ± 1.1 [S12D]GI 0.52 ± 0.07 12.0 ± 2.9 230 ± 50 SI 0.53 ± 0.06 4.0 ± 1.2 58 ± 25 [S12R]SI 0.59 ± 0.04 1.0 ± 0.3 8.2 ± 3.6 SIA 0.32 ± 0.06 3.5 ± 1.3 440 ± 150 [D12S]SIA 0.46 ± 0.03 0.13 ± 0.02 6.6 ± 0.6 [D12K]SIA 0.52 ± 0.05 0.10 ± 0.05 1.7 ± 0.7 MI 0.55 ± 0.04 0.26 ± 0.07 6.6 ± 0.7 [H5K]MI 0.53 ± 0.04 9.1 ± 1.8 130 ± 60 [A7K]MI 0.83 ± 0.06 54 ± 4 54 ± 4 [N11K]M 0.68 ± 0.06 0.24 ± 0.16 3.7 ± 0.7 [A10L,D14K]PnIA – – 100 [A10L]PnIA – – 100 Novel a-conotoxin analogs I. E. Kasheverov et al. 4474 FEBS Journal 273 (2006) 4470–4481 ª 2006 The Authors Journal compilation ª 2006 FEBS weakening of the binding capacity as seen for a series of Lys-analogs of a-conotoxin MI (Fig. 2D; Table 2). Our experiments revealed a new site in the muscle- type a-conotoxins, wherein the presence of a charge considerably affects the efficiency of interaction with the Torpedo nAChR: a positive charge in the C-termi- nus increases the affinity for both binding sites, whereas a negative charge drastically decreases it. In contrast to muscle-type a-conotoxins, negatively charged amino acid residues can be found in the C-ter- minus of many a-conotoxins acting on neuronal nAChRs, namely in AuIA, AuIB, AnIA ⁄ C, EpI, Vc1.1, PnIA ⁄ B [43]. Substitutions in the neuronal a-conotoxins were used to modify their selectivity [9– 11]: A10L mutation in PnIA enhanced its affinity for rat and chicken a7 nAChR and weakened it for a3b2, converting the parent peptide from a3b2-preferring to a7-preferring [10,11,44]. However, no studies on the role of the above-mentioned negative charges in the C-terminus were performed earlier. Based on [A10L]PnIA, we recently synthesized a new analog that bears an additional D14K substitution [19]. This sub- stitution increased the affinity of the ‘double mutant’ for the chicken a7 nAChR and for L. stagnalis AChBP, but not for A. californica AChBP [19]. In this study we show that [A10L,D14K]PnIA exhibits high affinity for one subtype of a7-like nAChRs in L. stag- nalis neurons, discriminating two nAChRs. a-Conotoxin blockade of Cl – currents elicited by ACh in identified neurons of L. stagnalis PnIA analogs were tested on the identified neurons (LP1–3, RP2,3) from left and right parietal ganglia of L. stagnalis. The responses to ACh of these neurons result from an increase in only Cl – conductance, as revealed by I–V relationship determination at various Cl – concentrations in the internal solution (C.A. Vulf- ius et al. unpublished results). The AChRs in parietal Lymnaea neurons resemble a7 nAChRs of vertebrates in terms of the efficacy of choline, cytisine, and nico- tine (all of them are full agonists) and their high sensi- tivity to a-conotoxin ImI [23]. Two groups of cells distinct in terms of desensitization kinetics and sensi- tivity to ImI (IC 50 288 ± 27 and 10.3 ± 1.3 nm, respectively) have been recognized [23]. Both PnIA mutants inhibited the ACh-elicited cur- rent but had a weaker potency than a-conotoxin ImI (Fig. 3), in contrast to their much higher affinity for Lymnaea AChBP [19]. The residual unblocked current was of approximately the same amplitude in the pres- ence of saturating concentrations of ImI or either of the two PnIA mutants. The relative potency of the PnIA variants differed significantly in two types of neurons. There was no large distinction between [A10L]PnIA and [A10L,D14K]PnIA on cells with low sensitivity to a-conotoxin ImI (Fig. 3A; IC 50 30 and 15 lm, respectively). However, in neurons with high sensitivity to ImI, the [D14K] mutation increased the affinity 50-fold (Fig. 3B; IC 50 400 nm compared with 20 lm for [A10L]-variant). Average IC 50 ratios for ImI ⁄ [A10L,D14K]PnIA ⁄ [A10L]PnIA were 1 : 60 : 120 and 1 : 13 : 670 in two groups of cells. Thus, only those nAChRs which can be blocked by a-conotoxin ImI at very low concentrations discriminate between [A10L]PnIA and [A10L,D14K]PnIA. These results support our previous suggestion about the existence of two distinct populations of ImI-sensitive nAChRs in the Lymnaea neurons [23]. Enhancement of the affinity of the [D14K] mutant for nAChRs in a group of cells with high sensitivity to a-conotoxin ImI is comparable with the increase in the affinity for Lymnaea AChBP and chicken a7 nAChR [19], but the effect in the case of Lymnaea nAChRs is much more pronounced. In contrast, introduction of a positive charge at position 14 does not seem important for the interaction with nAChRs in neurons with low sensitivity to ImI just as for the interaction with Aply- sia AChBP [19]. Thus, a positive charge seems to be important for the interaction with some but not all neuronal nAChRs. Twelve nAChR subunits (A–L) have recently been identified in the CNS of L. stagnalis and three (A, B, and I) have been expressed in Xenopus laevis oocytes yielding functional homopentameric nAChRs [25,26]. It is interesting to compare the heterologously expressed nAChRs with nAChRs in the Lymnaea neurons differing in the affinity for a-conotoxins ImI and [A10L,D14K]PnIA. Pharmacological profiles of heterologously expressed nAChR-A and native nAChRs are very similar, but the A-homomer mediates cation conductance [25]. Anion-selective nAChR-B can be activated by choline, nicotine and cytisine (all three drugs being full agonists) [25], and blocked slightly by 100 nm a-conotoxin ImI (the maximal concentration used). Therefore, nAChR-B might be a candidate for native nAChR which has low sensitivity to a-conotox- in ImI and does not discriminate two PnIA variants. However, more probably, nAChRs with low or high sensitivity to ImI in parietal neurons may be formed with the participation of some other subunits. Alter- natively, some unidentified factors can influence a-conotoxin pharmacology on o ocyte-express ed nAChRs as has been earlier suggested from the comparison of a-conotoxin EpI and AuIB effects on the recombinant and native a3- and a7-containing nAChRs [45]. I. E. Kasheverov et al. Novel a-conotoxin analogs FEBS Journal 273 (2006) 4470–4481 ª 2006 The Authors Journal compilation ª 2006 FEBS 4475 Modeling a-conotoxin complexes with T. californica nAChR X-Ray structures of Aplysia AChBP in complexes with [A10L,D14K]PnIA [19] and ImI [20,21] provide the basis for modeling the a-conotoxin complexes with those nAChRs that are blocked specifically by the respective a-conotoxins. Using these X-ray crystal structures and the cryoelectron microscopy structure of 4A ˚ resolution for the Torpedo marmorata nAChR [22], we performed computer modeling of the [D12K]SIA complex with the T. californica nAChR (Fig. 4). The aim was to envisage the structure of mus- cle a-conotoxins with their target receptors and to explain a dramatic increase in the affinity contributed by the D12K substitution. The NMR structure of a homologous a-conotoxin SI [46] was used for docking experiments. We modeled only complexes with an a–c interface of the receptor because the structure of some fragments of the d subunit still remains unsolved and the reliability of the complexes of ligands bound to the a–d interface is lower. According to our calculations, the fold of the a-conotoxin analog remains practically unchanged when serine or lysine are substituted for D12. The averaged rmsd is 0.19–0.22 A ˚ . Increased flexibility in the N-ter- minus in [D12S]SIA and especially in [D12K]SIA, compared with native SIA, was detected. The main dif- ference is seen in the C- and N-termini. In the case of native toxin, the position of the C-terminus is stabil- ized by the ionic pair (salt bridge) between the N-ter- minus and the side chain of the aspartate D12. In the case of [D12S]SIA, the ionic link is changed to the H-bond, which provides slightly more flexibility to the N-terminus. The introduced lysine side chain is orien- ted mainly to the C-terminus, forming H-bond with it, being also directed to the aromatic ring of Y1. How- ever, in general, the conformation of the mutant toxins is very similar to that of the wild-type molecule. Docking and fast molecular dynamics simulations demonstrated a similar position for SIA and its analogs in the binding pocket. We found that all a-conotoxins are kept in the binding pocket mostly by Van der Waals’ interactions and by stacking of their disulfide bridges with aromatic residues of the receptor (Table 3), similarly to what has been demonstrated for Fig. 3. Comparison of blocking activity of three a-conotoxins on Lymnaea neurons with low (A) and high (B) sensitivity to ImI. The insets show the ACh-elicited currents recorded from 4 neurons in control (solid lines) and after 5 min pretreatment with a-conotoxins ImI, [A10L]PnIA or [A10L,D14K]PnIA (dotted lines). Concentrations of a-conotoxins (in lM) are marked left to the corresponding traces. Calibrations are the same for all oscillograms. The plots are inhibi- tion curves for three a-conotoxins. Uninhibited currents were nor- malized by the control response just before treatment with the a-conotoxin. The points are either the mean ± SE from 3 to 9 experiments or the mean from duplicates. The curves were fitted to the Hill equation. The IC 50 and Hill coefficient (n H ) values are 0.25 l M and 1.16 (n ¼ 9 cells) for ImI, 15 lM and 0.62 (n ¼ 6) for [A10L,D14K]PnIA, 30 l M and 0.64 (n ¼ 7) for [A10L]PnIA in cells with low sensitivity to ImI (A); 0.03 l M and 0.81 (n ¼ 7) for ImI, 0.4 l M and 0.49 (n ¼ 2) for [A10L,D14K]PnIA, 20 lM and 0.71 (n ¼ 5) for [A10L]PnIA in cells with high sensitivity to ImI (B). Novel a-conotoxin analogs I. E. Kasheverov et al. 4476 FEBS Journal 273 (2006) 4470–4481 ª 2006 The Authors Journal compilation ª 2006 FEBS a-conotoxins [A10L,D14K]PnIA and ImI bound to AChBP [19–21]. The main difference between SIA and its analogs was found for the mutated residue of the toxin. The D12 side chain plays no role in binding, at least its side chain forms no bonds with the receptor residues. In the case of [D12S]SIA, the abovementioned increased flexibility of the N-terminus permits [D12S]SIA to enter deeper into the pocket and to form closer and stronger contacts (mainly Van der Waals) with the receptor. The [D12K]SIA occupies the position in the nAChR pocket similar to that of [D12S]SIA, but in addition a new ionic interaction is observed: K12 is directly interacting with E57 of the nAChR c-subunit, whereas several amino acid residues (Q59, Y117 and some other) facilitate the formation of this bond (Fig. 4). The reason why both SIA mutants have identi- cal affinities for the a ⁄ c site (Table 2) may be that, according to docking experiments, the [D12S] analog is entering the binding site somewhat deeper and is form- ing stronger Van der Waals’ contacts, which may give a potential energy gain comparable with that of the ionic bond formed by the [D12K] variant. In summary, our results show that introduction of a positive charge to the C-terminus of a-conotoxins gives new analogs of distinct selectivity whose mode of action, with the purpose of future design of novel antagonists, can be rationalized in the light of the available X-ray data. Experimental procedures Materials nAChR-enriched membranes from the electric organ of T. californica used in the radioligand assays [47] were kindly provided by Prof F. Hucho (Free University of Berlin, Germany). All iodinations of conotoxins were per- formed using chloramine T (Serva, Heidelberg, Germany) and Na [ 125 I] (Izotop, Moscow, Russia). Monoiodinated (3-[ 125 I]iodotyrosyl 54 )-a-bungarotoxin (~ 2000 CiÆmmol )1 ) was from Amersham Biosciences (Little Chalfont, UK). a-Cobratoxin was purified from crude venom of Naja kaou- thia as described previously [48]. Synthesis of a-conotoxin analogs All peptides except for PnIA variants were synthesized on Rink-resin using Fmoc-strategy and trityl protection of the cysteine thiol groups. Coupling of amino acids was carried out with the hydroxybenzotriazole–carbodiimide procedure. Deprotection with simultaneous cleavage of the peptides from resin was achieved using a mixture of trifluoro- acetic acid, ethanedithiol, m-cresole, and dimethylsulfide Table 3. Amino acid interactions between nAChR (a–c interface) and bound a-conotoxins SIA, [D12S]SIA and [D12K]SIA. The addi- tional interactions for the analogs are placed in square brackets. Designations by type: normal, direct Van der Waals interactions; underlined, H-bond (toxin residue atom ID-receptor residue atom ID); bold, ionic pair; in parentheses – doubtful or weak. Toxin residue Receptor residue a-subunit c-subunit Tyr1 Trp170(OH-NE1) Cys2–Cys7 Tyr190, Tyr198 Cys3–Cys13 Trp55 His4 Tyr93(ND1-OH) (Tyr190) Pro5 Trp149, Thr150 Arg79(O-NH1 ⁄ 2) Ala6 Thr150, Asp152(O-N) Arg79(O-NH1 ⁄ 2) Lys9 Asp76, (Leu109), Tyr111(NZ-OH) Asn10 Cys192–Cys193 Phe11 Tyr117(O-OH), Leu119 Asp12 Tyr117 [Ser12] [Trp55, Tyr117] [Lys12] [Tyr117, Glu57(NZ-OE1 ⁄ 2)] Cys13 Thr36(SG-OG1) Fig. 4. A model for complexes of a-conotoxin SIA and its [D12K]- analog with the Torpedo nAChR extracellular domain. The extracel- lular domains of a- (left) and c- (right) subunits are in pink and tan. a-Conotoxin SIA and [D12K]SIA molecules are shown by green and blue sticks (-C-S-S-C-bridges in yellow), respectively. Aromatic amino acid residues of the Torpedo nAChR forming its ligand-bind- ing site at the a–c interface are colored with orange. Some resi- dues of the c-subunit close to the second loop of the toxin molecule (Table 3) are numbered. Ionic pair between analog Lys12 and c-subunit Glu57 side chains is in red. The side chain of the Lys9 residue and the N- and C-termini of toxins are marked in green. I. E. Kasheverov et al. Novel a-conotoxin analogs FEBS Journal 273 (2006) 4470–4481 ª 2006 The Authors Journal compilation ª 2006 FEBS 4477 (9 : 0.3 : 0.3 : 0.3 v ⁄ v ⁄ v ⁄ v) for 40 min at 25 °C. The crude linear peptides were dissolved in 50% isopropanol, titrated to pH 9.0 with N-ethyldiisopropylamine, and left at 25 °C [27]. The oxidation process, as monitored by reaction with Ellman’s reagent, was complete in 18 h, and then the pH was decreased to 5.0 with acetic acid. RP-HPLC on a semi- preparative C 18 column was used to purify one predomin- ant peak or in some cases of several isomers; each of them was characterized by CD spectra and tested for ability to bind to the membrane-bound T. californica nAChR (see below). [A10L]PnIA and [A10L,D14K]PnIA were synthesized on a Rink polymer using the O-benzothiazol-1-yl-N,N,N¢,N¢- tetramethyluronium tetrafluoroborate ⁄ N,N-diisopropyleth- ylamine method for activation of Fmoc-amino acids. In the [A10L]PnIA synthesis, all thiols were protected by a Trt group. In the [A10L,D14K]PnIA synthesis, Trt was used for Cys3 and Cys16, and tBu for Cys2 and Cys8. Deblocking of peptides was carried out with trifluoroacetic acid as des- cribed above. Linear peptides were purified by RP-HPLC on a Reprosil-Pur C 18 column (250 · 10 mm) using an acetonit- rile gradient from 10 to 40% in 30 min. Two disulfide brid- ges in [A10L]PnIA were closed simultaneously in 0.1 m NH 4 CO 3 solution [30]. The required product was isolated by HPLC and characterized with the aid of MALDI MS. When synthesizing [A10L,D14K]PnIA, disulfide bridges were formed selectively. First, after removal of the peptide from the polymer, oxidation on air at pH 8.5 in the isopropa- nol ⁄ water mixture was used to form a disulfide between Cys3 and Cys16. Then, using a silyl chloride ⁄ sulfoxide method [49], tBu protection was removed from Cys2 and Cys8 with simultaneous formation of the respective disulfide. MALDI-TOF analysis was carried out on a Reflex III mass spectrometer (Bruker, Bremen, Germany) using 2,5- dihydroxybenzoic acid as a matrix. CD spectroscopy CD spectra were recorded on a JASCO J-810 spectropola- rimeter (JASCO International Co., Tokyo, Japan). The results were expressed as molar ellipticity, [Q] (degÆcm 2 Æ dmol )1 ), determined as [Q] ¼ Q·100 · MRW ⁄ (c · L), where Q is the measured ellipticity in degrees at a wave- length k, MRW is the mean amino acid residue weight cal- culated for each a-conotoxin as the division of peptide molecular mass by the number of amino acid residues, c is the peptide concentration in mgÆmL )1 , and L is the light path length in cm. The instrument was calibrated with (+))10-camphorsulfonic acid, assuming [Q] 291 ¼ 7820 degÆcm 2 Ædmol )1 [50]. Radioligand assays 125 I-Iodination of a-conotoxins GI and MI was carried out by the chloramine T method as described previously [41]. For competition binding assays, suspensions of nAChR- rich membranes (230 nm a-bungarotoxin binding sites pre- pared in 50 mm Tris ⁄ HCl buffer, pH 8.0, containing 1mgÆmL )1 of BSA) were incubated for 1 h with various amounts of the a-conotoxin analogs, followed by an addi- tional 35 min incubation with 280 nm 125 I-labeled a-cono- toxin GI or 125 I-labeled a-conotoxin MI. Nonspecific binding was determined by preincubation of the membranes with a 200-fold excess of a-cobratoxin. The membrane sus- pensions were applied to glass GF ⁄ F filters (Whatman, Maidstone, UK) presoaked in 0.25% polyethylenimine, and the unbound radioactivity was removed from the filter by washes (3 · 3 mL) with 50 mm Tris ⁄ HCl buffer, pH 8.0. The inhibition curves obtained are presented in Fig. 2, the IC 50 values given in Table 2. Data analyses were performed using origin 6.1 (Micro- Cal Software Inc, Northampton, MA). The competition curves of 125 I-labeled GI ⁄ MI binding inhibition with a-conotoxin analogs were fit both to one-site or two-site models. Electrophysiology Experiments were carried out on identified giant neurons (LP1–3, RP2,3) isolated from L. stagnalis right or left pari- etal ganglia after mild enzymatic digestion (protease from Streptomyces griseus, Sigma, St Louis, MO, 2 mgÆmL )1 , 50 min at room temperature). Neurons were internally per- fused and voltage-clamped at )60 mV. The composition of the internal and external solutions, techniques of ACh application and cell incubation with the toxins were as des- cribed previously [23]. ACh-induced currents were digitized and sampled online on a Pentium PC via a home-made operational amplifier supplying a virtual ground and a Digidata1200 B interface (Axon Instruments Inc., Foster City, CA). Acquisition and analysis of the data were made using pclamp6 (Axon Instruments Inc.). IC 50 values were determined as the toxin concentration required to reduce by half the current fraction sensitive to this toxin. Model building The model of the extracellular domains of the T. californica nAChR subunits was constructed using modeller 7v7 (http://www.salilab.org/modeller) with the sequence align- ment from LGIC database (http://www.ebi.ac.uk/compneur- srv/LGICdb/LGICdb.php) on the basis of the crystal structures of L. stagnalis AChBP complexes with nicotine (1UW6) and carbamylcholine (1UV6), the X-ray crystal structure of the complex of A. californica AChBP with a-conotoxin [A10L,D14K]PnIA [19] and the T. marmorata nAChR cryo-electron microscopy structure (2BG9), as will be published in more detail elsewhere. The models of the SIA, [D12S]SIA and [D12K]SIA were built using the X-ray crystal structure of a-conotoxin SI Novel a-conotoxin analogs I. E. Kasheverov et al. 4478 FEBS Journal 273 (2006) 4470–4481 ª 2006 The Authors Journal compilation ª 2006 FEBS (1HJE). All crystal structures were from the Protein Data Bank (http://www.rcsb.org/pdb). Point mutations were introduced in the molecule with spdbviewer 3.7 sp5 (http:// swissmodel.expasy.org/spdbv/) mutation instrument. The structure verification was carried out with what_check (http://swift.cmbi.kun.nl/swift/whatcheck/). Then the struc- tures were relaxed (300 steps of steepest descent with cutoff 10 A ˚ ) with tinker (http://dasher.wustl.edu/tinker/) using AMBER¢99 force field [51] during minimization and molecular dynamics simulations. Rather short (100 pico- second) trajectories were calculated at the temperature 300 K and dielectric permittivity e ¼ 1. Time step of integ- ration procedures were taken as small as 1 femtosecond. Radius of truncation for Coulomb interactions was 20 A ˚ . No periodic boundaries were applied. Lennard–Jones inter- actions were calculated only up to 16 A ˚ (at that, from 15 to 16 A ˚ a polynomial switch function was applied). Berend- sen thermostat was applied [52]. Docking simulations and selection of solutions Docking simulations were performed under hex 4.2b (http://www.csd.abdn.ac.uk/hex/). Thus flexible ligand was docked to the rigid receptor. Visual analysis in the spdb viewer followed to reject false-positive solutions. The posi- tion of the toxin in the binding pocket proposed by the program was considered valid if there was a contact of toxin Lys9 residue with cTyr111 found by the pair-wise mutagenesis studies [40]. Molecular dynamics procedures were run over the solutions after this selection using the same parameters as was described in the previous section. Acknowledgements This research was supported by the Russian Founda- tion for Basic Research (06-04-49198; 05-04-48932), partially by the Civilian Research and Development Foundation grant RB1-2028, and by a grant of RFBR- NWO (047.015.016) to ABS and VIT, grant MCB RAN to VIT. We also express our thanks to Prof N. 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