Actions of sea anemone type 1 neurotoxins on voltage-gated sodium channel isoforms

Toxicon 54 (2009) 1102–1111

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Toxicon journal homepage: www.elsevier.com/locate/toxicon

Actions of sea anemone type 1 neurotoxins on voltage-gated sodium channel isoforms Enzo Wanke a, *, Andre´ Junqueira Zaharenko b, Elisa Redaelli a, Emanuele Schiavon a a b

` di Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy Dipartimento di Biotecnologie e Bioscienze, Universita ˜o Paulo, Rua do Mata ˜o, Travessa 14, n 101, 05508-900 Sa ˜o Paulo, Brazil Departamento de Fisiologia do Instituto de Biocieˆncias, Universidade de Sa

a r t i c l e i n f o

a b s t r a c t

Article history: Available online 23 April 2009

As voltage-gated Naþ channels are responsible for the conduction of electrical impulses in most excitable tissues in the majority of animals (except nematodes), they have become important targets for the toxins of venomous animals, from sea anemones to molluscs, scorpions, spiders and even fishes. During their evolution, different animals have developed a set of cysteine-rich peptides capable of binding different extracellular sites of this channel protein. A fundamental question concerning the mechanism of action of these toxins is whether they act at a common receptor site in Naþ channels when exerting their different pharmacological effects, or at distinct receptor sites in different Nav channels subtypes whose particular properties lead to these pharmacological differences. The a-subunits of voltage-gated Naþ channels (Nav1.x) have been divided into at least nine subtypes on the basis of amino acid sequences. Sea anemones have been extensively studied from the toxinological point of view for more than 40 years. There are about 40 sea anemone type 1 peptides known to be active on Nav1.x channels and all are 46–49 amino acid residues long, with three disulfide bonds and their molecular weights range between 3000 and 5000 Da. About 12 years ago a general model of Nav1.2-toxin interaction, developed for the a-scorpion toxins, was shown to fit also to action of sea anemone toxin such as ATX-II. According to this model these peptides are specifically acting on the type 3 site known to be between segments 3 and 4 in domain IV of the Naþ channel protein. This region is indeed responsible for the normal Naþ currents fast inactivation that is potently slowed by these toxins. This fundamental ‘‘gain-of-function’’ mechanism is responsible for the strong increase in the action potential duration. They constitute a class of tools by means of which physiologists and pharmacologists can study the structure/function relationships of channel proteins. As most of the structural and electrophysiological studies were performed on type 1 sea anemone sodium channel toxins, we will present a comprehensive and updated review on the current understanding of the physiological actions of these Na channel modifiers. Ó 2009 Published by Elsevier Ltd.

Keywords: Sea anemone Neurotoxins VGSC Ion channels Inactivation

1. Introduction Sodium channels consist of the principal a-subunit (w260 kDa), without or with a noncovalently attached b1or b3-subunit, and a disulfide-linked b2- or b4-subunit * Corresponding author. Tel.: þ39 026448 3303; fax: þ39 026448 3565. E-mail address: [email protected] (E. Wanke). 0041-0101/$ – see front matter Ó 2009 Published by Elsevier Ltd. doi:10.1016/j.toxicon.2009.04.018

(Isom, 2002; Catterall et al., 2005). The a-subunit is composed of four homologous domains (DI-DIV), each containing six transmembrane segments (S1–S6), forms the ion-pore and let to define the five distinct toxin binding sites, including site 1 for tetrodotoxin (TTX), saxitoxin and m-conotoxins, site 2 for veratridine (and batrachotoxin, aconitine and grayanotoxin), site 3 for a-scorpion toxin

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(and sea anemone toxins), site 4 for b-scorpion toxin and site 5 for d-Conotoxins (Ceste`le and Catterall, 2000; Oliveira et al., 2004; Catterall et al., 2002, 2005; Schiavon et al., 2006). The nine a-subunit isoforms (Nav1.1–Nav1.9) arise from nine different genes (SCN1A–SCN5A and SCN8A– SCN11A) (Catterall et al., 2005). In this context, it should be noted that b-subunits normally accelerate gating kinetics in the oocyte expression system (Ji et al., 1994; Wallace et al., 1998). On the contrary, more recent data obtained in mammalian expression systems, suggest other forms of modulating the biophysical properties of these channels, for instance, by altering the voltage-dependence of the steady-state inactivation (Meadows et al., 2002; Mantegazza et al., 2005a,b). Sea anemone toxins are peptides that share their binding sites with a-scorpion toxins and their actions involve almost completely and selectively a particular ion channel protein conformational change called inactivation (transition from the open to the shut state induced by an inner face particle-block) as opposed to the early process of activation (opening of the Naþ-selective pore). This inactivated state, is distinct from the closed state and there are many different methods to manipulate it from the intracellular side, either by using enzymes (originally discovered by Armstrong et al., 1973) or drugs and point mutations (for a review see Ulbricht, 2005). A crucial characteristic of these sea anemone toxins actions is that they are able to produce their effects by acting from the extracellular side of the plasma membrane. The conformational change of voltage-gated ion channel proteins called ‘‘inactivation’’ is a very general property common to all the VGSC families, to some Ca2þ channels (so called low-voltage-activated (LVA) channels, more recently known as Cav3.x channels) and Kþ (so called A-currents, also known as Kv1.4, Kv3.3–4, Kv4.x) ion channels. The inactivation process has time constants that are always much longer as compared to those observed in the activation–deactivation transition, but the Kv channels of the EAG superfamily are exceptions (Bauer & Schwarz, 2001). These comprise ERG (also known more recently as Kv11.x, Wanke & Restano-Cassulini, 2007) and ELK (also known more recently as Kv12.x, Becchetti et al., 2002) channels.

2. Phenomena The typical voltage-gated sodium channel currents elicited on depolarization can be shut at their peak by a sudden repolarization, showing a very fast exponential decrease called deactivation which represents the closure of the channels. On the contrary, on sustained depolarization, the same current can be let to decrease physiologically much more slowly. The latter process is termed ‘‘inactivation’’ and leaves the channel refractory for some time after repolarization. It is the main functional representation of a typical physiological property, the ‘‘refractoriness’’ present in all firing neurons. Since the effect of the majority of purified sea anemone peptides acting on VGSC are that of modifying the ‘‘inactivation’’ process, we will here focus mainly on the biophysical characterization of this process.

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In the classical study of the squid giant axon, Hodgkin and Huxley (HH, Hodgkin and Huxley, 1952) described the sodium conductance (channel gating) being proportional to m3[t, V] $ h[t, V], where m (activation, represents the transition between closed and open states) and h (inactivation, represents the transition from open to the inactivated states) are variables, describing kinetics changes, that are function of time, t, and membrane potential V. h can assume values between 1 and 0 when V ranges between physiologically relevant membrane potentials from 90 to þ60 mV. From the phenomenological point of view, it is easy to describe the h stationary curve (on a time scale of less of a second, to be distinguished from the ‘‘slow inactivation’’, see below), which is commonly called ‘‘steadystate inactivation’’ (also hN[V]) and functionally represents the fractional ‘‘availability’’ of channels to be opened on depolarization at each V value. Since it was discovered that the inactivation process is characterized also by slow time courses, the term ‘‘slow’’ refers always to changes during times larger than 500 ms up to minutes and in which the number of functional channels can change (Brismar, 1977; Almers et al., 1983). The actions here reviewed refer only to the fast inactivation that represents protein conformational changes of the supposedly constant number of channels present in the cell. The HH description defines that h[t, V] changes, between different membrane potentials, with exponential time courses that depend on voltage-dependent time constants called sh[V]. This curve has a bell-shaped form with its maximum around the V½ value of hN[V]. In a simplified form, the left- and right-sided parts of sh[V], respectively, represent the process of recovery (to the closed states) from the inactivated states and the process of development of the inactivated states of the channels (from either open or closed states). It should be stressed here that a critical feature of this model is the recovery from inactivation that does not seem to pass through the open state as no ionic current is usually seen during this period. An exception is observed in Purkinje cerebellar neurons that produce a ‘‘resurgent’’ current on direct repolarization from fully inactivated states (Raman and Bean, 1997). This current was assumed to reflect unblocking of an open channel block in a portion of channels (the isoform Nav1.6) by a hypothetical particle during strong depolarization, constituting an additional mechanism of inactivation. It thus reduces recovery from inactivation, which enables these neurons to fire at high frequency. Interestingly, the resurgent current can be produced in the Nav1.6 isoform by a b-scorpion peptide acting extracellularly (Cn2, Schiavon et al., 2006). In general, the inactivation time constants sh[V] are small at hyperpolarized (w1/5 ms at 90/70 mV) and at depolarized (w0.1/1 at þ30/þ40 mV) membrane potentials, but they are strictly isoform-dependent because they are related not only to the specific role of each isoform in the excitable tissue but also on specific channelopathies (see Herzog et al., 2003; Cummins et al., 2004). Most models of ion channel gating (Cha et al., 1999) describe the depolarization-induced inactivation process as an interaction involving both outward movements of S4 charged segments (opening of the channel) and the

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cytoplasmic-positioned inactivation particle (also called the IFM motif from the initials of the involved amino acids) which links DIII-S6 and DIV-S1 segments from the intracellular side. Specifically, the binding of the inactivation particle immobilizes S4 segments of domains III and IV (but not DI-S4 and DII-S4) in the outward position (Yang and Kuo, 2003). Repolarization-induced recovery from inactivation starts with the detachment of the inactivation particle from the intracellular side of the S4 segment which regain its final resting position. Since this detachment is a slow process and repositioning of DI-S4 and DII-S4 is a fast process (deactivation), no ion current (except for the singular ‘‘resurgent current’’ present in Nav1.6 channels, Raman and Bean, 1997; Schiavon et al., 2006) is seen immediately after repolarization because the channels are virtually closed. Indeed this observation fits the conventional mechanism of sodium channel inactivation, from which recovery is thought to proceed through closed states (Kuo and Bean, 1994). As already mentioned, the mechanism of action produced by sea anemone toxins is very similar to those produced by a-scorpion toxins. Indeed, it has been suggested that probably there exists a group of amino acids where these very different types of peptides can bind, namely site 3 (Kem, 1988; Norton, 1991; Rogers et al., 1996; Possani et al., 1999). It consists mainly in the appearance of a component of the inactivating current characterized by a much slower time constant as compared to the normal fast inactivation (El Sherif et al., 1992). This is illustrated in Fig. 1A–C, where the effects of different concentrations of the sea anemone toxin ATX-II are shown together with the control currents. Data can be fitted with two exponential terms, one identical to the control currents and the other much slower (Fig. 1D). It can be seen that the amplitudes of these components show two complementary doseresponse curves, suggesting that the process can be interpreted with the presence of two types of ion channel proteins, those unaffected and those bound by the toxin. Sometimes, in the toxin action also a constant term can be

observed, suggesting that a third component is present showing currents which apparently do not inactivate (see below Fig. 2).

3. Sea anemone toxin type 1 VGSC isoformdependence The studies that try to link the amino acid sequence similarities observed in various VGSC isoforms with the different effects exerted by the different peptides can be very useful for understanding the site 3 binding mechanisms. Other studies use site-directed mutagenesis either on the ion channel protein or on the toxin peptide or both to specifically investigate the origin of the structure–function relationships between biophysical properties and 3D peptide structure. These studies started many years ago when only few VGSC isoforms were known and it was completely unknown how the VGSC a- and b-subunits interactions were linked to their biophysical properties. Several pioneering groups working before 1994–1995, instead of using the today’s standard mammalian transfection system (i.e. HEK-expressing system), made use either of the oocyteexpressing system or of neuroblastoma cell lines in which not only different species (rat and mouse) were used to establish the line, but, more important, the lines were used independently from knowing the differentiated state of the cells. Indeed, it is well known that these immortalized cells can be easily induced to change their set of genes by simply changing the culture medium. Thus, many results obtained under these different conditions, not only make comparisons with more recent data difficult, but they are also hard to interpret because they were obtained under conditions in which the recorded current was not originating from only one isoform, and it is no longer possible to establish the possible contaminations from other channel isoforms. Moreover, currents originating from species-different VGSCs were compared and at present it is not clearly

Fig. 1. Effects of the sea anemone toxin ATX-II on Nav1.5 channels. A–C) Superimposed recordings of inward currents in control (A) and during the application of 10 nM (B) and 200 nM ATX-II (C) to a single cell (voltage command protocol shown in the inset to Fig. 1A, one single cell). D) The fitting procedure for the first panel B episode (from 130 mV to 20 mV), showing the two exponentially decaying components (triangles for the slow component and circles for the fast component) and the control trace (continuous line). E) The relative increase and decrease in these components are plotted for different concentrations (same symbols as in D, n ¼ 4), showing that the contribution of slow and fast amplitudes is related to the bound and unbound channels in relation to the toxin concentration. Reproduced, with permission, from Oliveira et al., 2004.

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Fig. 2. ATX-II, AFT-II and Bc-III toxins dose-response relationships of the increase in the amplitude of the slow component in the different channel types. Graphs show the plots of the fractional increases in the slow component observed in the different isoforms. The different toxins are shown with the indicated symbols. Reproduced, with permission, from Oliveira et al., 2004.

known if there are biophysically relevant differences at this level. This is the reason why the best present procedure is to use a cell line where the amount of VGSC currents should be less than 1% of the currents recorded under transfected conditions. Second, the data should be always corrected for the unwanted currents present in the recordings by using high concentrations of TTX. Third, and not less important, the experiments should be done under controlled cytoplasmic conditions that guarantee: i) the complete exchange of the pipette solution because voltage-dependent changes are always present during the first 10–15 min after the whole-cell recording condition is achieved, ii) the minimal interference of the b-subunits in determining the amount of VGSC inactivation caused by the interaction of G-protein betagamma subunits with various VGSC isoforms (Mantegazza et al., 2005b), and iii) to exclude, in very long experiments, that the slow and very-slow inactivation takes place. Early experiments, primarily in the laboratory of Catterall, were carried out with neuronal and cardiac channels (Rogers et al., 1996). The results of these site-directed mutagenesis studies suggested that the sea anemone toxin ATX-II (and the a-toxins purified from scorpions that induce similar actions) interacts with a glutamic acid residue (Glu-1613 in Nav1.2) on the extracellular S3–S4 loop of the domain IV of the rat neuronal channel Nav1.2 (see Table 1). These data also indicated that non identical

amino acids of the IVS3–S4 loop participate in a-scorpion and sea anemone toxin binding to overlapping sites and that neighboring amino acid residues in the IVS3 segment contribute to the difference in a-scorpion toxin binding affinity between cardiac and neuronal VGSCs. Up to now, few studies have determined the effects exerted by the same toxin on various VGSC isoforms (Oliveira et al., 2004; Schiavon et al., 2006). An analysis of the EC50s for the increase in the slow component caused by the action of three sea anemone toxins indicates that for one peptide (ATX-II) the observed EC50s range can be of the order of 100 or more among six isoforms. Moreover, the efficacy of the response can vary from 0.4 to 2. Interestingly, the same isoform can show not only one order of magnitude difference in EC50 but also a five-fold change in efficacy. These data are shown in Fig. 2 for the Nav1.1 to Nav1.6 isoforms and in Fig. 3 for the Nav1.7 isoform. In general some sea anemone toxins produce the slow component increase at low concentration, but are also able to add a persistent component if used at high concentrations. This effect is isoform-dependent, for example ATX-II never produces large persistent components in Nav1.4 and Nav1.5 (in which it produces considerable slow component). On the contrary, AFT-II is able to produce a huge persistent component only in Nav1.3 and Nav1.6 isoforms (Oliveira et al., 2004). However, when tested on insect VGSCs transfected in Xenopus laevis oocytes, ATX-II (Av2) simply causes the channel not to inactivate (Moran

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Table 1 Position and amino acid sequences of Nav1.1–1.7 channels in the segment 3 and 4 extracellular loop of the IV domain. Identical amino acids have a white background; homologous amino acids have a gray background; and different amino acids have a black background.

Fig. 3. ATX-II, AFT-II and Bc-III toxins effects on Nav1.7 channels. A–C) Superimposed traces elicited from 110 to 10 mV under control conditions and at the indicated concentrations (different symbols, a single cell for each toxin). D) Concentration response curves of the slow component increase for the three toxins. Continuous lines best fit the data (n ¼ 4) with the following EC50s(mM) and Hill coefficients: ATX-II, 1.8  0.02, 1.6  0.02; AFT-II 5.8  0.25, 1  0.05; Bc-III 5.7  0.3, 1.4  0.02. E–G) Steady-state fast inactivation for the action of the three toxins at the indicated concentrations. Continuous lines are Boltzmann curves which best fit the data points (n ¼ 3) with the following V½ and slopes (mV) in control (parenthesis) and in toxin: ATX-II, (79  1, 9.3  0.8) 77  0.9, 9.6  0.8; AFT-II (75.2  0.3, 8.6  0.5) 74  0.25, 8.7  0.7; Bc-III (74.7  0.7, 8.3  0.6) 72  0.9, 8.4  0.8. The insets show the superimposed traces in control (upper) and in toxin (lower), notice the increase in the peak current and in the slow component. No significant effects were seen at the level of the persistent component.

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Table 2 Sequence alignment of type 1 sea anemone neurotoxins. Vertical and horizontal bars denote the disulphide bonds (1–5, 2–4 and 3–6). Letter O in Am-III, Rc-I, CgNa and CpI peptides denotes the occurrence of hydroxyproline in 3rd position. Black, gray and white backgrounds show identical, homologous and different amino acids, respectively.

et al., 2006). Interestingly, ATX-II and AFT-II have only two amino acids different (see Table 2). On the whole, sea anemone toxins have a large spectrum of responses that can be always interpreted as gain-of-function effects because they affect the action potential duration. Depending on the specific isoform expression in excitatory or inhibitory neurons, these actions always produce large changes in the activity of the excitable tissue. 4. Sea anemone toxin type 1 sequence-dependence It has been 40 years since sea anemone toxins began to be studied in vitro (Shapiro, 1968). In the mid 1970s the first isolated type 1 polypeptides were reported from the Mediterranean species Anemonia sulcata (now referred to as Anemonia viridis), in the classical papers of La´szlo Be´ress’ group in Germany (Alsen et al., 1976; Beress and Beress, 1975). Later, it was observed that both sea anemone peptides and a-scorpion toxins shared the same binding site on sodium channels (Catterall and Beress, 1978; Couraud et al., 1978), drastically prolonging the neuronal action potential by delaying the inactivation process of sodium channels. After these initial discoveries, several other distinct peptides, the so called type 2 neurotoxins, were also isolated from sea anemones (Zykova et al., 1985; Schweitz et al., 1985; Metrione et al., 1987; Kem et al., 1989), especially from species belonging to the family Stichodactylidae. On the contrary, most of the type 1 toxins come from the Actiniidae family, even though some exceptions may occur (Shiomi et al., 2003; Honma et al., 2005). The type 2 toxins possess the same cysteine pairing of type 1 peptides, even though they are distinct in terms of primary sequence. Type 2 toxins were also reported to be active upon Naþ channels, competing by the same binding site of the type 1 peptides and also delaying the inactivation process of the

Naþ currents (Sorokina et al., 1984; Schweitz et al., 1985; Salgado and Kem, 1992). Indeed, only a few detailed studies have been conducted with this class of polypeptides, (Pennington et al., 1990a,b) and certainly they are still a promising group to be investigated and characterized more in detail. For a comprehensive and detailed review of this category of peptides, some recent articles are recommended (Honma and Shiomi, 2005; Bosmans and Tytgat, 2007; Shiomi, 2009). Turning back to the type 1 toxins, the topic of this review, the investigations concerning the crucial amino acids involved in their binding to the sodium channels started in the very beginning of 1980s. The group of prof. Michel Lazdunski in France showed that removal of positive charges from the cationic residues K37 and R14 of the toxin ATX-II, from A. sulcata, completely abolished its toxic action (Barhanin et al., 1981). Also, the same authors demonstrated that modifications of the carboxylate functions of D7, D9 and of COOH-terminal Q47 abolished the toxicity of ATX-II. Nevertheless, more recently the D7A substitution by site-directed mutagenesis was not demonstrated to be so critical for its activity (Moran et al., 2006). More than one decade after the first experiments of Lazdunki’s group, another study confirmed the role of K37 and K48 of anthopleurin-A (ApA, see Table 2), another type 1 peptide selective for cardiac sodium channels, in its cardiotonic action (Gould and Norton, 1995). On the other hand R14, a conservative residue in almost all type 1 toxins (see Table 2), was not essential for the activity of ApA. Consistently, similar positively charged amino acids of anthopleurin-B (ApB), a peptide active over neuronal and cardiac channels, were demonstrated to be important either to the activity or to the affinity of ApB in these channels subtypes (Khera et al., 1995). In the meantime, the 3D structures of both ApA and ApB were determined in solution by NMR (Monks et al., 1995; Pallaghy et al., 1995), confirming the overall arrangement of

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these type 1 toxins as all beta peptides, composed of 4 betastrands interconnected by antiparallel beta-sheets. Until some years ago, ApB was the sea anemone sodium channel toxin most extensively characterized by site-directed mutagenesis, and other amino acid residues were proved to be essential for its activity. As an example, D7 was supposed to be essential for the proper folding of the molecule during recombinant production, and truncation of the side chain in D9A substitution results in a further decrease in activity, especially in the cardiac channel (Khera and Blumenthal, 1996), confirming previous data with the ATX-II toxin (Barhanin et al., 1981). On the other hand, Moran et al. (2006) did a complete alanine scan in all amino acids of ATX-II (called as Av2 in that work), turning ATX-II as the only representative of type 1 sodium channel toxins fully studied by this approach. Also, the D7A mutant folded properly in this recent work, in contradiction to the results obtained by Khera and Blumenthal, 1996. It is interesting to note in Table 2 that almost all the peptides possess an aspartic acid at 7th and 9th positions, as ApB and ATX-II, reflecting their possible fundamental role in interaction with sodium channels. However, AeI (from Actinia equina) and AETX-I (from Anemonia erythraea) do not present negative charges in the corresponding positions (Lin et al., 1996; Shiomi et al., 1997), especially at 7th position. In addition, both toxins were not electrophysiologically assayed over VGSC, but only by in vivo injections in crabs, and in that case their specific and detailed potencies were not assessed yet. Moreover, some hydrophobic positions were determined as being crucial for activity of ApB, suggesting that a hydrophobic contact cluster is involved in toxin-induced stabilization of the open conformation of the cardiac sodium channel, as observed by W33F and W45A/S/F mutants (Dias-Kadambi et al., 1996a). Other hydrophobic position shown to be essential in the ApB activity is the L18. According to another site-directed mutagenesis screening performed by the same researchers, L18A displayed a marked loss in affinity (34-fold and 328-fold) for the neuronal and cardiac isoforms, suggesting an increased preference of the L18A mutant for the closed state of the neuronal channel (Dias-Kadambi et al., 1996b). The complete mutagenesis scan of ATX-II (Av2) corroborates the role of these amino acids (Moran et al., 2006). Interestingly, all these hydrophobic amino acids are extremely conserved amongst all the type 1 sodium channel toxins, as shown in Table 2. Nevertheless, the peptides Am-III (Antheopsis maculata), Rc-I (Heteractis crispa), CgNa (Condylactis gigantea), CpI (Condylactis passiflora) and Gigantoxin-II (Stichodactyla gigantea) do not present the tryptophan at 45th position, but a glutamic acid and a threonine instead. As for AeI and AETX-I mentioned in the above paragraph, most of them were not characterized in detail by electrophysiology experiments, so their potencies still remain to be determined. An exception is CgNa, described and characterized by patch-clamp recording in TTX-sensitive currents expressed in primary cultures of DRG neurons (Standker et al., 2006). Recently CgNa was electrophysiologically characterized in more detail and its 3D solution structure was determined by NMR (Salceda et al., 2007). Indeed, this is an interesting

example of a toxin which is poorly active on sodium channels probably because of the presence of a negatively charged patch in the 35th position, revealed by the occurrence of an aspartic acid and a glutamic acid at 36th position. According to Salceda et al. (2007), the occurrence of a strong negative region in CgNa disrupts a surface-exposed cluster of hydrophobic residues present in all the toxins, including the tryptophans discussed above. As verified in the vast majority of these toxins, this region is composed of critical positively charged residues, such as ‘‘35 KKH 37’’ in ATX-II and the corresponding ‘‘KAH’’ in ApB (see Table 2). As discussed in the above paragraphs, positively charged amino acids in this area are important for high-affinity binding of type 1 toxins to sodium channels. As a good example, it was shown that among Bc-III, AFT-II and ATX-II, the latter is the most potent overall Nav1.1–1.6 clones when assayed in patch-clamp experiments (Oliveira et al., 2004), having also the most dense positively charged surface in these positions. This data corroborates previous experiments which determined possible interactions of ATX-II with E1613 of the S3–S4 extracellular segment of domain IV in the a-subunit of sodium channels (Rogers et al., 1996). In addition, ATX-II and AFT-II differ by only two amino acids positions: one extra glycine at the N-terminal and K36A in AFT-II. This substitution at the ATX-II ‘‘KKH’’ corresponding region makes AFT-II less potent in all the Nav1.1–1.6 clones but, interestingly, much more specific at the Nav1.3 channel, inducing a dramatic increase in the persistent currents (Oliveira et al., 2004). On the whole, there is strong evidence that positive charges are important for highaffinity binding of the toxins over their targets, but the CgNa example clearly shows that even negatively charged amino acid residues in this region not necessarily impede the binding of the toxin. In that case, a definite contribution by full characterization of CgNa, Am-III, Rc-I, CpI and Gigantoxin-II over different clones of sodium channels should be performed in order to shed some light into these toxin–channel interactions. Another very important and critical conservative amino acid relies on the asparagine at the 16th position. As shown in Table 2, all the peptides display N except BgIII, from the Caribbean species Bunodosoma granulifera. BgIII differs from BgII by only this N16D substitution, which is enough to decrease its potency by near 100 times less compared to BgII (Goudet et al., 2001; Loret et al., 1994; Salceda et al., 2002). On the other hand, when both BgII and BgIII were characterized over the Drosophila paraTip/E and human Nav1.2, 1.4 and 1.5 channels, expressed in X. laevis oocytes and assayed by two-electrode voltage-clamp, their potencies reached around 100–200 times higher for BgII and around 4–12 times higher for BgIII over the insect channel. Furthermore, in this assay some of the currents simply do not deactivate, and the peptides keep the channels locked in the open state. This clearly shows a preference for a target of a phylogenetically closer group of their prey (crustaceans) (Bosmans et al., 2002) and suggests that sea anemone type 1 toxins might be used as lead compounds for the development of insecticides in transgenic plants (Bosmans and Tytgat, 2007). Obviously, more sea anemone peptides should be assayed over insect channels, to compare their potencies with BgII and BgIII and even serve

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Aslow / (Aslow+ Afast)

1.0

0.8 0.6 0.4 0.2

0.0 xin-II, cangito 1.9 µM xin-II, cangito 50 nM

Na

1.1

Na

1.2

Na

1.3

Na

1.4

Na

1.5

Na

1.6

Na

1.7

Fig. 4. Specificity of cangitoxin-II at different concentrations in the TTXsensitive VGSC isoforms. 3D plots of the fractional increase in the slow component amplitude for the seven known TTX-sensitive VGSC isoforms (n ¼ 4 for each isoform). Data are shown at two toxin concentrations.

as a richest source of potential insecticides. An example that reinforces this proposition is the result shown for the Nv1 peptide, expressed by the sea anemone Nematostella vectensis (Moran et al., 2008). The toxin is inactive in mammalian Nav channels at a concentration up to 25 mM, but keeps the insect VGSC locked in open state (channels do not inactivate) at 1 mM. An interesting finding is the recent publication of a paper by our group where two isoforms of the cangitoxin peptide (Cunha et al., 2005), named as cangitoxin-II and III (CGTX-II and CGTX-III), were isolated from the sea anemone Bunodosoma cangicum (Zaharenko et al., 2008). CGTX-II has the single N16D substitution in relation to cangitoxin (similarly to BgII and BgIII), while CGTX-III has the N16D and R14H substitutions. Interestingly, both peptides are as active as Bc-III and AFT-II over Nav1.1. In addition, the data compilated in Fig. 4 show that CGTX-II is as potent as other type 1 sea anemone toxins in Nav1.1–1.7, suggesting that for this molecule the N16D substitution is not as critical as in BgII and BgIII. In this way, the actual importance of the N16D substitution for this class of molecules remains to be determined. It suggests that in some cases a single mutation may affect the potency of a toxin, but the same position may not be crucial for other peptides from the same group. On the whole, the amount of accumulated knowledge on the type 1 sea anemone toxins field suggests that most of the peptides should be characterized by electrophysiology as much as possible. As some site-directed mutagenesis experiments were performed in a very few toxins, generalized extrapolations to the contributions of each amino acid should be carefully considered. In addition, the occurrence of differentially charged amino acids in crucial regions, such as in the comparison of CgNa with ATX-II, may not reflect a general rule, but novel perspectives on the structure–activity relationships. The recent cited paper of Zaharenko et al. (2008) also contributes to the importance of N16D substitution in these sodium channel toxins, as

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mentioned above and shown in Fig. 4. Moreover, in the recent years some groups attempted to compare the dipole moment orientation of several structurally similar toxins which target different channels, such as some scorpion peptides which target potassium channels (Jouirou et al., 2004) and the sea anemone toxins APETx1 and APETx2, which target ‘‘Human Ether-a-gogo Related Gene’’ (hERG) potassium channels and ‘‘acid-sensing ion channels’’ (ASIC3) (Chagot et al., 2005). Interestingly, what these authors proposed is that very subtle variations in primary sequences may completely alter the dipole orientation of the peptide. As a consequence, this may be a determinant to orientate a peptide towards a docking to its specific channel target. In case of type 1 sea anemone peptides, it would be reasonable that different distribution of positively and negatively charged residues would drastically change the dipole orientation of the toxins, and as a consequence they might dock through different residues and at distinct spatial orientations over their different specific sodium channel subtypes. We suggest that future molecular modeling analyses and structure determinations may elucidate a role for the dipole moment in the type 1 sea anemone toxins, shedding light on how the observed differences in primary sequence contribute to distinct specificities and potencies profiles. Acknowledgments This study was supported by grants from Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica (MURST-COFIN 2005–2007; FISR-Neurobiotecnologie: Fisiopatologia del sistema nervoso; FIRB-2001) to E.W. E.R and E.S. are post-doc students in Physiology at MilanoBicocca University, Department of Biotechnology and Biosciences. A.J.Z. is a post-doc at Dept. of Physiology, Institute of Biosciences at University of Sa˜o Paulo and financial support from FAPESP is acknowledged. Conflict of interest The authors declare having no conflict of interest. References Almers, W., Stanfield, P.R., Stuehmer, W., 1983. Slow changes in currents through sodium channels in frog muscle membrane. J. Physiol. 339, 253–271. Alsen, C., Beress, L., Fischer, K., Proppe, D., Reinberg, T., Sattler, R.W., 1976. The action of a toxin from the sea anemone Anemonia sulcata upon mammalian heart muscles. Naunyn Schmiedebergs Arch. Pharmacol. 295, 55–62. Armstrong, C.M., Bezanilla, F., Rojas, E., 1973. Destruction of sodium conductance inactivation in squid axons perfused with pronase. J. Gen. Physiol. 62, 375–391. Barhanin, J., Hugues, M., Schweitz, H., Vincent, J.P., Lazdunski, M., 1981. Structure–function relationships of sea anemone toxin II from Anemonia sulcata. J. Biol. Chem. 256, 5764–5769. Bauer, C.K., Schwarz, J.R., 2001. Physiology of EAG Kþ channels. J. Membr. Biol. 182, 1–15. Beress, L., Beress, R., 1975. Purification of three polypeptides with neuroand cardiotoxic activity from the sea anemone Anemonia sulcata. Toxicon 13, 359–367. Becchetti, A., De Fusco, M., Crociani, O., Cherubini, A., Restano-Cassulini, R., Lecchi, M., Masi, A., Arcangeli, A., Casari, G., Wanke, E., 2002. The functional properties of the human ether-a`-go-go-like (HELK2) Kþ channel. Eur. J. Neurosci. 16, 415–428.

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