©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Kalicludines and Kaliseptine
TWO DIFFERENT CLASSES OF SEA ANEMONE TOXINS FOR VOLTAGE-SENSITIVE K CHANNELS (*)

(Received for publication, May 18, 1995; and in revised form, August 3, 1995)

Hugues Schweitz Thomas Bruhn (1) Eric Guillemare Danielle Moinier Jean-Marc Lancelin (2) László Béress (1) Michel Lazdunski (§)

From the  (1)Institut de Pharmacologie Moléculaire et Cellulaire, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France, the Klinikum der Christian-Albrechts-Universität, Abteilung Toxikologie, Brunswiker Strabetae 10, 2300 Kiel, Federal Republic of Germany, and the (2)Institut de Biologie Structurale Jean-Pierre Ebel CEA-CNRS, 41 avenue des Martyrs, 38027 Grenoble, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

New peptides have been isolated from the sea anemone Anemonia sulcata which inhibit competitively the binding of I-dendrotoxin I (a classical ligand for K channel) to rat brain membranes and behave as blockers of voltage-sensitive K channels. Sea anemone kalicludines are 58-59-amino acid peptides cross-linked with three disulfide bridges. They are structurally homologous both to dendrotoxins which are snake venom toxins and to the basic pancreatic trypsin inhibitor (Kunitz inhibitor) and have the unique property of expressing both the function of dendrotoxins in blocking voltage-sensitive K channels and the function of the Kunitz inhibitor in inhibiting trypsin. Kaliseptine is another structural class of peptide comprising 36 amino acids with no sequence homology with kalicludines or with dendrotoxins. In spite of this structural difference, it binds to the same receptor site as dendrotoxin and kalicludines and is as efficient as a K channel inhibitor as the most potent kalicludine.


INTRODUCTION

Potassium channels have an essential role in repolarization phases of action potentials and in the fine regulation of the resting potential. Molecular cloning has recently led to the identification of a large number (over 15) of genes for voltage-sensitive, non inward-rectifier, K (Kv) channels (1, 2) which, when expressed in Xenopus oocytes, generate a variety of K channel activities with different kinetics, voltage dependences, conductances, and regulation properties. Surprisingly, only a relatively small number of toxins active on these channels has yet been discovered(3, 4) . They are MCD peptide from bee venom(5, 6) , charybdotoxin and analogs from different scorpion species (7, 8, 9, 10, 11, 12, 13, 14) , beta-bungarotoxin(15, 16) , and dendrotoxins from mamba venoms (3, 5, 17, 18, 19, 20, 21, 22) .

These different toxins only block the expression of four of the cloned Kv channels (Kv1.1, Kv1.2, and Kv1.6 for MCD peptide and dendrotoxin, Kv1.1, Kv1.2, Kv1.3, and Kv1.6 for charybdotoxin) (reviewed in (23) ). Binding studies using radioiodinated derivatives of these toxins have been essential for the identification, purification, and determination of the subunit structure (6, 24, 25, 26) of these Kv channels. These toxins have also been important for the first brain localizations of Kv channels (16, 27) and are particularly interesting inducers of long term potentiation(28) .

Sea anemones produce toxins with which they paralyze their prey. They are particularly important as sources of toxins active on voltage-dependent Na channel which have been essential tools for studying the structure, the mechanism, and the diversity of this channel type(29, 30, 31, 32, 33, 34, 35, 36, 37, 38) .

This paper reports the isolation, structure, and properties of a series of new toxins from Anemonia sulcata which behave as blockers of Kv channels.


EXPERIMENTAL PROCEDURES

Materials

Trypsin, the Kunitz trypsin inhibitor (BPTI), (^1)and N-benzoyl-DL-arginine p-nitroanilide (BAPNA) were obtained from Sigma. Sephadex G-25, Sephadex G-50, SP Sephadex C-25 were obtained from Pharmacia, Fractogel TSK HW-50 (F), Fractogel EMD SO(3)-650 (M), and RP18 Lichrocart were from Merck. For HPLC columns, TSK SP 5PW was from Toyosoda. Ultrasphere ODS was from Beckman, Hypersil BDS was from SFCC Shandon, and Alltima was from Alltech. HPLC purifications were performed with a Waters system.

Purification of Anemonia Sulcata Peptides

The first steps of this purification were performed with slight modifications of a method previously described for the isolation of Na channel toxins of A. sulcata(39) . In this procedure 12 g of the crude sea anemone toxin ((39) ; Fig. 2B1) was dissolved in 120 ml of NaCl 1 M and regelfiltered in two parts on a Sephadex G-50 medium column (7 times 140 cm) equilibrated in 1 M NaCl. The crab paralyzing fractions of these gel filtrations were combined, dialyzed in a Visking Dialysis tube (molecular weight cutoff 12,000-14,000) for 5 h, concentrated at reduced pressure, and desalted on a Sephadex G-25 column (7 times 70 cm) equilibrated with 0.3 M acetic acid. After a concentration at reduced pressure and lyophilization, this crude sea anemone toxins fraction (6 g) was separated into its main components by ion-exchange chromatography on a SP-Sephadex C-25 column (2.5 times 140 cm) as described in Fig. 1A. Fractions which were potentially active on K channels (30, 34, and 38) are shaded. These fractions were identified by their capacity to inhibit binding of the I derivative of dendrotoxin I (I-DTX(I)) one of the most potent blocker of Kv channels. The identified fractions contain peptides designated as AsKS, AsKC1, and AsKC2. They were concentrated and desalted on Sephadex G-25 (not shown) in 0.3 M acetic acid and lyophilized. The last purification steps for each peptide were carried out by HPLC as described below.


Figure 2: Sequence homologies of A. sulcata kalicludines with DTX(I) (top) and the basic pancreatic trypsin inhibitor, BPTI (bottom).




Figure 1: A, purification on SP Sephadex C-25 of 6 g of crude sea anemone toxic material obtained from A. sulcata as described under ``Experimental Procedures.'' The column (2.5 times 140 cm) was equilibrated with 0.01 M ammonium acetate buffer, pH 4.5, the crude toxic fraction was loaded and eluted in this buffer, followed by a stepwise gradient as indicated in the figure. The final elution was carried out with 1 M sodium chloride (not shown). Elution was monitored by measuring the absorbance at 278, flow rate, 225 ml/h. Fraction size, 8 ml. Collected fractions 30, 34, and 38 are shaded; B-D, last purification step of AsKS, AsKC1, and AsKC2, respectively, as described under ``Experimental Procedures.'' Collected fractions are shaded. E, last purification step of AsKC3. The peptide is indicated by the arrow. F, elution profile of AsKC3 in the same conditions as in E.



Solvents used for HPLC were a linear gradient between solution A = 1% acetic acid and solution B = 1 M ammonium acetate passed in 50 min at a flow rate of 1 ml/min for the cation exchanger column TSKSP 5PW (7.5 times 75 mm), and different gradients between solution C = 0.5% trilfluoroacetic acid plus 0.85% triethylamine, plus 10 µl/liter beta-mercaptoethanol in water, and solution D = the same components in acetonitrile, for the three different RP18 reverse phase columns used. At each chromatographic step, all the eluted fractions were checked for their ability to inhibit I-DTX(I) binding to its receptor in rat brain P3 membranes. After the last purification step, pure peptides were lyophilized and desalted on RP18 Lichrocart) with mixtures of 0.1% trifluoroacetic acid in water and acetonitrile. The peptides were first absorbed in 2% acetonitrile and then eluted with 50% acetonitrile and lyophilized.

Purification of Anemonia Sulcata Kaliseptine (AsKS)

Fraction 30 from Fig. 1A (100 mg) was further purified by ion-exchange chromatography in equilibrium conditions on a Fractogel SO(3) EMD 650 (1 times 40 cm) column with 0.5 M ammonium acetate at pH 4.5 as eluting buffer. The eluted material appeared in three peaks, the first one was desalted on Sephadex G-25 and lyophilized. A 11.3-mg fraction of it was loaded in an Ultrasphere ODS (10 times 250 mm) 5 µm column and eluted at 3 ml/min with a linear gradient from 15 to 40% D in 60 min. Activity was found in the first important peak eluting at 17% D between 5.0 and 6.0 mn. This peak was lyophilized and loaded on the same column. A linear gradient from 5 to 20% D in 60 min was applied at the same flow rate of 3 ml/min. AsKS eluted at 14% D, between 34 and 36 min as shown in Fig. 1B (recovery, 1.45 mg).

Purification of Anemonia Sulcata Kalicludine 1 (AsKC1)

Fraction 34 from Fig. 1A was further purified by gel filtration on fractogel TSK HW50 in 0.9 M NaCl with 0.1 M ammonium acetate, pH 5.5, as eluting solvent. Three peaks were observed in the eluted material. The first one was desalted on Sephadex G-25 and lyophilized. Then the following step was performed as for AsKS: 12.7 mg of the product being chromatographed on the Ultrasphere ODS column. Activity was found in the main peak eluting at 23% D between 16 and 19 min. This fraction was lyophilized and chromatographed on the TSK SP 5PW column. Activity was found in the main peak eluting at 59% B between 28 and 34 min. After lyophilization this fraction was chromatographed on a Hypersil BDS column (4.6 times 250 mm), C18, 5 µm, but with a concave gradient (curve 8) between 15% and 20% D in 30 min. AsKC1 eluted as shown in Fig. 1C at 20% D, between 30.5 and 35.5 min (recovery, 1.13 mg).

Purification of Anemonia Sulcata Kalicludine 2 (AsKC2)

Fraction 38 from Fig. 1A was first purified, like AsKC1, on Fractogel. Here also the first of the three peaks was kept. The following step was performed as for AsKS with 7.9 mg of the product being chromatographed on the Ultrasphere ODS column. Activity was found in the main peak eluting at 23.5% D between 17 and 20.5 min. This fraction was lyophilized and chromatographed on the TSK SP 5PW column. Activity was found in the main peak eluting at 85% B between 38 and 45 min. After lyophilization, this fraction was chromatographed on the Hypersil BDS column under the same conditions as for the last purification step of AsKC1. AsKC2 eluted as shown in Fig. 1D at 20% D between 31 and 37.5 min. (recovery, 0.78 mg).

Purification of Anemonia Sulcata Kalicludine 3 (AsKC3)

The starting material used in this purification is the last eluted and most basic fraction (chromatography on SP-Sephadex C25 with 40 mM sodium phosphate buffer, pH 6, and a NaCl gradient between 80 and 380 mM) in the previously described isolation of Na channel toxins from A. sulcata(40) . A part of this fraction (9.8 mg) was chromatographed on the TSK SP 5PW column. Activity was found in the main fraction eluting at 57% B, between 27 and 34 min. The corresponding material was lyophilized, redissolved in 2 ml of water, and chromatographed by fractions of 500 µl on an Alltima C18, 5 µm, metal-free (4.6 times 250 mm) column. Elution was performed at a flow rate of 1 ml/min with a linear gradient from 15 to 40% D in 30 min as shown in Fig. 1E. AsKC3 eluted at 28.5% D, between 16 and 16.8 min as shown by the arrow (recovery, 0.18 mg). The elution profile of AsKC3 is given in Fig. 1F.

Primary Structure of the Peptides

Peptide structures ( Fig. 2and Fig. 3) were determined by Edman degradation using an Applied Biosystems model 477A microsequencer. Before sequencing, S-pyridyl ethylation of cysteine residues was performed according to Tarr et al.(41) . The NH(2)-terminal sequence was completed for AsKC1, AsKC2, and AsKC3 by sequence determination of the peptides obtained by endoproteinase Glu-C cleavage. Samples of 0.2 nmol of S-pyridylated peptides were digested in 50 mM ammonium bicarbonate, pH 7.8, at 37 °C for 24 h with 1 pmol of endoproteinase Glu-C (Promega). The reaction mixture was evaporated by speed-vac centrifugation and redissolved in 0.1% trifluoroacetic acid. All peptide hydrolysates were then fractionated onto a C18 reverse phase column (220 times 2.1 mm Brownlee columns) by using an Applied Biosystems model 140 A apparatus. Initial chromatographic conditions were 0.1% trifluoroacetic acid in water with a flow rate of 100 µl/min at room temperature, and elution was performed by increasing the acetonitrile concentration to 70% with 0.1% trifluoroacetic acid and using a linear gradient of 0.5%/min. Definitive confirmation of the structures was obtained by mass spectrometry analysis using a laser desorption technique (Finnigan laser mat). It gave the following values for the molecular weights, very close to values obtained from the sequences, in parentheses: AsKS, 3834(3835); AsKC1, 6685(6690); AsKC2, 6772(6781); and AsKC3, 6732(6738).


Figure 3: Sequence homologies of AsKS with the sea anemone B. granulifera (BgK) toxin.



Binding to Membranes

Iodination of DTX(I) from Dendroaspis polylepis at a specific radioactivity of 2000 Ci/mmol and binding of I-DTX(I) to rat brain P3 membranes were performed as described previously(27) .

Electrophysiological Experiments

Procedures for Xenopus laevis oocyte injection with the cloned channel, cRNA preparation, and electrophysiological method have been previously described(42) . The standard saline solution ND96 containing 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl(2), 2 mM MgCl(2), and 5 mM HEPES adjusted at pH 7.4 was used in all procedures.

Assay of Trypsin Activity

The enzymatic activity of trypsin and its inhibition by the Kunitz-type inhibitor BPTI and by A. sulcata peptides were measured spectrophotometrically. Aliquots of trypsin at the final concentration of about 3 µM were incubated for 3 h at 25 °C in 50 mM Tris-HCl, pH 7.8, plus 5 mM CaCl(2) in the presence of various concentrations of the peptides in a final volume of 45 µl. After incubation, the free remaining trypsin was determined by addition of 5 µl of 5 mM BAPNA in anhydrous ethanol. Kinetics of paranitroaniline release were measured at 405 nm.

Molecular Modeling

Molecular modeling was done using the ensemble of modeling programs from Biosym Technologies. For DTX(I) and BPTI, respectively, we used coordinates 1DEM (43) and 4PTI (44) taken from the Protein Data Bank(45) . The peptide sequence of AsKC2 was assigned to the coordinates of dendrotoxin without changes in the protein conformation. Non-bonded atomic contacts induced were then analyzed and manually removed by rotation around the side chain carbons-carbons bonds. No energy minimization was further applied.


RESULTS

We have purified from the sea anemone A. sulcata four peptides for their ability to prevent I-DTX(I) binding to its receptors in rat brain. Since DTX(I) is a well-known blocker of K channels and since these A. sulcata peptides not only inhibit I-DTX(I) binding but also inhibit K channel activity it has been decided to designate them under the name of A. sulcata kaliseptine and kalicludines (AsKS and AsKC). Fig. 2and 3 present the sequences of the four peptides. Three of them have sequence similarities with DTX(I) and the basic bovine trypsin inhibitor (Kunitz inhibitor, BPTI) which is a well-known homolog of DTX(I)(17) . These toxins were named A. sulcata kalicludines (AsKC1, AsKC2, and AsKC3) because they also have structural homologies with calcicludine (CaC) (32-35%), another homolog of the Kunitz inhibitor and of DTX(I)(43, 46) which is a blocker of voltage-sensitive Ca channels. The last purified peptide (Fig. 3) is not homologous to DTX(I). It was named A. sulcata kaliseptine (AsKS).

Fig. 4shows the concentration dependence of the inhibition of I-DTX(I) binding by the different sea anemone toxins. Values of IC are 27 nM for AsKS, 60 nM for AsKC2, 375 nM for AsKC1, and 500 nM for AsKC3. These values were 2-4 orders of magnitude higher than the IC for DTX(I) inhibition of I-DTX(I) binding which is 0.14 nM. Scatchard plots presented in Fig. 5show that both kaliseptine (AsKS) and kalicludines (AsKC2) inhibit I-DTX(I) binding in a competitive way. Plots of the apparent values K(d)(app)/K(d)versus the toxins concentrations provide the true values of inhibition constants K(i) which are 10 nM for AsKS and 20 nM for AsKC2 (Fig. 5, insets). None of the three kalicludines (nor AsKS) prevented I-calcicludine (^2)binding to its receptors in rat brain up to the concentration of 5 µM, although they are also structurally homologous to this Ca channel blocking toxin (46) .


Figure 4: Inhibition by DTX(I), the Kunitz trypsin inhibitor, and different peptides from A. sulcata of the specific I-DTX(I) binding to rat brain microsomes. Unlabeled DTX(I) and the different peptides were first incubated at different concentrations with the membranes (20 µg/ml) and then I-DTX(I) (3 pM) was added and membranes were incubated for 1 h at 25 °C. Results are mean of two experiments. Nonspecific I-DTX(I) binding was below 2% and was subtracted. bullet, AsKS; , AsKC1; up triangle, AsKC2; circle, AsKC3; , DTXI; , the Kunitz inhibitor BPTI.




Figure 5: Binding of I-DTX(I) to rat brain membranes in the presence of different concentrations of AsKS (A) and AsKC2 (B). Membranes (7 µg of protein/ml) were incubated for 1 h at 25 °C with I-DTX(I) (2-100 pM). Main panels, Scatchard plots for I-DTX(I)-specific binding obtained after subtraction of nonspecific I-DTX(I) binding determined by including 0.1 µM DTX(I) in the incubation medium. Results are means of two determinations. Bound/free is expressed as pmol/(mg of protein times nM). A, concentrations of AsKS: circle, 0 nM; bullet, 2 nM; up triangle, 8 nM; , 27 nM. B, concentrations of AsKC2: bullet, 10 nM; up triangle, 30 nM; , 60 nM. Insets, effects of increasing concentrations of A. sulcata peptides on the K(d)(app)/K(d) ratio where K(d)(app) is the value obtained in the presence of toxins.



Electrophysiological measurements presented in Fig. 6show that these peptides from A. sulcata which recognize DTX(I) receptors also inhibit the Kv1.2 K channel expressed in Xenopus oocytes as DTX(I) does. The IC for inhibition of the Kv1.2 current ranged from 140 nM for AsKS to 1.1 µM for AsKC2 to 1.3 µM for AsKC3 to 2.8 µM for AsKC1. Under these experimental conditions, DTX(I) itself has an IC of 2.1 nM. Peptides which inhibit the Kv1.2 current with the best efficiency are also those which have the highest affinity for the DTX(I) receptors.


Figure 6: Inhibition of the K current in Xenopus oocytes expressing the Kv1.2 channels. The oocytes were injected with 0.2 ng of Kv1.2 cRNA. In these experiments, the holding potential was -80 mV, and current amplitudes were measured at +30 mV (n = 3). The inset shows current traces recorded in control and in the presence of AsKS peptide (600 nM). bullet, AsKS; , AsKC1; up triangle, AsKC2; circle, AsKC3; , DTX(I); , BPTI.



Since the three kalicludines have extensive homologies with BPTI which is a very potent blocker of trypsin activity, it was checked whether kalicludines also could have an ability to inhibit trypsin. Trypsin at the concentration of about 3 µM was first incubated at room temperature for 3 h with different concentrations of the kalicludines to be sure to reach equilibrium. After incubation, the free trypsin was measured by its ability to release paranitroaniline from BAPNA. Fig. 7shows that all three kalicludines inhibit trypsin. Inhibition profiles of trypsin by AsKC1, AsKC2, AsKC3, and BPTI are very similar. Total inhibition of trypsin was reached by addition of a stoichiometric amount (1:1) of the kalicludines. The profile of inhibition observed with AsKC1, AsKC2, and AsKC3 (which indicates a stoichiometric 1:1 inhibition) is encountered when the K(d) for the interaction is below 1/100th of the concentration of trypsin in the incubation medium(47) , here 3 µM. Then, the K(d) of interaction of these sea anemone peptides with trypsin has to be below 30 nM. Conversely, Fig. 7also shows that a large excess of DTX(I), another Kv channel inhibitior of similar structure, is unable to inhibit trypsin. This lack of trypsin inhibition was also observed for toxin K from D. polylepis, another dendrotoxin toxin for K channels (17) which is structurally more closely related to the Kunitz inhibitor than DTX(I) because it has a lysine in position 15 corresponding to the essential lysine 15 at the active site of the trypsin inhibitor (48) (the corresponding residue is a tyrosine in DTX(I)). Finally, Fig. 7shows that A. sulcata kaliseptine AsKS which has no structural homology with BPTI does not inhibit trypsin even at a molecular excess of 7 to 1.


Figure 7: Concentration dependence of trypsin inhibition by different concentrations of BPTI, A. sulcata peptides, and dendrotoxins. bullet, AsKS; , AsKC1; up triangle, AsKC2; circle, AsKC3; , DTX(I); down triangle, toxin K from D. polylepis.; , BPTI. Trypsin activity is plotted against the molecular ratios of the different peptides to trypsin in the incubation medium.




DISCUSSION

The first category of peptides isolated in this work has been designated A. sulcata kalicludines. They inhibit I-DTX binding to Kv channel proteins and block the Kv1.2 channel expressed in the Xenopus oocyte. Sea anemone kalicludines 1, 2, and 3 are 57-60 amino acid peptides homologous to serine protease inhibitors of the Kunitz type and to dendrotoxins (Fig. 2). They have from 40 to 41% homologies with the Kunitz inhibitor and from 38 to 42% homologies with DTX(I). These sequence homologies led us to compare more closely their properties with those of these two peptides. DTX(I) is the most potent blocker of the Kv1.2 channel(42) . Affinity for its receptor in rat brain is very high, 10M as also shown in Fig. 4. On the other hand, BPTI is the most potent inhibitor of trypsin with a dissociation constant of 6.10M(47) but is not a blocker of Kv channels.

Fig. 4, Fig. 6, and Fig. 7show that A. sulcata kalicludines are molecules with dual types of activity. They have both the properties of DTX(I) and BPTI, they prevent I-DTX(I) binding to Kv1.2 channel in a competitive manner (Fig. 5B), and they inhibit trypsin in a stoichiometric manner like BPTI. From titration curves presented in Fig. 7, one can deduce that K(d) of these trypsin inhibitions is below 30 nM. Conversely and as expected, the other class of sea anemone K channel toxins, kaliseptine, which is structurally different from the Kunitz-type proteinase inhibitors, does not inhibit trypsin. It is the first time that both functions, i.e. blockade of a K channel and a potent inhibition of a protease, have been found in a single molecule of about 60 amino acids and structurally related to BPTI. Dendrotoxins do not inhibit trypsin, and protease inhibitors do not display dendrotoxin-like activity even at high concentrations(49) .

Sea anemones appeared at least 800 millions years ago and it may be that bifunctional molecules such as kalicludines are survivors of a remote past, and that evolution has gradually given rise, for efficiency, to molecules fully specialized either for trypsin inhibition or for blockade of the voltage-sensitive K channel.

The molecular structure basement of dendrotoxins and BPTI is shown in Fig. 8a. This basement corresponds to pear-shaped molecules of 35 Å length and 25 Å of grand diameter. The replacement of the peptide sequence of AsKC2 in the coordinates of the mean NMR structure of DTX(I) only induced a few unfavorable non-bonded atomic contacts between side chain atoms of Asn^2 and Asp^4, Arg and Arg, Lys and Asn. All could be removed by the only change of the (1) angle by rotation of the side chain around C-C bonds of Asn^2, Arg, and Lys. All the residues forming the hydrophobic interior of both BPTI and DTX(I) are well conserved and could be easily adapted at the same place. These amino acids are tyrosine at positions 21, 22, 23, and 35, phenylalanine at positions 33 and 45. AsKC2 (net charge of +8) is less charged than DTX(I) (+11) and more charged than BPTI (+6) (Fig. 8b).


Figure 8: Main chain folding of the averaged-minimized NMR structure of dendrotoxin I drawn using MOLSCRIPT (53) and the 1DEM entry of the Protein Data Bank(43, 45) . Disulfide bridges are represented in a ``ball-and-stick'' style. The trypsin binding site in BPTI is located in the loop comprizing residues 15-19 in dendrotoxin I. = NH(2)-terminal position, Ct = COOH-terminal position 60. b, space-filling atomic models of (left to right in each panels) AsKC2, dendrotoxin I and BPTI. Basic amino acids Arg, His, and Lys are colored in blue, acidic amino acids Asp and Glu in white, and the others in red. In the upper left panel, the models are shown in the same orientation as in a. In the upper right panel, the structure were rotated by 90 °C and are shown on the Ct and Nt side. The lower left panel is obtained by a further 90° rotation and shows the opposite side of the orientation given in a (up-and-down). The lower right panel is an additional rotation by 90° showing the apical trypsin binding side described in a.



A recent analysis explaining why dendrotoxins are unable to inhibit trypsin has ben given(50) . Replacement of both the Lys of BPTI by a Tyr, and Ala by a longer amino acid (aspartate in dendrotoxin alpha and glutamine in DTX(I)), produce a highly unfavorable complex with the trypsin active site. Moreover, the presence in DTX(I) of a proline in position 21 that is occupied by an isoleucine in BPTI has been seen as an additional unfavorable feature for good atomic contact with Tyr of trypsin. Among these three positions necessary for a good inhibition of trypsin by BPTI, two central ones, Arg and Ala, are conserved in AsKC2. Pro which is the equivalent to Pro in dendrotoxins is retained. Thus, the prediction would be that AsKC2 has indeed, as it is found, a trypsin inhibitory property but with an efficacy which would be lower than that of BPTI.

It has been recently proposed (43) that the most prominent structural difference between BPTI and dendrotoxins concerned surface electrostatics which could explain differences between the two types of molecules in their capacity to block K channels. The space-filling models presented in Fig. 8b show that the major surface-electrostatic differences existing between DTX(I) and BPTI in the COOH-terminal and NH(2)-terminal regions (that are packed together due to the peptide fold) are in part reproduced when comparing AsKC2 and BPTI. For instance, the cluster Arg, Arg, Arg, and Lys in DTX(I) is replaced by another four-charges cluster made of Lys, Lys, Lys, and Arg in AsKC2 (upper right panel of Fig. 8b). Negative charges in the COOH-terminal alpha-helix are slightly modified in AsKC2 relative to DTX(I) with a Glu-Cys-Glu sequence instead of Glu-Glu-Cys. The negative charge on Glu in DTX(I) I is conserved in AsKC2 (Glu) as well as the positive charge on Lys^5 of DTX(I) (Lys^3 in AsKC2).

The structure of kaliseptine (AsKS) is different from that of kalicludines. It is also a much smaller peptide (only 36 amino acids). It has no homology with proteinase inhibitors of the Kunitz type but presents 49% homologies with a 37-amino acid peptide isolated from another sea anemone, Bunodosoma granulifera (BgK, Fig. 3) which is also a probable blocker of the voltage-dependent K channel(s) sensitive to DTX(I) since it inhibits I-DTX(I) binding(51) . However these two structures have a striking difference in the position of one of their Cys residues (Cys and Cys in the COOH-terminal part). This difference is surprising since Cys residues, as for all other short polypeptide toxins, are expected to form disulfide bridges. It might result from a mistake in the sequence of the B. granulifera peptide. Both AsKS and BgK also have limited homologies with a 110-amino acid protease inhibitor from the eggwhite of a red sea turtle (51, 52) which itself has some homologies with BPTI. However homologies with AsKS are limited to the sequence 58-93 of the sea turtle inhibitor and not to the sequence 3-58 which is homologous to BPTI. There could exist other homologs of kaliseptines with a trypsin inhibitor activity but they have not been identified yet.

DTX(I) blocks with a high affinity not only Kv1.2 channels but also Kv1.1 and Kv1.6 channels when they are expressed in Xenopus oocytes as well as channels formed by heterologous association of different subunits corresponding to these different types of channels(1, 2) . Therefore measuring the capacity of sea anemone kalicludines and kaliseptines to inhibit I-DTX(I) binding to neuronal membranes is not equivalent to measuring their capacity to inhibit oocyte expression of a particular class of K channel (in this paper Kv1.2). I-DTX(I) binding identifies a multiplicity of types of voltage-sensitive K channels(14, 23, 24) . For all these reasons a very systematic study of the different toxins described in this work will have to be made on different types of Kv channels, as previously done for DTX(I)(23) . This will require larger quantities of these peptides which are in relatively low amount but could now be prepared by total synthesis or in a recombinant way.

Also, it has been shown before that a particularly interesting property of sea anemone venom is that it not only contains toxins that can disciminate between different types of voltage-sensitive Na channels in different tissues of a given animal but also between Na channels in different animal species(35, 37) . A good number of the sea anemone toxins for Na channels have only a very small activity toward mammals but a very high activity toward crustaceans(40) . When larger quantities of the peptides described in this work become available, it will be particularly interesting to assay their activity in crustaceans and also in insects which often have nervous system properties similar to those of crustaceans. From a biological point of view it would make sense that sea anemones make toxins more oriented toward K channels normally found in their preys (fish, crustaceans, etc.). On the other hand, if these new peptides, or analogs of these new peptides, were particularly active on insects, this could provide new ways of thinking toward the development of new insecticides.

Finally the mixture of ion channel toxins found in sea anemone venom and comprising both Na channel toxins tending to ``activate'' the Na channels (37) and K channel toxins tending to block K channels is expected to have devastating neurotoxic effects by producing very massive release of neurotransmitters as well as very potent effects on heart, muscle, and endocrine cells.


FOOTNOTES

*
This work was supported by the Centre National de la Recherche Scientifique (CNRS) and the Ministère de la Défense Nationale (grant DRET 93/122). This work is dedicated to Prof. Dr. Hans Fritz, Institut für Klinische Chemie und Biochemie, Nussbaum Strasse 20, München. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 33-93-95-77-00; Fax: 33-93-95-77-04; ipmc@unice.fr.

(^1)
The abbreviations used are: BPTI, bovine pancreatic trypsin inhibitor also called aprotinine when extracted from bovine lung; DTX(I), dendrotoxin I from D. polylepis; I-DTX(I), the iodinated toxin; HPLC, high performance liquid chromatography; BAPNA, N-benzoyl-DL-arginine p-nitroanilide.

(^2)
H. Schweitz, unpublished results.


ACKNOWLEDGEMENTS

We are very grateful to Dr. J. C. Doury and to Dr. H. Tolou (Institut de Médecine Tropicale du Service de Santé des Armées, CERMT, Marseille, France) for their very generous and essential help with mass spectrometry analyses. We thank Dr. J.-P. Vincent and G. Lambeau for fruitful discussions, and C. Roulinat and F. Aguila for skilful technical assistance. Thanks are due to Bristol-Myers Squibb Company for an ``Unrestricted Award.''


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