Bougis1 and Marie-France Martin-Eauclaire1 1 UMR 6560 CNRS and2UMR 6150 CNRS, Universite´ de la Me´diterrane´e, Faculte´ de Me´decine secteur Nord, IFR Jean Roche, Marseille, France;3Ins
Trang 1Expanding the scorpion toxin a-KTX 15 family with AmmTX3
He´le`ne Vacher1, Meriem Alami3, Marcel Crest2, Lourival D Possani4, Pierre E Bougis1
and Marie-France Martin-Eauclaire1
1
UMR 6560 CNRS and2UMR 6150 CNRS, Universite´ de la Me´diterrane´e, Faculte´ de Me´decine secteur Nord, IFR Jean Roche, Marseille, France;3Institut Pasteur du Maroc, Casablanca, Morocco;4Biotechnology Institute, UNAM, Cuernavaca, Mexico
A novel toxin, AmmTX3 (3823.5 Da), was isolated from the
venom of the scorpion Androctonus mauretanicus It showed
94% sequence homology with Aa1 from Androctonus
aus-tralisand 91% with BmTX3 from Buthus martensi which,
respectively, block A-type K+current in cerebellum granular
cells and striatum cultured neurons Binding and
displace-ment experidisplace-ments using rat brain synaptosomes showed that
AmmTX3 and Aa1 competed effectively with125I-labelled
sBmTX3 binding They fully inhibited the 125I-labelled
sBmTX3 binding (Ki values of 19.5 pM and 44.2 pM,
respectively), demonstrating unambiguously that the three molecules shared the same target in rat brain The specific binding parameters of125I-labelled AmmTX3 for its site were determined at equilibrium (Kd¼ 66 pM, Bmax¼ 22 fmol per mg of protein) Finally, patch-clamp experiments on striatal neurons in culture demonstrated that AmmTX3 was able to inhibit the A-type K+current (Ki¼ 131 nM) Keywords: scorpion toxins; A-type potassium current; stri-atum neurons; patch clamp; binding
An increasing number of toxins blocking the activity of K+
channels are isolated from various animal venoms and
become key molecular probes for the characterization of
these channels They are usually small basic polypeptides
(between 30 and 70 amino acids), cross-linked by three or
four disulphide bridges, reviewed in [1] They recognize
principally voltage-dependent (Kv) channels (in particular
Kv channels of the Kv1 family, which generate sustained
K+ current) and some Ca2+-activated channels of big,
intermediate or small conductance (BKCa, IKCaor SKCa)
The binding sites of the most studied toxins purified from
scorpion venoms, such as charybdotoxin (ChTX), agitoxin
(AgTX) and kaliotoxin (KTX), have been described in
detail These toxins occlude channels by binding to the outer
opening of the conduction pore, at the centre of symmetry
of the channel [2–6]
Two new toxins blocking transient A-currents were
recently isolated from scorpion venoms: Aa1, from
Andr-octonus australis, which was shown to block an A-type K+
channels in cerebellar granular cells [7] and BmTX3, from
Buthus martensiKarch, which was found to block an A-type
current in striatal neurons in culture These toxins revealed a
new class of scorpion toxin binding sites in rat brain [8]
In this study, a third component (AmmTX3) of this new
toxin family is identified from the venom of the scorpion
Androctonus mauretanicus.Its biochemical and pharmaco-logical features are depicted and we show that AmmTX3 share with Aa1 and BmTX3 high sequence homologies as well as the same binding site on rat brain synaptosomes
M A T E R I A L S A N D M E T H O D S
Materials The venom from Androctonus mauretanicus scorpions obtained by manual stimulation was generously provided
by the Pasteur Institute at Casablanca, Morocco Aa1 was obtained from Androctonus australis venom bought from Latoxan as previously described [7] Synthetic kaliotoxin (sKTX), P05 and BmTX3 (sBmTX3) were chemically synthesized as previously described [8–10] IbTX and ChTX were from Bachem Laboratory Apamin, BSA and a-cyano-4-hydroxycinnamic acid were from Sigma a-Den-drotoxin (DTX) was obtained as described [11] UV grade acetonitrile was from Fisons Scientific, trifluoroacetic acid, from Baker, and all other analytical reagents, from Merck The pyroglutamate aminopeptidase was from Boerhinger The water used for the preparation of solutions and buffers was purified with the Milli Ro/Milli Q system from Millipore
HPLC Androctonus mauretanicus venom was purified by a two-step reverse-phase HPLC procedure at 25C: the first step
on a Merck semipreparative column prepacked with Ultrasphere 5 lm 100 RP-8; the second stepon an analytical column Lichrosphere 5 lm 100 RP-18 The system used was a Waters Associate System, as previously described [10,11] Additional details of the chromatographic procedure are given later in the text and in the figure legends
Correspondence to M F Martin-Eauclaire, UMR 6560 CNRS,
Faculte´ de Me´decine secteur Nord, Bd Pierre Dramard,
F-13916, Marseille Cedex 20, France.
Fax: 33 4 9169 8839, Tel.: 33 4 9169 8914,
E-mail: eauclaire.mf@jean-roche.univ-mrs.fr
Abbreviations: AgTX, agitoxin; ChTX, charybdotoxin; DTX,
a-dendrotoxin; KTX, kaliotoxin.
(Received 6 August 2002, revised 30 September 2002,
accepted 7 October 2002)
Trang 2Amino acid analysis and sequence determination
Amino acid analysis and sequence determination of
AmmTX3 (5 nmoles), S-alkylated with 4-vinyl-pyridine,
were as previously described [11] Treatment with
pyroglu-tamate aminopeptidase unblocked the N-terminal glupyroglu-tamate
residue [12–14] An Applied Biosystems 476A sequencer and
the recommended programme cycles were used for
automa-ted Edman degradation Phenylthiohydantoin derivatives
were characterized by HPLC on RP-18
MS
Electrospray MS (ES/MS) was performed on a Quatro II
mass spectrometer (Micromass), as previously described
[9,15] MALDI-TOF/MS was performed on a Perseptive
DE-RP (Applied Biosystem) using
a-cyano-4-hydroxycin-namic acid as matrix
Lethality assay in mice
The in vivo toxicity of venoms, HPLC fractions or purified
toxins was tested in male C57 Bl/6 mice by
intracerebro-ventricular injections Experiments were carried out in
accordance with the European Communities Council
Directive
Radioiodination of toxins
The toxins sBmTX3 and native AmmTX3 were
radioiodi-nated by the lactoperoxidase method, as previously
des-cribed [8] MALDI-TOF/MS was used to check that the
derivatives were monoiodinated 125I-labelled sKTX was
obtained as previously described [9] Specific radioactivities
of 2000 CiÆmmol)1were routinely obtained
Pharmacological tests
Rat brain synaptic nerve ending particles (P2fraction) were
obtained as described elsewhere [8,9] We carried out
competition assays with native AmmTX3 and125I-labelled
AmmTX3 or125I-labelled sBmTX3 bound to their receptor
sites on P2(90 lg per assay, in a total volume of 200 lL) as
previously described [8] The binding buffer used was
20 mM Tris/HCl, pH 7.4, 50 mMNaCl, 0.1% (w/v) BSA
Identical conditions were used for binding and competition
assays with 125I-labelled AmmTX3 Nonspecific binding
was determined in the presence of 100 nM unlabeled
BmTX3 or AmmTX3 Incubation was 1 h at 25C The
reaction was stopped by dilution [4 mL of washing buffer,
20 mM Tris/HCl, pH 7.4, 150 mM NaCl, 1% (w/v) BSA]
and the solution was immediately filtered through a GF/C
filter Whatman soaked in 0.1% (v/v) poly(ethylenimine)
Filters were washed twice and the radioactivity was
determined by c-counting Each experiment was in
dupli-cate Data were analyzed withPRISMsoftware (GraphPad)
Patch recording of striatal neurons in culture
For primary culture of striatal neurons, striata were
dissected from 18-day-old Sprague–Dawley rat embryos
and cultured according to [16] Neurons were studied using
the whole-cell patch-clamp technique The bath solution,
designed to suppress Na+and Ca2+currents and to reduce the sustained delayed rectifier K+ current, contained (in
mM): 135 NaCl, 2.5 KCl, 1 MgCl2, 1.8 CaCl2, 0.2 CdCl2, 5 tetraethylammonium, 0.01 tetrodotoxin, 10 Hepes and 10 glucose, pH 7.35, with an osmolarity of 290–300 mosM AmmTX3 was applied under pressure with a broken pipette
or directly added in the chamber containing 300 lL of bath solution Experiments were carried out at room temperature (20–24C) Patch pipettes were filled with (in mM): 90 KF,
30 KCl, 5 NaCl, 2 MgCl2, 2 EGTA, 10 Hepes and 30 glucose, pH 7.35, with an osmolarity of 290–300 mosM Capacity transient compensation was routinely performed
in the cell-attached mode before patch membrane rupture
In the whole-cell voltage-clampconfiguration, capacitive transients and leakage currents were subtracted using a factored hyperpolarizing pulse, without additional transient
or series resistance compensation
R E S U L T S A N D D I S C U S S I O N
Purification and determination of the amino acid sequence of AmmTX3
Previous studies on the fractionation of Androctonus mauretaniusvenom obtained by manual stimulation led to the identification of five fractions (P01, P02, P04, P05 and P06) that inhibited binding of125I-labelled apamin (a SK2 and SK3 channel blocker from bee venom) to rat brain synaptosomes [10] P01 and P05 were extensively studied [17,18] Fractions P02, P04 and P06, which were heteroge-neous after the first HPLC step, remained to be character-ized P03 was identified as KTX [19], a high-affinity blocker
of Kv1.1 and KV1.3 channels [2,6,20]
Fraction P06 (Fig 1A) completely displaced125I-labelled sBmTX3, but not125I-labelled sKTX, from their respective binding sites on rat brain synaptosomes The injection of P06 into mice (approximately 8 lg for a 20-g mouse by intracerebroventricular injections) caused epileptiform behaviour before death P06 contained a major low molecular mass component (3823.5 Da) After a second HPLC step, this major peptide was completely homoge-neous according to biochemical criteria (Fig 1B) This toxin, which accounted for 0.06% of the dry mass of the venom, was named AmmTX3 Its amino acid composition gave the following: 1.93 Asx (2); 0.96 Thr (1); 1.59 Ser (2); 3.0 Glx (3); 1.1 Pro (1); 4.7 Gly (5); 3.1 Ala (3); 5.6 VP-Cys (6); 3.48 Val (4); 2.47 Ile (3); 0.9 Tyr (1); 4.02 Lys (4); 2.1 Arg (2)
No phenylthiohydantoin derivatives was detected in the first stepin the Edman sequencing of AmmTX3, suggesting that this peptide was blocked at its N-terminal extremity The molecular mass, determined by ES/MS of the native peptide, was 3823.5 Da (Fig 1B, inset) This was 16 Da less than the mass deduced from amino acid composition (3839.5 Da) This difference is consistent with the presence
of a pyroglutamic acid residue at the N-terminus, as in Aa1 and BmTX3 The S-alkylated AmmTX3, unblocked at its N-terminus after treatment with pyroglutaminase, was further sequenced in a single run AmmTX3 consists of a single chain of 37 amino acid residues cross-linked by three disulphide bridges (Fig 1C) The amino acid sequences of Aa1, BmTX3 and AmmTX3 were aligned on the basis of their cysteine residues (Fig 2) AmmTX3 has 94% sequence homology with Aa1 [7] and 91% with BmTX3 [8]
Trang 3AmmTX3 interacts with the125I-labelled sBmTX3
receptor site on rat brain neuronal membranes
To analyse the pharmacological properties of the AmmTX3
target, we first performed competition experiments with
AmmTX3 and Aa1 against125I-labelled sBmTX3 bound to
its receptor site in rat brain P2fraction (Fig 3A) AmmTX3
and Aa1 fully inhibited the binding of 125I-labelled
sBmTX3, with Ki values of 19.5 ± 1.95 pM and
44.2 ± 40 pM, respectively (n¼ 3), indicating that all these
molecules bind to the same target in rat brain The affinity
of AmmTX3 for its binding site was higher than that of125 I-labelled sBmTX3 [8], and that observed for Aa1 in the competition experiments reported here The affinity for the binding site seems to increase with the number of positively charged residues in the N-terminal half of these toxins (six for AmmTX3 and Aa1, five in BmTX3) and with the hydrophobicity of certain residues (Ile2 in AmmTX3 instead of the Asn2 observed in Aa1)
We also studied the competition between 125I-labelled AmmTX3 bound to its receptor site and increasing concentrations of native AmmTX3 (Fig 3B) A Kivalue
of 8.4 ± 18 pMwas obtained These values are consistent with those obtained in competition experiments with125 I-labelled sBmTX3 To further compare the binding proper-ties of AmmTX3 and sBmTX3, we examined the direct binding of 125I-labelled AmmTX3 to rat brain neuronal membranes by means of saturation experiments (Fig 3C) Specific binding was saturable A Kdof 66 ± 19 pMand a
Bmaxof 22 ± 0.18 fmol per mg of protein were obtained (n¼ 2) Nonspecific binding accounted for approximately 40% of the total binding Finally, in order to characterize further the pharmacological properties of 125I-labelled AmmTX3 receptor sites in rat brain, other K+ channel peptide blockers were tested for their ability to modulate the
125I-labelled AmmTX3 binding (Fig 3D) The following were tested upto 1 lM: (a) the Kv1 family blockers KTX, ChTX and a-DTX, (b) the BKCachannel blockers ChTX and IbTX and (c) the SKCablockers P05 and apamin All were unable to displace 125I-labelled AmmTX3 from its binding site
Whole-cell patch recording of striatal neurons
in culture Performing whole cell patch recording using primary striatal neurons in cell culture assessed that AmmTX3 blocked the transient K+current In experimental condi-tions voltage steps between )40 and +30 mV from a holding potential of)90 mV elicited a large transient K+ current and a small sustained delayed rectifier The presence of tetraethylammonium in the external medium blocked approximately 40% of the sustained K+current Figure 4A shows that AmmTX3 at 10 lM almost com-pletely blocked the transient K+ current without modi-fying the sustained component at all the voltages tested Application of AmmTX3 at various concentrations ranging from 0.1 nM to 10 lM induced an increasing percentage of block (measured at the current peak) and the best fit of the experimental values gave a Kiof 131 nM with a Hill coefficient of 0.90 (Fig 4B) Toxin effect reverse with a K of 1.1· 10)3Æs)1 (Fig 4C) This K
Fig 1 Purification and amino acid sequence determination of
AmmTX3 (A) Androctonus mauretanicus venom profile (800 lg) in
reverse-phase (RP) HPLC on a C-8 column Solvent A, 0.1% (v/v)
trifluoroacetic acid; solvent B, acetonitrile/0.1% (v/v) trifluoroacetic
acid; linear gradient from 5–45% B in 100 min; flow rate 5 mLÆmin)1.
The fraction used for subsequent purification (P06) is indicated (B)
Final purification of AmmTX3 (fraction 6 from A) by RP-C18 HPLC.
Solvent A was 0.1% (v/v) trifluoroacetic acid A linear gradient of
0.1% (v/v) trifluoroacetic acid in acetonitrile was applied from 0–100%
over 30 min, at a flow rate of 1 mLÆmin)1; AUFS at 230 nm ¼ 1 (a)
and at 280 nm (b)
3 Inset: electrospray mass spectrum of AmmTX3 (C)
Amino acid sequence of AmmTX3 The reduced and S-alkylated
AmmTX3 was first treated with pyroglutaminase to remove the
py-roglutamic acid residue (Z) blocking the N-terminus of the peptide.
Fig 2 Amino acid sequence similarities between AmmTX3, Aa1 and BmTX3 Sequences were aligned according to cysteine residues (bold), with the ALIGN programme of SBDS Aa1 [7] BmTX3 [8] and AmmTX3, this work Z is pyroglutamate Shadowed amino acids indicate positions of non-identical residues.
Trang 4value is much higher than the binding Kdvalue (66 pM).
Differences between the affinities found in binding or
electrophysiological experimemts were frequently observed
by others [20–22], and could proceed from differences in
either ionic strengths of the media or between the channel
subtypes found in the primary striatum neurons (as used
in electrophysiological experiments) vs brain homogenate
C O N C L U S I O N S
Two toxins, Aa1 and BmTX3, with very similar primary structures, were recently described [7,8] It has been shown that Aa1 blocks the A-type K+ currents in cerebellar granular cells (Ki 150 nM) and BmTX3 blocks an A-type
K+ current in striatum neurones in primary culture
Fig 3 Characterization of the pharmacologi-cal binding site of AmmTX3 on rat brain P 2 (A) Competitive binding of 125 I-labelled sBmTX3 (200 p M ) with increasing concentra-tions of sBmTX3 (j), native AmmTX3 (m) and Aa1 (.) (B) Competitive binding of
125 I-labelled AmmTX3 (40 p M ) with increas-ing concentrations of native AmmTX3 (m) (C) Equilibrium isotherm of 125 I-labelled AmmTX3 binding to rat membrane vesicles incubated in the presence of increasing con-centrations (10–300 p M ) of 125 I-labelled AmmTX3 (j, total binding) Nonspecific binding (m) was determined in the presence of 0.1 l M unlabelled AmmTX3 Specific binding (.) was assessed from the difference between total and nonspecific binding K d ¼ 66 ±
19 p M and B max ¼ 22 ± 0.18 fmolÆmg)1of protein (D) Percentage of 125 I-labelled AmmTX3 displaced by some K + channel peptide (up to 1 l M ).
Fig 4 AmmTX3 blocks the A-type current in striatal neurones in culture (A) Transient and sustained K+current recorded in control conditions and
at the steady-state effect of AmmTX3 (10 l M ) Currents were elicited by successive voltage steps from )40 to +30 mV from a holding potential of )90 mV Currents were recorded in the presence of tetradoxin (10 l M ), CdCl 2 (0.2 m M ) and tetraethylammonium (5 m M ) (B) Dose–response curve
of the effect of various concentrations of AmmTX3 One test corresponds to the effect of one concentration applied to one neurone Each concentration was tested three to six times The experimental points were fitted with a hyperbolic curve and the best-fit values correspond to a K i of
131 n M and a Hill coefficient of 0.90 (C) Time-course of current recovery from block The time constant for recovery was determined by plotting the percentage of block [(Ic–It)/(Ic–Iss)] · 100 as a function of the time of toxin wash-out Ic: current in control conditions before toxin application; It: current amplitude during recovery, Iss: current at the steady-state of block before recovery.
Trang 5(Ki 54 nM) Here, we unambiguously demonstrate that
Aa1 and BmTX3 recognize the same binding site in rat
brain Yet, this target is not clearly identified at the
molecular level In addition, AmmTX3, a third related
peptide, competing with125I-labelled sBmTX3 for binding,
was identified in the venom of Androctonus mauretanicus
mauretanicus The number of K+channel blockers purified
from scorpion venom are ever expanding and several new
subfamilies have been added to the classification formally
proposed by Tytgat and collaborators [5] Therefore,
according to the pharmacological criteria and sequence
homologies, we propose that Aa1, BmTX3 and AmmTX3
constitute the members of a new subfamily of short-chain
scorpion toxins active on K+channels, which may
corres-pond to the a-KTX 15 subfamily
A C K N O W L E D G E M E N T S
We thank the Pasteur Institute from Morocco and Professors A.
Benslimane for generously providing venoms of Androctonus
maure-tanicus mauremaure-tanicus obtained by manual stimulation We also thank
Dr B Ce´ard, R Ouguideni S Canarelli and F Coronas for technical
assistance and Dr P Mansuelle for expert interpretation of amino acid
sequence and ES/MS data Dr Alami was supported by the World
Health Organization and by the Socie´te´ de Secours des Amis des
Sciences H Vacher was supported by the De´le´gation Ge´ne´rale pour
l’Armement.
R E F E R E N C E S
1 Strong, P.N (1990) Potassium channel toxins Pharmacol Ther.
46, 137–162.
2 Aiyar, J., Withka, J.M., Rizzi, J.P., Singleton, D.H., Andrews,
G.C., Lin, W., Boyd, J., Hanson, D.C., Simon, M., Dethlefs, B.
et al (1995) Topology of the pore-region of a K+ channel
revealed by the NMR-derived structures of scorpion toxins.
Neuron 15, 1169–1181.
3 Naini, A.A & Miller, C (1996) A symmetry-driven search for
electrostatic interaction partners in charybdotoxin and a
voltage-gated K+ channel Biochemistry 35, 6181–6187.
4 Ranganathan, R., Lewis, J.H & MacKinnon, R (1996) Spatial
localization of the K+ channel selectivity filter by mutant
cycle-based structure analysis Neuron 16, 131–139.
5 Tytgat, J., Chandy, K.G., Garcia, M.L., Gutman, G.A.,
Martin-Eauclaire, M.F., van der Walt, J.J & Possani, L.D (1999) A
unified nomenclature for short-chain peptides isolated from
scorpion venoms: alpha-KTX molecular subfamilies Trends
Pharmacol Sci 20, 444–447.
6 Legros, C., Pollmann, V., Knaus, H.G., Farrell, A.M., Darbon,
H., Bougis, P.E., Martin-Eauclaire, M.F & Pongs, O (2000)
Generating a high affinity scorpion toxin receptor in KcsA-Kv1.3
chimeric potassium channels J Biol Chem 275, 16918–16924.
7 Pisciotta, M., Coronas, F.I., Bloch, C., Prestipino, G & Possani,
L.D (2000) Fast K(+) currents from cerebellum granular cells are
completely blocked by a peptide purified from Androctonus
aus-tralis Garzoni scorpion venom Biochim Biophys Acta 1468, 203–
212.
8 Vacher, H., Romi-Lebrun, R., Mourre, C., Lebrun, B., Kourrich,
S., Masmejean, F., Nakajima, T., Legros, C., Crest, M., Bougis,
P.E & Martin-Eauclaire, M.F (2001) A new class of scorpion
toxin binding sites related to an A-type K+ channel:
pharmaco-logical characterization and localization in rat brain FEBS Lett.
501, 31–36.
9 Romi, R., Crest, M., Gola, M., Sampieri, F., Jacquet, G.,
Zerrouk, H., Mansuelle, P., Sorokine, O., Van Dorsselaer, A.,
Rochat, H et al (1993) Synthesis and characterization of kalio-toxin Is the 26–32 sequence essential for potassium channel re-cognition? J Biol Chem 268, 26302–26309.
10 Zerrouk, H., Mansuelle, P., Benslimane, A., Rochat, H & Martin-Eauclaire, M.F (1993) Characterization of a new leiurotoxin I-like scorpion toxin PO5 from Androctonus mauretanicus mauretanicus FEBS Lett 320, 189–192.
11 Laraba-Djebari, F., Legros, C., Crest, M., Ceard, B., Romi, R., Mansuelle, P., Jacquet, G., van Rietschoten, J., Gola, M., Rochat,
H et al (1994) The kaliotoxin family enlarged Purification, characterization, and precursor nucleotide sequence of KTX2 from Androctonus australis venom J Biol Chem 269, 32835– 32843.
12 Romi-Lebrun, R., Lebrun, B., Martin-Eauclaire, M.F., Ishiguro, M., Escoubas, P., Wu, F.Q., Hisada, M., Pongs, O & Nakajima,
T (1997) Purification, characterization, and synthesis of three novel toxins from the Chinese scorpion Buthus martensi, which act
on K+ channels Biochemistry 36, 13473–13482.
13 Gimenez-Gallego, G., Navia, M.A., Reuben, J.P., Katz, G.M., Kaczorowski, G.J & Garcia, M.L (1988) Purification, sequence, and model structure of charybdotoxin, a potent selective inhibitor
of calcium-activated potassium channels Proc Natl Acad Sci USA 85, 3329–3333.
14 Galvez, A., Gimenez-Gallego, G., Reuben, J.P., Roy-Contancin, L., Feigenbaum, P., Kaczorowski, G.J & Garcia, M.L (1990) Purification and characterization of a unique, potent, peptidyl probe for the high conductance calcium-activated potassium channel from venom of the scorpion Buthus tamulus J Biol Chem 265, 11083–11090.
15 Hassani, O., Loew, D., Van Dorsselaer, A., Papandreou, M.J., Sorokine, O., Rochat, H., Sampieri, F & Mansuelle, P (1999) Aah VI, a novel, N-glycosylated anti-insect toxin from Androcto-nus australis hector scorpion venom: isolation, characterisation, and glycan structure determination FEBS Lett 443, 175–180.
16 Kowalski, C., Crest, M., Vuillet, J., Pin, T., Gola, M & Nieoullon,
A (1995) Emergence of a synaptic neuronal network within pri-mary striatal cultures seeded in serum-free medium Neuroscience
64, 979–993.
17 Zerrouk, H., Laraba-Djebari, F., Fremont, V., Meki, A., Darbon, H., Mansuelle, P., Oughideni, R., van Rietschoten, J., Rochat, H.
& Martin-Eauclaire, M.F (1996) Characterization of PO1, a new peptide ligand of the apamin-sensitive Ca2+ activated K+ channel Int J Pept Protein Res 48, 514–521.
18 Sabatier, J.M., Zerrouk, H., Darbon, H., Mabrouk, K., Bensli-mane, A., Rochat, H., Martin-Eauclaire, M.F & Van Rietscho-ten, J (1993) P05, a new leiurotoxin I-like scorpion toxin: synthesis and structure- activity relationships of the alpha-amidated analog,
a ligand of Ca(2+)-activated K+ channels with increased affinity Biochemistry 32, 2763–2770.
19 Crest, M., Jacquet, G., Gola, M., Zerrouk, H., Benslimane, A., Rochat, H., Mansuelle, P & Martin-Eauclaire, M.F (1992) Kaliotoxin, a novel peptidyl inhibitor of neuronal BK-type Ca(2+)-activated K+ channels characterized from Androctonus mauretanicus mauretanicus venom J Biol Chem 267, 1640–1647.
20 Mourre, C., Chernova, M.N., Martin-Eauclaire, M.F., Bessone, R., Jacquet, G., Gola, M., Alper, S.L & Crest, M (1999) Distri-bution in rat brain of binding sites of kaliotoxin, a blocker of Kv1.1 and Kv1.3 alpha-subunits J Pharmacol Exp Ther 291, 943–952.
21 Park, C.S & Miller, C (1992) Interaction of charybdotoxin with permeant ions inside the pore of a K+channel Neuron 9, 307–313.
22 Vazquez, J., Feigenbaum, P., King, V.F., Kaczorowski, G.J & Garcia, M.L (1990) Characterization of high affinity binding sites for charybdotoxin in synaptic plasma membranes from rat brain Evidence for a direct association with an inactivating, voltage-dependent, potassium channel J Biol Chem 265, 15564–15571.