Expanding the scorpion toxin a-KTX 15 family with AmmTX3
from
Androctonus mauretanicus
He
´
le
`
ne Vacher
1
, Meriem Alami
3
, Marcel Crest
2
, Lourival D. Possani
4
, Pierre E. Bougis
1
and Marie-France Martin-Eauclaire
1
1
UMR 6560 CNRS and
2
UMR 6150 CNRS, Universite
´
de la Me
´
diterrane
´
e, Faculte
´
de Me

molecules shared the same target in rat brain. The specific
binding parameters of
125
I-labelled AmmTX3 for its site were
determined at equilibrium (K
d
¼ 66 p
M
, B
max
¼ 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 (K
i
¼ 131 n
M
).
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

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.
MATERIALS AND METHODS
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 25 °C: the first step
on a Merck semipreparative column prepacked with
Ultrasphere 5 lm 100 RP-8; the second step on 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

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.
125
I-labelled sKTX was
obtained as previously described [9]. Specific radioactivities
of 2000 CiÆmmol
)1
were routinely obtained.
Pharmacological tests
Rat brain synaptic nerve ending particles (P
2
fraction) were
obtained as described elsewhere [8,9]. We carried out
competition assays with native AmmTX3 and
125
I-labelled
AmmTX3 or
125
I-labelled sBmTX3 bound to their receptor
sites on P
2

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 Ca
2+
currents and to reduce
the sustained delayed rectifier K
+
current, contained (in
m
M
): 135 NaCl, 2.5 KCl, 1 MgCl
2
,1.8CaCl
2
,0.2CdCl
2
,5
tetraethylammonium, 0.01 tetrodotoxin, 10 Hepes and 10
glucose, pH 7.35, with an osmolarity of 290–300 mos
M
.
AmmTX3 was applied under pressure with a broken pipette
or directly added in the chamber containing 300 lLofbath
solution. Experiments were carried out at room temperature
(20–24 °C). Patch pipettes were filled with (in m
M
): 90 KF,

sBmTX3, but not
125
I-labelled sKTX, from their respective
binding sites on rat brain synaptosomes. The injection of
P06 into mice (approximately 8 lgfora20-gmouseby
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 step in 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

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
125
I-labelled
AmmTX3 bound to its receptor site and increasing
concentrations of native AmmTX3 (Fig. 3B). A K
i
value
of 8.4 ± 18 p
M
was obtained. These values are consistent
with those obtained in competition experiments with
125
I-
labelled sBmTX3. To further compare the binding proper-
ties of AmmTX3 and sBmTX3, we examined the direct
binding of
125
I-labelled AmmTX3 to rat brain neuronal
membranes by means of saturation experiments (Fig. 3C).
Specific binding was saturable. A K
d
of 66 ± 19 p
M
and a

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 l
M
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 n
M
to 10 l
M
induced an increasing
percentage of block (measured at the current peak) and
the best fit of the experimental values gave a K
i
of 131 n
M

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.
Ó FEBS 2002 Expanding the scorpion toxin a-KTX 15 family (Eur. J. Biochem. 269) 6039
value is much higher than the binding K
d
value (66 p
M
).
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.
CONCLUSIONS
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 (K
i
 150 n
M
) and BmTX3 blocks an A-type
K

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
)1
of
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

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 Ôshort-chainÕ
scorpion toxins active on K
+
channels, which may corres-
pond to the a-KTX 15 subfamily.
ACKNOWLEDGEMENTS
We thank the Pasteur Institute from Morocco and Professors A.
Benslimane for generously providing venoms of Androctonus maure-
tanicus mauretanicus 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
´

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

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.
Ó FEBS 2002 Expanding the scorpion toxin a-KTX 15 family (Eur. J. Biochem. 269) 6041