RESEARCH ARTICLE

Inhibition of human Kv3.1 current expressed in Xenopus oocytesby the toxic venom fraction of Androctonus australis hector

Amani Cheikh • Rym Benkhalifa • Zied Landoulsi •

Imen Chatti • Mohamed El Ayeb

Received: 29 January 2013 / Accepted: 3 June 2013

� The Pharmaceutical Society of Korea 2013

Abstract AahG50, the toxic fraction of Androctonus aus-

tralis hector venom, was studied on human Kv3.1 channels

activation, stably expressed in Xenopus oocytes using the two-

electrode voltage clamp technique. AahG50 reduced Kv3.1

currents in a reversible concentration-dependent manner, with

an IC50 value and a Hill coefficient of 40.4 ± 0.2 lg/ml and

1.3 ± 0.05, respectively. AahG50 inhibited IKv3.1 without

modifying the current activation kinetics. The AahG50-

induced inhibition of Kv3.1 channels was voltage-dependent,

with a gradual increase at lower concentrations and over the

voltage range of channels opening. However, at higher con-

centrations, the inhibition exhibited voltage dependence only

in the first range of channels opening from -20 to ?10 mV,

but demonstrates a low degree of voltage-dependence when

channels are fully activated. In the literature, toxins have

previously been isolated from AahG50, KAaH1 and KAaH2

and were reported not to have any effect on IKv3.1. The

present article’s findings suggest that AahG50 may contain a

peptidic component active on Kv3.1 channels, which inhibits

IKv3.1 in a selective manner.

Keywords Potassium channel � Kv3.1 � Toxins �Androctonus australis hector � Kv3.1channel blocker

Introduction

Potassium channels form a large family of transmembrane

proteins that are present in almost living cells. They play

crucial roles in many different cellular functions, including

cell excitability, neurotransmitter release, signal transduction

and neuronal integration (Coetzee et al. 1999). Their wide

distribution in many tissues is associated with important

functions in excitable and non excitable cells that are now

better understood thanks to the discovery of animal toxins.

Kv3.1, one of the Shaw-type K? channel, is abundantly

expressed in neurons that have the ability to fire at high fre-

quencies (Perney et al. 1992; Wang et al. 1998). It is

expressed at particularly high levels in brainstem. Kv3.1 has

a number of features that distinguish it from other potassium

channels: a high activation threshold and very rapid activa-

tion and deactivation kinetics (Kanemasa et al. 1995). On the

other hand, the lack of Kv3.1 channel subunits is mainly

responsible for behavioral alterations that include impaired

motor performance, hyperactivity and sleep loss (McMahon

et al. 2004). Besides, potassium-channel-blocking scorpion

toxins have been shown to be valuable tools for the study of

potassium channels and so to understand many physiological

and pathophysiological processes (Salkoff et al. 1992). Sev-

eral toxins target Kv1 subunits and are useful in identifying

the function of low threshold channels (Geiger and Jonas

2000; Southan and Robertson 2000; Dodson et al. 2003).

There is a shortage of Kv3 subfamily specific-toxins, but

Diochot et al. (1998) reported that sea anemone toxins BDS-I

and BDS-II (blood depressing blockers) were highly specific

blockers for Kv3.4 in expression systems. According to Ye-

ung et al. (2005), these toxins are also able to inhibit Kv3.1

and Kv3.2 subunits. Nevertheless, some chemical molecules

have been reported to be active on Kv3.1 channels and used

in different therapeutic fields. Among which, the Prozac is

Imen Chatti—Former member in the laboratory.

A. Cheikh (&) � R. Benkhalifa (&) � Z. Landoulsi � I. Chatti �M. E. Ayeb

Laboratoire des Venins et Molecules Therapeutiques, Institut

Pasteur de Tunis, BP 74, 1002 Tunis, Tunisia

e-mail: cheikh_amani@yahoo.com

R. Benkhalifa

e-mail: rym.benkhalifa@pasteur.rns.tn

123

Arch. Pharm. Res.

DOI 10.1007/s12272-013-0176-5

known by its antidepressant activity (Choi et al. 1999) and the

psoralen by its anticonvulsant activity (Sung et al. 2009).

They are all blocking Kv3.1 channel, but also other different

channels subtypes, such as sibutramine (Kim et al. 2007) and

Riluzole (Ahn et al. 2005). In our laboratory, we are inter-

ested by the biochemical characterization of bioactive sub-

stances in scorpion venoms that have a potentially therapeutic

value. As a matter of fact, scorpion venoms are mixtures of

many polypeptides active on ion channels, in which only a

small proportion is lethal to mammals. Previous studies on

Androctonus australis hector (Aah) scorpion venom allowed

us to isolate toxins that were active on Kv1 subunit types,

such as KAaH1 active on Kv1.1 and Kv1.3 channels (Srairi-

Abid et al. 2005).

In the present work, we have studied the effect of the

toxic fraction of Aah venom (AahG50) on Kv3.1 channel

activity, expressed in Xenopus oocytes. We reported that

this fraction inhibited the Kv3.1 currents in a potent manner,

without modifying the kinetics of activation. The same

fraction was also shown to block Kv1.1 and Kv1.3 currents,

at lower concentrations. Since AahG50 is a mixture of

neurotoxins, we reported the toxins active on Kv3.1 chan-

nels to be different from those acting on Kv1.1 and Kv1.3.

Materials and methods

Purification of Aah venom

The venoms of Androctonus australis hector (Aah) was

collected from Beni Khedach (Tunisia) by the veterinary

department of Pasteur Institute—Tunisia and kept frozen at

-20 �c, in its crude form until used. The crude venom was

dissolved in water and loaded on Sephadex G50 gel filtration

chromatography column (K26/100, Pharmacia) to separate

the toxic fraction named AahG50. The column was equili-

brated and eluted at 15 �C with 0.1 M acetic acid buffer (pH

4.7), with a flow rate of 0.5 ml/min. Following (Miranda

et al. 1970), the detection was carried out at 280 nm, by a

spectrophotometer, namely BECKMAN DU 640. After

freeze-drying, the toxic fraction is stored at -20 �C until use.

Synthesis of Kv3.1 mRNA

The plasmid containing cDNA encoding for the Kv3.1 wild

type channel was graciously provided by Dr. Marcel Crest

(Research Center in Neurobiology and Neurophysiology of

Marseille). The DNA matrix was linearized by HpaI, and

capped cRNA was transcribed in vitro, using the SP6

mMessage mMachine kit (Ambion).

Electrophysiological measurements

Mature Xenopus laevis female were anaesthetized with

0.17 % solution of Tricaine (Ethyl 3-aminobenzoate

methanesulfonate salt, Sigma) during 45 min. Experiments

on Xenopus were approved by the Institutional Research

Board of Pasteur Institute—Tunis and carried out follow-

ing the European Community Council Directive (86/609/

EEC), for experimental animal care and procedures. Parts

of ovaries were then surgically removed from the abdom-

inal cavity and bathed in a calcium-free modified Barth’s

Solution (MBS: NaCl 88 mM, KCl 1 mM, MgCl20.41 mM, NaHCO3 2.4 mM, MgSO4 0.82 mM, HEPES

10 mM, pH 7.4). Oocytes were defolliculated enzymati-

cally by incubation in collagenases A and B (2 mg/ml,

Boehringer Roche), then stages V and VI oocytes were

selected by mechanical defolliculation, using forceps.

Individual oocytes were injected with 5–8 ng of mRNA,

using an automatic nano-injector (Nanoject, Drummond);

then incubated in MBS sterile solution: calcium-free MBS

added with CaCl2 2.4 mM, Ca (NO3)2 0.33 mM, Genta-

mycin (Sigma) 50 mg/l), pH 7.4, during 48 h at 18 �C and

before electrophysiological measurements were performed.

The MBS was changed once a day.

Electrophysiological measurements of Kv3.1 current

were recorded with the two electrodes voltage-clamp

method using a Gene Clamp 500 amplifier (Molecular

Devices, USA). Oocytes were immersed in Ca2? free MBS

and impaled with 2 intracellular glass electrodes filled with

3 M KCl. The resistance of the pulled electrodes (P-97

puller; Sutter Instruments, Novato, CA, USA) was 1–2 MW.

The perfusion system was controlled by a Manifold Solution

Changer (MSC-200; Bio-Logic; Grenoble, France). Data

acquisition and analysis were performed using Clampex and

Clampfit from pclamp8 software (Molecular Devices, Sun-

nyvale, CA, USA). Leak and capacitive currents were sub-

tracted during analysis. The study of AahG50 effect (10, 25,

50 and 100 lg/ml) was carried out on the Kv3.1 currents

recorded, using 250 ms depolarizing pulses from -40 to

?40 mV.

Electrophysiological results analyses

Obtained results are recorded and analyzed by the Pclamp 8.2

software (Molecular Devices). Activation curve is estimated

in terms of the relative conductance g/gmax (g = I/V - Vinv).

With Vinv as the inversion potential, curves are fitted by

Boltzmann equation: g/gmax = 1/(1 ? exp[(E0.5 - Em)/k],

where Em is the membrane potential, E0.5 is the half-activa-

tion potential and k is the slope factor.

A. Cheikh et al.

123

Results

Sephadex gel filtration of scorpion venoms

Sephadex gel filtration of scorpion venoms is carried out

according to Miranda et al. (1970) protocol. The profile of

purification of the venom of Androctonus australis hector

highlights the existence of four non-toxic fractions (M1–

M4) and one toxic fraction named AahG50 (Fig. 1).

Concentration-dependent effects on Kv3.1 currents

by AahG50

The effect of the toxic fraction of Androctonus australis

hector venom (AahG50) was tested on Kv3.1 channels

stably expressed in Xenopus oocytes and studied by means

of the two electrodes voltage-clamp technique. Figure 2a

shows the superimposed control Kv3.1 current traces under

control conditions and in the presence of different con-

centrations of AahG50 (5, 10, 25, 50 and 100 lg/ml),

obtained after a depolarizing step of ?20 mV, during

250 ms. AahG50 (5 lg/ml) was applied for *2 min and

the Kv3.1 currents were decreased to ?20 mV from 3.2 to

2.8 lA, whereas a more pronounced effect was obtained

with higher concentrations. The current amplitude

decreased from 3.2 to 2.2 lA (10 lg/ml) and from 3.2 to

1.7 lA (25 lg/ml) respectively; then reached 0.9 lA after

the perfusion of 50 lg/ml of AahG50.

The inhibition of Kv3.1 currents induced by AahG50

affect the current amplitude in a concentration-dependent

manner. Figure 2b shows the concentration dependence of

Kv3.1 current inhibition by AahG50. The concentration–

response curve was obtained by plotting the decrease of

currents at the end of a 250 ms pulse of ?40 mV in terms

of AahG50 concentration. The normalized currents were

fitted with the Hill equation, which yielded an IC50 value of

40.4 ± 0.2 lg/ml and a Hill coefficient of 1.3 ± 0.05

(n = 3–8).

To study the evolution of current inhibition as a time

function, the time-course of AahG50 (50 lg/ml) effect was

generated as shown in Fig. 2c. The inhibition appeared

after 10 s of the toxic fraction perfusion and the current

decreased gradually and reached a steady state within

3 min. Following a 1-min washout by means of MSB

perfusion, the current recovered nearly the control value

with a faster time course, indicating that the effect of

AahG50 is largely reversible upon washout.

Voltage-dependent effect on Kv3.1 currents,

by AahG50

The inhibitory effect of AahG50 was estimated according

to the variation of the outward potassium current by gen-

erating current–voltage (I–V) relationships in the range

-40 to ?40 mV. Currents were induced from a holding

potential of -80 mV, with potentials steps in 10 mV

increments, during 250 ms. Under control conditions, the

Kv3.1 expressing oocytes display outward fast-activating

potassium currents that rapidly deactivate (Fig. 3a left

panel). The perfusion of AahG50 50 lg/ml in the oocyte

bath strongly reduces the Kv3.1 current (Fig. 3a right

panel).

The voltage dependence of steady-state activation was

studied in both the absence and the presence of AahG50

(50 lg/ml) (Fig. 3b). Conductance–voltage (G–V) relation-

ships for Kv3.1 were calculated and the activation curve

obtained under control conditions was drawn by fitting the

normalized conductance to the Boltzmann equation. The

current activation threshold was approximately -20 mV, the

potential for half-maximum activation (V1/2) and the slope

factor value (k) were respectively 8.4 ± 0.5 and

8.3 ± 0.4 mV (n = 16). After the perfusion of AahG50

(50 lg/ml), the threshold of activation did not yield any

change. The activation curve shifted to more positive

potentials, the value of V1/2 was 13.7 ± 1.3 mV and k was

9.8 ± 1.2 mV (n = 9). The shift in the depolarizing direc-

tion in activation is about 5 mV even if the change in the

slope factor is not significant. In presence of AahG50, the

G–V curve saturated at a lower conductance level. In Fig. 3c,

the K? currents carried by Kv3.1 channels, displayed acti-

vation kinetics, which were best fitted by biexponential

function (Grissmer et al. 1992). The activation time constant

s was slightly voltage-dependent in potential range values

ranging from -10 to ?40 mV. In presence of AahG50

(50 lg/ml), the slowing of activation rate was more pro-

nounced at potentials ranging from -10 mV (s = 70 ±

9.7 ms) to ?10 mV (s = 28 ± 3.3 ms). At potentials higher

than ?10 mV, the slowing of activation rate is less noted

Fig. 1 Purification of Androctonus australis hector scorpion venom.

Dialyzed venom was loaded into a Sephadex G50 column and five

fractions were collected (M1, M2, AahG50, M3 and M4)

Inhibition of human Kv3.1 current

123

(s varies from 28 ± 3.3 ms at ?10 mV to 21.16 ± 4.4 ms at

?40 mV).

Figure 3d showed relative normalized current (IAahG50/

Icontrol) as a function of potential. The toxic fraction

decreased the current amplitude starting at 0 mV, when

applied at lower concentrations (10 and 25 lg/ml). From

-20 to 0 mV, the percentages of inhibition variations vary

not significantly, as a function of potentials. At these

concentrations, AahG50 decreased Kv3.1 current in a

voltage dependent manner at potential positive to ?10 mV.

At 10 lg/ml, the maximal inhibition rates observed at ?10

and ?40 mV were respectively 15.5 ± 3 and 31.6 ± 4 %,

when currents are fully activated.

At higher concentration (50 lg/ml), this inhibitory effect

includes the whole voltage range of channel opening

(Fig. 3d), but not in the same manner. The curve shape pre-

sents a biphasic evolution. From -20 to ?10 mV the

inhibitory effect increased in a rapid manner. Between -10

and ?10 mV, the current inhibited by 47.5 % from 14.5 ± 3

to 62 ± 5 %, which corresponds to a fourfold decrease.

However, in the range of ?10 to ?40 mV, we noted that the

inhibitory effect reached a steady state. The current inhibited

by 6.8 %, from 65.2 ± 4 % at ?20 mV to 72.2 ± 3 % at

?40 mV. These results suggested that the inhibition of

IKv3.1 by AahG50 was found to be clearly voltage-and

concentration-dependent. At low concentrations, the toxic

fraction acted at potentials positive to ?10 mV, while at

50 lg/ml its effect was observed in the whole activating

voltage range. The inhibition occurred in a biphasic manner

with a faster effect at potentials less than ?10 mV to reach

then, a steady state with a maximal effect about 72.2 %. The

AahG50 observed effect did not require fully activated

channel.

AahG50 inhibitory effect on Kv1.1 and Kv1.3 currents

In order to characterize the specificity of AahG50 inhibi-

tory effect on Kv3.1, we performed experiments on Kv1.1

and Kv1.3 channels. Figure 4a, b shows the effect of

AahG50 (10 lg/ml) on Kv1.1 current–voltage relationship.

Kv1.1 current traces were elicited from a holding potential

of -80 mV, by potentials ranging from -40 to ?40 mV,

in 10 mV increments during 250 ms in control conditions

(upper panel) and after the application of 10 lg/ml of

AahG50 (lower panel). The Kv1.1 current was activated at

pulses greater than -60 mV, and the steady state I–V

Fig. 2 Concentration dependence for AahG50 block of human Kv3.1

currents expressed in Xenopus oocytes. a Superimposed currents were

obtained by applying 250 ms pulses from a holding potential of -80

to ?20 mV every 3 s under control conditions and after the blockade

by 5, 10, 25, 50 and 100 lg/ml of AahG50. The inhibition levels were

respectively 13.9 % (±8.8 %, n = 3), 27.03 % (±7.2 %, n = 10),

56.81 % (±16.26 %, n = 5), 84.31 % (±7.2 %, n = 3) and 92.1 %

(±5.5, n = 2). b Dose–response curve for the inhibition of Kv3.1 by

various concentrations of AahG50. Currents amplitude were measured

at the end of the depolarizing pulse (?40 mV), the percentage of

inhibition was plotted against respective concentrations of AahG50.

The curve was fitted by the Hill equation which yielded an IC50 value

of 40.4 ± 0.25 lg/ml and a Hill coefficient of 1.3 ± 0.05 (n = 3–8

for each concentration). c Time course of inhibition in the presence of

AahG50, 50 lg/ml. Maximal effect occurs about 3 min after the

beginning of the perfusion. A complete recovery from the inhibitory

effect was observed after the washout of the toxic fraction

b

A. Cheikh et al.

123

relationship curve showed a linear shape between -40

and ? 60 mV (Fig. 4b). The inhibition by AahG50 was

observed in the whole voltage range over which current is

activated and a steady state inhibition was reached at

?20 mV, with 83.9 % of maximum of inhibition. The

same experiments were applied on Kv1.3 currents expres-

sed on Xenopus oocytes (Fig. 4c, d). The current inhibition

by AahG50 was shown at all potentials but did not reach

the saturation as observed with Kv1.1 channels. The steady

state I–V relationship curve showed a similar linear shape

as in the control conditions and the maximum of inhibition

was about 70,4 % at ?40 mV (Fig. 4d).

Effect of KAaH1 and KAaH2 on Kv3.1 channels

Given that KAaH1 and KAaH2 are two toxins that are active

on Kv1.1 and Kv1.3 channels and present in AahG50 (Srairi-

Abid et al. 2005), we tested them on Kv3.1 channels. Currents

were induced by a step to ?20 mV from a holding potential of

-80 mV, during 250 ms. Figure 5a (left and right panels) did

not show any significant effects on the control current traces

before and after the application of 100 nM of KAaH1 and

KAaH2. Histograms in Fig. 5b summarize data deduced from

different oocytes (n = 3–8). These results suggest that

AahG50 may contain other toxic compounds active on Kv3.1

channels and different from the toxin showing already spec-

ificity to Shaker potassium channels.

Discussion

The activity of Kv channels is crucial in maintaining the

resting membrane potential, regulating action potential

duration and frequency, and determining the pacemaker

activity in a variety of excitable cells (Jan and Jan 2012).

Besides, toxins from a variety of species have been used to

characterize and pharmacologically profiling different ion

channel types. The effect of toxins on specific ion channels

Fig. 3 Voltage dependent effect of the toxic venom fraction AahG50

on Kv3.1 currents. a Typical voltage activated Kv3.1 currents

expressed in Xenopus oocytes. Current traces are shown in the

absence (left panel) and the presence (right panel) of 50 lg/ml

AahG50. Currents were elicited from a holding potential of -80 mV

by pulses applied in 10 mV increments to potentials ranging from

-40 to ?40 mV (above the figure). b Effect of AahG50 on the

steady-state activation of Kv3.1 currents. The normalized conduc-

tance-voltage relationship of Kv3.1 currents in Xenopus oocytes were

plotted under control conditions and after the perfusion of 50 lg/ml

AahG50. Data were normalized and fitted to the Boltzmann equation.

The V1/2 and the slope factor were respectively 8.3 ± 0.5 mV and

8.4 ± 0.4 (n = 16) for control conditions, and 13.6 ± 1.3 mV and

9.8 ± 1.2 (n = 9) under 50 lg/ml AahG50 application. c Activation

rate of Kv3.1 in absence and presence of AahG50 (50 mV) venom

fraction. Activation kinetics was fitted by biexponential function for

potentials positive to -10 mV. d Voltage-dependant inhibition of

Kv3.1 peak current by 10, 25 and 50 lg/ml of AahG50. Normalized

inhibition was expressed as a relative current (IAaHG50/Icontrol) for

potentials positive to -20 mV

Inhibition of human Kv3.1 current

123

has had a huge impact on the understanding of the physi-

ological as well as pathophysiological role of different ion

channels in cell functions (Grissmer and Tytgat 2004).

The present work describes the selective inhibition of

Androctonus australis hector toxic venom fraction (AahG50)

on cloned neuronal human Kv3.1 expressed in Xenopus

oocytes, using the two electrodes voltage clamp technique.

We were interested by studying whether AahG50 acts on

IKv3.1, and in which manner. AahG50 is composed of

several neurotoxins. Some ones are active on voltage gated

sodium channels such as AaHI, a scorpion a-toxin, con-

sidered as a channel gating-modifier toxin (Bosmans and

Tytgat 2007). In the literature, rarely are toxins reported

active on Kv channels and their mechanism of action is

usually not described (Srairi-Abid et al. 2005). In order to

investigate the AahG50 action mode on Kv3.1 channel, the

toxin classification suggested by Miller in 1995 seems

more suitable to discuss our results. According to this

classification, toxins are grouped into a first class of pore-

blocking toxins that interact with the external vestibule of

the ion conduction pore and a second class that interacts

with the voltage sensing domains of voltage-gated ion

channels and modifies the gating behavior of the channel,

through allosteric mechanisms (Miller 1995).

This study demonstrates that AahG50, inhibited IKv3.1

in a dose-dependent manner, within the concentration

range of 1–100 lg/ml. The inhibition rate of Kv3.1 current

amplitude is about 93 % at 100 lg/ml.

The characteristic of the AahG50-induced inhibition is

based on the following deductions: (1) AahG50 inhibited

IKv3.1, with an IC50 value of 40.4 lg/ml and a Hill coef-

ficient of 1.3. The washout allows the initial current

amplitude recovery. (2) IKv3.1 is inhibited without any

effect on the kinetics of channel activation. (3) In the con-

centration range of 5–50 lg/ml, we have noted that AahG50

effect is characterized by an acceleration of the rate decay.

Previous works, investigating the effect of scorpion

venom fractions, on in vitro expressed ion channels are

very rare. The dialyzed venom of Androctonus mauritani-

cus mauritanicus (Amm) totally blocked KCa current of

Helix U cells at 45 lg/ml (Crest et al. 1992). In a previous

work, we have reported the effect of the non toxic fraction

Fig. 4 AahG50 venom fraction inhibitory effect on Kv1.1 and Kv1.3

currents expressed in Xenopus oocytes. a Voltage-activated Kv1.1

current traces are shown in the absence (upper panel) and the

presence of 10 lg/ml AahG50 (lower panel). Currents were activated

by a voltage step protocol from a holding potential of -80 mV

followed by steps to voltages ranging from -40 to ?40 mV in 10 mV

increments during 250 ms. b The I–V relationship of Kv1.1. Control

currents are shown with empty circles and AahG50 inhibited currents

are shown with filled circles. The inhibition rate was 83.9 % at

?40 mV. c Voltage-activated Kv1.3 current traces are shown in the

absence (upper panel) and the presence of 10 lg/ml AahG50 (lower

panel). Currents were recorded using the same protocol described in

A. d The I–V relationship of Kv1.3. Control currents are shown with

empty circles and AahG50 inhibited currents are shown with filled

circles. The inhibition rate was 70.4 % at ?40 mV

A. Cheikh et al.

123

of Buthus occitanus tunetanus scorpion venom (M1 Bot)

on rat heart contraction and L-type calcium current (ICaL).

Studies with the whole cell patch clamp technique showed

that M1 decreased the ICaL in a dose-dependent manner

with an IC50 of 0.36 lg/ml. Besides, at 10 lg/ml this

fraction also induced a decrease in both heart tension and

rate (Cheikh et al. 2006).

The activation kinetics in presence of AahG50 (50 lg/ml)

demonstrates an inhibitory effect from the threshold poten-

tial (-20 mV) and the channel saturation is reached at

earlier potentials. In the same manner as gambierol and

subitramine, AahG50 doesn’t require the open channel state

to bind to Kv3.1 channels (Kim et al. 2007; Kopljar et al.

2009). AahG50 shifts slightly the V1/2 by about 5 mV

contrary to BDS toxins, which shift the V1/2 of the steady-

state activation curve by 10–20 mV to more positive

potentials (Yeung et al. 2005), and don’t induce a significant

change in the slope factor (1 mV).

The gambierol, belonging to the group of ciguatera

toxins, is active on both voltage-gated sodium and

potassium channels with different potencies (Perez et al.

2012). It inhibits Kv3.1 channels in a potent and reversible

manner, by stabilizing the closed state of the channel,

which results in a ‘‘loss of function’’ (Kopljar et al. 2009).

AahG50 increases the activation rate decay, s is 70.9 ms

instead of 43.5 ms, but the slowing down is not regular and

reaches a steady state at ?20 mV. Following the study of

the voltage-dependent inhibition of IKv3.1 by AahG50 at

different concentrations, we noted some differences. In

fact, at lower concentrations the inhibition increases

gradually with voltages. At higher concentrations, the

voltage-dependence is clearly marked in the range of -20

to ?10 mV, corresponding to the beginning of current

activation. From ?10 to ?40 mV, the inhibition produced

by AahG50 demonstrates a low degree of inhibition,

despite Kv3.1 being fully activated at this voltage range.

Sung and colleagues have explained this behavior in

presence of fluoxetine as a consequence of the effects of

the transmembrane electrical field on the interaction

between fluoxetine and Kv3.1 (Sung et al. 2008). Given

that the presence of several neurotoxins in AahG50 doesn’t

allow concluding the mode of interaction with Kv3.1, the

biochemical purification program for isolating the active

molecule is settled up.

Like fluoxetine, the psoralen inhibits Kv3.1 channel

without modifying the kinetics of current activation (Sung

et al. 2009). The psoralen-induced inhibition is voltage-

dependent with a steep increase over the voltage range of

channel opening. However, the inhibition exhibits voltage

independence over the voltage range, in which channels are

fully activated (Sung et al. 2008) reminding the AahG50

effect. Besides, AahG50 is also able to reduce Shaker

potassium current Kv1.1 and Kv1.3 subtypes with more

potency. At 10 lg/ml, AahG50 inhibits 27 % of IKv3.1 but

83.9 % of IKv1.1 and 70.4 % of IKv1.3. We have previously,

characterized two toxins; KAaH1 and KAaH2. KAaH1

inhibits Kv1.1 and Kv1.3 channels with IC50 of 5 and 50 nM

respectively, while KAaH2 (100 nM) blocks 20 % of Kv1.1

and is inactive on Kv1.3. Interestingly, 100 nM of each toxin

was applied on Kv3.1. They both neither affected the current

amplitude nor the kinetics. This observed results parallel

reported data by Cotton et al. (1997), that Shaker-related Kvl

channels are equally affected by Bunodosoma granulifera

(BgK) (Kd = 6 nM for Kvl.1, 15 nM for Kv1.2, 10 nM for

Kv1.3), while Kv3.1 is insensitive up to 0.125 lM toxin

Commonly, compounds are considered as potential

pharmacological tools when they are active at a standard

concentration level. In fact, they lack their efficiency at

high concentrations and could be toxic. We therefore

deduced that AahG50, contains an other active component

on Kv3.1 channels different from KAaH1 and KAaH2,

previously described. When isolated, this component

should be tested on Kv1.1 and Kv1.3.

Fig. 5 a Current traces following the same protocols as Fig. 2 are

shown in the absence and in the presence of 100 nM of KAaH1

(upper panel) and KAaH2 (lower panel). The protocol used for

currents recordings is represented in the figure. b Histograms

representing the effect of 100 nM of KAaH1 (n = 3) and KAaH2

(n = 8) on Kv3.1 currents at ?20 mV

Inhibition of human Kv3.1 current

123

According to our results, we could consider that this

component is able to inhibit IKv3.1, by blocking the

channel pore without affecting its conformation. As sug-

gested for psoralen, the block of Kv3.1 by AahG50 may

have significant pharmacological relevance to its possible

anticonvulsant activity (Sung et al. 2009).

Evidently, biochemical purification and characterization

of such toxin in scorpion venom would be useful not only

to carry on structure–function relationship studies, but

should enhance the development of new therapeutic tools

for the treatment of a variety of neurodegenerative diseases

including Kv3.1 channel hyperactivity.

Acknowledgments Thanks are addressed to Professor Hechmi

Louzir, General Director of Pasteur Institute for constant encour-

agements. The authors thank Dr. Zakaria Belasfer, for taking care of

animals and providing us venom; Thouraya Chagour, for venom fil-

tration. Dr. Romain Guinamard, Professor in the University of Caen,

for his valuable remarks and Salaheddine Mnasri for English cor-

rections. This work was supported by Pasteur Institute, the Ministry of

Public Health and the Ministry of Research and Development (lmvt).

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