1. Biochemical and Biophysical Research Communications 375 (2008) 571–575
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Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
Some effects of the venom of the Chilean spider Latrodectus mactans on
endogenous ion-currents of Xenopus laevis oocytes
Jorge Parodi a,b,*, Fernando Romero a, Ricardo Miledi b, Ataúlfo Martínez-Torres b
a
Laboratorio de neurociencias-CEBIOR, Departamento de Ciencias Preclínicas, Facultad de Medicina, Universidad de la Frontera,
Av. Francisco Salazar 01145, P.O. Box 54-D, Temuco, Chile
b
Laboratorio Neurobiología Molecular y Celular II, Departamento de Neurobiología Molecular y Celular, Instituto de Neurobiología, Campus Juriquilla-Querétaro, UNAM, Mexico
a r t i c l e i n f o a b s t r a c t
Article history: A study was made of the effects of the venom of the Chilean spider Latrodectus mactans on endogenous
Received 11 August 2008 ion-currents of Xenopus laevis oocytes. 1 lg/ml of the venom made the resting plasma membrane poten-
Available online 24 August 2008 tial more negative in cells voltage-clamped at À60 mV. The effect was potentially due to the closure of
one or several conductances that were investigated further. Thus, we determined the effects of the venom
on the following endogenous ionic-currents: (a) voltage-activated potassium currents, (b) voltage-acti-
Keywords: vated chloride-currents, and (c) calcium-dependent chloride-currents (Tout). The results suggest that
Venom
the venom exerts its action mainly on a transient outward potassium-current that is probably mediated
Latrodectus mactans
Electrophysiology
by a Kv channel homologous to shaker. Consistent with the electrophysiological evidence we detected the
Oocytes expression of the mRNA coding for xKv1.1 in the oocytes.
Potassium channels Ó 2008 Elsevier Inc. All rights reserved.
Xenopus laevis
.
a-Latrotoxin (LTX) is the main component of the venom of the Material and methods
euroasiatic black widow spider Latrodectus mactans [1]. LTX is a po-
tent synaptic modulator that induces the release of neurotransmit- Spider retrieval. Female adult L. mactans spiders from Chile were
ters from vertebrate nerve terminals by inhibiting potassium captured during the summer months (December 2005 and January
channels and blocking L-type calcium channels [2–4]. In addition, 2006) from the area of Alto Bio-Bio in the VIII region (72°160 5100 W,
this toxin assembles in multimeric complexes in the plasma mem- 7°450 2400 S). The specimens were maintained separate in individual
brane forming a cation permeable channel [5]. jars for 30 days without food and given only water in order to stim-
In contrast to the high concentration of LTX in spider populations ulate the production and concentration of venom in the glands [7].
from Europe and Asia, this toxin has not been detected in the Chilean Venom retrieval. The spiders were immersed in liquid nitrogen
L. mactans, although the symptoms of patients after spider bitting and after 1 min transferred to a phosphate buffer saline (PBS:
are very similar. The most common symptoms include muscle con- NaH2PO4 0.1 M, Na2HPO4 0.01 M, NaCl 1.35 M, pH 7.4) at 4 °C.
traction, vomiting, and cephalea. The rate of survival of patients at- The glands were removed and placed in a tube containing PBS
tacked by the South American species is higher [6], most probably (25 pairs of glands for 100 ll of PBS) and homogenized. The
due to the low concentration or total absence of LTX. homogenate was immediately centrifuged at 1000g for 15 min
Although L. mactans from Chile apparently does not posses LTX and the supernatant was subsequently aliquoted and frozen at
in the venom, this contains several oligopeptides which have been À20 °C. The total protein concentration was determined by the
shown to induce muscle contraction and inhibit sperm motility; ef- method of Bradford [9] and dissolved in Ringer to making the final
fects that are suspected to be due to the modulation or blocking of concentration 1 lg/ml in the experimental solutions.
ion channels [7,8]. In order to determine the targets for the modu- Oocyte recordings. Oocytes were collected from several frogs
latory actions of the venom, we report here an examination of the (Nasco), manually dissected from the ovary and treated with colla-
effect of whole extracts of the venom of the Chilean L. mactans on genase (0.5 mg/ml, 30 min), then kept at 15–16 °C in Barth’s solu-
several native ion currents of X. laevis oocytes. tion supplemented with gentamicin (0.1 mg/ml). Recordings were
performed in oocytes superfused with frog Ringer at room temper-
ature (20–25 °C). Membrane currents were recorded from oocytes
* Corresponding author. Address: Laboratorio de neurociencias-CEBIOR, Departa- voltage-clamped at À60 mV and then voltage stepped using four
mento de Ciencias Preclínicas, Facultad de Medicina, Universidad de la Frontera, Av. protocols to activate an hyperpolarization-activated current [25],
Francisco Salazar 01145, P.O. Box 54-D, Temuco, Chile.
a calcium-dependent chloride-current [10], an outwrdly rectifying
E-mail address: jparodi@ufro.cl (J. Parodi).
0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2008.08.070

2. 572 J. Parodi et al. / Biochemical and Biophysical Research Communications 375 (2008) 571–575
Fig. 1. Curents gated by L. mactans venom. (A) Sample records from an oocyte exposed to the spider venom at 1 or 5 lg/ml. (B) Plot of averaged currents generated by 100 or
500 nM (6 oocytes). (C) Currents generated by 20 mV increments in the absence or presence of 1 lg/ml venom gave the I/V shown in (D) (9 oocytes). Data from experiments
using 0.01 and 10 lg/ml venom are also shown. *p < 0.05 vs control values.
chloride current [16] or a transient-outward potassium-current any effect on the calcium-dependent chloride-current activated
[12]. In addition, serum was perfused onto some oocytes to see through the lysophosphatidic acid (LPA) receptor [13]. TEA (0.03–
Fig. 2. Voltage-and calcium-activated chloride channels. (A) Tout recorded by stepping the voltage fom À100 mV to either 0 or +20 mV in the absence or presence of 1 lg/ml
of venom. (B) Oscillatory chloride-currents generated by serum (1:1000) were not affected by the venom.

3. J. Parodi et al. / Biochemical and Biophysical Research Communications 375 (2008) 571–575 573
2 mM), 4-AP (0.03–2 mM) or low calcium Riger were perfused onto tion at a membrane potential of about À20 mV, which is near the
several oocytes to observe the potential role of potassium-, so- equilibrium potential for chloride in oocytes; furthermore this
dium- and calcium-channels respectively. Moreover, several oo- reversal potential was independent of the venom concentration
cytes were frozen in liquid nitrogen to isolate RNA [14] and RT- at all tested concentrations (Fig. 1D). This result was contrary to
PCR using primers designed to amplify xKv 1.1 (GenBank Accession a previous report [8] suggesting that the venom modulates mainly
No. M94258) [15]. voltage dependent calcium-channels. Therefore we decided to
Data analysis. All the recordings were acquired using a Warner investigate further other venom effects on endogenous currents.
amplifier (OC-765C) and an AC/DC converter (Digidata 1322A,
Axon) and stored in winWCP. Files were analyzed and plotted Calcium-dependent chloride-currents
using pClampfit 9 (Axon, molecular devices) and Origin 6.0
respectively. Frog oocytes normally possess chloride-channels that are acti-
vated by either calcium ions permating through voltage-operated
Results channels (Tout) [11] or calcium released from intracellular stores
due to the activation of G-protein coupled receptors such as the
Activation of a smooth slow-activating ionic-current LPA receptor that is effectively activated with serum [13].
Perfussion of 1 lg/ml of the venom extract in 9 oocytes and 3
Perfusion of the venom generated a slow-activating non-desen- frogs, did not produce any evident effect on the Tout current
sitizing current in oocytes held at À60 mV (Fig. 1A). Those currents (962 ± 240 vs 820 ± 216 nA Fig. 2A), thus discounting its ability to
were clearly evident from 0.01 lg/ml and saturated at about 5 lg/ block either voltage-dependent calcium-channels or the calcium-
ml, this concentration gave rise to currents that averaged dependent chloride-current. In addition, the oscillatory chloride
60 ± 20 nA from 9 oocytes and 3 frogs. The venom gave an EC50 currents elicited by activation of the LPA receptor did not show
of about 1 lg/ml and therefore this concentration was used for any indication of being affected by the venom (Fig. 2B).
the following experiments (Fig. 1B).
We tried to define the ion responsible for the current by con- Voltage-dependent chloride-currents
structing an I/V curve, stepping the membrane voltage of the
oocyte from the holding potential (À60 mV) to À100 mV and then Two main conductances carried out by chloride ions are known
to 40 mV in 20 mV steps while in the absence or presence of 0.01, in the oocyte: (1) an outward rectifying current, probably due to
0.1 or 10 lg/ml of the venom (Fig. 1C). The current reversed direc- the expression of ClC5 [16] and an hyperpolarizing inward current
Fig. 3. Voltage-activated chloride-currents. (A) Sample traces of the outwardly rectifying chloride current. (B) This conductance was not affected by the venom, and was also
evidenced by substituting ClÀ with IÀ. (C) Hyperpolarizing currents generated by stepping the oocyte membrane potential from À60 to À140 mV were potentiated by 1 lg/ml
of venom with an EC50 of 0.3 lg/ml (D). (E) The potentiation was evident at all tested membrane potentials as shown in I/V curves. *p < 0.05 vs control values.

4. 574 J. Parodi et al. / Biochemical and Biophysical Research Communications 375 (2008) 571–575
whose molecular identity is unknown and is a mixture of anionic 0.5 s to +40 mV, brought back to À80 mV and finally returned to
and cationic conductances [25]. À60 mV. This protocol of activation evidenced an outward rectify-
Fig. 3A shows that up to 1 lg/ml the venom did not have an evi- ing potassium current which is sensitive to TEA. Thus, we con-
dent effect on the outwardly-rectifying chloride-current. To see trasted the effect of 5 mM TEA with that of the venom during the
more clearly this current, chloride was substituted by iodide in peaks of hyperpolarization and depolarization and plotted the re-
the bathing solution. This did not alter the results found in normal sults in Fig. 4B and C. In addition, the venom is more potent block-
´
Ringers (Fig. 3B). ing the currents than TEA at all tested voltages in Fig. 4 D and E.
On the other hand, hyperpolarizing the membrane potential of Considering the strong blocking of the venom on the potassium
the oocyte from À60 to À140 mV revealed a current that is mainly current we tested the effect of several concentrations of TEA (0.1–
due to a mixture of conductances. Fig. 3C shows sample traces of 5 mM) to try to learn about the molecular identity of the channel.
this current in the presence or absence of the venom which poten- 1 mM TEA blocked considerably the current, suggesting that a Kv
tiates the current up to 100% in a dose-dependent manner with an channel may be involved [17]. Therfore, we tried to detect the
EC50 of 0.3 lg/ml (Fig. 3D). The potentiation was evident at all the expression of the xKv 1.1 gene in the oocyte. This channel has some
hyperpolarizing voltages tested (Fig. 3E); but when the membrane of the properties and TEA sensitivity that we illustrate in Fig. 4F.
was depolarized the venom partially blocked the current. This part Fig. 4G shows the result of an RT-PCR detecting the transcipt for
of the protocol suggested the blocking of conductances other than xKv 1.1 in RNA isolated from defolliculated oocytes and from heart,
chloride which could be due to potassium channels. where it is well know that Kv1.1 is expressed.
Blocking of an outward-rectifying potassium-current Discussion
Fig. 4A shows sample recordings of an oocyte whose membrane The venom of the Euroasiatic black widow affects synaptic func-
potential was changed from À60 to À80 mV then depolarized for tion due to the effect of the LTX [18–20]. However, some reports
Fig. 4. Potassium current. (A) The outward-rectifying potassium-current is sensitive to 5 mM TEA and 1 lg/ml venom also blocks strongly the current. (B,C) Plots of the
blocked current by either 5 mM TEA or 1 lg/ml venom for voltage steps from À70 to 40 (B) and from 40 to À90 mV (C). (D) Plot from sample traces control and a venom
current from (E). (F) TEA sensitivity of the potassium current suggests the expression of a Kv channel in oocytes. (G) RT-PCR identified the mRNA of xKv 1.1 in samples from
heart and oocytes. Tubulin was also present in both samples. *p < 0.05 vs control values.

5. J. Parodi et al. / Biochemical and Biophysical Research Communications 375 (2008) 571–575 575
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postdoctoral fellow from CTIC-UNAM and from MIDEPLAN-Chile. binding protein consists of a family of related high molecular weight
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