Biochemical and Biophysical Research Communications 375 (2008) 571–575

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Biochemical and Biophysical Research Communications

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Some effects of the venom of the Chilean spider Latrodectus mactans onendogenous 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, Chileb 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:Received 11 August 2008Available online 24 August 2008

Keywords:VenomLatrodectus mactansElectrophysiologyOocytesPotassium channelsXenopus laevis.

0006-291X/$ - see front matter � 2008 Elsevier Inc. Adoi:10.1016/j.bbrc.2008.08.070

* Corresponding author. Address: Laboratorio de nemento de Ciencias Preclínicas, Facultad de Medicina, UFrancisco Salazar 01145, P.O. Box 54-D, Temuco, Chil

E-mail address: jparodi@ufro.cl (J. Parodi).

A study was made of the effects of the venom of the Chilean spider Latrodectus mactans on endogenousion-currents of Xenopus laevis oocytes. 1 lg/ml of the venom made the resting plasma membrane poten-tial more negative in cells voltage-clamped at �60 mV. The effect was potentially due to the closure ofone or several conductances that were investigated further. Thus, we determined the effects of the venomon the following endogenous ionic-currents: (a) voltage-activated potassium currents, (b) voltage-acti-vated chloride-currents, and (c) calcium-dependent chloride-currents (Tout). The results suggest thatthe venom exerts its action mainly on a transient outward potassium-current that is probably mediatedby a Kv channel homologous to shaker. Consistent with the electrophysiological evidence we detected theexpression of the mRNA coding for xKv1.1 in the oocytes.

� 2008 Elsevier Inc. All rights reserved.

a-Latrotoxin (LTX) is the main component of the venom of theeuroasiatic black widow spider Latrodectus mactans [1]. LTX is a po-tent synaptic modulator that induces the release of neurotransmit-ters from vertebrate nerve terminals by inhibiting potassiumchannels and blocking L-type calcium channels [2–4]. In addition,this toxin assembles in multimeric complexes in the plasma mem-brane forming a cation permeable channel [5].

In contrast to the high concentration of LTX in spider populationsfrom Europe and Asia, this toxin has not been detected in the ChileanL. mactans, although the symptoms of patients after spider bittingare very similar. The most common symptoms include muscle con-traction, vomiting, and cephalea. The rate of survival of patients at-tacked by the South American species is higher [6], most probablydue to the low concentration or total absence of LTX.

Although L. mactans from Chile apparently does not posses LTXin the venom, this contains several oligopeptides which have beenshown to induce muscle contraction and inhibit sperm motility; ef-fects that are suspected to be due to the modulation or blocking ofion channels [7,8]. In order to determine the targets for the modu-latory actions of the venom, we report here an examination of theeffect of whole extracts of the venom of the Chilean L. mactans onseveral native ion currents of X. laevis oocytes.

ll rights reserved.

urociencias-CEBIOR, Departa-niversidad de la Frontera, Av.

e.

Material and methods

Spider retrieval. Female adult L. mactans spiders from Chile werecaptured during the summer months (December 2005 and January2006) from the area of Alto Bio-Bio in the VIII region (72�160510 0W,7�450240 0S). The specimens were maintained separate in individualjars for 30 days without food and given only water in order to stim-ulate the production and concentration of venom in the glands [7].

Venom retrieval. The spiders were immersed in liquid nitrogenand after 1 min transferred to a phosphate buffer saline (PBS:NaH2PO4 0.1 M, Na2HPO4 0.01 M, NaCl 1.35 M, pH 7.4) at 4 �C.The glands were removed and placed in a tube containing PBS(25 pairs of glands for 100 ll of PBS) and homogenized. Thehomogenate was immediately centrifuged at 1000g for 15 minand the supernatant was subsequently aliquoted and frozen at�20 �C. The total protein concentration was determined by themethod of Bradford [9] and dissolved in Ringer to making the finalconcentration 1 lg/ml in the experimental solutions.

Oocyte recordings. Oocytes were collected from several frogs(Nasco), manually dissected from the ovary and treated with colla-genase (0.5 mg/ml, 30 min), then kept at 15–16 �C in Barth’s solu-tion supplemented with gentamicin (0.1 mg/ml). Recordings wereperformed in oocytes superfused with frog Ringer at room temper-ature (20–25 �C). Membrane currents were recorded from oocytesvoltage-clamped at �60 mV and then voltage stepped using fourprotocols to activate an hyperpolarization-activated current [25],a calcium-dependent chloride-current [10], an outwrdly rectifying

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 or500 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 experimentsusing 0.01 and 10 lg/ml venom are also shown. *p < 0.05 vs control values.

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chloride current [16] or a transient-outward potassium-current[12]. In addition, serum was perfused onto some oocytes to see

Fig. 2. Voltage-and calcium-activated chloride channels. (A) Tout recorded by stepping thof venom. (B) Oscillatory chloride-currents generated by serum (1:1000) were not affec

any effect on the calcium-dependent chloride-current activatedthrough the lysophosphatidic acid (LPA) receptor [13]. TEA (0.03–

e voltage fom �100 mV to either 0 or +20 mV in the absence or presence of 1 lg/mlted by the venom.

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 ontoseveral oocytes to observe the potential role of potassium-, so-dium- and calcium-channels respectively. Moreover, several oo-cytes were frozen in liquid nitrogen to isolate RNA [14] and RT-PCR using primers designed to amplify xKv 1.1 (GenBank AccessionNo. M94258) [15].

Data analysis. All the recordings were acquired using a Warneramplifier (OC-765C) and an AC/DC converter (Digidata 1322A,Axon) and stored in winWCP. Files were analyzed and plottedusing pClampfit 9 (Axon, molecular devices) and Origin 6.0respectively.

Results

Activation of a smooth slow-activating ionic-current

Perfusion of the venom generated a slow-activating non-desen-sitizing current in oocytes held at �60 mV (Fig. 1A). Those currentswere clearly evident from 0.01 lg/ml and saturated at about 5 lg/ml, this concentration gave rise to currents that averaged60 ± 20 nA from 9 oocytes and 3 frogs. The venom gave an EC50

of about 1 lg/ml and therefore this concentration was used forthe following experiments (Fig. 1B).

We tried to define the ion responsible for the current by con-structing an I/V curve, stepping the membrane voltage of theoocyte from the holding potential (�60 mV) to �100 mV and thento 40 mV in 20 mV steps while in the absence or presence of 0.01,0.1 or 10 lg/ml of the venom (Fig. 1C). The current reversed direc-

Fig. 3. Voltage-activated chloride-currents. (A) Sample traces of the outwardly rectifyingevidenced by substituting Cl�with I�. (C) Hyperpolarizing currents generated by steppingof venom with an EC50 of 0.3 lg/ml (D). (E) The potentiation was evident at all tested m

tion at a membrane potential of about �20 mV, which is near theequilibrium potential for chloride in oocytes; furthermore thisreversal potential was independent of the venom concentrationat all tested concentrations (Fig. 1D). This result was contrary toa previous report [8] suggesting that the venom modulates mainlyvoltage dependent calcium-channels. Therefore we decided toinvestigate further other venom effects on endogenous currents.

Calcium-dependent chloride-currents

Frog oocytes normally possess chloride-channels that are acti-vated by either calcium ions permating through voltage-operatedchannels (Tout) [11] or calcium released from intracellular storesdue to the activation of G-protein coupled receptors such as theLPA receptor that is effectively activated with serum [13].

Perfussion of 1 lg/ml of the venom extract in 9 oocytes and 3frogs, did not produce any evident effect on the Tout current(962 ± 240 vs 820 ± 216 nA Fig. 2A), thus discounting its ability toblock either voltage-dependent calcium-channels or the calcium-dependent chloride-current. In addition, the oscillatory chloridecurrents elicited by activation of the LPA receptor did not showany indication of being affected by the venom (Fig. 2B).

Voltage-dependent chloride-currents

Two main conductances carried out by chloride ions are knownin the oocyte: (1) an outward rectifying current, probably due tothe expression of ClC5 [16] and an hyperpolarizing inward current

chloride current. (B) This conductance was not affected by the venom, and was alsothe oocyte membrane potential from �60 to �140 mV were potentiated by 1 lg/mlembrane potentials as shown in I/V curves. *p < 0.05 vs control values.

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whose molecular identity is unknown and is a mixture of anionicand cationic conductances [25].

Fig. 3A shows that up to 1 lg/ml the venom did not have an evi-dent effect on the outwardly-rectifying chloride-current. To seemore clearly this current, chloride was substituted by iodide inthe bathing solution. This did not alter the results found in normalRingers (Fig. 3B).

On the other hand, hyperpolarizing the membrane potential ofthe oocyte from �60 to �140 mV revealed a current that is mainlydue to a mixture of conductances. Fig. 3C shows sample traces ofthis current in the presence or absence of the venom which poten-tiates the current up to 100% in a dose-dependent manner with anEC50 of 0.3 lg/ml (Fig. 3D). The potentiation was evident at all thehyperpolarizing voltages tested (Fig. 3E); but when the membranewas depolarized the venom partially blocked the current. This partof the protocol suggested the blocking of conductances other thanchloride which could be due to potassium channels.

Blocking of an outward-rectifying potassium-current

Fig. 4A shows sample recordings of an oocyte whose membranepotential was changed from �60 to �80 mV then depolarized for

Fig. 4. Potassium current. (A) The outward-rectifying potassium-current is sensitive toblocked current by either 5 mM TEA or 1 lg/ml venom for voltage steps from �70 to 4current from (E). (F) TEA sensitivity of the potassium current suggests the expression ofheart and oocytes. Tubulin was also present in both samples. *p < 0.05 vs control values

0.5 s to +40 mV, brought back to �80 mV and finally returned to�60 mV. This protocol of activation evidenced an outward rectify-ing potassium current which is sensitive to TEA. Thus, we con-trasted the effect of 5 mM TEA with that of the venom during thepeaks of hyperpolarization and depolarization and plotted the re-sults in Fig. 4B and C. In addition, the venom is more potent block-ing the currents than TEA at all tested voltages in Fig. 4 D and E.

Considering the strong blocking of the venom on the potassiumcurrent we tested the effect of several concentrations of TEA (0.1–5 mM) to try to learn about the molecular identity of the channel.1 mM TEA blocked considerably the current, suggesting that a Kvchannel may be involved [17]. Therfore, we tried to detect theexpression of the xKv 1.1 gene in the oocyte. This channel has someof the properties and TEA sensitivity that we illustrate in Fig. 4F.Fig. 4G shows the result of an RT-PCR detecting the transcipt forxKv 1.1 in RNA isolated from defolliculated oocytes and from heart,where it is well know that Kv1.1 is expressed.

Discussion

The venom of the Euroasiatic black widow affects synaptic func-tion due to the effect of the LTX [18–20]. However, some reports

5 mM TEA and 1 lg/ml venom also blocks strongly the current. (B,C) Plots of the0 (B) and from 40 to �90 mV (C). (D) Plot from sample traces control and a venoma Kv channel in oocytes. (G) RT-PCR identified the mRNA of xKv 1.1 in samples from.

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indicate other effects due to several minor components of the ve-nom, in particular those that cause the systemic effects whichmay be generated by low molecular weight peptides [21,22]. Inthe Chilean L. mactans, several experiments suggested that the no-cive effects are also mediated by the impact of low molecularweight components of the venom on the function of ion channelsand the results described here explain in part the previously re-ported modulatory actions of the venom on cells such as the spermcell [8].

To try to explain the effects of the venom on ion channels,endogenous conductances of the frog oocyte were investigated[11, 12, 23–25]. The voltage-operated calcium-and chloride-cur-rents were not sensitive to the concentrations of venom tested.In addition, the calcium-dependent chloride-currents, which aremainly generated by the bestrophins [26] were not affected whenapplying up to 5 lg/ml of the venom. Therefore, in the venom ex-tract used for these experiments we have no evidence of blockingor modulatory effects on either voltage gated calcium channels orthe calcium gated -chloride currents. In contrast, a previous reportexplained the blocking of calcium channels with the L. mactans ve-nom [8]. The discrepancy with the present results may be due todifferences in the molecular identities of voltage-activated cal-cium-channels of frog oocytes and sperm cells [8]. Unfortunately,thus far, the calcium channels present in the oocyte have not beencloned.

Potassium currents of the oocyte were also investigated [27].The genes coding for the channels responsible for these currentshave not been fully characterized, but due to their functional prop-erties and TEA sensitivity shown in Fig. 4 we assumed they may bepart of the Kv family. The L. mactans venom efficiently and revers-ibly blocked the transient-outward potassium-current, whereas noapparent effects were found in other potassium conductances. Inorder to confirm the expression of a Kv channel in the oocytes,we searched for the mRNA coding for the xKv 1.1 [17] channelwhich generates currents similar to those of the endogenous potas-sium channel of the oocyte. RT-PCR indirectly evidenced theexpression of this channel (Fig. 4G). In conclusion, the results pre-sented here suggest that the L. mactans venom blocks voltage acti-vated potassium channels. Nevertheless, more studies will benecessary to determine: (a) the active oligopeptide(s) of the venomand (b) their molecular target(s).

Acknowledgments

This work was partially supported by grants from Fondef-Con-icyt Chile No. DO5I10416 (to F.R. and J.P.) and by CONACYT55025 and UNAM-PAPIIT 204806 (to A.M.-T and R.M.). J.P. is apostdoctoral fellow from CTIC-UNAM and from MIDEPLAN-Chile.We are in debt with E. Ruiz Alcíbar and E. Pérez for technical assis-tance and to Dr. R. Arellano (INB-UNAM) for suggesting someexperiments. WinWCP was kindly provided by Dr. J. Dempster,University of Strathclyde in Glasgow, UK.

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