Biochemical and Biophysical Research Communications 375 (2008) 571–575 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrcSome effects of the venom of the Chilean spider Latrodectus mactans onendogenous ion-currents of Xenopus laevis oocytesJorge Parodi a,b,*, Fernando Romero a, Ricardo Miledi b, Ataúlfo Martínez-Torres ba 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, Mexicoa r t i c l e i n f o a b s t r a c tArticle history: A study was made of the effects of the venom of the Chilean spider Latrodectus mactans on endogenousReceived 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 thatVenom the venom exerts its action mainly on a transient outward potassium-current that is probably mediatedLatrodectus mactansElectrophysiology by a Kv channel homologous to shaker. Consistent with the electrophysiological evidence we detected theOocytes 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 methodseuroasiatic black widow spider Latrodectus mactans . LTX is a po-tent synaptic modulator that induces the release of neurotransmit- Spider retrieval. Female adult L. mactans spiders from Chile wereters from vertebrate nerve terminals by inhibiting potassium captured during the summer months (December 2005 and Januarychannels 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 individualbrane forming a cation permeable channel . 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 .from Europe and Asia, this toxin has not been detected in the Chilean Venom retrieval. The spiders were immersed in liquid nitrogenL. 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 PBStacked by the South American species is higher , most probably (25 pairs of glands for 100 ll of PBS) and homogenized. Thedue 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 atin the venom, this contains several oligopeptides which have been À20 °C. The total protein concentration was determined by theshown to induce muscle contraction and inhibit sperm motility; ef- method of Bradford  and dissolved in Ringer to making the ﬁnalfects 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 frogslatory 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 fourmento de Ciencias Preclínicas, Facultad de Medicina, Universidad de la Frontera, Av. protocols to activate an hyperpolarization-activated current ,Francisco Salazar 01145, P.O. Box 54-D, Temuco, Chile. a calcium-dependent chloride-current , an outwrdly rectifying E-mail address: email@example.com (J. Parodi).0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved.doi:10.1016/j.bbrc.2008.08.070
572 J. Parodi et al. / Biochemical and Biophysical Research Communications 375 (2008) 571–575Fig. 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.chloride current  or a transient-outward potassium-current any effect on the calcium-dependent chloride-current activated. In addition, serum was perfused onto some oocytes to see through the lysophosphatidic acid (LPA) receptor . 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/mlof venom. (B) Oscillatory chloride-currents generated by serum (1:1000) were not affected by the venom.
J. Parodi et al. / Biochemical and Biophysical Research Communications 375 (2008) 571–575 5732 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 theseveral oocytes to observe the potential role of potassium-, so- equilibrium potential for chloride in oocytes; furthermore thisdium- and calcium-channels respectively. Moreover, several oo- reversal potential was independent of the venom concentrationcytes were frozen in liquid nitrogen to isolate RNA  and RT- at all tested concentrations (Fig. 1D). This result was contrary toPCR using primers designed to amplify xKv 1.1 (GenBank Accession a previous report  suggesting that the venom modulates mainlyNo. M94258) . 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-currentsusing pClampﬁt 9 (Axon, molecular devices) and Origin 6.0respectively. Frog oocytes normally possess chloride-channels that are acti- vated by either calcium ions permating through voltage-operatedResults channels (Tout)  or calcium released from intracellular stores due to the activation of G-protein coupled receptors such as theActivation of a smooth slow-activating ionic-current LPA receptor that is effectively activated with serum . 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 currentsitizing current in oocytes held at À60 mV (Fig. 1A). Those currents (962 ± 240 vs 820 ± 216 nA Fig. 2A), thus discounting its ability towere 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 chloride60 ± 20 nA from 9 oocytes and 3 frogs. The venom gave an EC50 currents elicited by activation of the LPA receptor did not showof 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-currentsstructing an I/V curve, stepping the membrane voltage of theoocyte from the holding potential (À60 mV) to À100 mV and then Two main conductances carried out by chloride ions are knownto 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 to0.1 or 10 lg/ml of the venom (Fig. 1C). The current reversed direc- the expression of ClC5  and an hyperpolarizing inward currentFig. 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 alsoevidenced 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/mlof 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.
574 J. Parodi et al. / Biochemical and Biophysical Research Communications 375 (2008) 571–575whose molecular identity is unknown and is a mixture of anionic 0.5 s to +40 mV, brought back to À80 mV and ﬁnally returned toand cationic conductances . 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 themore 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 potassiumthe 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 Kvtiates the current up to 100% in a dose-dependent manner with an channel may be involved . Therfore, we tried to detect theEC50 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 somehyperpolarizing 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 forof 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 reportsFig. 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 theblocked 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 venomcurrent 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 fromheart and oocytes. Tubulin was also present in both samples. *p < 0.05 vs control values.
J. Parodi et al. / Biochemical and Biophysical Research Communications 375 (2008) 571–575 575indicate other effects due to several minor components of the ve-  H. Scheer, L. Madeddu, N. Dozio, G. Gatti, L.M. Vicentini, J. Meldolesi, Alpha latrotoxin of black widow spider venom: an interesting neurotoxin and a toolnom, in particular those that cause the systemic effects which for investigating the process of neurotransmitter release, J. Physiol. (Paris) 79may be generated by low molecular weight peptides [21,22]. In (1984) 216–221.the Chilean L. mactans, several experiments suggested that the no-  G. Schiavo, M. Matteoli, C. Montecucco, Neurotoxins affecting neuroexocytosis,cive effects are also mediated by the impact of low molecular Physiol. Rev. 80 (2000) 717–766.  E.V. Orlova, M.A. Rahman, B. Gowen, K.E. Volynski, A.C. Ashton, C. Manser, M.weight components of the venom on the function of ion channels van Heel, Y.A. Ushkaryov, Structure of alpha-latrotoxin oligomers reveals thatand the results described here explain in part the previously re- divalent cation-dependent tetramers form membrane pores, Nat. Struct. Biol. 7ported modulatory actions of the venom on cells such as the sperm (2000) 48–53.  H. Schenone, L.E. Correa, Some practical knowledge of the biology of the spidercell . Latrodectus mactans and the latrodectism syndrome in Chile, Bol Chil Parasitol To try to explain the effects of the venom on ion channels, 40 (1985) 18–23.endogenous conductances of the frog oocyte were investigated  F. Romero, E. Altieri, M. Urrutia, J. Jara, Venom of Latrodectus mactans from Chile (Araneae, Theridiidae): effect on smooth muscle, Rev. Biol. Trop. 51[11, 12, 23–25]. The voltage-operated calcium-and chloride-cur- (2003) 305–312.rents were not sensitive to the concentrations of venom tested.  F. Romero, M.A. Cunha, R. Sanchez, A.T. Ferreira, N. Schor, M.E. Oshiro, EffectsIn addition, the calcium-dependent chloride-currents, which are of arachnotoxin on intracellular pH and calcium in human spermatozoa, Fertil. Steril. 87 (2007) 1345–1349.mainly generated by the bestrophins  were not affected when  C.V. Sapan, R.L. Lundblad, N.C. Price, Colorimetric protein assay techniques,applying up to 5 lg/ml of the venom. Therefore, in the venom ex- Biotechnol. Appl. Biochem. 29 (Pt 2) (1999) 99–108.tract used for these experiments we have no evidence of blocking  I. Parker, C.B. Gundersen, R. Miledi, A transient inward current elicited by hyperpolarization during serotonin activation in Xenopus oocytes, Proc. R. Soc.or modulatory effects on either voltage gated calcium channels or Lond. B. Biol. Sci. 223 (1985) 279–292.the calcium gated -chloride currents. In contrast, a previous report  R. 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(1987) 156–159.The genes coding for the channels responsible for these currents  A.B. Ribera, D.A. Nguyen, Primary sensory neurons express a Shaker-like potassium channel gene, J. Neurosci. 13 (1993) 4988–4996.have not been fully characterized, but due to their functional prop-  J.P. Reyes, C.Y. Hernandez-Carballo, P. Perez-Cornejo, U. Meza, R. Espinosa-erties and TEA sensitivity shown in Fig. 4 we assumed they may be Tanguma, J. Arreola, Novel outwardly rectifying anion conductance in Xenopuspart of the Kv family. The L. mactans venom efﬁciently and revers- oocytes, Pﬂugers Arch. 449 (2004) 271–277.  G.A. Gutman, K.G. Chandy, J.P. Adelman, J. Aiyar, D.A. Bayliss, D.E. Clapham, M.ibly blocked the transient-outward potassium-current, whereas no Covarriubias, G.V. Desir, K. Furuichi, B. Ganetzky, M.L. Garcia, S. Grissmer, L.Y.apparent effects were found in other potassium conductances. In Jan, A. Karschin, D. Kim, S. Kuperschmidt, Y. Kurachi, M. Lazdunski, F. 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