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Toxicon 57 (2011) 60–67

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Toxicon

journal homepage: www.elsevier .com/locate/ toxicon

Electrophysiological characterization of a novel small peptidefrom the venom of Conus californicus that targets voltage-gatedneuronal Ca2þ channels

Johanna Bernaldez b,1, Omar López a,1, Alexei Licea b, Emilio Salceda a, Rogelio O. Arellano c,Rosario Vega a, Enrique Soto a,*

a Instituto de Fisiología, Benemérita Universidad Autónoma de Puebla, Mexicob Laboratorio de Inmunología Molecular y Biotoxinas, Departamento de Biotecnología Marina (C.I.C.E.S.E.), Ensenada, B.C., Mexicoc Instituto de Neurobiología, Universidad Nacional Autónoma de México, Querétaro, Mexico

a r t i c l e i n f o

Article history:Received 8 June 2010Received in revised form 7 September 2010Accepted 25 September 2010Available online 7 October 2010

Keywords:Conus californicusConotoxinsCalcium channelElectrophysiologyXenopus laevisDorsal-root ganglion neurons

* Corresponding author. Instituto de Fisiología, B406, 72000 Puebla, Pue, Mexico. Tel.: þ52 222 229222 2295500x7301.

E-mail addresses: esoto@siu.buap.mx, esoto24@g1 These two authors contributed equally to this w

0041-0101/$ – see front matter � 2010 Elsevier Ltddoi:10.1016/j.toxicon.2010.09.015

a b s t r a c t

Conus californicusbelongs to a genus ofmarine gastropodswithmore than 700 extant species.C. californicushasbeen shown tobedistantly related to allConus species, but showing unusualbiological features. We report a novel peptide isolated from C. californicus with a significantinhibitory action over neuronal voltage-gated calcium channels. The new toxin is formed by13-amino acid residues with two disulfide bonds, whose sequence (NCPAGCRSQGCCM) isstrikingly different from regularu-conotoxins. In theHPLC purificationprocedure, the venomfraction eluted in the first 10–15 min produced a significant decrease (54% � 3%) of the Ca2þ

current inXenopus laevis oocytes transfectedwith purified rat-brainmRNA. A specific peptideobtained from the elution at 13 min decreased the Ca2þ current in the adult rat dorsal-rootganglion neurons in a primary culture by 34% � 2%. The cysteine pattern of this peptidecorresponds to the framework XVI described for the M-superfamily of conopeptides and isunprecedented among Conus peptides acting on Ca2þ channels.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Over 50 million years of evolution, the predatory marinesnails of the genus Conus have evolved from primitivegastropods, developing specific toxins used to hunt a remark-ably wide phylogenetic range of prey species includingbivalves, cephalopods, crustacea, other gastropods, hemi-chordata, polychaete worms, echiuroids, and other fast-moving prey, such as fish predator species (Nybakken, 1990).

The genus Conus is the largest single genus of venemousanimals known (Kohn, 1990) with ca. 700 species, and each

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species could express between 100 and 200 venompeptides (Olivera, 2006). Several studies of the genus Conusshow that the venom composition of cone snails possessesa sophisticated biochemical arsenal of conotoxins to rapidlyimmobilize their prey (Jones and Bulaj, 2000). Due toa rapid divergence in venom peptide sequences as specia-tion occurs, the sequences of peptides are notoriouslydifferent when compared different Conus species (Olivera,2006). Therefore, it has been estimated that the numberof different peptides that can be expressed in the venom ofliving Conus species is around 70,000 (Olivera, 2006). Manyof these peptides have specific actions on ion channel andreceptor subtypes with a high binding affinity (Jones andBulaj, 2000).

Conopeptides have been divided into two groupsaccording to the number of disulfide bridges present in itsstructure: the non-disulfide-rich peptides, which lack

J. Bernaldez et al. / Toxicon 57 (2011) 60–67 61

multiple disulfide bridges, and the disulfide-rich peptideswith two to five disulfide cross-links. Attending to its signalpeptide sequence the latter group has been classified intoat least ten superfamilies (Biggs et al., 2010; Imperial et al.,2006; Olivera, 2006; Peng et al., 2006; Terlau and Olivera,2004). Characteristically, the signal sequence is highlyconserved in every superfamily; however, the inter-cysteine loops in the mature peptide are hypermuted.Peptides belonging to the same superfamily share itscysteine arrangement. To date, a total of 20 patterns ofcysteine scaffold have been described.

Conus californicus is the only Conus species occurring inthe eastern Pacific Ocean from San Francisco Bay, California,to Cabo San Lucas, Baja California Sur, Mexico. Conse-quently, they are adapted to cooler water and a lack ofcompetition from congener species (Hanna and Strong,1949). C. californicus has an unspecialized diet, consumingup to 56 different organisms (Kohn, 1966), includinga variety of worms, mollusks, fish (Saunders and Wolfson,1961; Stewart and Gilly, 2005) and crustaceans (Biggset al., 2010). The activity of the components of the venomof C. californicus has been poorly studied (Cottrell andTwarog, 1972; Elliot and Kehoe, 1978; Elliot and Raftery,1979), and only recent molecular data have established itsremarkable phylogenetic divergence from other Conusspecies (Biggs et al., 2010).

In this work we provide electrophysiological evidencesindicating that CalTx, a toxin from the venom of the marinesnail C. californicus, exerts its action on voltage-activatedcalcium channels (VACC). To identify the conotoxin com-ponents of the venom of C. californicus, we used twoelectrophysiological-model systems. The 13-amino acidconopeptide (NCPAGCRSQGCCM) isolated fromC. californicus(Biggs et al., 2010) showed considerable sequencedivergencefrom theu-conotoxins isolated from the other Conus speciesknown to have inhibitory actions over the VACC.

2. Material and methods

2.1. Crude venom extraction

Specimens of C. californicus (average length 2 cm) werecollected on the coastal region of Baja California, Mexico.The venom apparatus of the snails were dissected and thevenom duct was homogenized in 1 mL of buffer A (99.9%H2O: 0.1% trifluoroacetic acid (TFA) by using this volume forevery 20 organisms. The homogenate was centrifugedtwice at 10,000 g for 5 min at room temperature. Aftercentrifugation, the supernatant was separated, lyophilized,and stored at �20 �C before use.

2.2. Isolation of native peptide

The lyophilized venom extract was dissolved in buffer A.After centrifugation at 10,000 g, the supernatant was addedto a reverse-phase C18 analytical column (Grace-Vydac218TP54, 4.6 � 250 mm, 300 Å). The HPLC elution wasmade by using a linear gradient increasing the buffer Bconcentration (99.88% acetonitrile (AcN): 0.12% TFA) from0 to 60% in 60 min at room temperature. Fractions of 5 mLwere collected using a flow rate of 1 mL/min. Further

purification steps of the eluant at minute 13 were doneusing a similar procedure. All fractions were lyophilized.The detection of peptides was made by monitoring theabsorbance at 230 nm.

2.3. Cellular models of the expressed Ca2þ channels

The analysis of the activity of the venom fractions on theCa2þ channels was done using two cellular models. The firstbiological model was a heterologous expression system ofpurified rat-brain-mRNA ion channels in Xenopus laevisoocytes.We used a two-electrode voltage-clamp technique.The standard voltage protocols were used to producecalcium currents caused by the activation of a Ca2þ-dependent Cl� conductance by the influx of Ca2þ throughvoltage-dependent Ca2þ channels in the plasmamembrane(Miledi, 1982; Barish, 1983). The measurement of the Cl�

current is an indirect way to quantify the current generatedby the activation of the Ca2þ channels. For the other bio-logical model, we used dorsal-root ganglion neurons ina primary culture. Here, we used voltage-clamp techniquesin the whole-cell configuration. The standard voltageprotocols were used to generate calcium currents.

2.4. Xenopus oocytes transfection

The RNA of rat brain was isolated using a guanidiniumthiocyanate–phenol–chloroform extraction as described(Chomczynski and Sacchi, 1987). The poly (A) mRNA wasisolated from total RNA by means of oligo (dT) cellulosechromatography using the standard binding and elutionprotocols (Sambrook and Russell, 2001).

Stage V and VI oocytes were isolated from adult X. laevisfrogs as described before (Arellano et al., 1996). Briefly, theovary lobules were surgically removed under sterileconditions from frogs anesthetized by hypothermia andkilled by decapitation and pithing, and then maintainedovernight at 18 �C in normal Barth’s solution (containing inmM: 88 NaCl; 1 KCl; 2.4 NaHCO3; 0.33 Ca(NO3)2; 0.41 CaCl2;0.82 MgSO4; 5 HEPES, pH 7.4 (NaOH), supplemented with70 mg/ml gentamicin). The next day, a group of oocytes wasinjected with 50 nl containing 50 ng of mRNA and incu-bated at 18 �C. Uninjected oocytes served as the control.After 24 h the normally appearing injected and uninjectedoocytes were enzymatically treated in normal Ringersolution (NR, containing: 115 mM NaCl, 2 mM KCl, 1.8 mMCaCl2, 5 mM HEPES, pH 7) with 0.5-mg mL�1 collagenase(type I from Sigma) for 40 min at room temperature toremove all follicular layers. After defoliculation, oocyteswere extensively rinsed with Ringer solution and finallymaintained in a Barth’s solution at 17 �C for 24 h beforerecording. Oocytes were used 4–5 days after isolation.

2.5. Current recording in oocytes

The recordingsweremade at room temperaturewith theoocytes bathed in NR solution. The measurements of themembrane currents were obtained using a two-electrodevoltage clamp with 3 M KCl-filled microelectrodes withresistance of 1–1.2 MU. The Ca2þ-dependent Cl� currentsactivated by depolarization (transient outward current, Tout)

J. Bernaldez et al. / Toxicon 57 (2011) 60–6762

were measured by applying square pulses in increments of20 mV steps between �80 mV and þ60 mV during 4 s anda holding potential of �100 mV. By using this protocol thecurrent of the Ca2þ-dependent Cl� channels is caused by theinflux of Ca2þ through voltage-dependent Ca2þ channels(Miledi, 1982).

2.6. Solutions

Three extracellular Ringer solutions with different Ca2þ

concentrationswere used in these experiments; NR solutioncontaining 1.8 mM Ca2þ, R5Ca solution containing 5 mMCaCl2, and R0Ca solution where CaCl2 was substituted by5 mM MgCl2. The measurable Ca2þ-dependent Cl� currentsappeared 3–5 days after the mRNA microinjection.

The maximum current was expected in injected oocytesperfused in R5Ca solution. All Tout currents were indeedtested for their complete dependency on extracellular Ca2þ

applying R0Ca solution. Tout current–voltage relationshipswere built subtracting to the peak current in each potentialrecorded in R5Ca solution that obtained during superfusionof R0Ca. The currents were recorded before, during, andafter the addition of each fraction.

2.7. Dorsal-root ganglion neurons

Wistar rats at postnatal day 7–10 of either gender wereused for the experiments. The animal care and procedureswere in accordance with the National Institutes of HealthGuide for the Care and Use of Laboratory Animals. The ratswere anesthetized and killed with an overdose of sevo-fluorane. The dorsal-root ganglia were isolated from thevertebral columnand incubated (30minat37 �C) inLeibovitzL-15 medium (L15) (Invitrogen, Carlsbad, CA) containing1.25 mg/mL trypsin and 1.25 mg/mL collagenase (both fromSigma–Aldrich, St. Louis, MO, USA). After the enzyme treat-ment, the gangliawerewashed 3 timeswith sterile L15. Cellswere mechanically dissociated using a Pasteur pipette andthen plated on 12-mm � 10-mm glass coverslips (Corning,Corning, NY) pretreated with poly-D-lysine (Sigma–Aldrich)and placed onto 35-mm culture dishes (Corning). Neuronswere incubated 4–8 h in a humidified atmosphere (95% air,5% CO2, at 37 �C) using a CO2 water-jacketed incubator(Nuaire, Plymouth, MN) to allow the isolated cells to settleand adhere to the coverslips. The plating medium was L15,with added 15.7 mM NaHCO3 (Merck, Naucalpan, Mexico),10% fetal bovine serum, 2.5 mg/mL fungizone (both fromInvitrogen), 100 U/mL penicillin (Lakeside, Toluca, Mexico),and 15.8 mM HEPES (Sigma–Aldrich).

2.8. Electrophysiological recording in DRG neurons

The culture dishwith attached cells wasmounted on thestage of an invertedphase-contrastmicroscope (TMS,NikonCo. Tokyo, Japan). The cell–voltage responses were studiedby standard protocols of current-clamp techniques at roomtemperature (23–25 �C) using an Axopatch 1D amplifier(Molecular Devices, Union City, CA). The command-pulsegeneration and data sampling were controlled by pClamp8.0 software (Molecular Devices) using a 16-bit data-acquisition system (Digidata 1320, Molecular Devices).

Signals were low-pass filtered at 5 kHz and digitized at10 kHz. Patch pipettes were pulled from borosilicate glasscapillaries (TW120-3; WPI, Sarasota, FL) using a Flaming–Brown electrode puller (80/PC; Sutter Instruments, SanRafael, CA). They typically had a resistance of 1–3MUwhenfilled with an internal solution. The pipette solutioncontains 0.134mMCaCl2,130mMCsCl, 5 mM TEA-Cl, 5 mMHEPES, 10 mM EGTA, 2 mM Mg-ATP, 1 mM Na-GTP. Theexperiments indicated were made with a perforated patchusing 350 mM nystatin in the pipette solution. The seriesresistance was z80% electronically compensated. In thetime-course of an experiment, seal and series resistanceswere continuouslymonitored to guarantee stable recordingconditions. The recordingwas not included in the analysis ifthe access resistance changed >10%. The digital data werestored in a compatible PC computer for off-line analysis.

2.9. Solutions, drugs, and experimental design

The cells were bath-perfused with an extracellularsolution containing 10mMCaCl2, 5mMCsCl,1.2mMMgCl2,125 mM TEA-Cl, 10 mM 4-AP, and 10 mM HEPES to isolatethe calcium current. The recording chamber was perfusedby a gravity-driven perfusion system that maintained theexternal solution flowing at a rate of about 100 mL/min.Additionally, by using two square-tube rapid-solutionchanger (SF-77BWarner Inst. Hamden, CT), each coupled toan independent syringe driven by a Baby Bee pump (BAS,West Lafayette, IN), the neuron was continuously micro-perfused (10 mL/min) with an external solution or with anexternal solutionplus toxin. To study total Ca2þ current, cellswere voltage clamped at a holding potential of�80 mV andthe current was activated using a depolarizing pulse to10 mV for 400 ms. To study the LVA component of the Ca2þ

current the cells were voltage camped at �100 mV witha �40 mV test pulse for 400 ms. In all the experimentscontrol currents were recorded before any experimentalmanipulation was done to guarantee that cells had a stablevoltage-dependent Ca2þ current.

2.10. Data analysis

The recordings were analyzed using Clampfit 9.2(Molecular Devices) and Origin software (Microcal Soft-ware, Northampton, MA). The statistical differences weredetermined using a Student’s t-test with P < 0.05. Curve-fitting routines were made using a nonlinear least-squaresmethod. The numerical data are given as the mean � SE forat least four measurements.

The current–voltage relationships and availabilitycurves were constructed using a standard double-pulseprotocol. From a holding potential of�80mV, a 100-ms testpulse to 10 mV was preceded by 200-ms prepulsesbetween �80 and 70 mV (8-s interval between sweeps).The current density was calculated by dividing the currentamplitude by the capacitance of the corresponding cell,which value was then plotted against the applied voltage.

The peak amplitudes of the currents were measured atthe prepulse and converted to calcium conductance byusing the equation

J. Bernaldez et al. / Toxicon 57 (2011) 60–67 63

GCa ¼ ICa;

Fig. 1. Purification of the components from Conus californicus venom. Crudevenom was applied to an analytical C18 Vydac column as described. Thepeak highlighted by the arrow was further purified using same HPLCconditions.

Vtest � ECa

where GCa is the calcium conductance, ICa is the calciumcurrent peak amplitude, Vtest is the test potential, and ECa isthe reversal potential for the calcium current. Thenormalized conductance curves were then plotted and fitby a Boltzmann distribution with the function

GCa

Gmax¼ 1

1þ eðVtest�V1=2Þ=k;

where Gmax is the maximum calcium conductance, V1/2 isthe test potential that activates 50% of the calcium chan-nels, k is the slope of the curve, and the other terms are asabove.

The steady-state inactivation parameter (hN) wascalculated by dividing the current achieved followinga given prepulse by the maximum current achieved in thetest pulse. The results of such an operation were plotted asa function of the prepulse potential and were fitted by theBoltzmann function

hN ¼ 1

1þ eðVpre�V1=2inactÞ=k;

where Vpre is the prepulse potential, V1/2 inact is the prepulsepotential at which hN is 0.5, and k is the slope of the curveat this midpoint.

3. Results

3.1. Isolation and sequence of the native peptide

The C. californicus venom extract was initially fraction-ated by liquid chromatography on a reverse-phase column,using previously described conditions. Each fraction wastested by measurement of the Ca2þ current indirectlyexamined in the heterologous expression in X. laevisoocytes. The active component eluting between 10- and 15-min retention time produced a significant modulation ofthe Ca2þ-dependent Cl� current. Therefore, we isolatedeach of the individual peaks in this fraction and all weretested to detect which of them produced the modulation ofthe Ca2þ current. The activity was detected on the peakwith a retention time of 13 min, which peak was the mostabundant in this fraction and was named CalTx (Fig. 1). TheCalTx comprises approximately 4% of the total venomcomponents determined by HPLC (230 nm).

The determination of the amino acid sequence of theCalTx peptide was previously reported by Biggs et al.(2010), showing that CalTx is a 13-amino acid peptidewith the sequence NCPAGCRSQGCCM. In this work we haveanalyzed the activity of the whole venom activity in thecalcium currents.

3.2. Transfected Xenopus oocytes

The aimof the experimentsmade using the oocytemodelwas to explore ona sample of differentmembranemolecules(expressed through the brain mRNA injection) a possibleeffect on a hypothetical targetmolecule (e.g. VACC) or others

not previously considered. The perfusion of fraction 10–15(21 mg/mL) had not any effect on control non-injectedoocytes, thus, the effect observed on injected oocytes wasdependent on the expression of molecules from the braintissue throughmRNA injection. Recordings with transfectedoocytes showed that no other currents apart from Tout weresignificantly affected by fraction 10–15 application.

The Ca2þ-dependent Cl� conductance was used as anindex of the action of the CalTx on the Ca2þ current inoocytes. The oocyteswere held at�100mV and the currentswere caused by positive voltage increments of 20 mV. Thedepolarization of Xenopus oocytes to potentials more posi-tive than0mVgenerates a transient, outward currentnamedTout (Fig. 2). Tout amplitude has a clear-cut dependence on theextracellular Ca2þ concentration, indicating it was caused bythe activation of a Ca2þ-dependent Cl� conductance by theinfluxof Ca2þ through the voltage-dependent Ca2þ channelsin the plasmamembrane, as described (Miledi, 1982; Barish,1983). This Cl� current was undetectable in uninjectedoocytes but its expression increased several times inmRNA-injected oocytes after 3–5 days in incubation. In mRNA-injected oocytes the perfusion of fraction 10–15 (21 mg/mL)consistently decreased the Tout current 54% � 3% ina reversible form with 67% recovery of the control current(n¼ 3) (Fig. 3), suggesting that it will inhibit the Cl� channeldirectly or the Ca2þ current. To discern between these twopossibilities the Ca2þ current was studied in isolated dorsal-root ganglion (DRG) neurons that in addition allowed amoredetailed study of the effects caused by the toxin.

3.3. Rat dorsal-root ganglion neurons

Based on the results obtained by studying the effect offraction 10–15 of C. californicus on the oocyte ionic currents,we decided to characterize the action of this fraction on theCa2þ current isolated from the DRG neurons.

The perfusion of fraction 10–15 (25 mg/mL) produceda significant decrease of the Ca2þ current of 26.4% � 4.2%(P < 0.01) (n ¼ 12) (Fig. 4). The washout produced a 92%recovery of the control current. The analysis of the chro-matographic peaks obtained every minute showed that in

Fig. 2. Voltage- and Ca2þ-dependent Cl� current in oocytes, Tout current. The cells were transfected with rat-brain mRNA, voltage-clamped at �100 mV and testedwith depolarizing steps from �80 to þ60 mV. The activation of voltage-dependent Cl� current is seen as an outward current that had a clear-cut dependence onthe extracellular-Ca2þ concentration (Tout current). All Tout currents were indeed tested for their complete dependency on extracellular Ca2þ that wassubstituted by Mg2þ in the R0Ca solution and which did not allow Tout current activation.

J. Bernaldez et al. / Toxicon 57 (2011) 60–6764

the 10-min to 15-min segment the peak at 13 min was theonly significant one. Consequently we decided to study theaction of the peak at 13 min, called CalTx, on the Ca2þ

current in the DRG neurons. The perfusion of the CalTx(20 mg/mL; n ¼ 16) produced a significant decrease of thetotal Ca2þ current of 34% � 2% (P < 0.001) within the firstw40 s of its perfusion (Fig. 4A, C). The inhibition producedby CalTx was 91% reversible after a w2 min washout withnormal Ringer solution. We tested CalTx (20 mg/mL) on LVACa2þ current (n¼ 3); no effect of CalTxwas observed on thistype of current (Fig. 4B).

To further characterize the effect of CalTx on the Ca2þ

current from the DRG neurons, we used the perforated-patch clamp technique (see methods) that allowed havinga longer-lasting stable-current recording. Under theseconditions the Ca2þ current from the DRGs activated above�40 mV attained its maximum at 10 mV and inverted at57 � 4 mV. The use of 20 mg/mL CalTx from C. californicussignificantly reduced the current density to 41%� 5% of thecontrol value (n ¼ 6; P < 0.001 paired Student’s t-test). TheCalTxnon-significantlymodified theCa2þ-current inversion

Fig. 3. Effect of fraction 10–15 (21 mg/mL) on Tout currents generated in rat-brain mcondition (black, recorded in R5Ca solution) and after fraction 10–15 application (grvoltage relationships, measured before, during, and after application of fraction 10–

displacing it by 11 mV to the left to 46 � 6 mV (P ¼ 0.08).Other current parameters also remain unchanged (Fig. 5).

The Ca2þ channel conductance was calculated (n ¼ 6;see Material and methods) and a Boltzmann function fit tothe conductance showed that CalTx did not significantlymodify the Ca2þ-current half activation (control V1/

2¼�4.1�1.5mV; CalTx V1/2¼�5.3�1.6mV) nor the slopeof the curves (control¼ 5.2� 0.7mV; CalTx¼ 4.9� 0.4mV)(Fig. 6A–C).

The voltage dependence of the inactivation was alsostudied using a two-pulse voltage-clamp protocol, andmeasuring the tail currents produced at 10mVas a function oftheprepulse voltages (seematerial andmethods). The steady-state inactivation of the Ca2þ current was not significantlymodified by the use of 20 mg/mL CalTx (n¼ 6). Although therewas a small reduction in the V1/2 of the inactivation curve, itwas not significant (control ¼ �13.3 � 1.2 mV;CalTx ¼ �13.14 � 1.7 mV; P > 0.05 paired Student’s t-test)(Fig. 6D–F). The slopes of the curves were also similar incontrol and after CalTx use (control ¼ 9 � 0.9 mV;CalTx ¼ 8.6� 1.2 mV).

RNA-transfected oocytes. In A, typical Tout current at þ20 mV under controlay). Decrease of Tout was 55% in the case shown. In B, complete Tout current–15 (n ¼ 3), Ca2þ-independent currents were subtracted using R0Ca solution.

Fig. 4. The effect of CalTx (20 mg/mL) on the Ca2þ currents from the DRGneurons. In A, a typical record of the Ca2þ current in control (black) and afterCalTx perfusion (gray). Decrease of the Ca2þ current was 34% in the caseshown. The current was elicited with the protocol shown in the inset. In B,LVA Ca2þ current obtained with the protocol in the inset, under control(black) and after CalTx perfusion (gray), there was no significant effect onthe LVA component of Ca2þ current (n ¼ 3). In C, a bar graph of the percentreduction of the control Ca2þ current produced by fraction 10–15 (n ¼ 12)and by CalTx (n ¼ 16). Both were significant with P < 0.001 using a pairedStudent’s t-test.

Fig. 5. Action of CalTx on the Ca2þ current density of DRG neurons. In A,a typical record of the Ca2þ currents obtained at various voltages undercontrol conditions (black) and after the use of CalTx (gray). In B, currentdensity versus voltage curve for the Ca2þ current under control conditionsand after the use of 20 mg/mL CalTx (n ¼ 6). The current density changeswere significant with P � 0.05 (*) or P � 0.01 (**). The use of CalTx alsoproduced a non-significant (P ¼ 0.08) displacement of the current inversion.

J. Bernaldez et al. / Toxicon 57 (2011) 60–67 65

4. Discussion

Here we report the purification and the electrophysio-logical action of a Ca2þ channel toxin, CalTx, obtained fromthe C. californicus venom. The peptide CalTx reversiblyblocked calcium current in the dorsal-root ganglionneurons of the rat.

CalTx is a low molecular-weight peptide formed by 13-amino acids,with 4 cysteines forming 2 disulfide bonds. Theamino acid sequence of the peptide (NCPAGCRSQGCCM)was previously reported (Biggs et al., 2010). In contrast withCalTx, u-conotoxins typically include peptides of 12–30

amino acids, with 6 cysteines forming a frame C–C–CC–C–C(frameworkVI/VII) that forms three disulfide bonds (Oliveraet al., 1984; Lewis et al., 2000; Terlau and Olivera, 2004). Asimilar,13-aminoacid toxin that blocks thepresynaptic Ca2þ

influx has been reported (e-TxIX, isolated from Conustextile); however, the frame formed by the cysteines isdifferent from CalTx (Rigby et al., 1999).

CalTx has the framework XVI of the M-superfamilypreviously reported by Pi et al. (2006), however noprevious reports of blocking activity on voltage-gated ionicchannels has been reported for this family of peptides, andit has been proposed that C. californicus did not have toxinsof the M-superfamily (Biggs et al., 2010).

Initial experiments using oocyte recordings with rat-brain RNA transfection hint to voltage-dependent Ca2þ

currents as the main target of CalTx. Studies of the action ofCalTx on the low-voltage activated (LVA) and high-voltageactivated (HVA) components of the Ca2þ current in dorsal-root ganglion neurons confirmed that the Ca2þ channelswere significantly affected by the toxin. We dissected thetwo components (LVA and HVA) by means of a voltageprotocol (Lewis et al., 2000; Scroggs and Fox, 1992; Hille,2001). Our results showed a clear block of the HVAcalcium currents by CalTx (20 mg/mL). The HVA currentscorrespond to L, N, P-Q, and R- type Ca2þ currents, all ofwhich are present in dorsal-root ganglion neurons of therat (Kostyuk et al., 1993; Yuaf et al., 2001). In contrast, therewas no significant effect on the LVA current, correspondingto the T-type Ca2þ current. We still need to determine if

Fig. 6. Effect of the CalTx on the Ca2þ-current activation and steady-state inactivation. In A, control record of the Ca2þ current obtained with the voltage protocolshown in the inset, from which the conductance was measured (at the peak current). In B, a recording obtained after perfusion of 20 mg/mL of CalTx. In C, theconductance curves obtained under control condition (black) and after the perfusion of fraction 13 (white) (n ¼ 6). In D, control recordings of the tail currentobtained at 10 mV after prepulses at various voltages (inset). In E, after perfusion of 20 mg/mL of CalTx. In F, curve of the steady-state inactivation of the Ca2þ

current in control (black) and after perfusion of the CalTx (white) (n ¼ 6). Continuous line is the Boltzmann fit to the data.

J. Bernaldez et al. / Toxicon 57 (2011) 60–6766

CalTx is selective to the components of the HVA Ca2þ

current. Themean capacitance of the recorded neurons was47� 1.5 pF, which according to the study of Scroggs and Fox(1992) indicates that the neurons we recorded are largeneurons, which exhibit almost no LVA current. The LVAcurrent was observed in z25% of the recorded cells,although most of the total current was the HVA type. Withour experiments we cannot differentiate between thecomponents of the HVA current so that future experimentsusing pharmacological agents to selectively block each ofits components are needed (McDonough et al., 2002).

Similar to other calcium-channel pore-blocking toxins(McDonough, 2007; McDonough et al., 2002; Ellinor et al.,1994; Lew et al., 1997), CalTx did not modify the voltagedependency of activation nor of the inactivation. The u-con-otoxins isolated from molluscivorous and piscivorous Conusspecies, selectively inhibit neuronal types of VACC found ininvertebrates and vertebrates (Fainzilber et al., 1996; Oliveraet al., 1987). The interaction of u-conotoxins with differenttypes of the VACC is strong and specific, with their site ofaction unequivocally extracellular. The u-conotoxins areuseful tools inneuroscience to characterizedifferent calcium-channel subtypes. The u-conotoxins family is 24–29 aminoacids long. They have in their structure three intramoleculardisulfide bonds known as the four-loop Cys scaffold (Uchitel,1997). A striking diversity of the amino acid sequence isa feature of u-conotoxins, with the only common features of

the sequences the six half-cysteine residues and the Gly atposition 5 (Uchitel, 1997). CalTx has the cysteine frameworkC–C–CC reported for the M-superfamily of conopeptides(Pi et al., 2006). However, its belonging to a superfamilyremains to be defined since the corresponding signal peptidehas not been identified. Interestingly, CalTx exhibits a Glyresidue at position 5. C. californicus is a species so evolution-arily distant from themajor Conus clade that, even in the casein which the signal peptide may exclude CalTx from theM-superfamily, itmaystill have the samecysteine frameworkand target similar receptors. This would make the genethat codifies for CalTx orthologue to the major-cladeM-superfamily.

CalTx non-significantly modified the reverse potentialof the calcium current. The selectivity for monovalentcations through the calcium channel in Ca2þ-free solutionsis Naþ > Kþ > Rbþ > Csþ (Sather and McCleskey, 2003). Inthe experimental conditions of our work, the recordingsolutions do not have Naþ. But because we recorded usingthe perforated patch with nystatin, it only allows themonovalent cation passage through nystatin pores (Akaineand Harata, 1994), meaning that the ionic concentrations inthe cellular interior would be the physiological ones, withlow Naþ and high Kþ, with the ratio of permeation for thecalcium channel of these two ions being 1:0.8 (Akaine andHarata, 1994; Sather and McCleskey, 2003), so it could befeasible that Kþ passes through the channel, causing of the

J. Bernaldez et al. / Toxicon 57 (2011) 60–67 67

non-significant displacement of the reversal potential tohyperpolarizing values. Also the decrease in the calciumcurrent will uncover other remnant Kþ currents.

This is the first report demonstrating that C. californicusvenom possesses a short 13-amino acid peptide withsignificant action on the HVA Ca2þ current in the DRGneurons. Given the number of amino acids and theframework of Cys-paired, CalTx is a novel Conus calcium-channel toxin and may be a member of a new family ofCa2þ-blocking peptides whose characterization seemsworthwhile given the physiological relevance and potentialapplications of Ca2þ-blocking molecules.

Acknowledgements

The authors wish to thank Dr. Ellis Glazier for the editingof the English-language manuscript, and to Dr. Edith GarayRojas for her invaluable help with mRNA-injected oocytesprotocol. This work was submitted in partial fulfillment ofthe requirements for the PhD degree of Omar López Ram-írez at the Doctorado en Ciencias Fisiológicas, BeneméritaUniversidad Autónoma de Puebla, México. OLR was sup-ported by CONACyT fellowships 175945.

Conflict of interest statementThe authors declare that there are no conflicts of interest.

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