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Crotamine Inhibits Preferentially Fast-Twitching Muscles but Is Inactive on Sodium Channels$

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ARTICLE IN PRESS Toxicon 50 (2007) 553–562 www.elsevier.com/locate/toxicon Crotamine inhibits preferentially fast-twitching muscles but is inactive on sodium channels$ Carina T. Rizzia,d, Joa˜o Luı´s Carvalho-de-Souzab, Emanuele Schiavonc, Antoˆnio Carlos Cassolab, Enzo Wankec, Lanfranco R.P. Tronconea,Ã aLaboratory of Pharmacology, Instituto Butantan, Av. Vital Brasil 1500, Sao Paulo SP-05503-900, Brazil bDepartment of Pharmacology and Physiology, Institute of Biomedical Sciences, University of Sa˜o Paulo, Brazil cDepartment of Biotechnology and Biosciences, Laboratory of Neurophysiology, University of Milano, Bicocca, 20126 Milano, Italy dDepartment of Physiology, Biosciences Institute, University of Sa˜o Paulo, Brazil Received 15 March 2007; received in revised form 27 April 2007; accepted 30 April 2007 Available online 18 May 2007 Abstract Crotamine is a peptide toxin from the venom of the rattlesnake Crotalus durissus terrificus that induces a typical hind- limb paralysis of unknown nature. Hind limbs have a predominance of fast-twitching muscles that bear a higher density of sodium channels believed until now to be the primary target of crotamine. Hypothetically, this makes these muscles more sensitive to crotamine and would explain such hind-limb paralysis. To challenge this hypothesis, we performed concentration vs. response curves on fast (extensor digitorum longus (EDL)) and slow (soleus) muscles of adult male rats. + Crotamine was tested on various human Na channel isoforms (Nav1.1–Nav1.6 a-subunits) expressed in HEK293 cells in patch-clamp experiments, as well as in acutely dissociated dorsal root ganglion (DRG) neurons. Also, the behavioral effects of crotamine intoxication were compared with those of a muscle-selective sodium channel antagonist m-CgTx- GIIIA, and other sodium-acting toxins such as tetrodotoxin a- and b-pompilidotoxins, sea anemone toxin BcIII, spider toxin Tx2-6. Results pointed out that EDL was more susceptible to crotamine than soleus under direct electrical + stimulation. Surprisingly, electrophysiological experiments in human Nav1.1 to Nav1.6 Na channels failed to show any significant change in channel characteristics, in a clear contrast with former studies. DRG neurons did not respond to crotamine. The behavioral effects of the toxins were described in detail and showed remarkable differences. We conclude that, although differences in the physiology of fast and slow muscles may cause the typical crotamine syndrome, sodium channels are not the primary target of crotamine and therefore, the real mechanism of action of this toxin is still unknown. r 2007 Elsevier Ltd. All rights reserved. Keywords: Crotalus durissus terrificus; Crotamine; Sodium channels; Toxin; Behavior; Muscle $Ethical statement: We hereby certify that the Commission for the ethical use of animals in experimentation—Instituto Butantan 1. Introduction has evaluated the experimental protocol entitled ‘‘Crotamine- induced hind limb paralysis—what is behind it?’’ and found it in Crotamine is a small basic peptide of 42 amino accordance with the principles adopted by the Brazilian College acids and 3 disulfide bonds, considered to be a of Animal Experimentation and registered under the authoriza- tion number 283/06. myotoxin by several authors. Its three-dimensional ÃCorresponding author. structure has been proposed and several similarities E-mail address: [email protected] (L.R.P. Troncone). with other small peptide toxins were described 0041-0101/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2007.04.026 ARTICLE IN PRESS 554 C.T. Rizzi et al. / Toxicon 50 (2007) 553–562 (Beltran et al., 1985, 1990; Endo et al., 1989; Hampe Energe´ticas e Nucleares (IPEN) at Sa˜o Paulo. Puri- et al., 1978; Laure, 1975; Nicastro et al., 2003). The fication was performed as described (Boni-Mitake most remarkable sign of crotamine intoxication in et al., 2001), with the following modifications: rodents is the typical hind-limb paralysis character- 150 mg of crude lyophilized Crotalus durissus ized by an extended posture with the hind limbs terrificus venom was dissolved in 2 ml of ammonium completely immobilized, resembling the effects of a formiate buffer (100 mM, pH 3.0) and centrifuged medullar transaction (Cheymol et al., 1971; Teno at 200g for 10 min. The supernatant was then et al., 1990). Some authors proposed that crotamine applied to a Superdex 75 gel filtration column might act through changes in voltage-gated sodium previously equilibrated with the same buffer. channels (Chang and Tseng, 1978; Matavel et al., Elution was monitored by UV detection at 280 nm 1998). In an attempt to explain the crotamine’s and collected with an automatic fraction collector. typical behavioral syndrome, we proposed a working Fractions with the main peaks were pooled hypothesis based on the fact that rodents have a clear and freeze-dried under vacuum. The crotamine predominance of fast-twitching muscles in the hind fraction was dissolved in phosphate buffer 50 mM, limbs that bear a much larger number of sodium pH 7.8, and applied to a Resource S FPLC channels than slow-twitching fibers (Bewick et al., column and eluted by a gradient of NaCl from 0 2004; Wang and Kernell, 2001a). This implies that to 2 M. Elution was monitored by automatic UV fast-twitching fibers rely heavily on functional sodium detection at 280 nm, followed by dialysis against channels and therefore would be more susceptible to distilled/deionized water through a membrane sodium channel toxins than slow-twitching muscles. of nominal cutoff of 3000 Da, and, finally, lyophi- The rat slow-twitching muscle soleus, and the fast- lized. Purity was checked by mass spectrometry in a twitching muscle extensor digitorum longus (EDL) Q-TOF (Micromass) and showed the characteristic were tested for crotamine blockade and the results 4884 Da. partially support this hypothesis. This observation prompted us to evaluate whether crotamine has any 2.2. Muscle contraction experiments preference for the muscle-type sodium channel, further supporting our hypothesis. The selectivity of Male Wistar rats weighing 180–200 g were sacri- crotamine for the different isoforms of voltage- ficed in a CO2 chamber and both soleus and EDL dependent sodium channels was assayed in a whole- were dissected and transferred to a Petri dish cell voltage clamp using transfected HEK cells containing regular Krebs–Henseleit solution with expressing selectively human Nav1.1–6 channels. the following salts (in mM): NaCl 115, KCl 3.5, The toxin was also assayed on dorsal root ganglion NaHCO3 25, NaH2PO4 1, MgCl2 6, glucose 12, (DRG) cells in order to have a typical neuron gassed with O2/CO2, 95%/5%, and pH ¼ 7.2. expressing all the ancillary molecular machinery of Muscles were mounted on isometric transducers sodium channels. Finally, the behavioral syndrome of a Powerlab isolated organ system in a 5 mL induced by crotamine was compared with those organ bath with constant gassing. Direct stimula- induced by injections of the prototypical muscle- tion was performed with a pair of platinum selective sodium channel blocker m-conotoxin-GIIIa, wires running parallel and equally spaced to the the classical blocker tetrodotoxin, the wasp toxins muscles, connected to a Grass 88 stimulator a-andb-pompilidotoxin, the sea anemone toxin delivering square wave pulses of 80 V and 8 ms BcIII, and the Phoneutria nigriventer toxin Tx2-6 duration at 0.2 Hz. Data acquisition was performed known to delay sodium channel inactivation, in order using the software Chart 4.1 running on an iMac to establish a pattern of behavioral signs that could computer. Aliquots of crotamine were added at mirror the action of this class of toxin and shed some regular intervals in order to perform a cumulative light on the effects of crotamine. concentration vs. response curve. Single-dose ex- periments were performed by adding a single 2. Methods selected amount of toxin to muscles. Two soleus and two EDL muscles were assembled simulta- 2.1. Toxin purification neously and one of each pair was assigned the role of control, to which only vehicle was added at the Crotamine has been purified and kindly supplied same time schedules as crotamine to experimental by Dr. P. Spencer from the Instituto de Pesquisas muscles. ARTICLE IN PRESS C.T. Rizzi et al. / Toxicon 50 (2007) 553–562 555 2.3. Electrophysiology of Nav1.1– 1.6 human sodium from À110 to À10 mV in 10 mV increments for channels 450 ms, immediately followed by a test pulse to À10 mV. The peak current amplitudes during the Human embryonic kidney 293 (HEK293) cell tests were normalized to the amplitude of the first lines stably expressing human Nav1.1, 1.2, 1.3, 1.5, pulse and plotted against the potential of the and 1.6 a-subunits (generously donated by Dr. J.J. conditioning pulse. Voltage-dependent steady-state Clare of GlaxoSmithKline, Stevenage, UK) were activation currents were elicited by step depolariza- cultured in Dulbecco’s modified Eagle’s medium tion to test potentials ranging from À80 (or À100) supplemented with 10% fetal bovine serum as to 10 mV from a holding of À120 mV. Peak described in Oliveira et al. (2004).Nav1.4-expressing conductances were calculated and plotted against cells were obtained after transient transfection of a test potentials. Steady-state activation and inactiva- plasmid containing the hNav1.4 construct (a kind tion curves were fitted with Boltzmann equations. gift from Prof. Diana Conti-Camerino, University pClamp 8.2 (Axon Instruments) and Origin 7 of Bari). (Microcal Inc.) software were routinely used during The standard extracellular solution contained (in data acquisition and analysis. mM): NaCl 70, NMDG 67, CaCl2 2, MgCl2 2, HEPES-NaOH 10, and pH ¼ 7.40. The standard 2.4. Sodium currents in neurons of the DRG pipette solution contained (in mM): K+-aspartate 130, NaCl 10, MgCl2 2, EGTA-KOH 10, Hepes- DRGs were dissected from newborn Wistar rats.
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