Vol 444 | 9 November 2006 | doi:10.1038/nature05285 LETTERS Spider toxins activate the capsaicin receptor to produce inflammatory pain Jan Siemens1, Sharleen Zhou2, Rebecca Piskorowski3{, Tetsuro Nikai4, Ellen A. Lumpkin3{, Allan I. Basbaum4, David King2 & David Julius1 Bites and stings from venomous creatures can produce pain and molecular masses (observed monoisotopic masses were 4,008.5, inflammation as part of their defensive strategy to ward off pre- 3,842 and 4,179 Da, respectively). Indeed, Edman sequencing re- dators or competitors1,2. Molecules accounting for lethal effects of vealed a family of three closely related peptides, which we have venoms have been extensively characterized, but less is known named vanillotoxins (VaTx) 1, 2 and 3. Each peptide consisted of about the mechanisms by which they produce pain. Venoms from 34 or 35 amino acid residues plus an amidated carboxy terminus spiders, snakes, cone snails or scorpions contain a pharmacopoeia (Fig. 1b), with calculated molecular masses (4,008.5, 3,842.5 of peptide toxins that block receptor or channel activation as a and 4,179.6 Da, respectively) matching the observed masses noted means of producing shock, paralysis or death3–5. We examined above. whether these venoms also contain toxins that activate (rather Vanillotoxins are new members of the extended family of ICK than inhibit) excitatory channels on somatosensory neurons to peptides from spiders and cone snails10. ICK toxins are widely recog- produce a noxious sensation in mammals. Here we show that nized as blockers of cationic channels, perhaps best characterized by venom from a tarantula that is native to the West Indies contains the interaction of hanatoxins (HaTx1 and HaTx2) with voltage-gated three inhibitor cysteine knot (ICK) peptides that target the (Kv) potassium channels7,11. The typical ICK toxin is a 30–40-residue capsaicin receptor (TRPV1), an excitatory channel expressed by peptide in which three disulphide bridges form a cysteine knot, con- sensory neurons of the pain pathway6. In contrast with the pre- straining the molecule into a rigid and compact structure. A short dominant role of ICK toxins as channel inhibitors5,7, these pre- (five to seven residues) hypervariable b-hairpin turn protruding viously unknown ‘vanillotoxins’ function as TRPV1 agonists, from this compact core is believed to be critical for specifying providing new tools for understanding mechanisms of TRP chan- toxin–ion-channel interactions12–14. Interestingly, VaTx1 and nel gating. Some vanillotoxins also inhibit voltage-gated pot- VaTx2 are identical at five of the six residues in this hypervariable assium channels, supporting potential similarities between TRP turn, whereas VaTx3 shows significant diversity within this region and voltage-gated channel structures. TRP channels can now be (Fig. 1b). VaTx1 and VaTx2 show greatest identity to heteroscodra- included among the targets of peptide toxins, showing that ani- toxin 1 (HmTx1) (Fig. 1c), an inhibitor of Kv2-type voltage-gated mals, like plants (for example, chilli peppers), avert predators by potassium channels15. VaTx3 is more closely related to the spider activating TRP channels on sensory nerve fibres to elicit pain and toxin, huwentoxin 5 (HwTx-V), from Ornithoctonus huwena, for inflammation. which a molecular target is not known16. Intrigued by the striking To identify previously unknown activators of sensory neurons, we similarity between these latter two toxins, we examined whether examined venoms from 22 spider and scorpion species whose bites crude venom from O. huwena could activate the capsaicin receptor. are known to produce pain. Screening targets included three mem- Indeed, application of highly diluted (1:4,000) venom to TRPV1- bers of the TRP ion-channel family whose activation leads to the expressing HEK-293 cells produced large calcium responses that were direct depolarization of primary afferent neurons, consequently pro- attenuated by ruthenium red, a non-selective blocker of numerous ducing irritation or pain. These include TRPV1, TRPA1 and TRPM8, TRP channels (Fig. 1d). In contrast, no response was observed with which are activated by plant-derived irritants such as capsaicin, TRPA1-expressing cells. These results show that P. cambridgei is not mustard oil and menthol, respectively8,9. Using calcium imaging as unique in targeting TRPV1, indicating that VaTx-like peptides might a functional readout, we found that robust and reproducible signals contribute to the pain-producing actions of other spider venoms. were obtained when crude venom from Psalmopoeus cambridgei,a To show unequivocally that VaTx peptides account for TRPV1 tarantula native to the West Indies, was applied to TRPV1-expressing activation by P. cambridgei venom, we examined whether synthetic HEK-293 cells. In contrast, no activity was observed with cells expres- VaTx1 peptide could recapitulate the actions of native material. sing TRPA1 or TRPM8 (Fig. 1a), nor did the venom inhibit activa- Synthetic VaTx1 was denatured, refolded in glutathione-based redox tion of these channels by their cognate chemical agonists (not buffer and subjected to C8 reverse-phase chromatography, revealing shown). a series of overlapping peaks whose most prominent species had a Reverse-phase chromatographic methods were used to purify the retention time matching that of native VaTx1 peptide (Supple- active component(s) from P. cambridgei venom. Three fractions with mentary Fig. 2). Refolded synthetic material elicited robust TRPV1 TRPV1 agonist activity were identified (Supplementary Fig. 1) and activation, confirming the identity of VaTx1 as an active constituent presumed to consist of peptides on the basis of retention times and of P. cambridgei venom. 1Department of Cellular and Molecular Pharmacology, University of California–San Francisco, 600 16th Street, San Francisco, California 94143-2140, USA. 2Howard Hughes Medical Institute Mass Spectrometry Laboratory, University of California–Berkeley, California 94720-3202, USA. 3Department of Physiology, University of California–San Francisco, 600 16th Street, San Francisco, California 94143-2280, USA. 4Departments of Anatomy and Physiology and W. M. Keck Center for Integrative Neuroscience, University of California–San Francisco, 1550 4th Street, San Francisco, California 94143-2722, USA. {Present addresses: Center for Neurobiology and Behavior, HHMI Columbia University, 1051 Riverside Drive, PI Annex, Room 633 New York, New York 10032, USA (R.P.); Department of Neuroscience, Baylor College of Medicine, One Baylor Plaza, Room S636-A, Houston, Texas 77030, USA (E.A.L.). 208 © 2006 Nature Publishing Group NATURE | Vol 444 | 9 November 2006 LETTERS As observed with crude venom, each of the purified vanillotoxins Fig. 4a). Thermosensitive TRP channels, such as TRPV1 and selectively activated HEK-293 cells expressing TRPV1, but not TRPM8, also show modest voltage dependence, a property that TRPA1 or TRPM8 (Supplementary Fig. 3). Dose–response analy- might contribute to gating by chemical or thermal stimuli17. In this sis by calcium imaging revealed a rank order potency of regard, we analysed tail currents from TRPV1-expressing HEK-293 VaTx3 . VaTx2 . VaTx1, with half-maximal effective concentra- cells (Supplementary Fig. 4b) and found that conductance– tions (EC50) of 0.45 6 0.04, 1.35 6 0.15 and 9.9 6 0.97 mM (means voltage relations for capsaicin and VaTx3 were essentially super- 6 s.e.m.), respectively (Fig. 2a). We found that vanillotoxins are imposable except for a small but significant difference in slope present in crude venom at concentrations greatly exceeding their (0.6 6 0.0 and 0.9 6 0.2 for capsaicin and VaTx3, respectively; potency at TRPV1, such that toxin concentration and potency are P 5 0.0017, Student’s t-test) (Fig. 2b). Thus we conclude that these inversely correlated, with VaTx3 being least abundant (180 mM) and agonists gate TRPV1 by a similar mechanism, although the toxin may VaTx1 most prevalent (1,700 mM). Because TRPV1 is a heat-sensitive have a somewhat greater effect on voltage dependence of gating. channel6, the most physiologically relevant measure of toxin potency Capsaicin binds to residues on TRPV1 that face the cytoplasm or may be that assessed at skin temperature, corresponding to the site of reside within the inner leaflet of the lipid bilayer18–20. In contrast, ICK venom injection. Under these conditions (34 uC), the EC50 for VaTx3 toxins have been proposed to interact with their targets by binding to was substantially enhanced, to 0.2 6 0.03 mM. Taken together, our an exposed extracellular loop21 or partitioning into the outer leaflet of findings demonstrate that TRPV1 is a specific and physiologically the plasma membrane to contact residues within the bilayer11,22,23.It relevant target for vanillotoxins. Whole-cell patch-clamp analysis of TRPV1-transfected HEK-293 a cells showed that vanillotoxin-evoked currents are characterized by 120 pronounced outward rectification, resembling that observed with 100 capsaicin and other TRPV1 agonists (Fig. 2b, and Supplementary 80 a 60 CAP MO ME PC GR 40 TRPV1 20 Percentage of maximal response of maximal response Percentage TRPA1 0 10–8 10–7 10–6 10–5 10–4 10–3 VaTx concentration (M) TRPM8 High Ca2+ b Low Ca2+ 1.0 b 1.0 VaTx1: S E C RWFMGGC DSTLDCCC C KHLSC KMGLYYC AWDGTF* 0.5 0.5 VaTx2: G A C RWFLGGC KSTSDCCC C EHLSC KMGLDYC AWDGTF* VaTx3: - E C RWYLGGC KEDSECCCC EHLQC HSYWEWC LWDGSF* current Normalized Normalized HwTx-V: - E C RWYLGGC SQDGDCCCC KHLQC HSNYEWC VWDGTFS conductance 0 HmTx1: - E C RYLFGGC SSTSDCCCC KHLSC RSDWKYC AWDGTFS SGTx1: - T C RYLFGGC KTTADCCCC KHLAC RSDGKYC AWDGTF 0 100 200 HaTx1: - E C RYLFGGC KTTSDCCCC KHLGC KFRDKYC AWDFTFS –100 0 100 200 JzTx-Xi:- E C RKMFGGC SVDSDCCCC AHLGC KPTLKYC AWDGTF Voltage (mV) Voltage (mV) c d c O. huwena Inside out Outside out Outside out 2+ 2+ Control O. huwena +RR Ext.: Ca Ext.: Ca Ext.: EGTA Identity (%) ICK pair Similarity (%) VaTx1/VaTx2 82 88 TRPV1 400 pA 700 pA VaTx1/VaTx3 53 73 100 pA 100 s 100 s 100 s VaTx2/VaTx3 59 76 ControlO.