a Absorbance (mau) 5 2 5 3 4 5 6 7 8 9 6 2 3 4 5 6 VaTx2 High Ca 2+ Low Ca 2+ b 38.2 min Absorbance (mau) 3 2 3 4 5 3 2 VaTx2 39.3 min 3 4 5 3 2 4. min 3 4 5 Supplementary Figure. Toxin Purification For purification, an equivalent of μl lyophylized P. cambridgei venom was resuspended in ml of water containing.% trifluoroacetic acid (TFA). The suspension was filtered thru.2 μm spin filters (Amicon) and fractionated by HPLC (Prostar 2, Varian) on a semi-preparative reverse-phase C8 column (Vydac, mm x 25 mm) employing a 9 minute linear gradient from % to 5% acetontrile with.% TFA (flow rate 3 ml/min). a, Elution was monitored at a wavelength of 28nm (left) and numbered peaks were tested for activity by calcium imaging of -expressing HEK293 cells (right). b, Purity of (2 nmol), VaTx2 (.8 nmol), and (.75 nmol) was assessed on a Vydac C8 analytical column (4.6 mm x 25 mm) using a linear gradient from % to 6% acetonitrile in hour (flow rate: ml/min). Retention times specific to each vanillotoxin are given above each peak. Fractions were lyophilized and taken up in water containing % acetonitrile for sequencing/mass spectrometric analysis, or in physiological buffer for calcium imaging and electrophysiological recordings. Similar elution profiles and activities were obtained from 9 different and independent batches of P. cambridgei venom. For cysteine modification, approximately nmol of peptide was denatured in 6M guanidine-hcl (Sigma) for 3 minutes at 65 C buffered to ph 7.5 with mm N-methyl morpholine (Sigma). Subsequently, TECP (Calbiochem, mm) and 4 μl of 2% 4-vinyl pyridine (Sigma) in ethanol were added and the reaction incubated for 3 hours at room temperature in the dark. The reaction mixture was acidified with TFA to ph 4 and the derivatized peptide isolated by HPLC using a Vydac C8 analytical column (4.6 mm x 25 mm) with a linear gradient from % to 5% acetonitrile in hour (flow rate ml/min). Native, derivatized, or digested peptides were analyzed by mass spectrometry on a Bruker Apex III FT-ICR 9.4T mass spectrometer or sequenced on an Applied Biosystems 492 Procise Sequencer. For digestion, 6 pmol to 8 pmol of peptide was reduced with mm DTT at 55 C for 5 min, and digested in 25 mm ammonium bicarbonate with Lysyl Endopeptidase (Wako), or sequencing grade Endoproteinase Glu-C (Boehringer Mannheim) at 37 C overnight. Digested fragments were separated on an Aquapore x25 mm reversed-phase column (Perkin Elmer) using an Applied Biosystems 72A microbore HPLC.
Control syn syn + RR A 23 (mau) 4 syn 2 4 2 26 32 38 44 5 TRPA Control syn MO Supplementary Figure 2. Refolded synthetic vanillotoxin (syn) recapitulates the chromatographic and pharmacological properties of the native peptide. Synthesis of was performed on an Applied Biosystems 43A peptide synthesizer using Fmoc chemistry. For reconstitution/oxidation of the linear peptide, mg of peptide was taken up in ml of 5 mm Tris buffer ph 8 containing 8 M guanidine HCl and incubated at 6 C for 2 minutes. After cooling down to room temperature, 25 mm reduced glutathione (Sigma) and 2.5 mm oxidized glutathione (Sigma) were added and the solution stirred at room temperature overnight. Subsequently, the solution was acidified with TFA to ph 4 and oxidized peptides separated from the unreacted, reduced precursor by HPLC using a Vydac C8 analytical column (4.6 mm x 25 mm). (Left) Similar retention times were observed for synthetic and native toxins when examined by C8 reverse-phase hydrophobic chromatography. (Right) syn elicited robust calcium increases in - expressing HEK293 cells that were blocked by ruthenium red (+RR, μm). Cells expressing TRPA (bottom) were insensitive to syn, but responded to mustard oil (MO). At least 4 cells were imaged per visual field
Control VaTx2 Control Agonist + RR TRPA TRPM8 MO ME High Ca 2+ Low Ca 2+ Supplementary Figure 3. Purified vanillotoxins activate the capsaicin receptor,, but not the menthol or mustard oil receptors, TRPM8 and TRPA. HEK293 cells expressing, TRPM8 or TRPA were monitored by calcium imaging during treatment with purified ( μm), VaTx2 ( μm), ( μm), or appropriate control agonist, capsaicin (, 4 nm), allyl isothionate (mustard oil, MO, μm) or menthol (ME, 75 μm), respectively. responses were inhibited by the pore blocker ruthenium red (RR, μm). Bar depicts pseudo-color code indicating relative intracellular calcium levels. Graph shows representative experiment from 8 independent vanillotoxin purifications. A minimum of 4 cells were imaged per visual field.
a Normalized G.2.8.6.4.2-8 -4 4 Voltage (mv).2.8.6.4.2 HM -8-4 4 Voltage (mv) b Vanillotoxin Concentration (µm) Fold Excess over EC at 5 Kv2. Inhibition (% at mv) VaTx2 VaTx2 4.4 3.8 38 7.5 23.7 6.6 2.8.37x.2x 2x 3x x 2x 65x 3.3 ± 7.4 58.8 ± 2. 79. ±.8 24.7 ± 3. 4.4 ± 3.8 undetectable 8.2 ± 2.6 Supplementary Figure 5. Effect of vanillotoxins on Kv channels. a, Kv2. tail conductance amplitudes were measured at indicated voltages before (black traces) and after (red traces) application of (top) or H. maculata venom (HM, bottom). Conductances were fitted with the Boltzmann equation (G = Gmax / (+exp[- zf(v-v/2)/rt])) and normalized to the steady-state response. For reference see: Hille, B. Ionic Channels of Excitable Membranes 2nd edition (Sinauer, Sunderland, Massachusettes, 992). b, Inhibitory effects of all vanillotoxins at Kv2. are shown in the table, noting relative potency at. Errors are standard error of the mean with n 3 for all VaTx concentrations.
a Current ( µ A) 2..5..5 VaTx2 5 5 2 Time (s) 25 b 8 HM HM + Current ( µ A) 6 4 2 5 Time (s) 5 2 Supplementary Figure 6. Heteroscodra toxins, closely related to vanillotoxins, do not activate or inhibit. a, Xenopus oocytes expressing were stimulated with (2 μm), VaTx2 (5 μm), (.75 μm) or capsaicin (;.2 μm), as indicated, and currents recorded at +8 mv holding potential (n=4 cells per agonist). Uninjected control oocytes showed no response to these toxins (not shown). b, Xenopus oocyte expressing was challenged with capsaicin (;.5 μm), Heteroscodra Maculata (HM) venom (:2), or a mixture, as indicated. Current was recorded at +8 mv holding potential. HM venom neither inhibited, nor blocked its activation by capsaicin (n=4 per trial).
A 34 / A 38 5 4 3 2 Venom K 4 8 Time (s) 2 Supplementary Figure 7. Crude P. Cambridgei venom elicits calcium responses in mouse trigeminal neurons that are mostly (but not entirely) -dependent. Dissociated mouse trigeminal neurons were challenged with crude P. Cambridgei venom (:5), followed by wash-out and subsequent challenge with capsaicin (; 3 μm) as indicated. High potassium buffer (K; mm) was apllied last to activate excitable cells. Responses were analyzed by ratiometric calcium imaging. Average responses from capsaicin-sensitive (black) or -insensitive (blue) wild type neurons, or -deficient neurons (red) are shown. Venom-evoked peak ratiometric responses were 3.3±.,.77±.5, and.78 ±.5, respectively (n 3 cells per category).