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	Figure 1. Cloning, synthesis and folding of α-conopeptide Eu1.6. (a) The cDNA sequence and predicted translation product of Eu1.6 (above). The signal peptide and mature toxin are shaded. The signal peptide is shown in gray, the propeptide is underlined, and the mature peptide is shown with white text on a blackground. HPLC analyses of the folded products of linear Eu1.6 and its Acm derivatives. Photograph of C. eburneus shell (right). (b) One-step oxidative folding of Eu1.6 (left). Traces from bottom to top: (a) linear peptide; (b) one-step oxidized products; (c) purified product. Determination of the disulfide bond connectivity of Eu1.6 (right). Traces from bottom to top: (a) Linear peptide with Acm modification at Cys 2 and Cys 4; (b) the first oxidized product; (c) The second oxidized product and (d) Co-elution of the two-step folding products and one-step folding products. Samples were applied to a Calesil ODS-100 C18 column (4.6 mm × 250 mm) and eluted with a linear gradient of 0~1 min, 5–10% B (B is acetonitrile (0.1% TFA); 1~25 min, 10~50% B; 25~28 min, 50~95% B at a flow rate of 1 ml/min, 214 nm of wavelength. (c) Comparison of the primary structure of Eu1.6 with other α-conotoxins that act on nAChRs and GABAB receptor/CaV2.2.
	Figure 2. NMR solution structure of Eu1.6. (a) Backbone ensemble of 20 lowest energy structures. The side chains of Cys residues are shown in red. (b) Ribbon representation of the closest-to-mean structure.
	Figure 3. Analgesic activity of Eu1.6 in rat PNL and CCI pain models. (a,b,e,f) PNL and CCI rats (n = 8/group) were treated with sterile saline (i.m.), Eu1.6 (0.5, 2.49 and 24.9 μg/kg, i.m.), Vc1.1 (27.2 μg/kg, i.m.), or a combination of morphine (5 mg/kg), subcutaneous injection (sc) and gabapentin (100 mg/kg, intragastric administration (i.g.). Sham rats (n = 8) were treated with sterile saline (i.m.). (c,d,g,h) PNL and CCI rats (n = 8/group) were treated with sterile saline (i.v.), Eu1.6 (1, 4.98 and 49.8 μg/kg, i.v.) or the combination of morphine (5 mg/kg, sc) and gabapentin (100 mg/kg, i.g.). The LHS graphs shows the mean pain threshold at pre-injection and 2, 4 and 6 h following treatments (a,c,e,g), whereas the RHS bar  graphs show the corresponding elevation percentage at 2 h and 4 h following treatments (b,d,f,h). The different groups were analyzed by one-way ANOVA followed by the LSD test for multiple comparison tests at a 0.05 level of significance. *p < 0.05 vs. saline, #p < 0.05 Eu1.6 (low dose group) vs. Vc1.1 or the combined group of morphine and gabapentin.
	Figure 4. Eu1.6 inhibition of nicotinic acetylcholine receptor (nAChR) subtypes. Eu1.6 (10 µM) was applied by perfusion to oocytes expressing nAChRs as described in Methods. The error bars denote the S.E.M. of the data from four to six oocytes for each determination. (a) Representative ACh-evoked currents mediated by α9α10 and α7 nAChRs in the absence (control) and presence of 10 µM Eu1.6. (b) Bar graph of the mean ACh-evoked current amplitude of nAChR subtypes in the presence of 10 μM  Eu1.6. Data are presented as Mean ± SEM, Statistical significance was determined with One-way ANOVA, *p < 0.05, **p < 0.001. (c) A summary of the effect of Eu1.6 on various neuronal nAChR subtypes.
	Figure 5. Eu1.6 inhibition of depolarization-activated Ba2+ currents is independent of GABAB receptor activation in mouse DRG neurons. (a) Superimposed representative time plots of IBa inhibition by 1 µM Eu1.6 in the presence of 1 µM CGP55845in mouse DRG neurons. Bars indicate the bath application of CGP55845, Eu1.6 in the presence of CGP55845, and ω-conotoxin CVIE. Superimposed inward Ba2+ currents (bottom insets) obtained in the presence of 1 µM CGP 55845 (a), Eu1.6 in the presence of CGP 55845 (b), and 300 nM CVIE (c), are shown at the times indicated by lowercase letters. Dotted lines indicate zero-current level. (b) Time course of IBa inhibition by 1 µM Eu1.6 Eu1.6 in the presence of CGP55845 in mouse DRG neurons. Bars indicate the bath application of Eu1.6, Eu1.6 in the presence of CGP55845, and ω-conotoxin CVIE. Application of CGP 55845 did not antagonize the effect of Eu1.6 on HVA calcium channel. (c) Time course of the inhibition of IBa by 300 nM CVIE (b) and 1 µM Eu1.6 in the presence of 300 nM CVIE (c) in mouse DRG neurons. Inward IBa elicited by step depolarization applied at 0.1 Hz from a HP of −80 mV to −5 mV or 0 mV (as indicated in bottom inserts for each representative time course) plotted as a function of time. (d) Bar graph summary of inhibition of peak current amplitude by 1 µM Eu1.6, 1 µM Eu1.6 in the presence of 1 µM CGP55845, 300 nM CVIE, and 300 nM CVIE + 1 μM Eu1.6. Data are presented as Mean ± SEM. No significant difference was observed between inhibition of HVA calcium channel current by application of Eu1.6 and Eu1.6 + CGP55845 (P > 0.05, paired two-tailed t-test) and the inhibition of HVA calcium current by CVIE and CVIE + Eu1.6 (P > 0.05, paired two-tailed t-test). Number of experiments is indicated in parentheses.
	Figure 6. Eu1.6 concentration-dependent inhibition of human CaV2.2 expressed in HEK293 cells. (a) Concentration- response relationship obtained for Eu1.6 inhibition of CaV2.2 giving an IC50 of 1.1 nM and a maximum block of <50% (n = 4). (b) Ba2+current amplitude plotted as a function of time in the absence and presence of 1 µM Eu1.6 and 100 nM CVIE. Cells were activated by a depolarizing pulse from a holding potential of −80 mV to a test potential of +10 mv for 200 ms at 0.2 Hz, after reaching a plateau (max. block) cells were superfused (washout) with external bath solution for several minutes for complete recovery. (c) Voltage protocol used to determine the I-V relationship under control conditions and in the presence of 1 µM Eu1.6. Representative current traces recorded over a series of depolarizing testing potentials from a holding potential of −100 mV to −40 mV to +55 mV (∆5 mV increments) under control conditions and after >2 min. exposure to 1 µM Eu1.6 in the external bath solution. (d) I-V relationship recorded at certain time points in the absence (filled circle) and presence of Eu1.6 (open circle). (e) Activation kinetics (tau) measured in the absence (control, filled circle) and presence of 1 µM Eu1.6 (open circle) was determined by a mono-exponential function (inset) for a series of testing potentials derived from the same I-V traces.
	Figure 7. Eu1.6 slows deactivation kinetics of CaV2.2 channel. (a) Tail currents were determined at a repolarizing potential (−50 mV) for 10 ms in the absence (control) and presence of Eu1.6. Insert: expanded time scale of superimposed tail currents obtained in the absence (control, black line; washout, dashed line) and presence of Eu1.6 (grey line). (b) Conductance-voltage (G-V) relationships determined from tail currents recorded in the absence (control, filled circle) and presence of Eu1.6 (open circle). The current was normalized to its maximum current and fitted with a double Boltzmann function. (c) Current density plot was derived from the current amplitude (pA) divided by the cell capacitance (pF). See Supp. Tables S2 and S3 for parameter and significance testing. (d) Deactivation kinetics (tau) was determined by a mono-exponential fit of tail currents. The scatter plot depicts the deactivation kinetics in the absence (filled circle) and presence of Eu1.6 (open circle) for a series of experiments at a single prepulse potential. Statistical significance was determined with an unpaired t-test, *p < 0.05. (e) Representative deactivation kinetics of currents traces recorded in the absence (control, filled circle), after incubation (>2 min.) with Eu1.6 (open circle) and upon washout (open diamond).
	Figure 8. Eu1.6 shifts the voltage-dependence of steady-state inactivation (SSI) of the CaV2.2 channel. The steady-state inactivation was determined by a triple pulse protocol. A long conditioning pulse over a series of membrane potentials (holding potentials), starting from −110 mV to 0 mV (10 mV increments, 10 s), was recorded immediately after a 50 ms prepulse to +10 mV (P1). Another brief pulse after the preconditioning pulse was recorded for 50 ms at +10 mV. (a) Representative current traces (P2) are shown in the absence and presence of 1 µM Eu1.6. Currents were normalized to the maximum current and plotted over a series of holding potentials. Data points were fitted with a single Boltzmann function as exemplified in (b). Insert: bar graph shows half-maximum inactivation (V0.5,inact) was shifted by more than 10 mV to more hyperpolarized potentials. (c) Bar graph of P2/P1 as a function of holding potential represents mean ± SEM values. Statistical significance for each holding potential was determined with an unpaired t-test, *p < 0.05, **p < 0.01 and ***p < 0.001.

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