Christensen SB , Hone AJ , Roux I , Kniazeff J , Pin JP , Upert G , Servent D , Glowatzki E , McIntosh JM .

R01 DC006476 NIDCD NIH HHS , R01 GM103801 NIGMS NIH HHS , P01 GM048677 NIGMS NIH HHS , R01 DC012957 NIDCD NIH HHS , R03 DC013374 NIDCD NIH HHS

	Figure 1. (A) Amino acid sequence alignment of the extracellular domains of mouse α9 and α10 subunits. Numbering complies with the numbering of the α9 nAChR subunit presented in the crystal structure of the human α9 extracellular domain (Zouridakis et al., 2014). Arrow heads point at residues thought to interact with α-CT× RgIA4: T61 and D121 (α9) and E197, P200, and D201 (α10), based on comparison with the binding of rat α9 and α10 subunits to α-conotoxin RgIA (Perez et al., 2009; Azam et al., 2015). Conserved residues are highlighted, colors were chosen depending on the chemical properties of the residues. (B) Sequence alignments of the α-conotoxin RgIA–interacting domains of mouse, rat and human α9, and α10 nAChR subunits.
	Figure 2. Functional characterization of mouse α9α10 nAChRs heterologously expressed in Xenopus laevis oocytes. Oocytes expressing mouse α9α10 nAChRs were subjected to two-electrode voltage clamp electrophysiology as described in Materials and Methods. The EC50for activation of α9α10 nAChRs by ACh was 30.1 (23.5–38.7) μM and the Hill slope was 1.1 (0.8–1.4) (n = 5). The EC50 for activation by choline was 601 (311–1160) μM; Hill slope 1.3 (0.5–2.1) maximal efficacy was 50 ± 5% relative to ACh (n = 4). Nicotine failed to evoke currents in all oocytes tested (0.4 ± 0.2%) (n = 4). The values in parenthesis denote the 95% confidence interval.
	Figure 3. RgIA4 selectively blocks mouse α9α10 nAChRs. nAChRs were expressed in Xenopus laevis oocytes. (A) The concentration response curves for α9α10 and α7 are shown. The IC50 for α9α10 was 1.2 (0.95–1.6) nM with a Hill slope of 1.6 (0.91–2.2). The IC50 for α7 was 4.5 (3.1–6.4) μM with a Hill slope of 2.0 (0.84–3.2). The values in parenthesis denote the 95% confidence intervals; n = 3–7 for each condition. (B) Recovery from toxin block of α9α10 is relatively slow. Example trace of block by 33 nM RgIA4. Recovery after 15 min toxin washout was ~2%. Arrows indicate application of 10 μM ACh. (C) At all other indicated nAChR subtypes, RgIA4 (10 μM) blocked less than 50% of the ACh-evoked response; the percent of the ACh-response for each subtype was α1β1δϵ, 95.0 ± 3.8; α1β1δγ, 86.2 ± 3.8; α3β2, 106 ± 2.9; α3β4, 105 ± 0.7; α4β2, 102 ± 3.3; α4β4, 74.3 ± 3.0. n = 3–4 for each condition; ± is S.E.M. Example traces for α4β2, α3β4, and α3β2 are shown. Results are summarized in Table 1.
	Figure 4. RgIA4 prevents oxaliplatin-induced cold allodynia. Mice were injected once per day with oxaliplatin (3.5 mg/kg i.p) or 0.9% saline (vehicle) for control animals. In addition, mice were injected once per day with RgIA4 (40 μg/kg, s.c.) or vehicle. During the treatment period, mice were injected for 2 days, followed by a 2 day break, followed by injection for another 3 days. Twenty-four hours after last injection, mice were assessed for cold allodynia using a cold-plate as described in Materials and Methods. This injection schedule was repeated for two additional weeks. Treatments were stopped on day 21; cold-plate assessment was performed 24 h later on day 22; subsequent testings were performed once a week for three additional weeks. Time to respond to decreasing temperature was measured and indicated on the y axis as the mean ± SEM (n = 8) for each experimental group. Experimenters were blinded to treatment conditions. Data was analyzed using a one-way ANOVA with Dunnett's Multiple Comparison Test, P-values were indicated as: *P < 0.05, **P < 0.01, and ***P < 0.001 for significant difference from vehicle/vehicle control. And 0P < 0.05, 000P < 0.001, and indicating significant difference compared with oxaliplatin/vehicle mice.
	Figure 5. (A) α-Conotoxins do not displace the orthosteric GABABR antagonist CGP54626-Red. GABA, CGP54626, and the indicated α-Ctxs (at 1 μM) were assessed for the ability to displace CGP54626-Red binding from GABABR heterologously expressed in HEK293 cells as described in Material and Methods. Three replicates were obtained for each value and the experiment was repeated three times. (B) α-Conotoxins do not activate GABABR. The indicated α-conotoxins were tested at 1 μM for their ability to activate GABABR heterolgously expressed together with fluorescently tagged G protein subunits in HEK293 cells. At resting state, the G-protein subunits are in close proximity resulting in a strong basal bioluminescence resonance energy transfer (BRET) signal. Activation of GABABR was assessed by monitoring dissociation of Gαi1-Rluc (top) or GαoA-Rluc (bottom) from β2γ2-Venus as measured by the change in BRET. The results after 10 min of toxin incubation are expressed as a percentage of GABA-induced BRET change from basal fluorescence levels. Three replicates were obtained for each value and the experiment was repeated three times.

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