Stürzebecher AS , Hu J , Smith ES , Frahm S , Santos-Torres J , Kampfrath B , Auer S , Lewin GR , Ibañez-Tallon I .

	Figure 1. Generation of BAC transgenic mice encoding membrane-tethered MrVIa toxinA, diagram showing t-MrVIa (green ribbon) including a FLAG epitope for immunodetection, a flexible poly (asparagine-glycine) linker (grey) and a GPI tether to the cell membrane. B, schematic overview of the BAC modification by two-step homologous recombination. The modified shuttle vector (SV) contains the t-toxin cassette (green), flanked by a secretion signal (sec) and a polyA (pA), inserted between two recombination boxes (A and B in red) corresponding to the sequences flanking the initiator methionine of the Scn10a gene encoding the Nav1.8 VGSC subunit. Recombination (red lines) with the Scn10a BAC results in the modified BAC and the free shuttle vector. Scn10a gene promoter (black arrow), Scn10a exons (grey boxes), selection markers (dark green boxes), NcoI restriction sites for Southern blot (N), PI-SceI restriction site for BAC linearisation (P). C, Southern blot of transgenic founder lines. Lines 2 and 27 showed the highest relative ratio of the transgene band (∼2.3 kb) in comparison with the endogenous Scn10a allele (∼1.5 kb) indicative of high BAC copy number.
	Figure 2. Cell-autonomous inhibition of sodium currents in nociceptors of Tg-t-MrVIa miceA, co-expression of Scn10a and t-MrVIa transcripts in DRGs of Tg-t-MrVIa mice detected by RT-PCR analysis (Sk, skin; Bs, brain stem; Co, cortex. B, in situ hybridisation on spinal cord sections of mouse embryos (E15) show strong t-MrVIa signal in DRGs but not in spinal cord (Sc) of Tg-t-MrVIa mice. Vc, vertebral column. Scale bar: 100 μm. C, t-MrVIa transcripts were detected by in situ hybridisation in nociceptors (arrows) but not mechanoreceptors (arrowheads) of Tg-t-MrVIa mice (2–4 weeks old). Scale bar: 50 μm. D–H, voltage-gated currents were evoked by step depolarisations from −50 mV to +40 mV preceded by a hyperpolarizing prepulse from −60 to −120 mV to prevent inactivation. D and E, representative traces and current–voltage relationships of inward currents indicate a significant reduction of sodium currents in nociceptors of Tg-t-MrVIa mice in comparison to wild-type littermates. F, current–voltage relations of outward currents are not affected in nociceptors of Tg-t-MrVIa mice. G and H, representative traces and current–voltage relations of inward currents, evoked by the same protocol shown in D, indicate no differences in mechanoreceptors of Tg-t-MrVIa mice and wild-type littermates. I, peak VGSC current densities are significantly reduced in nociceptors of Tg-t-MrVIa mice (243.5 ± 37.2 pA pF−1) with respect to wild-type mice (413.7 ± 37.2 pA pF−1) (n= 32 per group) and unchanged in mechanoreceptors (n= 9 per group). P < 0.05 two-way ANOVA in E and H; P > 0.05 two-way ANOVA in F; P < 0.05 t test in I. J and K, representative traces (J) and current–voltage relations (K) of voltage-gated calcium currents, evoked by step depolarisations from −90 mV to +40 mV, indicate no differences in nociceptors of Tg-t-MrVIa mice and wild-type littermates (n= 10 per group). L, representative action potentials used to identify nociceptors and mechanoreceptors.
	Figure 3. t-MrVIa acts at the cell membrane and specifically blocks TTX-R currents with no compensatory upregulation of other VGSCsA, t-MrVIa (FLAG: red) colocalizes with the membrane marker WGA (blue) in DRG neurones co-electroporated with t-MrVIa and cytoplasmic EGFP (upper panel). PI-PLC treatment eliminates FLAG immunoreactivity showing specific cleavage of the GPI-anchored toxin from the membrane (lower panel). Scale bars: 5 μm. B, mammalian cells transfected with t-MrVIa show expression of the toxin in the membrane fraction by FLAG immunoprecipitation. C, bar graph indicating the quantification of VGSC peak currents in nociceptors of wild-type (Wt) and Tg-t-MrVIa mice (n= 32 cells per group) before and after TTX and PI-PLC treatment (n= 12 cells). The current densities of total VGSC and TTX-R currents are significantly reduced in Tg-t-MrVIa mice compared to wild-type mice, and significantly recover from t-toxin inhibition after PI-PLC treatment. TTX-S currents are not significantly affected in Tg-t-MrVIa mice. D, current–voltage relationships of TTX-R currents indicate a significant inhibition of sodium currents in nociceptors of Tg-t-MrVIa mice in comparison to Wt littermates (P < 0.05 two-way-ANOVA), and no significant inhibition, with respect to Wt, in Tg-t-MrVIa mice after PI-PLC treatment (P > 0.05 two-way ANOVA). E, peak current measurements in nociceptors treated with TTX and live-labelled with isolectin B4 indicate that IB4+ve nociceptors display more TTX-R than IB4–ve nociceptors in Wt and Tg mice and that t-MrVIa inhibition is significant in both subpopulations but more pronounced in IB4+ve nociceptors (n= 10–22 cells per group). F, the mean amplitude of action potentials is significantly reduced in IB4+ve nociceptors (Wt: 89.2 ± 2.3 mV, Tg-t-MrVIa: 74.3 ± 3.9 mV) but not in IB4–ve nociceptors and mechanoreceptors (t test). (n= 9–20 per group.) G, immunodetection of substance P (red) and isolectin B4 (green) in dorsal spinal cord indicate no differences in afferent innervation between Wt and Tg-t-MrVIa adult mice. Scale bar: 50 μm, t test in C and E.
	Figure 4. Tg-t-MrVIa adult mice show reduced inflammatory hyperalgesia and insensitivity to cold painA, no differences in inflammatory thermal hyperalgesia induced by intraplantar injection of carrageenan were observed between wild-type (Wt) and Tg-t-MrVIa mice (n= 10 mice). B, induced inflammation causes mechanical hyperalgesia in Wt mice (peak at 4 h) and significantly reduced hyperalgesia in Tg-t-MrVIa (n= 10 mice). Dashed line indicates baseline pain response. C, nocifensive cold responses scored during 90 s on a 0°C-cooled plate showed significantly reduced responses to noxious cold in Tg-t-MrVIa mice (n= 10 mice). D, temperature preference tests quantifying the time mice spent in either of two plates kept at 30°C and 10°C (n= 8 mice). E, histogram indicating the firing frequency of cold-sensitive C-fibres with a threshold >10°C over a cooling gradient from 30 to 0°C. The firing frequency did not differ between Wt and Tg-t-MrVIa mice (fibres: n= 8–12 fibres per group). F, histogram indicating the firing frequency of cold-sensitive C-fibres with a threshold < 10°C responding to noxious cold stimuli over a cooling gradient from 30 to 0°C. The firing frequency of cooling units with a threshold below 10°C is significantly reduced in Tg-t-MrVIa compared to Wt mice (fibres: n= 4–8 fibres per group). The lower trace corresponds to the cold ramp stimuli used in both cases. P > 0.05 two-way ANOVA in A and E; P < 0.05 two-way ANOVA in B and F; P < 0.05 t test in C.

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