J Physiol 588.10 Pain modulation by tethered toxins 1699 indicated that t-MrVIa transcripts were present during embryonic development (Fig. 2B). In adult mice, t-MrVIa transcripts were detected in small-diameter nociceptors, but not in large-diameter sensory neurones (Fig. 2C). An important feature of membrane t-toxins is their cell-autonomous action. To assess, whether this property was maintained in vivo, we prepared DRG primary cultures, which contain a mixture of sensory neurone types e.g. nociceptors and mechanoreceptors. Nociceptors were discriminated from mechanoreceptors by measuring AP conﬁguration using the whole-cell patch clamp technique (Fig. 2L; AP half-peak width >3.1 ms for nociceptors and <0.8 ms for mechanoreceptors; see online Supplemental Table 2) and cell soma size (<25 μm soma diameter for nociceptors and >25 μm for mechano- receptors; Supplemental Table 2) as previously described (Stucky & Lewin, 1999; Lawson, 2002). Representative traces of VGSC currents, evoked by step depolarisation in voltage clamp mode, in nociceptors and mechano- receptors of both Tg-t-MrVIa mice and wild-type littermates are shown in Fig. 2D and G. Current–voltage relations indicated a signiﬁcant reduction of VGSC currents in nociceptors of Tg-t-MrVIa mice compared to wild-type littermates (Fig. 2E). In contrast, VGSC currents in mechanoreceptors of Tg-t-MrVIa mice were indistinguishable from those measured in mechano- receptors of wild-type (Wt) littermates (Fig. 2G and H ). Normalisation of peak VGSC currents to cell capacitance indicated a 41.1 ± 9% reduction of peak current densities in nociceptors of Tg-t-MrVIa mice compared to controls (Wt: 413.7 ± 37.2 pA pF−1, Tg-t-MrVIa: 243.5 ± 37.2 pA pF−1, n = 32, P < 0.05). However, no difference was observed in mechanoreceptors (Wt: 306.9 ± 57.4 pA pF−1, Tg-t-MrVIa: 332.8 ± 26.8 pA pF−1, n = 9) (Fig. 2I ; Supplemental Table 2). To exclude the possibility that the t-MrVIa toxin could be affecting other ion channels present in nociceptors besides VGSCs, we tested the functionality of VGCC (Fig. 2J and K ) and TRP channels (Fig. 1 in online Supplemental material). Patch-clamp recordings of VGCCs performed by step depolarisations indicated no differences between nociceptors of Tg-t-MrVIa mice compared to wild-type littermates (Fig. 2J ). Current–voltage relationships did not differ between nociceptors of Tg-t-MrVIa and wild-type mice with currents measured with increasing voltage steps from −50 to +40 mV, and maximal peak currents obtained at a voltage depolarisation from −50 to −10 mV (Wt: −1.11 ± 0.9 nA, Tg-t-MrVIa: −1.13 ± 0.8 nA, n = 10 each) (Fig. 2K ). The functionality and incidence of the TRP channels: TRPV1, TRPM8 and TRPA1 was measured by fura-2-based imaging of increases in [Ca2+]i in DRG neurones through the sequential application of the speciﬁc activators capsaicin, menthol and cinnamaldehyde (Supplemental Fig. 1A and C). No signiﬁcant differences in the C 2010 The Authors. Journal compilation C 2010 The Physiological Society percentage of DRG neurones responding to each agonist were observed between Tg-t-MrVIa mice compared to wild-type littermates (capsaicin: Wt 35.8 ± 8.1%, Tg-t-MrVIa 35.5 ± 5.1; menthol: Wt 15.3 ± 5.6%, Figure 1. Generation of BAC transgenic mice encoding membrane-tethered MrVIa toxin A, 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.