J Physiol 588.10 Pain modulation by tethered toxins 1701 Tg-t-MrVIa 13.8 ± 4.0; cinnamaldehyde: Wt 10.1 ± 6.6%, Tg-t-MrVIa 11.6 ± 6.2, n = 4 for both genotypes) (Supplemental Fig. 1B and D). These results show that these ion channels are not non-speciﬁcally affected by genetic expression of the t-toxin in these neurones. Other parameters, such as voltage-gated potassium outward currents (Fig. 2F) and the resting membrane potential (Wt −51.9. ± 1.95 mV, Tg-t-MrVIa −52.7 ± 1.1 mV, n = 32; Supplemental Table 2), were also unaltered in nociceptors of Tg-t-MrVIa mice compared to controls. These results indicate that t-MrVIa is cell autonomous and speciﬁc for VGSCs in vivo. Nociceptors, electroporated with t-MrVIa and EGFP, showed FLAG immunolabelling at the cell membrane, which was abolished after treatment with phosphatidylinositol-speciﬁc phospholipase C (PI-PLC) that cleaves GPI-anchored proteins from the cell surface (Fig. 3A). Immunoprecipitation assays indicated that t-MrVIa is enriched in membrane fractions and cannot be detected in the culture medium (Fig. 3B). VGSC currents were measured before and after treatment with PI-PLC. Total peak current densities in nociceptors of Tg-t-MrVIa mice were restored to 87.5 ± 8.6% of control currents after PI-PLC (Fig. 3C). Taken together, these data demonstrate that t-MrVIa acts at the cell surface to block VGSCs. A number of studies have reported that native MrVIa and its close relative MrVIb preferentially block TTX-R VGSCs, but at higher concentrations a subset of TTX-S channels are also affected by these toxins (Daly et al. 2004; online Supplemental Table 1). In Supplemental Table 1 we have presented an overview of the reported VGSC blocking capabilities of the native soluble forms of μO-conotoxins MrVIa and MrVIb in different experimental systems. These studies show that while the native conotoxins MrVIa/b are not highly selective for only one type of VGSC, they can discriminate between TTX-R channels present in nociceptors over TTX-S. Although the relative extent of the inhibition of VGSC current depends on the assay, and there is no direct comparison of all α-subunits within one experimental set of data, it would seem that native MrVIa/b preferentially blocks Nav1.8 followed by Nav1.7, Nav1.4 and Nav1.2. To determine whether the modiﬁed MrVIa used in this study maintains the characteristics of the native soluble MrVIa form, we performed oocyte electrophysiological assays with t-MrVIa and different VGSC α-subunits. These experiments show signiﬁcant block of Nav1.2, but not of Nav1.1, Nav1.3 and Nav1.6 VGSCs by t-MrVIa (Supplemental Fig. 2) in agreement with the reported afﬁnities of the soluble toxin (Supplemental Table 1). Thus our experimental evidence suggests that the tether does not change the speciﬁcity of the MrVIa toxin. We next used TTX to discriminate between TTX-R and TTX-S currents present in nociceptors. TTX-R currents in nociceptors of t-MrVIa transgenic mice show a 44 ± 7% reduction compared to wild-type mice (Wt: 284.3 ± 34.1 pA pF−1, n = 30; Tg-t-MrVIa: 158.6 ± 20.4 pA pF−1, n = 31, P < 0.5). This block was reversed by PI-PLC treatment thus demonstrating that the tethered toxin was responsible for this inhibition (PI-PLC: 243.6 ± 31.5 pA pF−1, n = 12) (Fig. 3C and D). We observed a non-statistically signiﬁcant inhibition (31 ± 13%; Wt: 129.6 ± 22.1 pA pF−1, Tg-t-MrVIa: 89.3 ± 16.6 pA pF−1, P = 0.23) of TTX-S currents in nociceptors with a very large sample size (n = 32 nociceptors recorded) (Fig. 3C). In addition the recovery of TTX-S currents after PI-PLC enzymatic cleavage of the tether was not statistically signiﬁcant as it was for TTX-R currents. Thus the changes in TTX-S currents observed in transgenic mice might be indicative of partial (but not statistically signiﬁcant) antagonistic activity of the t-MrVIa on these channels (Fig. 3C). Furthermore, we used a pulse protocol that avoids any substantial contamination by Nav1.9, which undergoes ultraslow inactivation such that clamping the membrane potential for several minutes to at least −100 mV is required to release it from inactivation (Cummins et al. 1999; Amaya et al. 2006). Given that we used a holding potential of −60 mV while measuring TTX-R currents, similar to the experiments performed with the soluble toxin (Daly et al. 2004), our results demonstrate a block of Nav1.8 TTX-R channels by t-MrVIa in vivo. We next tested the effect of t-MrVIa on isolectin-B4 positive (IB4+ve) nociceptors, which have a higher density of TTX-R currents (Dib-Hajj et al. 1998; Stucky & Lewin, 1999; Snape et al. 2010). Voltage clamp analyses in DRG neurones live-labelled with ﬂuorescently coupled IB4 indicated that the t-MrVIa-mediated block was 48 ± 8% in the IB4+ve neurone population (Wt: 422.7 ± 57.0 pA pF−1, n = 10, Tg-t-MrVIa: 221.8 ± 33.2 pA pF−1, n = 12) and 48 ± 9% in the IB4–ve population (Wt: 215.1 ± 33.6 pA pF−1, n = 20; 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 mechano- receptors. C 2010 The Authors. Journal compilation C 2010 The Physiological Society