J Physiol 588.10 (2010) pp 1695–1707 R A P I D R E P O R T 1695 An in vivo tethered toxin approach for the cell-autonomous inactivation of voltage-gated sodium channel currents in nociceptors Annika S. Stu¨ rzebecher1, Jing Hu2, Ewan St John Smith2, Silke Frahm1, Julio Santos-Torres1, Branka Kampfrath1, Sebastian Auer1, Gary R. Lewin2 and Ine´s Iban˜ez-Tallon1 1Molecular Neurobiology group, 2Molecular Physiology of Somatic Sensation, Department of Neuroscience, Max-Delbru¨ck Center for Molecular Medicine, Robert-Ro¨ssle Strasse 10, 13125 Berlin, Germany Understanding information ﬂow in sensory pathways requires cell-selective approaches to manipulate the activity of deﬁned neurones. Primary afferent nociceptors, which detect painful stimuli, are enriched in speciﬁc voltage-gated sodium channel (VGSC) subtypes. Toxins derived from venomous animals can be used to dissect the contributions of particular ion currents to cell physiology. Here we have used a transgenic approach to target a membrane-tethered isoform of the conotoxin MrVIa (t-MrVIa) only to nociceptive neurones in mice. T-MrVIa transgenic mice show a 44 ± 7% reduction of tetrodotoxin-resistant (TTX-R) VGSC current densities. This inhibition is permanent, reversible and does not result in functional upregulation of TTX-sensitive (TTX-S) VGSCs, voltage-gated calcium channels (VGCCs) or transient receptor potential (TRP) channels present in nociceptive neurones. As a consequence of the reduction of TTX-R VGSC currents, t-MrVIa transgenic mice display decreased inﬂammatory mechanical hypersensitivity, cold pain insensitivity and reduced ﬁring of cutaneous C-ﬁbres sensitive to noxious cold temperatures. These data validate the use of genetically encoded t-toxins as a powerful tool to manipulate VGSCs in speciﬁc cell types within the mammalian nervous system. This novel genetic methodology can be used for circuit mapping and has the key advantage that it enables the dissection of the contribution of speciﬁc ionic currents to neuronal function and to behaviour. (Received 7 January 2010; accepted after revision 16 March 2010; ﬁrst published online 22 March 2010) Corresponding author I. Iban˜ez-Tallon: Max-Delbru¨ck Center for Molecular Medicine, Robert-Ro¨ssle Strasse 10, 13125 Berlin, Germany. Email: ibanezi@mdc-berlin.de Abbreviations AP, action potential; BAC, bacterial artiﬁcial chromosome; DRG, dorsal root ganglion; GPI, glycophosphatidylinositol; IB4, isolectin-B4; PI-PLC, phosphatidylinositol-speciﬁc phospholipase C; t-MrVIa, membrane-tethered conotoxin MrVIa; TRP, transient receptor potential; TTX, tetrodotoxin; TTX-R, tetrodotoxin resistant; TTX-S, tetrodotoxin sensitive; VGSC, voltage-gated sodium channel; VGCC, voltage-gated calcium channel. Introduction Neuronal communication relies on action potentials (APs) generated by the activity of voltage-gated sodium channels (VGSCs) following membrane depolarisation. The alkaloid toxin tetrodotoxin (TTX) has been exploited for more than 40 years due to its unique ability to block VGSCs and therefore to assess the contribution of these channels to cell excitability and AP propagation. Nociceptive sensory neurones (nociceptors) detect noxious peripheral stimuli; this information is then transmitted to the superﬁcial dorsal horn of the spinal cord, relayed to the brain, and perceived as pain (Lewin & Moshourab, 2004). Nociceptors express two unusual VGSCs, Nav1.8 and Nav1.9, which are resistant to TTX (Dib-Hajj et al. 1998; Akopian et al. 1999). Nav1.8 generates sodium currents with a high activation threshold (−40 mV) and slow inactivation (Sangameswaran et al. 1997; Akopian et al. 1999; Renganathan et al. 2002), whereas Nav1.9 produces a persistent current with a more hyperpolarised voltage dependence and ultraslow recovery from inactivation (Baker, 2005; Cummins et al. 2007). In addition nociceptors are enriched in the Nav1.7 TTX-S VGSC subtype (Nassar et al. 2004) which produces C 2010 The Authors. Journal compilation C 2010 The Physiological Society DOI: 10.1113/jphysiol.2010.187112