Abstract
At synaptic terminals, high voltage–activated Cav2.1 and Cav2.2 calcium channels have an essential and joint role in coupling the presynaptic action potential to neurotransmitter release. Here we show that membrane-tethered toxins allowed cell-autonomous blockade of each channel individually or simultaneously in mouse neurons in vivo. We report optimized constitutive, inducible and Cre recombinase–dependent lentiviral vectors encoding fluorescent recombinant toxins, and we also validated the toxin-based strategy in a transgenic mouse model. Toxins delivered by lentiviral vectors selectively inhibited the dopaminergic nigrostriatal pathway, and transgenic mice with targeted expression in nociceptive peripheral neurons displayed long-lasting suppression of chronic pain. Optimized tethered toxins are tools for cell-specific and temporal manipulation of ion channel-mediated activities in vivo, including blockade of neurotransmitter release.
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References
Tsien, R.W., Lipscombe, D., Madison, D.V., Bley, K.R. & Fox, A.P. Multiple types of neuronal calcium channels and their selective modulation. Trends Neurosci. 11, 431–438 (1988).
Catterall, W.A. Structure and regulation of voltage-gated Ca2+ channels. Annu. Rev. Cell Dev. Biol. 16, 521–555 (2000).
Catterall, W.A. & Few, A.P. Calcium channel regulation and presynaptic plasticity. Neuron 59, 882–901 (2008).
Terlau, H. & Olivera, B.M. Conus venoms: a rich source of novel ion channel-targeted peptides. Physiol. Rev. 84, 41–68 (2004).
Lewis, R.J. & Garcia, M.L. Therapeutic potential of venom peptides. Natl. Rev. 2, 790–802 (2003).
Olivera, B.M. et al. Neuronal calcium channel antagonists. Discrimination between calcium channel subtypes using omega-conotoxin from Conus magus venom. Biochemistry 26, 2086–2090 (1987).
Hillyard, D.R. et al. A new Conus peptide ligand for mammalian presynaptic Ca2+ channels. Neuron 9, 69–77 (1992).
Ibañez-Tallon, I. et al. Tethering naturally occurring peptide toxins for cell-autonomous modulation of ion channels and receptors in vivo . Neuron 43, 305–311 (2004).
Wu, Y., Cao, G., Pavlicek, B., Luo, X. & Nitabach, M.N. Phase coupling of a circadian neuropeptide with rest/activity rhythms detected using a membrane-tethered spider toxin. PLoS Biol. 6, e273 (2008).
Holford, M., Auer, S., Laqua, M. & Ibañez-Tallon, I. Manipulating neuronal circuits with endogenous and recombinant cell-surface tethered modulators. Front. Mol. Neurosci. 2, 21 (2009).
Dudanova, I. et al. Important contribution of {alpha}-neurexins to Ca2+-triggered exocytosis of secretory granules. J. Neurosci. 26, 10599–10613 (2006).
Cao, Y.-Q. Presynaptic Ca2+ channels compete for channel type-preferring slots in altered neurotransmission arising from Ca2+ channelopathy. Neuron 43, 387–400 (2004).
Cao, Y.Q. & Tsien, R.W. Effects of familial hemiplegic migraine type 1 mutations on neuronal P/Q-type Ca2+ channel activity and inhibitory synaptic transmission. Proc. Natl. Acad. Sci. USA 102, 2590–2595 (2005).
Poncer, J.C., McKinney, R.A., Gahwiler, B.H. & Thompson, S.M. Either N- or P-type calcium channels mediate GABA release at distinct hippocampal inhibitory synapses. Neuron 18, 463–472 (1997).
Szulc, J., Wiznerowicz, M., Sauvain, M.-O., Trono, D. & Aebischer, P. A versatile tool for conditional gene expression and knockdown. Nat. Methods 3, 109–116 (2006).
Peitz, M., Pfannkuche, K., Rajewsky, K. & Edenhofer, F. Ability of the hydrophobic FGF and basic TAT peptides to promote cellular uptake of recombinant Cre recombinase: A tool for efficient genetic engineering of mammalian genomes. Proc. Natl. Acad. Sci. USA 99, 4489–4494 (2002).
Pfeifer, A., Brandon, E.P., Kootstra, N., Gage, F.H. & Verma, I.M. Delivery of the Cre recombinase by a self-deleting lentiviral vector: Efficient gene targeting in vivo. Proc. Natl. Acad. Sci. USA 98, 11450–11455 (2001).
Ahn, K., Mishina, Y., Hanks, M.C., Behringer, R.R. & Crenshaw, E.B. III. BMPR-IA signaling is required for the formation of the apical ectodermal ridge and dorsal-ventral patterning of the limb. Development 128, 4449–4461 (2001).
Ungerstedt, U. & Arbuthnott, G.W. Quantitative recording of rotational behavior in rats after 6-hydroxy-dopamine lesions of the nigrostriatal dopamine system. Brain Res. 24, 485–493 (1970).
Zamponi, G.W., Lewis, R.J., Todorovic, S.M., Arneric, S.P. & Snutch, T.P. Role of voltage-gated calcium channels in ascending pain pathways. Brain Res. Rev. 60, 84–89 (2009).
Scroggs, R.S. & Fox, A.P. Calcium current variation between acutely isolated adult rat dorsal root ganglion neurons of different size. J. Physiol. (Lond.) 445, 639–658 (1992).
Benn, S.C., Costigan, M., Tate, S., Fitzgerald, M. & Woolf, C.J. Developmental expression of the TTX-resistant voltage-gated sodium channels Nav1.8 (SNS) and Nav1.9 (SNS2) in primary sensory neurons. J. Neurosci. 21, 6077–6085 (2001).
Snider, W.D. & McMahon, S.B. Tackling pain at the source: new ideas about nociceptors. Neuron 20, 629–632 (1998).
Malmberg, A.B., Chen, C., Tonegawa, S. & Basbaum, A.I. Preserved acute pain and reduced neuropathic pain in mice lacking PKCgamma. Science 278, 279–283 (1997).
Saegusa, H. et al. Suppression of inflammatory and neuropathic pain symptoms in mice lacking the N-type Ca2+ channel. EMBO J. 20, 2349–2356 (2001).
Todorov, B. et al. Conditional inactivation of the Cacna1a gene in transgenic mice. Genesis 44, 589–594 (2006).
Inchauspe, C.G., Martini, F.J., Forsythe, I.D. & Uchitel, O.D. Functional compensation of P/Q by N-type channels blocks short-term plasticity at the calyx of held presynaptic terminal. J. Neurosci. 24, 10379–10383 (2004).
Jeng, C.J., Sun, M.C., Chen, Y.W. & Tang, C.Y. Dominant-negative effects of episodic ataxia type 2 mutations involve disruption of membrane trafficking of human P/Q-type Ca2+ channels. J. Cell. Physiol. 214, 422–433 (2008).
Kerschensteiner, D., Morgan, J.L., Parker, E.D., Lewis, R.M. & Wong, R.O. Neurotransmission selectively regulates synapse formation in parallel circuits in vivo . Nature 460, 1016–1020 (2009).
Airan, R.D. et al. Integration of light-controlled neuronal firing and fast circuit imaging. Curr. Opin. Neurobiol. 17, 587–592 (2007).
Miljanich, G.P. Ziconotide: neuronal calcium channel blocker for treating severe chronic pain. Curr. Med. Chem. 11, 3029–3040 (2004).
Hu, J. & Lewin, G.R. Mechanosensitive currents in the neurites of cultured mouse sensory neurones. J. Physiol. (Lond.) 577, 815–828 (2006).
Gong, S., Yang, X.W., Li, C. & Heintz, N. Highly efficient modification of bacterial artificial chromosomes (BACs) using novel shuttle vectors containing the R6Kgamma origin of replication. Genome Res. 12, 1992–1998 (2002).
Hargreaves, K., Dubner, R., Brown, F., Flores, C. & Joris, J. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 32, 77–88 (1988).
Bennett, G.J. & Xie, Y.K. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain 33, 87–107 (1988).
Muller, T. et al. The bHLH factor Olig3 coordinates the specification of dorsal neurons in the spinal cord. Genes Dev. 19, 733–743 (2005).
Acknowledgements
We thank P. Osten (Cold Spring Harbor Laboratory) for providing the original lentiviral plasmid; R.Y. Tsien (University of California, San Diego) for providing mCherry cDNA; P. Aebischer (École Polytechnique Fédéral de Lausanne) for providing pLVUT-tTR-KRAB plasmid; I.M. Verma (Salk Institute) for providing LV-Cre-SD plasmid; J. Duyster (Technical University Munich) for floxed DsRed cDNA; M. Missler (Westfälische Wilhelms-Universität Münster) for providing HEK293-Cav2.2 cells; J. Meyer for support with the hippocampus culture protocol, and F. Rathjen, G.R. Lewin, C. Birchmeier, T. Kuner, C. Scharff and G. Dittmar for helpful discussions. This work was supported by Helmholtz Association (31-002), Sonderforschungsbereich (SFB 665) and by Deutsche Forschungsgemeinschaft (DFG RA 424/5-1).
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S.A. and A.S.S. performed and analyzed most of the experiments. R.J. and J.S.-T. performed electrophysiology and analyzed data. C.H. and S.F. assisted with experiments. B.L. helped design tethers and established lentivirus protocol. I.I.-T. conceived and supervised the project and wrote the paper with S.A.
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Auer, S., Stürzebecher, A., Jüttner, R. et al. Silencing neurotransmission with membrane-tethered toxins. Nat Methods 7, 229–236 (2010). https://doi.org/10.1038/nmeth.1425
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DOI: https://doi.org/10.1038/nmeth.1425
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