Silencing neurotransmission with membrane-tethered toxins

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Theodore Whitehead
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1 nature methods Silencing neurotransmission with membrane-tethered toxins Sebastian Auer, Annika S Stürzebecher, René Jüttner, Julio Santos-Torres, Christina Hanack, Silke Frahm, Beate Liehl & Inés Ibañez-Tallon Supplementary figures and text: Supplementary Figure 1 Supplementary Figure 2 Supplementary Figure 3 Supplementary Figure 4 Supplementary Table 1 Supplementary Table 2 Supplementary Table 3 Expression analysis of t-toxins in cultured mammalian cells. T-toxin expression does not interfere with sodium currents or action potential generation in neurons of rat hippocampus culture. T-toxin expression does not impair neuronal survival in infected rat hippocampus culture. RT-PCR analyses show expression of MVIIA-G transcripts exclusively in DRGs of Tg(Scn10a-MVIIA-G)109Iit mice. Overview of generated tethered toxins targeting voltage-gated Ca 2+ -channels. Passive membrane properties of t-toxin infected neurons remain unaltered. Used primers for RT-PCR and quantitative real-time PCR.

2 Supplementary Figure 1 Expression analysis of t-toxins in cultured mammalian cells. (a) HeLa cells transduced with MVIIA-PE (upper panel) or MVIIA-PC (lower panel) show direct fluorescence and are positive for flag immunostaining without cell permeabilization. Scale bars, 10 µm. (b) Western Blot analysis directed against the flag-tag of protein extracts from transduced HEK293T cells shows expected molecular weights of expressed t-toxins (PE: 36,5 kda, MVIIA-PE: 39,2 kda, AgaIVA-VG: 38 kda and 36 kda (double band indicates unprocessed peptide containing the hydrophobic signal sequence for GPI attachment and the mature cleaved GPI-form).

3 Supplementary Figure 2 T-toxin expression does not interfere with sodium currents or action potential generation in neurons of rat hippocampus culture. (a) Representative sodium currents induced by depolarizing pulses from -70 to +20 mv show no differences between control (noninfected) and MVIIA-PE + AgaIVA-VG infected cells. (b) Sodium current maximal amplitudes were unaltered between control (non-infected) and MVIIA-PE + AgaIVA-VG infected cells. p=0.48, n=10 cells each. (c) Representative example traces of spiking behavior in control (non-infected), no toxin- PE, MVIIA-PE + AgaIVA-VG infected cells and cells with application of soluble MVIIA + AgaIVA indicating unaltered action potential firing properties in current clamp recordings. (d) Spiking behavior by application of depolarizing current to infected hippocampal neurons shows no significant differences between no toxin-pe and MVIIA-PE + AgaIVA-VG expressing cells (n=7 for each).

with respect to control conditions (non-infected) (data of three independent experiments). (b) Fluorescence images showing NeuN stained neurons (red), GFP expression (green) and merge.

4 Supplementary Figure 3 T-toxin expression does not impair neuronal survival in infected rat hippocampus culture. (a) No neuronal cell death due to t-toxin expression was observed in hippocampal neurons by monitoring with the neuronal marker (NeuN) 6 days after infection and quantification of surviving neurons with respect to control conditions (non-infected) (data of three independent experiments). (b) Fluorescence images showing NeuN stained neurons (red), GFP expression (green) and merge. Scale bars, 50 µm. Supplementary Figure 4 RT-PCR analyses show expression of MVIIA-G transcripts exclusively in DRGs of Tg(Scn10a-MVIIA-G)109Iit mice. Scn10a transcripts, which encode for Na v 1.8, were detected only in dorsal root ganglia (DRG) of wildtype (wt) and transgenic (Tg) mice. Cacna1B transcripts, which encode for Ca v 2.2 were detected in all tissue but skin. Positive control: Actb transcripts, encoding for ß-Actin. sk: skin, sc: spinal cord, bs: brain stem, ctx: cortex.

5 Supplementary Table 1 Overview of generated tethered toxins targeting voltage-gated Ca 2+ - channels. The optimization process required a variety of combinations including the toxin domain 1-5, the fluorescent reporter proteins, the epitope tag, the transmembrane or GPI anchoring sequences and linker peptides for surface expression. Expression analyses of t-toxins were carried out by Western Blot and/or immunofluorescence in injected oocytes, transduced HEK293T and HeLa cells and rat hippocampus neuronal cultures. Subsequent electrophysiological recordings in Xenopus laevis oocytes, Ca v 2.2 expressing stably transfected HEK293 cells and rat hippocampus culture were used for assessing the toxin functionality. Three transmembrane domains were tested: the TM domain of NYD-SP16 6, the β1-subunit of Na v channels 7 and the TM domain of the PDGF receptor (PDGF-R). As flexible linker, a sequence with glycine-asparagine repeats (23 aa) was used. The sequence of the integrated export ER signal (EResc) used in some constructs was FCYENEV.

6 Supplementary Table 2 Passive membrane properties of t-toxin infected neurons remain unaltered. The capacitance as well as the membrane resistance of hippocampal neurons infected with the control lentivirus (no toxin-pe) or with the t-toxin lentiviruses MVIIA-PE and AgaIVA-VG, show no significant difference to non-infected control cells, indicating that t-toxin expression does not impair neuronal membrane properties.

7 Supplementary Table 3 Used primers for RT-PCR and quantitative real-time PCR.

8 SUPPLEMENTARY REFERENCES 1. Olivera, B.M., et al. Neuronal calcium channel antagonists. Discrimination between calcium channel subtypes using omega-conotoxin from Conus magus venom. Biochemistry 26, (1987). 2. Olivera, B.M., McIntosh, J.M., Cruz, L.J., Luque, F.A. & Gray, W.R. Purification and sequence of a presynaptic peptide toxin from Conus geographus venom. Biochemistry 23, (1984). 3. Hillyard, D.R., et al. A new Conus peptide ligand for mammalian presynaptic Ca2+ channels. Neuron 9, (1992). 4. Mintz, I.M., et al. P-type calcium channels blocked by the spider toxin [omega]-aga- IVA. Nature 355, (1992). 5. Mintz, I.M., Venema, V.J., Adams, M.E. & Bean, B.P. Inhibition of N- and L-type Ca2+ channels by the spider venom toxin omega-aga-iiia. Proc Natl Acad Sci U S A 88, (1991). 6. Cheng, L.J., et al. NYD-SP16, a novel gene associated with spermatogenesis of human testis. Biol Reprod 68, (2003). 7. Zimmer, T., Biskup, C., Bollensdorff, C. & Benndorf, K. The beta1 subunit but not the beta2 subunit colocalizes with the human heart Na+ channel (hh1) already within the endoplasmic reticulum. J Membr Biol 186, (2002).

Supplementary Table 1. List of primers used in this study

Supplementary Table 1. List of primers used in this study Gene Forward primer Reverse primer Rat Met 5 -aggtcgcttcatgcaggt-3 5 -tccggagacacaggatgg-3 Rat Runx1 5 -cctccttgaaccactccact-3 5 -ctggatctgcctggcatc-3