Poster Presentation 8th Venoms to Drugs 2023

Pain-causing stinging nettle toxins target tmem233 to modulate nav1.7 function (#106)

Sina Jami 1 , Jennifer R Deuis 1 , Tabea Klasfauseweh 1 , Sergy Kurdyukov 2 , Felicity Chung 2 , Andrei L Okorokov 3 , Shengnan Li 3 , Mathilde R Israel 4 , Lotten Ragnarsson 1 , Åsa Andersson 1 , David A Andersson 4 , John N Wood 3 , Jing Zhao 3 , James J Cox 3 , G Gregory Neely 2 , Thomas Durek 1 , Irina Vetter 1
  1. Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD, Australia
  2. Centenary Institute, University of Sydney, Camperdown, NSW, Australia
  3. Wolfson Institute for Biomedical Research, University College London, London, United Kingdom
  4. Wolfson Centre for Age-Related Diseases, King's College London, London, United Kingdom

Voltage-gated sodium (NaV) channels are pore-forming transmembrane proteins critical for the initiation and propagation of action potentials in excitable cells. The subtype NaV1.7 contributes to action potential generation in nociceptive or “pain-sensing” neurons. Recently, we identified a novel family of pain-causing toxins from Australian stinging trees that target NaV1.7. Surprisingly, these toxins are only active on NaV1.7 when endogenously expressed in neurons, and not on NaV1.7 in overexpression systems.

To identify what confers toxin-sensitivity in neurons, we performed a genome-scale lentivirus-CRISPR knockdown screen in TE-671 neuroblastoma cells, in which ExTxA inhibits the fast inactivation of endogenously expressed NaV1.7. ExTxA-induced cytotoxicity was prevented by the knockdown of SCN9A (the gene encoding NaV1.7) as well as ten other genes (RNF121, GPAA1, PIGT, CRELD1, PIGK, STT3B, TMEM233, PIGS, MMGT1, and LMAN2L). Of these 10 candidate genes, only over-expression of TMEM233 conferred ExTxA-sensitivity to HEK293 cells expressing NaV1.7 in a fluorescence membrane potential assay.

When TMEM233 was co-expressed with NaV1.7 in HEK293 cells, ExTxA inhibited fast inactivation of NaV1.7, recapitulating the effect seen in neurons. Conversely, the toxin effects on NaV inactivation were attenuated in DRG neurons derived from TMEM233 knockout mice and toxin-induced pain behaviours were significantly reduced in TMEM233 knockout mice. Flow cytometry analysis of cells treated with biotin-ExTxA showed an increased signal in TMEM233-HEK293 cells but not in NaV1.7-HEK293 cells, suggesting that ExTxA directly binds to TMEM233.

These findings identify TMEM233 as a previously unknown NaV1.7-interacting protein. The pain-causing stinging nettle toxin ExTxA is the first NaV modulator reported to require an accessory protein to exert its pharmacological effect.