The gympietides are a newly discovered class of peptide neurotoxins from the venom of Australian stinging nettles (Dendrocnide spp.), notable for their highly unusual mechanism of modulating voltage-gated sodium (NaV) channels. Unlike other venom-derived peptides that bind directly to the NaV α subunit, gympietides act via an indirect and previously uncharacterised route: they bind to TMEM233, a poorly understood member of the dispanin family of transmembrane proteins expressed in sensory neurons, and inhibit fast inactivation of NaV channels via a presumptive tripartite toxin-TMEM233-NaV channel interaction. This unconventional mode of action offers a rare opportunity to probe the interface between accessory membrane proteins and ion channels - an emerging frontier in neurobiology and pharmacology.
This study aimed to delineate the structure-activity elements contributing to the interaction of the gympietides with TMEM233 as well as NaV1.7, to gain first insights into the molecular basis of this unique complex. We used patch-clamp electrophysiology and fluorescent membrane potential assays to assess toxin effects on NaV1.7 inactivation, as well as HTRF and AlphaScreen assays to quantify direct toxin-TMEM233 interactions.
We identified ExTxA residues R3, R13, I14, and T22 as key mediators of TMEM233 interaction - likely engaging with TMEM233 residues N53 and Y50, located in the re-entrant intramembrane loop. Conversely, NaV1.7 interaction was shown to depend on ExTxA residues D35, T22, and I14, suggesting that the peptide binds at an interface between TMEM233 and NaV1.7 via a dual pharmacophore. These findings not only elucidate the molecular underpinnings of gympietide action but also establish the first structural framework for targeting dispanin–ion channel complexes.