Poster Presentation The 16th Australian Peptide Conference 2025

Identifying the Binding Site of an Australian Stinging Nettle Toxin on a Novel Voltage-Gated Sodium Channel Accessory Protein (#124)

Lucinda Walker 1 , Hue Tran 1 , Qing Xu 1 , Jennifer Deuis 1 , Mehdi Mobli2 2 , Irina Vetter 1 3
  1. Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia
  2. Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD, Australia
  3. School of Pharmacy and Pharmaceutical Sciences, The University of Queensland, Brisbane, QLD, Australia

Gympietides are a new family of pain causing peptide toxins derived from the Australian stinging tree (Dendrocnide excelsa). Gympietides modulate voltage gated sodium (NaV) channel function via binding to dispanins, a novel class of NaV channel interacting proteins. This study aimed to determine the binding site of the gympietide Excelsatoxin A (ExTxA) on the dispanin TMEM233.

To quantify ExTxA binding, we established a homogeneous time-resolved fluorescence resonance energy transfer (HTRF) assay. In this assay C-terminal HA-tagged TMEM233 constructs (wild-type and mutant) are incubated with anti-HA terbium cryptate (donor) and a range of concentrations of Alexa488-conjugated ExTxA (acceptor). Binding affinity is inferred from the FRET signal intensity, which reflects proximity-dependent energy transfer between the donor and acceptor fluorophores.

ExTxA was conjugated with Alexa-488 at the N-terminus using click-chemistry. Automated patch clamp electrophysiology of TMEM233-CHA and native or Alexa488 labelled ExTxA indicates the EC50 at TMEM233-CHA of Alexa-488-ExTxA is 7.2nM, and native ExTxA is 26nM. Thus ExTxA is still active with the Alexa-488 tag. Adding a HA tag to the C-terminus of TMEM233 did not change ExTxA potency in patch clamp experiments.

HTRF assay results revealed that at wild-type TMEM233, Alexa488-labeled ExTxA exhibited an apparent EC₅₀ of 77.6 nM, indicating high-affinity binding. TMEM233 has a short predicted extracellular C-terminus, a transmembrane domain, a re-entrant loop, and an intracellular N-terminus. Replacement of the re-entrant loop of TMEM233 with the re-entrant loop of TMEM91 (a dispanin that does not bind ExTxA) resulted in an EC50 of 5.2µM; a 67-fold shift compared to wildtype. Alanine scanning in this region identified N53 as a key mediator of binding, with TMEM233[N53A] causing a similar loss of potency with an apparent EC50 of 1.5µM. This suggests that ExTxA binds to TMEM233 via the asparagine at position 53, and that the surrounding residues support this interaction. This knowledge provides the molecular foundation for the design of therapeutics that modulate or target NaV channels via TMEM233.