Double-knot toxin (DkTx) is a 75 amino acid peptidyl toxin isolated from the venom of the Chinese bird spider, Ornithoctonus huwena¹. DkTx selectively activates the transient receptor potential vanilloid 1 (TRPV1) channel by targeting the outer pore domain. DkTx binds bivalently to TRPV1 in an open state-dependent manner¹.

DkTx shown to consist of two head-to-tail inhibitory cysteine-knot (ICK) motif repeats, referred to as knot1 (K1) and knot2 (K2), separated by a short 7-amino-acid linker. Each unit can exist as structurally and functionally independent entities which, when combined, synergize to produce a ligand of exceedingly high avidity^1,2. The two ICK motifs of DkTx bind two homologous binding sites in adjacent subunits of the tetrameric channel, while its linker adopts a taut and constrained conformation. The ICK lobes also interact with lipids in the membrane bilayer, forming a toxin–channel–lipid tripartite complex. DkTx partitions into the membrane and that its two knots bind to the outer pore region of TRPV1 leading to sustained channel activation^3-5.

TRPV1 is a non-selective cation channel expressed in nociceptive sensory neurons that plays important roles in the transduction of noxious stimuli as well as thermosensation.

In the peripheral nervous system, TRPV1 channel was found to be highly expressed in the spinal dorsal root ganglion neurons, the trigeminal ganglion and primary sensory neurons, which mainly mediate pain perception, transmission and regulation process. In the central nervous system, the TRPV channel was found mainly involved in the regulation of body temperature, release of synaptic neurotransmitters, synaptic transmission and apoptosis. In addition, TRPV1 is widely expressed in non-neuronal cells and has been shown to play an important role in the immune system.  As a receptor for multiple injurious stimuli, TRPV1 has emerged as a new and promising target for developing analgesic and anti-inflammatory drugs^6,7.