UCSF

The TRPV1 ion channel serves a critical role in sensory nerve fibers as an integrator of various painful stimuli, including noxious heat, vanilloid compounds, acids, and toxins from venomous creatures. Targeting TRPV1 has shown promising therapeutic potential, as both agonists and antagonists alike are being explored to treat pain across a variety of chronic conditions. A deeper understanding of TRPV1's activity at both structural and functional levels is essential to advancing drug discovery, which can ultimately lead to improved clinical outcomes. The two chapters of this dissertation will address fundamental, yet unresolved, questions in TRPV1 pharmacology.For one, the precise mechanism by which lipids modulate channel activity continues to be debated. Natural products like capsaicin and resiniferatoxin are known to bind to a site within the channel known as the vanilloid binding pocket. In the absence of applied ligand, this pocket is instead occupied by a resident phosphoinositide lipid. Competition for the vanilloid binding pocket is clearly recognized as a key aspect of ligand action. Removal of this resident lipid has been speculated to underlie temperature-dependent gating as well, however this has yet to be definitively demonstrated. Moreover, the vanilloid binding pocket appears capable of accommodating other lipidic species. This includes phosphatidylinositol 4,5-bisphosphate (PIP2), which is crucial in cellular signaling mechanisms. Despite this, the effects of PIP2 on TRPV1 remain controversial. In Chapter 1, we investigate how both the resident lipid, along with PIP2 and its derivatives, influence TRPV1 channel gating. Meanwhile, venoms from tarantulas, scorpions, and other venomous species are a rich source of pharmacological agents targeting TRPV1. However, the discovery of new ligands is often complicated by the heterogeneous nature of venoms. While venoms contain specialized toxin peptides that exquisitely target key host proteins, like TRPV1, they often comprise a variety of toxins and other signaling molecules. Specific toxins can be identified through mass spectrometry of separated venom components or by sequencing nucleic acids from the animal's venom glands. While effective, these methods are resource-intensive and do not readily provide insight into the structural activity of identified toxins. In Chapter 2, we introduce a novel pipeline rooted in cryo-electron microscopy (cryo-EM) to facilitate TRPV1 toxin discovery out of crude venoms and elucidate mechanisms of action.