Optical Control of TRPV1 Channels In Vitro with Tethered Photopharmacology.

Transient receptor potential vanilloid 1 (TRPV1) is a nonselective cation channel that is important for nociception and inflammatory pain and is activated by a variety of nociceptive stimuli─including lipids such as capsaicin (CAP) and endocannabinoids. TRPV1's role in physiological systems is often studied by activating it with externally perfused ligands; however, this approach is plagued by poor spatiotemporal resolution. Lipid agonists are insoluble in physiological buffers and can permeate membranes to accumulate nonselectively inside cells, where they can have off-target effects. To increase the spatiotemporal precision with which we can activate lipids on cells and tissues, we previously developed optically cleavable targeted (OCT) ligands, which use protein tags (SNAP-tags) to localize a photocaged ligand on a target cellular membrane. After enrichment, the active ligand is released on a flash of light to activate nearby receptors. In our previous work, we developed an OCT-ligand to control a cannabinoid-sensitive GPCR. Here, we expand the scope of OCT-ligand technology to target TRPV1 ion channels. We synthesize a probe, OCT-CAP, that tethers to membrane-bound SNAP-tags and releases a TRPV1 agonist when triggered by UV-A irradiation. Using Ca2+ imaging and electrophysiology in HEK293T cells expressing TRPV1, we demonstrate that OCT-CAP uncaging activates TRPV1 with superior spatiotemporal precision when compared to standard diffusible ligands or photocages. This study is the first example of an OCT-ligand designed to manipulate an ion-channel target. We anticipate that these tools will find many applications in controlling lipid signaling pathways in various cells and tissues.

Genetically-targeted photorelease of endocannabinoids enables optical control of GPR55 in pancreatic ß-cells.

Fatty acid amides (FAAs) are a family of second-messenger lipids that target cannabinoid receptors, and are known mediators of glucose-stimulated insulin secretion from pancreatic ß-cells. Due to the diversity observed in FAA structure and pharmacology, coupled with the expression of at least 3 different cannabinoid G protein-coupled receptors in primary and model ß-cells, our understanding of their role is limited by our inability to control their actions in time and space. To investigate the mechanisms by which FAAs regulate ß-cell excitability, we developed the Optically-Cleavable Targeted (OCT)-ligand approach, which combines the spatial resolution of self-labeling protein (SNAP-) tags with the temporal control of photocaged ligands. By linking a photocaged FAA to an o-benzylguanine (BG) motif, FAA signalling can be directed towards genetically-defined cellular membranes. We designed a probe to release palmitoylethanolamide (PEA), a GPR55 agonist known to stimulate glucose-stimulated insulin secretion (GSIS). When applied to ß-cells, OCT-PEA revealed that plasma membrane GPR55 stimulates ß-cell Ca2+ activity via phospholipase C. Moving forward, the OCT-ligand approach can be translated to other ligands and receptors, and will open up new experimental possibilities in targeted pharmacology.