A Biomimetic C-Terminal Extension Strategy for Photocaging Amidated Neuropeptides

[Image: see text] Photoactivatable neuropeptides offer a robust stimulus–response relationship that can drive mechanistic studies into the physiological mechanisms of neuropeptidergic transmission. The majority of neuropeptides contain a C-terminal amide, which offers a potentially general site for installation of a C-terminal caging group. Here, we report a biomimetic caging strategy in which the neuropeptide C-terminus is extended via a photocleavable amino acid to mimic the proneuropeptides found in large dense-core vesicles. We explored this approach with four prominent neuropeptides: gastrin-releasing peptide (GRP), oxytocin (OT), substance P (SP), and cholecystokinin (CCK). C-terminus extension greatly reduced the activity of all four peptides at heterologously expressed receptors. In cell type-specific electrophysiological recordings from acute brain slices, subsecond flashes of ultraviolet light produced rapidly activating membrane currents via activation of endogenous G protein-coupled receptors. Subsequent mechanistic studies with caged CCK revealed a role for extracellular proteases in shaping the temporal dynamics of CCK signaling, and a striking switch-like, cell-autonomous anti-opioid effect of transient CCK signaling in hippocampal parvalbumin interneurons. These results suggest that C-terminus extension with a photocleavable linker may be a general strategy for photocaging amidated neuropeptides and demonstrate how photocaged neuropeptides can provide mechanistic insights into neuropeptide signaling that are inaccessible using conventional approaches.