Department of Neurobiology, School of Biological Sciences, University of California San Diego, La Jolla, California 92093, United States.
J Am Chem Soc. 2023 Sep 13;145(36):19611-19621. doi: 10.1021/jacs.3c03913. Epub 2023 Aug 31.
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.
光可激活神经肽提供了一种强大的刺激-反应关系，可以推动神经肽传递的生理机制的机理研究。大多数神经肽都含有 C 端酰胺，这为安装 C 端笼状基团提供了一个潜在的通用位点。在这里，我们报告了一种仿生笼状策略，其中通过光可裂解氨基酸扩展神经肽的 C 端，以模拟在大致密核心囊泡中发现的前神经肽。我们用四种突出的神经肽：胃泌素释放肽（GRP）、催产素（OT）、P 物质（SP）和胆囊收缩素（CCK）来探索这种方法。C 端延伸大大降低了所有四种肽在异源表达的受体中的活性。在急性脑片的细胞类型特异性电生理记录中，紫外光的亚秒级闪光通过激活内源性 G 蛋白偶联受体产生快速激活的膜电流。随后用笼状 CCK 进行的机理研究表明，细胞外蛋白酶在塑造 CCK 信号的时间动态方面起着重要作用，并且在海马钙蛋白酶-1 阳性中间神经元中，短暂的 CCK 信号具有惊人的开关样、细胞自主的抗阿片效应。这些结果表明，用光可裂解接头进行 C 端延伸可能是光笼状酰胺化神经肽的一种通用策略，并展示了光笼状神经肽如何为神经肽信号提供常规方法无法获得的机理见解。
