Caged vanilloid ligands for activation of TRPV1 receptors by 1-and 2-photon excitation

摘要

Nociceptive neurons in the peripheral nervous system detect noxious stimuli and report the information to the central nervous system. Most nociceptive neurons express the vanilloid receptor, TRPV 1, a nonselective cation channel gated by vanilloid ligands such as capsaicin, the pungent essence of chili peppers. Here, we report the synthesis and biological application of two caged vanilloids: biologically inert precursors that, when photolyzed, release bioactive vanilloid ligands. The two caged vanilloids, Nb-VNA and Nv-VNA, are photoreleased with quantum efficiency of 0. 13 and 0.04 1, respectively. Under flash photolysis conditions, photorelease of Nb-VNA and Nv-VNA is 95% complete in similar to 40 mu s and similar to 125 mu s, respectively. Through 1 -photon excitation with ultraviolet light (360 nm), or 2-photon excitation with red light (720 nm), the caged vanilloids can be photoreleased in situ to activate TRPV1 receptors on nociceptive neurons. The consequent increase in intracellular free Ca2+ concentration ([Ca2+](i)) can be Visualized by laser-scanning confocal imaging of neurons loaded with the fluorescent Ca2+ indicator, fluo-3. Simulation results from TRPV1 receptor activation, because the response is blocked by capsazepine, a selective TRPV1 antagonist. In Ca2+-free extracellular medium, photoreleased vanilloid can still elevate [Ca2+](i), which suggests that TRPV1 receptors also reside on endomembranes in neurons and can mediate Ca2+ release from intracellular stores. Notably, whole-cell voltage clamp measurements showed that flash photorelease of vanilloid can activate TRPV1 channels in <4 ms at 22 degrees C. In combination with 1- or 2-photon excitation, caged vanilloids are a powerful tool for probing morphologically distinct structures of nociceptive sensory neurons with high spatial and temporal precision.