期刊
BIOCHEMISTRY
卷 45, 期 15, 页码 4915-4926出版社
AMER CHEMICAL SOC
DOI: 10.1021/bi052082f
关键词
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资金
- NIGMS NIH HHS [GM-56481, R01 GM056481-07, R01 GM056481] Funding Source: Medline
- NINDS NIH HHS [R01 NS022069, NS-22069] Funding Source: Medline
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.
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