title: Caged vanilloid ligands for activation of TRPV1 receptors by 1- and 2-photon excitation.
abstract: 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, TRPV1, 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 0.041, respectively. Under flash photolysis conditions, photorelease of Nb-VNA and Nv-VNA is 95% complete in approximately 40 micros and approximately 125 micros, 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 Ca(2+) concentration ([Ca(2+)](i)) can be visualized by laser-scanning confocal imaging of neurons loaded with the fluorescent Ca(2+) indicator, fluo-3. Stimulation results from TRPV1 receptor activation, because the response is blocked by capsazepine, a selective TRPV1 antagonist. In Ca(2+)-free extracellular medium, photoreleased vanilloid can still elevate [Ca(2+)](i), which suggests that TRPV1 receptors also reside on endomembranes in neurons and can mediate Ca(2+) 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.
authors.affiliation: Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, Maryland 21201, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Zhao
authors.forename: Tony D
authors.initials: TD
authors.lastname: Gover
authors.forename: Sukumaran
authors.initials: S
authors.lastname: Muralidharan
authors.forename: Darryl A
authors.initials: DA
authors.lastname: Auston
authors.forename: Daniel
authors.initials: D
authors.lastname: Weinreich
authors.forename: Joseph P Y
authors.initials: JP
authors.lastname: Kao
mesh_terms: D000818:Animals; D008024:Ligands; D008297:Male; D018613:Microscopy, Confocal; D009474:Neurons / Q000378:metabolism; D010782:Photolysis; D017785:Photons; D051381:Rats; D017207:Rats, Sprague-Dawley; D050916:TRPV Cation Channels / Q000378:metabolism* / Q000528:radiation effects; D014641:Vanillic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism
chemical_list: D008024:Ligands; D050916:TRPV Cation Channels; C005871:vanillyl-N-nonylamide; D014641:Vanillic Acid