title: Activation of TRPV4 channels (hVRL-2/mTRP12) by phorbol derivatives.
abstract: We have studied activation by phorbol derivatives of TRPV4 channels, the human VRL-2, and murine TRP12 channels, which are highly homologous to the human VR-OAC, and the human and murine OTRPC4 channel. 4alpha-Phorbol 12,13-didecanoate (4alpha-PDD) induced an increase in intracellular Ca(2+) concentration, [Ca(2+)](i), in 1321N1 cells stably transfected with human VRL-2 (hVRL-2.1321N1) or HEK-293 cells transiently transfected with murine TRP12, but not in nontransfected or mock-transfected cells. Concomitantly with the increase in [Ca(2+)](i), 4alpha-PDD activated an outwardly rectifying cation channel with an Eisenman IV permeation sequence for monovalent cations that is Ca(2+)-permeable with P(Ca)/P(Na) = 5.8. Phorbol 12-myristate 13-acetate also induced an increase in [Ca(2+)](i) but was approximately 50 times less effective than 4alpha-PDD. EC(50) for Ca(2+) increase and current activation was nearly identical (pEC(50) approximately 6.7). Similar effects were observed in freshly isolated mouse aorta endothelial cells which express TRP12 endogenously. By using 4alpha-PDD as a tool to stimulate TRP12, we showed that activation of this channel is modulated by [Ca(2+)](i); an increase in [Ca(2+)](i) inhibits the channel with an IC(50) of 406 nm. Ruthenium Red at a concentration of 1 microm completely blocks inward currents at -80 mV but has a smaller effect on outward currents likely indicating a voltage dependent channel block. We concluded that the phorbol derivatives activate TRPV4 (VR-OAC, VRL-2, OTRPC4, TRP12) independently from protein kinase C, in a manner consistent with direct agonist gating of the channel.
authors.affiliation: Department of Physiology, Campus Gasthuisberg, KU Leuven, B-3000 Leuven, Belgium.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Watanabe
authors.forename: John B
authors.initials: JB
authors.lastname: Davis
authors.forename: Darren
authors.initials: D
authors.lastname: Smart
authors.forename: Jeff C
authors.initials: JC
authors.lastname: Jerman
authors.forename: Graham D
authors.initials: GD
authors.lastname: Smith
authors.forename: Phil
authors.initials: P
authors.lastname: Hayes
authors.forename: Joris
authors.initials: J
authors.lastname: Vriens
authors.forename: William
authors.initials: W
authors.lastname: Cairns
authors.forename: Ullrich
authors.initials: U
authors.lastname: Wissenbach
authors.forename: Jean
authors.initials: J
authors.lastname: Prenen
authors.forename: Veit
authors.initials: V
authors.lastname: Flockerzi
authors.forename: Guy
authors.initials: G
authors.lastname: Droogmans
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Benham
authors.forename: Bernd
authors.initials: B
authors.lastname: Nilius
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D027682:Cation Transport Proteins; D002460:Cell Line; D002478:Cells, Cultured; D004396:Coloring Agents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D004730:Endothelium, Vascular / Q000378:metabolism; D006801:Humans; D007473:Ion Channels / Q000819:agonists / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D010704:Phorbols / Q000494:pharmacology*; D011955:Receptors, Drug / Q000819:agonists / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012430:Ruthenium Red / Q000494:pharmacology; D050916:TRPV Cation Channels; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D027682:Cation Transport Proteins; D004396:Coloring Agents; D007473:Ion Channels; D010704:Phorbols; D011955:Receptors, Drug; D011994:Recombinant Proteins; D050916:TRPV Cation Channels; C416986:TRPV4 protein, human; C486576:Trpv4 protein, mouse; D012430:Ruthenium Red; D002118:Calcium; C033085:phorbol