Activation of the TRPV4 ion channel is enhanced by phosphorylation.
The TRPV4 (transient receptor potential vanilloid 4) ion channel, a member of the vanilloid subfamily of the transient receptor potential channels, is activated by membrane stretch, by non-noxious warm temperatures, and by a range of chemical activators. In the present study we examined the role of phosphorylation in modulating the activation of TRPV4. We expressed TRPV4 in HEK293 cells and activated the channel by cell swelling in a hypotonic solution. TRPV4 channel activation and serine phosphorylation were enhanced by exposure to the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate or by application of bradykinin, which activates PKC via a G-protein-coupled mechanism. The enhancement was inhibited by the PKC inhibitors staurosporine, bisindolylmaleimide I, and rottlerin or by mutation of the serine/threonine residues Ser(162), Thr(175), and Ser(189). The adenylate cyclase activator forskolin also enhanced activation of TRPV4, and the enhancement was antagonized by the selective cyclic AMP-dependent protein kinase (PKA) inhibitor H89 or by mutation of serine residue Ser(824). Sensitization of TRPV4 by both PKC and PKA depended on the scaffolding protein AKAP79, because channel activation and phosphorylation were enhanced by co-transfection of AKAP79 and were antagonized by removal of AKAP79 using small interfering RNA. We conclude that the serine/threonine kinases PKC and PKA enhance activation of the TRPV4 ion channel by phosphorylation at specific sites and that phosphorylation depends on assembly of PKC and PKA by AKAP79 into a signaling complex with TRPV4.
Fan
Hueng-Chuen
HC
Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD, United Kingdom; Department of Pediatrics, Tri-Service General Hospital and National Defense Medical Center, Taipei 11490, Taiwan.
Zhang
Xuming
X
Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD, United Kingdom.
McNaughton
Peter A
PA
Department of Pharmacology, University of Cambridge, Cambridge CB2 1PD, United Kingdom. Electronic address: pam42@cam.ac.uk.
D054758:A Kinase Anchor Proteins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D010703:Phorbol Esters / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D050916:TRPV Cation Channels / Q000235:genetics / Q000378:metabolism*; D014665:Vasodilator Agents / Q000494:pharmacology
D054758:A Kinase Anchor Proteins; C487775:AKAP5 protein, human; D004791:Enzyme Inhibitors; D010703:Phorbol Esters; D034741:RNA, Small Interfering; D050916:TRPV Cation Channels; C416986:TRPV4 protein, human; D014665:Vasodilator Agents; C119222:phorbol-12-myristate; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D001920:Bradykinin