Regulation of Ca2+-dependent desensitization in the vanilloid receptor TRPV1 by calcineurin and cAMP-dependent protein kinase.
The vanilloid receptor TRPV1 is a polymodal nonselective cation channel of nociceptive sensory neurons involved in the perception of inflammatory pain. TRPV1 exhibits desensitization in a Ca2+-dependent manner upon repeated activation by capsaicin or protons. The cAMP-dependent protein kinase (PKA) decreases desensitization of TRPV1 by directly phosphorylating the channel presumably at sites Ser116 and Thr370. In the present study we investigated the influence of protein phosphatase 2B (calcineurin) on Ca2+-dependent desensitization of capsaicin- and proton-activated currents. By using site-directed mutagenesis, we generated point mutations at PKA and protein kinase C consensus sites and studied wild type (WT) and mutant channels transiently expressed in HEK293t or HeLa cells under whole cell voltage clamp. We found that intracellular application of the cyclosporin A.cyclophilin A complex (CsA.CyP), a specific inhibitor of calcineurin, significantly decreased desensitization of capsaicin- or proton-activated TRPV1-WT currents. This effect was similar to that obtained by extracellular application of forskolin (FSK), an indirect activator of PKA. Simultaneous applications of CsA.CyP and FSK in varying concentrations suggested that these substances acted independently from each other. In mutation T370A, application of CsA.CyP did not reduce desensitization of capsaicin-activated currents as compared with WT and to mutant channels S116A and T144A. In a double mutation at candidate protein kinase C phosphorylation sites, application of CsA.CyP or FSK decreased desensitization of capsaicin-activated currents similar to WT channels. We conclude that Ca2+-dependent desensitization of TRPV1 might be in part regulated through channel dephosphorylation by calcineurin and channel phosphorylation by PKA possibly involving Thr370 as a key amino acid residue.
Mohapatra
Durga Prasanna
DP
Department of Anesthesiology, Friedrich-Alexander-University Erlangen-Nuremberg, 91054 Erlangen, Germany.
Nau
Carla
C
D000409:Alanine / Q000378:metabolism; D000818:Animals; D019703:Calcineurin / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D002211:Capsaicin / Q000378:metabolism; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D021984:Cyclophilin A / Q000378:metabolism; D016572:Cyclosporine / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D006801:Humans; D007473:Ion Channels / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011522:Protons; D051381:Rats; D013449:Sulfonamides / Q000378:metabolism; D050916:TRPV Cation Channels
D002147:Calmodulin; D004791:Enzyme Inhibitors; D007473:Ion Channels; D011522:Protons; D013449:Sulfonamides; D050916:TRPV Cation Channels; C493922:Trpv1 protein, rat; C017967:W 7; D016572:Cyclosporine; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D019703:Calcineurin; D021984:Cyclophilin A; D000409:Alanine; D002211:Capsaicin; D002118:Calcium