Increased sensitivity of desensitized TRPV1 by PMA occurs through PKCepsilon-mediated phosphorylation at S800.
Important mechanisms that regulate inhibitory and facilitatory effects on TRPV1-mediated nociception are desensitization and phosphorylation, respectively. Using Ca2+-imaging, we have previously shown that desensitization of TRPV1 upon successive capsaicin applications was reversed by protein kinase C activation in dorsal root ganglion neurons and CHO cells. Here, using both Ca2+-imaging and patch-clamp methods, we show that PMA-induced activation of PKCepsilon is essential for increased sensitivity of desensitized TRPV1. TRPV1 has two putative substrates S502 and S800 for PKCepsilon-mediated phosphorylation. Patch-clamp analysis showed that contribution of single mutant S502A or S800A towards increased sensitivity of desensitized TRPV1 is indistinguishable from that observed in a double mutant S502A/S800A. Since S502 is a non-specific substrate for TRPV1 phosphorylation by kinases like PKC, PKA or CAMKII, evidence for a role of PKC specific substrate S800 was investigated. Evidence for in vivo phosphorylation of TRPV1 at S800 was demonstrated for the first time. We also show that the expression level of PKCepsilon paralleled the amount of phosphorylated TRPV1 protein using an antibody specific for phosphorylated TRPV1 at S800. Furthermore, the anti-phosphoTRPV1 antibody detected phosphorylation of TRPV1 in mouse and rat DRG neurons and may be useful for research regarding nociception in native tissues. This study, therefore, identifies PKCepsilon and S800 as important therapeutic targets that may help regulate inhibitory effects on TRPV1 and hence its desensitization.
Faculty of Pharmacy, University of Sydney, NSW 2006, Australia.
Sravan
S
Mandadi
Tomoko
T
Tominaga
Mitsuko
M
Numazaki
Namie
N
Murayama
Naoaki
N
Saito
Patricia J
PJ
Armati
Basil D
BD
Roufogalis
Makoto
M
Tominaga
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000918:Antibody Specificity; D016466:CHO Cells; D002118:Calcium / Q000032:analysis; D002211:Capsaicin / Q000494:pharmacology; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D005727:Ganglia, Spinal / Q000166:cytology; D006367:HeLa Cells; D006801:Humans; D007668:Kidney; D051379:Mice; D008969:Molecular Sequence Data; D009475:Neurons, Afferent / Q000187:drug effects / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010446:Peptide Fragments / Q000494:pharmacology; D010766:Phosphorylation; D010768:Phosphoserine / Q000737:chemistry*; D051744:Protein Kinase C-epsilon / Q000502:physiology*; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000502:physiology; D050916:TRPV Cation Channels / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014162:Transfection
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D050916:TRPV Cation Channels; C493922:Trpv1 protein, rat; D010768:Phosphoserine; C497257:Prkce protein, rat; D051744:Protein Kinase C-epsilon; D013755:Tetradecanoylphorbol Acetate; D002211:Capsaicin; D002118:Calcium