title: The TRPM8 channel forms a complex with the 5-HT(1B) receptor and phospholipase D that amplifies its reversal of pain hypersensitivity.
abstract: Effective relief from chronic hypersensitive pain states remains an unmet need. Here we report the discovery that the TRPM8 ion channel, co-operating with the 5-HT(1B) receptor (5-HT(1B)R) in a subset of sensory afferents, exerts an influence at the spinal cord level to suppress central hypersensitivity in pain processing throughout the central nervous system. Using cell line models, ex vivo rat neural tissue and in vivo pain models, we assessed functional Ca(2+) fluorometric responses, protein:protein interactions, immuno-localisation and reflex pain behaviours, with pharmacological and molecular interventions. We report 5-HT(1B)R expression in many TRPM8-containing afferents and direct interaction of these proteins in a novel multi-protein signalling complex, which includes phospholipase D1 (PLD1). We provide evidence that the 5-HT(1B)R activates PLD1 to subsequently activate PIP 5-kinase and generate PIP2, an allosteric enhancer of TRPM8, achieving a several-fold increase in potency of TRPM8 activation. The enhanced activation responses of synaptoneurosomes prepared from spinal cord and cortical regions of animals with a chronic inflammatory pain state are inhibited by TRPM8 activators that were applied in vivo topically to the skin, an effect potentiated by co-administered 5-HT(1B)R agonists and attenuated by 5-HT(1B)R antagonists, while 5-HT(1B)R agents alone had no detectable effect. Corresponding results are seen when assessing reflex behaviours in inflammatory and neuropathic pain models. Control experiments with alternative receptor/TRP channel combinations reveal no such synergy. Identification of this novel receptor/effector/channel complex and its impact on nociceptive processing give new insights into possible strategies for enhanced analgesia in chronic pain.
authors.affiliation: Centre for Integrative Physiology, School of Biomedical Sciences, University of Edinburgh, Hugh Robson Building, Edinburgh EH8 9XD, United Kingdom.
authors.forename: Ignacio
authors.initials: I
authors.lastname: Vinuela-Fernandez
authors.affiliation: Centre for Integrative Physiology, School of Biomedical Sciences, University of Edinburgh, Hugh Robson Building, Edinburgh EH8 9XD, United Kingdom.
authors.forename: Liting
authors.initials: L
authors.lastname: Sun
authors.affiliation: Centre for Integrative Physiology, School of Biomedical Sciences, University of Edinburgh, Hugh Robson Building, Edinburgh EH8 9XD, United Kingdom.
authors.forename: Helen
authors.initials: H
authors.lastname: Jerina
authors.affiliation: Centre for Integrative Physiology, School of Biomedical Sciences, University of Edinburgh, Hugh Robson Building, Edinburgh EH8 9XD, United Kingdom.
authors.forename: John
authors.initials: J
authors.lastname: Curtis
authors.affiliation: Centre for Integrative Physiology, School of Biomedical Sciences, University of Edinburgh, Hugh Robson Building, Edinburgh EH8 9XD, United Kingdom.
authors.forename: Andrew
authors.initials: A
authors.lastname: Allchorne
authors.affiliation: Centre for Integrative Physiology, School of Biomedical Sciences, University of Edinburgh, Hugh Robson Building, Edinburgh EH8 9XD, United Kingdom.
authors.forename: Hayley
authors.initials: H
authors.lastname: Gooding
authors.affiliation: Centre for Integrative Physiology, School of Biomedical Sciences, University of Edinburgh, Hugh Robson Building, Edinburgh EH8 9XD, United Kingdom.
authors.forename: Roberta
authors.initials: R
authors.lastname: Rosie
authors.affiliation: Centre for Integrative Physiology, School of Biomedical Sciences, University of Edinburgh, Hugh Robson Building, Edinburgh EH8 9XD, United Kingdom.
authors.forename: Pamela
authors.initials: P
authors.lastname: Holland
authors.affiliation: Centre for Integrative Physiology, School of Biomedical Sciences, University of Edinburgh, Hugh Robson Building, Edinburgh EH8 9XD, United Kingdom.
authors.forename: Basak
authors.initials: B
authors.lastname: Tas
authors.affiliation: Centre for Integrative Physiology, School of Biomedical Sciences, University of Edinburgh, Hugh Robson Building, Edinburgh EH8 9XD, United Kingdom. Electronic address: rory.mitchell@ed.ac.uk.
authors.forename: Rory
authors.initials: R
authors.lastname: Mitchell
authors.affiliation: Centre for Integrative Physiology, School of Biomedical Sciences, University of Edinburgh, Hugh Robson Building, Edinburgh EH8 9XD, United Kingdom. Electronic address: s.m.fleetwood-walker@ed.ac.uk.
authors.forename: Sue
authors.initials: S
authors.lastname: Fleetwood-Walker
mesh_terms: D000818:Animals; D015220:Calcium Channels / Q000378:metabolism; D002478:Cells, Cultured; D005727:Ganglia, Spinal / Q000187:drug effects / Q000503:physiopathology; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009437:Neuralgia / Q000188:drug therapy / Q000503:physiopathology; D010146:Pain / Q000188:drug therapy / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D044302:Receptor, Serotonin, 5-HT1B / Q000378:metabolism*; D058825:Serotonin 5-HT1 Receptor Agonists / Q000494:pharmacology; D058829:Serotonin 5-HT1 Receptor Antagonists / Q000494:pharmacology; D013116:Spinal Cord / Q000187:drug effects / Q000503:physiopathology; D000074025:TRPA1 Cation Channel; D050053:TRPM Cation Channels / Q000378:metabolism*; D050916:TRPV Cation Channels / Q000378:metabolism; D050051:Transient Receptor Potential Channels / Q000378:metabolism
chemical_list: D015220:Calcium Channels; D009419:Nerve Tissue Proteins; D044302:Receptor, Serotonin, 5-HT1B; D058825:Serotonin 5-HT1 Receptor Agonists; D058829:Serotonin 5-HT1 Receptor Antagonists; D000074025:TRPA1 Cation Channel; C473350:TRPA1 protein, human; D050053:TRPM Cation Channels; C425966:TRPM8 protein, human; D050916:TRPV Cation Channels; C494482:TRPV1 protein, human; D050051:Transient Receptor Potential Channels; C498947:Trpm8 protein, rat; D010739:Phospholipase D; C106801:phospholipase D1
keywords: 5-HT; Analgesia; Pain; Receptor:channel complex; Signalling; TRPM8