The TRPM8 channel forms a complex with the 5-HT1B receptor and phospholipase D that amplifies its reversal of pain hypersensitivity

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-HT1B receptor (5-HT1BR) 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 Ca2+ fluorometric responses, protein:protein interactions, immuno-localisation and reflex pain behaviours, with pharmacological and molecular interventions. We report 5-HT1BR 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-HT1BR 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-HT1BR agonists and attenuated by 5-HT1BR antagonists, while 5-HT1BR 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. (C) 2013 Elsevier Ltd. All rights reserved.