Protein kinase C phosphorylation sensitizes but does not activate the capsaicin receptor transient receptor potential vanilloid 1 (TRPV1)

Abstract

Protein kinase C (PKC) modulates the function of the capsaicin receptor transient receptor potential vanilloid 1 (TRPV1). This modulation manifests as increased current when the channel is activated by capsaicin. In addition, studies have suggested that phosphorylation by PKC might directly gate the channel, because PKC-activating phorbol esters induce TRPV1 currents in the absence of applied ligands. To test whether PKC both modulates and gates the TRPV1 function by direct phosphorylation, we used direct sequencing to determine the major sites of PKC phosphorylation on TRPV1 intracellular domains. We then tested the ability of the PKC-activating phorbol 12-myristate 13-acetate (PMA) to potentiate capsaicin-induced currents and to directly gate TRPV1. We found that mutation of S800 to alanine significantly reduced the PMA-induced enhancement of capsaicin-evoked currents and the direct activation of TRPV1 by PMA. Mutation of S502 to alanine reduced PMA enhancement of capsaicin-evoked currents, but had no effect on direct activation of TRPV1 by PMA. Conversely, mutation of T704 to alanine had no effect on PMA enhancement of capsaicin-evoked currents but dramatically reduced direct activation of TRPV1 by PMA. These results, combined with pharmacological studies showing that inactive phorbol esters also weakly activate TRPV1, suggest that PKC-mediated phosphorylation modulates TRPV1 but does not directly gate the channel. Rather, currents induced by phorbol esters result from the combination of a weak direct ligand-like activation of TRPV1 and the phosphorylation-induced enhancement of the TRPV1 function. Furthermore, modulation of the TRPV1 function by PKC appears to involve distinct phosphorylation sites depending on the mechanism of channel activation. P rotein kinase C (PKC) in peripheral sensory afferents plays a prominent role in hypersensitivity to thermal and mechanical stimuli after tissue injury. PKC sensitizes heat responses and potentiates peptide release from cultured dorsal root ganglion neurons (1, 2) and sensitizes nociceptive afferent neurons to thermal and mechanical stimuli in intact peripheral nerve preparations (3, 4). Diabetic neuropathic hyperalgesia and epinephrine-induced hyperalgesia are attenuated by PKC inhibitors in vivo (5, 6). Recently, several studies have focused on the role of the PKC isoform. Specific blockade of PKC diminishes PKCmediated enhancement of heat currents in sensory neurons and epinephrine-induced hypersensitivity in vivo (7,8). PKC knockout mice exhibit reduced hyperalgesia after intracutaneous injection of epinephrine and nerve growth factor (8). Whereas a role of PKC in peripheral sensitization is well established, PKC-mediated phosphorylation and modulation of specific substrates during peripheral sensitization is not fully understood.Transient receptor potential vanilloid 1 [TRPV1; formerly known as vanilloid receptor 1 (VR1)] is an attractive PKC effector in peripheral nociceptors. TRPV1 was cloned as a capsaicin receptor and is a ligand-gated ion channel, which ...

Activity-dependent targeting of TRPV1 with a pore-permeating capsaicin analog

“…Protein kinase C activation (23)(24)(25)(26) and oxidative modification (20,27,28) are major biochemical pathways that enhance TRPV1 sensitivity to chemical agonists. PDBu and PAO, chemical activators stimulating PKC or mimicking cellular oxidation, respectively, could elicit TRPV1-dependent intracellular Ca 2+ rise (20,29). They also dramatically shifted doseresponse curves of agonist-evoked YO-PRO-1 entry leftward ( Fig.…”

Cannabinoid WIN 55,212-2 Regulates TRPV1 Phosphorylation in Sensory Neurons

“…It is important to note that the concentrations of cannabinoids used in these experiments were between 1000-and 10,000-fold higher than those concentrations initially characterized to activate the cannabinoid G-protein-coupled receptors CB1 and CB2 (37,38), consistent with the involvement of non-CB1/CB2 receptors. Both serine and threonine residues have been reported as potential phosphorylation sites for various kinases that serve to regulate channel activity via post-translational modifications of the TRPV1 receptor channel (25,36,39). To determine whether serine or threonine residues of the TRPV1 amino acid sequence were modified following cannabinoid treatments, equal immunoprecipitates from treated TG neurons were probed with antibody specific for phosphoserine or phosphothreonine.…”

Human Sensory Neuron-specific Mas-related G Protein-coupled Receptors-X1 Sensitize and Directly Activate Transient Receptor Potential Cation Channel V1 via Distinct Signaling Pathways

“…Inflammatory mediators like bradykinin (BK) potentiate heat-or proton-induced TRPV1 activity via phosphorylation of the channel protein by protein kinase A or C (PKC), calmodulin-dependent kinase II, phosphoinositide 3-kinase (PI3K), sarcoma kinase (SRC), or extracellular signal-regulated kinases-1/2 (ERK-1/2) (10 -15). However, bona fide activation of TRPV1 caused by phosphorylation has been questioned (16,17). In contrast, release of the phospholipase C (PLC) product diacylglycerol (DAG) has been suggested to enhance TRPV1 activity via binding to the channel protein (18) and degradation of the PLC substrate phosphatidylinositol-4,5-bisphosphate (PIP 2 ) to relieve TRPV1 from inhibitory PIP 2 effects, thereby activating the ion channel (17,19).…”