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 ...

A-type potassium currents are important determinants of neuronal excitability. In spinal cord dorsal horn neurons, A-type currents are modulated by extracellular signal-regulated kinases (ERKs), which mediate central sensitization during inflammatory pain. Here, we report that Kv4.2 mediates the majority of A-type current in dorsal horn neurons and is a critical site for modulation of neuronal excitability and nociceptive behaviors. Genetic elimination of Kv4.2 reduces A-type currents and increases excitability of dorsal horn neurons, resulting in enhanced sensitivity to tactile and thermal stimuli. Furthermore, ERK-mediated modulation of excitability in dorsal horn neurons and ERK-dependent forms of pain hypersensitivity are absent in Kv4.2(-/-) mice compared to wild-type littermates. Finally, mutational analysis of Kv4.2 indicates that S616 is the functionally relevant ERK phosphorylation site for modulation of Kv4.2-mediated currents in neurons. These results show that Kv4.2 is a downstream target of ERK in spinal cord and plays a crucial role in pain plasticity.

Transmitter release in neurons is thought to be mediated exclusively by high-voltage-activated (HVA) Ca(2+) channels. However, we now report that, in retinal bipolar cells, low-voltage-activated (LVA) Ca(2+) channels also mediate neurotransmitter release. Bipolar cells are specialized neurons that release neurotransmitter in response to graded depolarizations. Here we show that these cells express T-type Ca(2+) channel subunits and functional LVA Ca(2+) currents sensitive to mibefradil. Activation of these currents results in Ca(2+) influx into presynaptic terminals and exocytosis, which we detected as a capacitance increase in isolated terminals and the appearance of reciprocal currents in retinal slices. The involvement of T-type Ca(2+) channels in bipolar cell transmitter release may contribute to retinal information processing.

In addition to its role as a CNS neurotransmitter, glutamate has been shown recently to be an important component of the peripheral inflammation response. We demonstrated previously that the group I metabotropic glutamate receptors (mGluRs) mGlu1 and mGlu5 are expressed in the peripheral terminals of sensory neurons and that activation of group I mGluRs in the skin increases thermal sensitivity. In the present study, we provide evidence suggesting that group I mGluRs increase thermal sensitivity by enhancing vanilloid (capsaicin) receptor function. We show that mGlu5 potentiates capsaicin responses in mouse sensory neurons by the phospholipase C pathway but not by activation of protein kinase C. Rather, the effects are mediated by the metabolism of diacylglycerol and the production of prostaglandins via the cyclooxygenase pathway, leading to activation of the cAMP-dependent protein kinase subsequent to prostanoid receptor activation. Behavioral thermal sensitization in mice induced by intraplantar injection of mGlu1/5 agonists was also blocked by inhibitors of protein kinase A and cyclooxygenase, suggesting that a similar signaling pathway operates in vivo. These results demonstrate a novel signaling pathway in sensory neurons and provide a plausible mechanism for the enhancement of thermal sensitivity that occurs with inflammation and after activation of mGluRs on peripheral sensory neuron terminals.

The transient outward potassium currents (also known as A-type currents or IA) are important determinants of neuronal excitability. In the brain, IA is modulated by protein kinase C (PKC), protein kinase A (PKA), and extracellular signal-related kinase (ERK), three kinases that have been shown to be critical modulators of nociception. We wanted to determine the effects of these kinases on IA in superficial dorsal horn neurons. Using whole cell recordings from cultured mouse spinal cord superficial dorsal horn neurons, we found that PKC and PKA both inhibit IA in these cells, and that PKC has a tonic inhibitory action on IA. Further, we provide evidence supporting the hypothesis that PKC and PKA do not modulate IA directly, but rather act as upstream activators of ERKs, which modulate IA. These results suggest that ERKs serve as signal integrators in modulation of IA in dorsal horn neurons and that modulation of A-type potassium currents may underlie aspects of central sensitization mediated by PKC, PKA, and ERKs.