Transient receptor potential melastatin 8 (TRPM8) and transient receptor potential vanilloid 1 (TRPV1) are ion channels that detect cold and hot sensations, respectively. Their activation depolarizes the peripheral nerve terminals resulting in action potentials that propagate to brain via the spinal cord. These receptors also play a significant role in synaptic transmission between dorsal root ganglion (DRG) and dorsal horn (DH) neurons. Here, we show that TRPM8 is functionally downregulated by activation of protein kinase C (PKC) resulting in inhibition of membrane currents and increases in intracellular Ca 2ϩ compared with upregulation of TRPV1 in cloned and native receptors. Bradykinin significantly downregulates TRPM8 via activation of PKC in DRG neurons. Activation of TRPM8 or TRPV1 at first sensory synapse between DRG and DH neurons leads to a robust increase in frequency of spontaneous/miniature EPSCs. PKC activation blunts TRPM8-and facilitates TRPV1-mediated synaptic transmission. Significantly, downregulation is attributable to PKC-mediated dephosphorylation of TRPM8 that could be reversed by phosphatase inhibitors. These findings suggest that inflammatory thermal hyperalgesia mediated by TRPV1 may be further aggravated by downregulation of TRPM8, because the latter could mediate the much needed cool/soothing sensation.
Transient receptor potential vanilloid 1 (TRPV1) is a Ca2+ permeable non-selective cation channel activated by physical and chemical stimuli. Resiniferatoxin (RTX), an ultrapotent agonist of TRPV1, is under investigation for treatment of urinary bladder hyper-reflexia and chronic pain conditions. Here, we have determined the characteristics of RTX-induced responses in cells expressing native and cloned rat TRPV1. Whole-cell currents increase with repeated application of submaximal concentrations of RTX until a maximal response is attained and do not deactivate even after prolonged washout. Interestingly, the rate of activation and block by capsazepine of RTX-induced currents are significantly slower than for capsaicin-induced currents. RTX-induced whole-cell currents are outwardly rectifying, but to a lesser extent than capsaicin-induced currents. RTX-induced single channel currents exhibit multiple conductance states and outward rectification. The open probability (P o ) of RTX-induced currents is higher at all potentials as compared to capsaicin-induced currents, which showed a strong voltage-dependent decrease at negative potentials. Single-channel kinetic analyses reveal that open-time distribution of RTX-induced currents can be fitted with three exponential components at negative and positive potentials. The areas of distribution of the longer open time constants are significantly larger than capsaicin-induced currents. The closed-time distribution of RTX-induced currents can be fitted with three exponential components as compared to capsaicin-induced currents, which require four exponential components. Current-clamp experiments reveal that low concentrations of RTX caused a slow and sustained depolarization beyond threshold while generating few action potentials. Concentrations of capsaicin required for the same extent of depolarization generated a significantly greater number of action potentials. These properties of RTX may play a role in its clinical usefulness.
Transient receptor potential (TRP) ankyrin 1 (TRPA1) is a Ca(2+)-permeant, nonselective cationic channel. It is predominantly expressed in the C afferent sensory nerve fibers of trigeminal and dorsal root ganglion neurons and is highly coexpressed with the nociceptive ion channel transient receptor potential vanilloid 1 (TRPV1). Several physical and chemical stimuli have been shown to activate the channel. In this study, we have used electrophysiological techniques and behavioral models to characterize the properties of TRPA1. Whole cell TRPA1 currents induced by brief application of lower concentrations of N-methyl maleimide (NMM) or allyl isothiocyanate (AITC) can be reversed readily by washout, whereas continuous application of higher concentrations of NMM or AITC completely desensitized the currents. The deactivation and desensitization kinetics differed between NMM and AITC. TRPA1 current amplitude increased with repeated application of lower concentrations of AITC, whereas saturating concentrations of AITC induced tachyphylaxis, which was more pronounced in the presence of extracellular Ca(2+). The outward rectification exhibited by native TRPA1-mediated whole cell and single-channel currents was minimal as compared with other TRP channels. TRPA1 currents were negatively modulated by protons and polyamines, both of which activate the heat-sensitive channel, TRPV1. Interestingly, neither protein kinase C nor protein kinase A activation sensitized AITC-induced currents, but each profoundly sensitized capsaicin-induced currents. Current-clamp experiments revealed that AITC produced a slow and sustained depolarization as compared with capsaicin. TRPA1 is also expressed at the central terminals of nociceptors at the caudal spinal trigeminal nucleus. Activation of TRPA1 in this area increases the frequency and amplitude of miniature excitatory or inhibitory postsynaptic currents. In behavioral studies, intraplantar and intrathecal administration of AITC induced more pronounced and prolonged changes in nociceptive behavior than those induced by capsaicin. In conclusion, the characteristics of TRPA1 we have delineated suggest that it might play a unique role in nociception.
The search for an endogenous ligand for the vanilloid receptor (VR or TRPV1) has led to the identification of N-arachidonyl dopamine (NADA). This study investigates the role of protein kinase C (PKC)-mediated phosphorylation on NADA-induced membrane currents in Xenopus oocytes heterologously expressing TRPV1 and in dorsal root ganglion (DRG) neurons. In basal state, current induced by 10 microM NADA is 5-10% of the current induced by 1 microM capsaicin or protons at pH 5. However, PKC activator, phorbol 12,13-dibutyrate (PDBu) strongly potentiated ( approximately 15-fold) the NADA-induced current. Repeated application of NADA at short intervals potentiated its own response approximately fivefold in a PKC-dependent manner. PKC inhibitor, bisindolylmaleimide (BIM, 500 nM), a mutant TRPV1 (S800A/S502A), and maximal activation of PKC abolished the potentiation induced by repeated application of NADA. As a further confirmation that NADA could stimulate PKC, pretreatment with NADA potentiated the response of protons at pH 5 (approximately 20 fold), which was dramatically reduced in the mutant TRPV1. In DRG neurons, capsaicin (100 nM) induced a approximately 15 mV depolarization and initiated a train of action potentials compared with 1 microM NADA that produced a approximately 5 mV response. Pretreatment with PDBu induced significantly larger depolarization and potentiated NADA-induced current. Furthermore, exposure of NADA to the intracellular surface of the membrane-induced larger currents suggesting inaccessibility to the intracellular binding site might contribute to its weaker action. These results indicate that NADA is a potent agonist of VR when the receptor is in the PKC-mediated phosphorylation state.
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