Lack of potentiating effect of increasing temperature on responses to chemical activators in vagal sensory neurons isolated from TRPV1-null mice

Our recent study (Ni D, Lee LY. Am J Physiol Lung Cell Mol Physiol 294: L563-L571, 2008) demonstrated that the responses of rat pulmonary sensory neurons to transient receptor potential vanilloid (TRPV)1 activators were enhanced by increasing temperature, but the role of the TPRV1 channel in this potentiating effect could not be definitively evaluated. In the present study, we used whole cell perforated patch-clamp technique to compare the responses of isolated nodose/jugular sensory neurons to chemical activators and increasing temperature between wild-type (WT) and TRPV1-null (TRPV1-/-) mice. Our results showed that, in voltage-clamp mode, the peak inward current evoked by hyperthermia was not different between WT and TRPV1-/- neurons; however, the inward current evoked by 2-aminoethoxydiphenyl borate (2-APB), a common activator of TRPV1-3 channels, was greatly potentiated by increasing temperature from 36 to 40.5 degrees C in WT neurons (n = 9; P < 0.01) but was not affected by the same change in temperature in TRPV1-/- neurons (n = 9; P = 0.54). Similarly, the inward current evoked by acid (pH 5.5), an activator of both TRPV1 channel and the acid-sensing ion channel, was enhanced by increasing temperature (n = 7; P < 0.05) in WT neurons, and this potentiating effect was absent in TRPV1-/- neurons (n = 13; P = 0.11). These results demonstrated that deletion of the TRPV1 channel does not significantly alter the stimulatory effect of hyperthermia on nodose/jugular neurons but eliminates the potentiating effect of increasing temperature on the responses of these neurons to nonselective TRPV1 channel activators. This study further suggests that a positive interaction between these chemical activators and increasing temperature at the TRPV1 channel is primarily responsible for the hyperthermia-induced sensitization of these neurons.     

2-aminoethoxydiphenyl borate stimulates pulmonary C neurons via the activation of TRPV channels

This study was carried out to determine the effect of 2-aminoethoxydiphenyl borate (2-APB), a common activator of transient receptor potential vanilloid (TRPV) type 1, 2, and 3 channels, on cardiorespiratory reflexes, pulmonary C fiber afferents, and isolated pulmonary capsaicin-sensitive neurons. In anesthetized, spontaneously breathing rats, intravenous bolus injection of 2-APB elicited the pulmonary chemoreflex responses, characterized by apnea, bradycardia, and hypotension. After perineural treatment of both cervical vagi with capsaicin to block the conduction of C fibers, 2-APB no longer evoked any of these reflex responses. In open-chest and artificially ventilated rats, 2-APB evoked an abrupt and intense discharge in vagal pulmonary C fibers in a dose-dependent manner. The stimulation of C fibers by 2-APB was attenuated but not abolished by capsazepine, a selective antagonist of the TRPV1, which completely blocked the response to capsaicin in these C fiber afferents. In isolated pulmonary capsaicin-sensitive neurons, 2-APB concentration dependently evoked an inward current that was partially inhibited by capsazepine but almost completely abolished by ruthenium red, an effective blocker of all TRPV channels. In conclusion, 2-APB evokes a consistent and distinct stimulatory effect on pulmonary C fibers in vivo and on isolated pulmonary capsaicin-sensitive neurons in vitro. These results establish the functional evidence demonstrating that TRPV1, V2, and V3 channels are expressed on these sensory neurons and their terminals.     

Activation properties of heterologously expressed mammalian TRPV2: evidence for species dependence

TRPV2 has been proposed as a potential pain target, in part due to its relatedness to the nociceptor TRPV1 and to its reported activation by noxious high temperatures (>52 degrees C). However, TRPV2 responses to heat as well as to the nonselective agonist 2-aminoethoxydiphenyl borate (2-APB) have not been universally reproduced in other laboratories, leading to debate about the activation properties of this channel. Here, we report the expression of rat, mouse, and human TRPV2 in HEK293 cells and the differential properties of their responses to heat and 2-APB. Expression of mouse or rat TRPV2 in HEK293 cells resulted in robust channel activation when induced by either temperature (>53 degrees C) or 2-APB. By contrast, expression of human TRPV2 did not lead to detectable activation by either of these stimuli. Human TRPV2 protein was expressed at levels comparable with those of rat TRPV2, exhibited similar surface localization and responded to a novelly identified TRPV2 agonist, Delta(9)-tetrahydrocannabinol, indicating that human TRPV2 is functionally expressed on the cell surface. Studies using deletion mutants and chimeras between rat and human TRPV2 indicated that both amino- and carboxyl-cytoplasmic termini of rat TRPV2 are important for responses to heat and 2-APB but can be supplied in trans to form an active channel. The present study not only confirms and extends previous reports demonstrating that rat and mouse TRPV2 respond to 2-APB and noxious heat but also indicates that further investigation will be required to elucidate TRPV2 activation and regulatory mechanisms.     

Conserved residues within the putative S4-S5 region serve distinct functions among thermosensitive vanilloid transient receptor potential (TRPV) channels

The vanilloid transient receptor potential channel TRPV1 is a tetrameric six-transmembrane segment (S1-S6) channel that can be synergistically activated by various proalgesic agents such as capsaicin, protons, heat, or highly depolarizing voltages, and also by 2-aminoethoxydiphenyl borate (2-APB), a common activator of the related thermally gated vanilloid TRP channels TRPV1, TRPV2, and TRPV3. In these channels, the conserved charged residues in the intracellular S4-S5 region have been proposed to constitute part of a voltage sensor that acts in concert with other stimuli to regulate channel activation. The molecular basis of this gating event is poorly understood. We mutated charged residues all along the S4 and the S4-S5 linker of TRPV1 and identified four potential voltage-sensing residues (Arg(557), Glu(570), Asp(576), and Arg(579)) that, when specifically mutated, altered the functionality of the channel with respect to voltage, capsaicin, heat, 2-APB, and/or their interactions in different ways. The nonfunctional charge-reversing mutations R557E and R579E were partially rescued by the charge-swapping mutations R557E/E570R and D576R/R579E, indicating that electrostatic interactions contribute to allosteric coupling between the voltage-, temperature- and capsaicin-dependent activation mechanisms. The mutant K571E was normal in all aspects of TRPV1 activation except for 2-APB, revealing the specific role of Lys(571) in chemical sensitivity. Surprisingly, substitutions at homologous residues in TRPV2 or TRPV3 had no effect on temperature- and 2-APB-induced activity. Thus, the charged residues in S4 and the S4-S5 linker contribute to voltage sensing in TRPV1 and, despite their highly conserved nature, regulate the temperature and chemical gating in the various TRPV channels in different ways.     

Mouse colon sensory neurons detect extracellular acidosis via TRPV1

Extracellular acidification contributes to pain by activating or modulating nociceptor activity. To evaluate acidic signaling from the colon, we characterized acid-elicited currents in thoracolumbar (TL) and lumbosacral (LS) dorsal root ganglion (DRG) neurons identified by content of a fluorescent dye (DiI) previously injected into the colon wall. In 13% of unidentified LS DRG neurons (not labeled with DiI) and 69% of LS colon neurons labeled with DiI, protons activated a sustained current that was significantly and reversibly attenuated by the transient receptor potential vanilloid receptor 1 (TRPV1) antagonist capsazepine. In contrast, 63% of unidentified LS DRG neurons and 4% of LS colon neurons exhibited transient amiloride-sensitive acid-sensing ion channel (ASIC) currents. The peak current density of acid-elicited currents was significantly reduced in colon sensory neurons from TRPV1-null mice, supporting predominant expression of TRPV1 in LS colon sensory neurons, which was also confirmed immunohistochemically. Similar to LS colon DRG neurons, acid-elicited currents in TL colon DRG neurons were mediated predominantly by TRPV1. However, the pH producing half-activation of responses significantly differed between TL and LS colon DRG neurons. The properties of acid-elicited currents in colon DRG neurons suggest differential contributions of ASICs and TRPV1 to colon sensation and likely nociception. visceral pain; dorsal root ganglion neurons; acid-sensing ion channel; capsaicin receptor; acid-evoked currents; transient receptor potential vanilloid receptor 1     

Mechanisms of hyperthermia-induced depression of GABAergic synaptic transmission in the immature rat hippocampus

Clinical observations and experimental studies have shown that hyperthermia can provoke febrile seizures, which are the most common type of pathological brain activity in children. We previously demonstrated that hyperthermia produced a depression of GABAergic neurotransmission in the hippocampus of immature rats in vitro. To investigate the possible mechanisms through which hyperthermia may modulate GABAergic neurotransmission in the hippocampus, whole-cell voltage clamp recordings were performed on CA1 pyramidal neurons in the immature rat brain slices. We found that hyperthermia (38.4-40 degrees C) when compared with baseline temperature of 32 degrees C reduced the frequency of both spontaneous inhibitory post-synaptic currents (sIPSCs) and miniature IPSCs (mIPSCs). Also, hyperthermia decreased the amplitudes of mIPSCs and reduced the mIPSC decay time constants and charge transfer. Non-stationary noise analysis of mIPSCs suggested that the number of open post-synaptic receptors but not single channel conductance was reduced during hyperthermia. Activation of adenylyl cyclase with forskolin prevented, whereas protein kinase A inhibitor N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide potentiated, the hyperthermia (40 degrees C)-induced depression of evoked IPSCs (evIPSCs). But protein kinase C activator phorbol 12, 13-dibutyrate (PDBu) did not significantly affect this depression of evIPSCs induced by hyperthermia. Furthermore, hyperthermia-induced depression of evIPSCs was attenuated by 4-aminopyridine, but not by BaCl(2). These results suggest that hyperthermia reduces GABA release from pre-synaptic terminals, in part by blocking the adenylyl cyclase-protein kinase A signaling pathway and activating pre-synaptic 4-aminopyridine-sensitive K(+) channels. Also, the changes in amplitude and decay time constant of the mIPSCs may suggest that hyperthermia also decreases post-synaptic GABA(A) receptor function.     

Evidence TRPV4 contributes to mechanosensitive ion channels in mouse skeletal muscle fibers

We recorded the activity of single mechanosensitive (MS) ion channels from membrane patches on single muscle fibers isolated from mice. We investigated the actions of various TRP (transient receptor potential) channel blockers on MS channel activity. 2-aminoethoxydiphenyl borate (2-APB) neither inhibited nor facilitated single channel activity at submillimolar concentrations. The absence of an effect of 2-APB indicates MS channels are not composed purely of TRPC or TRPV1, 2 or 3 proteins. Exposing patches to 1-oleolyl-2-acetyl-sn-glycerol (OAG), a potent activator of TRPC channels, also had no effect on MS channel activity. In addition, flufenamic acid and spermidine had no effect on the activity of single MS channels. By contrast, SKF-96365 and ruthenium red blocked single-channel currents at micromolar concentrations. SKF-96365 produced a rapid block of the open channel current. The blocking rate depended linearly on blocker concentration, while the unblocking rate was independent of concentration, consistent with a simple model of open channel block. A fit to the concentration-dependence of block gave k_on = 13 x 10⁶ M^?1s^?1 and k_off = 1609 sec^?1 with K_D = ~124 µM. Block by ruthenium red was complex, involving both reduction of the amplitude of the single-channel current and increased occupancy of subconductance levels. The reduction in current amplitude with increasing concentration of ruthenium red gave a K_D = ~49 µM. The high sensitivity of MS channels to block by ruthenium red suggests MS channels in skeletal muscle contain TRPV subunits. Recordings from skeletal muscle isolated from TRPV4 knockout mice failed to show MS channel activity, consistent with a contribution of TRPV4. In addition, exposure to hypo-osmotic solutions increases opening of MS channels in muscle. Our results provide evidence TRPV4 contributes to MS channels in skeletal muscle.     

Evidence TRPV4 contributes to mechanosensitive ion channels in mouse skeletal muscle fibers

We recorded the activity of single mechanosensitive (MS) ion channels from membrane patches on single muscle fibers isolated from mice. We investigated the actions of various TRP (transient receptor potential) channel blockers on MS channel activity. 2-aminoethoxydiphenyl borate (2-APB) neither inhibited nor facilitated single channel activity at submillimolar concentrations. The absence of an effect of 2-APB indicates MS channels are not composed purely of TRPC or TRPV1, 2 or 3 proteins. Exposing patches to 1-oleolyl-2-acetyl-sn-glycerol (OAG), a potent activator of TRPC channels, also had no effect on MS channel activity. In addition, flufenamic acid and spermidine had no effect on the activity of single MS channels. By contrast, SKF-96365 and ruthenium red blocked single-channel currents at micromolar concentrations. SKF-96365 produced a rapid block of the open channel current. The blocking rate depended linearly on blocker concentration, while the unblocking rate was independent of concentration, consistent with a simple model of open channel block. A fit to the concentration-dependence of block gave k_on = 13 x 10⁶ M⁻¹s⁻¹ and k_off = 1609 sec⁻¹ with K_D = ~124 µM. Block by ruthenium red was complex, involving both reduction of the amplitude of the single-channel current and increased occupancy of subconductance levels. The reduction in current amplitude with increasing concentration of ruthenium red gave a K_D = ~49 µM. The high sensitivity of MS channels to block by ruthenium red suggests MS channels in skeletal muscle contain TRPV subunits. Recordings from skeletal muscle isolated from TRPV4 knockout mice failed to show MS channel activity, consistent with a contribution of TRPV4. In addition, exposure to hypo-osmotic solutions increases opening of MS channels in muscle. Our results provide evidence TRPV4 contributes to MS channels in skeletal muscle.     

Warm temperatures activate TRPV4 in mouse 308 keratinocytes

Mammalian survival requires constant monitoring of environmental and body temperature. Recently, several members of the transient receptor potential vanilloid (TRPV) subfamily of ion channels have been identified that can be gated by increases in temperature into the warm (TRPV3 and TRPV4) or painfully hot (TRPV1 and TRPV2) range. In rodents, TRPV3 and TRPV4 proteins have not been detected in sensory neurons but are highly expressed in skin epidermal keratinocytes. Here, we show that in response to warm temperatures (>32 degrees C), the mouse 308 keratinocyte cell line exhibits nonselective transmembrane cationic currents and Ca2+ influx. Both TRPV3 and TRPV4 are expressed in 308 cells. However, the warmth-evoked responses we observe most closely resemble those mediated by recombinant TRPV4 on the basis of their electrophysiological properties and sensitivity to osmolarity and the phorbol ester, 4alpha-phorbol-12,13-didecanoate. Together, these data support the notion that keratinocytes are capable of detecting modest temperature elevations, strongly suggest that TRPV4 participates in these responses, and define a system for the cell biological analysis of warmth transduction.     

Aminoethoxydiphenyl Borate and Flufenamic Acid Inhibit Ca<Superscript>2+</Superscript> Influx Through TRPM2 Channels in Rat Dorsal Root Ganglion Neurons Activated by ADP-Ribose and Rotenone

Exposure to oxidative stress causes health problems, including sensory neuron neuropathy and pain. Rotenone is a toxin used to generate intracellular oxidative stress in neurons. However, the mechanism of toxicity in dorsal root ganglion (DRG) neurons has not been characterized. Melastatin-like transient receptor potential 2 (TRPM2) channel activation and inhibition in response to oxidative stress, ADP-ribose (ADPR), flufenamic acid (FFA) and 2-aminoethoxydiphenyl borate (2-APB) in DRG neurons are also not clear. We tested the effects of FFA and 2-APB on ADPR and rotenone-induced TRPM2 cation channel activation in DRG neurons of rats. DRG neurons were freshly isolated from rats and studied with the conventional whole-cell patch-clamp technique. Rotenone, FFA and 2-APB were extracellularly added through the patch chamber, and ADPR was applied intracellularly through the patch pipette. TRPM2 cation currents were consistently induced by ADPR and rotenone. Current densities of the neurons were higher in the ADPR and rotenone groups than in control. The time courses (gating times) in the neurons were longer in the rotenone than in the ADPR group. ADPR and rotenone-induced TRPM2 currents were totally blocked by 2-APB and partially blocked by FFA. In conclusion, TRPM2 channels were constitutively activated by ADPR and rotenone, and 2-APB and FFA induced an inhibitory effect on TRPM2 cation channel currents in rat DRG neurons. Since oxidative stress is a common feature of neuropathic pain and diseases of sensory neurons, the present findings have broad application to the etiology of neuropathic pain and diseases of DRG neurons.     

Thermosensation and pain

We feel a wide range of temperatures spanning from cold to heat. Within this range, temperatures over about 43 degrees C and below about 15 degrees C evoke not only a thermal sensation, but also a feeling of pain. In mammals, six thermosensitive ion channels have been reported, all of which belong to the TRP (transient receptor potential) superfamily. These include TRPV1 (VR1), TRPV2 (VRL-1), TRPV3, TRPV4, TRPM8 (CMR1), and TRPA1 (ANKTM1). These channels exhibit distinct thermal activation thresholds (>43 degrees C for TRPV1, >52 degrees C for TRPV2, > approximately 34-38 degrees C for TRPV3, > approximately 27-35 degrees C for TRPV4, < approximately 25-28 degrees C for TRPM8 and <17 degrees C for TRPA1), and are expressed in primary sensory neurons as well as other tissues. The involvement of TRPV1 in thermal nociception has been demonstrated by multiple methods, including the analysis of TRPV1-deficient mice. TRPV2, TRPM8, and TRPA1 are also very likely to be involved in thermal nociception, because their activation thresholds are within the noxious range of temperatures.     

Co-activation of P2Y2 Receptor and TRPV Channel by ATP: Implications for ATP Induced Pain

1. Extracellular ATP is recognized as a peripheral modulator of pain. Activation of ionotropic P2X receptors in sensory neurons has been implicated in induction of pain, whereas metabotropic P2Y receptors in potentiation of pain induced by chemical or physical stimuli via capsaicin sensitive TRPV1 channel. Here we report that P2Y2 receptor activation by ATP can activate the TRPV1 channel in absence of any other stimuli. 2. ATP-induced Ca2+ signaling was studied in Neuro2a cells. ATP evoked release of intracellular Ca2+ from ER and Ca2+ influx through a fast inactivating channel. The Ca2+ response was induced by P2Y receptor agonists in the order of potency ATP>or=UTP>or=ATPgammaS>ADP and was inhibited by suramin and PPADS. The P2X receptor agonist alpha beta methyl ATP was ineffective. 3. The Ca2+ influx was blocked by ruthenium red, an inhibitor of TRPV1 channel. Capsaicin, the most potent activator of the TRPV1 channel, evoked a fast inactivating Ca2+ transient suggesting the presence of endogenous TRPV1 channels in Neuro2a cells. NMS and PDBu, repressors of IP3 formation, drastically inhibited both the components of Ca2+ response. 4. Our data show co-activation of the P2Y2 receptor and capsaicin sensitive TRPV1 channel by ATP. Such functional interaction between endogenous P2Y2 receptor and TRPV1 channels could explain the ATP-induced pain.     

Modulation of TRPV1 by nonreceptor tyrosine kinase, c-Src kinase

The capsaicin receptor TRPV1 is a nonselective cation channel that is expressed in sensory neurons. In this study, we examined the role of the nonreceptor cellular tyrosine kinase c-Src kinase in the modulation of the rat TRPV1. Capsaicin-induced currents in identified colonic dorsal root ganglion neurons were blocked by the c-Src kinase inhibitor PP2 and enhanced by the tyrosine phosphatase inhibitor sodium orthovandate. PP2 also abolished currents in human embryonic kidney-293 cells transfected with rat TRPV1, whereas cotransfection of TRPV1 with v-Src resulted in fivefold increase in capsaicin-induced currents. In cells transfected with dominant-negative c-Src and TRPV1, capsaicin-induced currents were decreased by approximately fourfold. TRPV1 co-immunoprecipitated with Src kinase and was tyrosine phosphorylated. These studies demonstrate that TRPV1 is a potential target for cellular tyrosine kinase-dependent phosphorylation.     

Short-Term Ketamine Treatment Decreases Oxidative Stress Without Influencing TRPM2 and TRPV1 Channel Gating in the Hippocampus and Dorsal Root Ganglion of Rats

Calcium ions (Ca²⁺) are important second messengers in neurons. Ketamine (KETAM) is an anesthetic and analgesic, with psychotomimetic effects and abuse potential. KETAM modulates the entry of Ca²⁺ in neurons through glutamate receptors, but its effect on transient receptor potential melastatin 2 (TRPM2) and transient receptor potential vanilloid 1 (TRPV1) channels has not been clarified. This study investigated the short-term effects of KETAM on oxidative stress and TRPM2 and TRPV1 channel gating in hippocampal and dorsal root ganglion (DRG) neurons of rats. Freshly isolated hippocampal and DRG neurons were incubated for 24 h with KETAM (0.3 mM). The TRPM2 channel antagonist, N-(p-amylcinnamoyl)anthranilic acid (ACA), inhibited cumene hydroperoxide and ADP-ribose-induced TRPM2 currents in the neurons, and capsazepine (CPZ) inhibited capsaicin-induced TRPV1 currents. The TRPM2 and TRPV1 channel current densities and intracellular free calcium ion concentration of the neurons were lower in the neurons exposed to ACA and CPZ compared to the control neurons, respectively. However, the values were not further decreased by the KETAM + CPZ and KETAM + ACA treatments. KETAM decreased lipid peroxidation levels in the neurons but increased glutathione peroxidase activity. In conclusion, short-term KETAM treatment decreased oxidative stress levels but did not seem to influence TRPM2- and TRPV1-mediated Ca²⁺ entry.     

Molecular cloning and functional characterization of Xenopus tropicalis frog transient receptor potential vanilloid 1 reveal its functional evolution for heat, acid, and capsaicin sensitivities in terrestrial vertebrates

The functional difference of thermosensitive transient receptor potential (TRP) channels in the evolutionary context has attracted attention, but thus far little information is available on the TRP vanilloid 1 (TRPV1) function of amphibians, which diverged earliest from terrestrial vertebrate lineages. In this study we cloned Xenopus tropicalis frog TRPV1 (xtTRPV1), and functional characterization was performed using HeLa cells heterologously expressing xtTRPV1 (xtTRPV1-HeLa) and dorsal root ganglion neurons isolated from X. tropicalis (xtDRG neurons) by measuring changes in the intracellular calcium concentration ([Ca(2+)](i)). The channel activity was also observed in xtTRPV1-expressing Xenopus oocytes. Furthermore, we tested capsaicin- and heat-induced nocifensive behaviors of the frog X. tropicalis in vivo. At the amino acid level, xtTRPV1 displays ～60% sequence identity to other terrestrial vertebrate TRPV1 orthologues. Capsaicin induced [Ca(2+)](i) increases in xtTRPV1-HeLa and xtDRG neurons and evoked nocifensive behavior in X. tropicalis. However, its sensitivity was extremely low compared with mammalian orthologues. Low extracellular pH and heat activated xtTRPV1-HeLa and xtDRG neurons. Heat also evoked nocifensive behavior. In oocytes expressing xtTRPV1, inward currents were elicited by heat and low extracellular pH. Mutagenesis analysis revealed that two amino acids (tyrosine 523 and alanine 561) were responsible for the low sensitivity to capsaicin. Taken together, our results indicate that xtTRPV1 functions as a polymodal receptor similar to its mammalian orthologues. The present study demonstrates that TRPV1 functions as a heat- and acid-sensitive channel in the ancestor of terrestrial vertebrates. Because it is possible to examine vanilloid and heat sensitivities in vitro and in vivo, X. tropicalis could be the ideal experimental lower vertebrate animal for the study of TRPV1 function.     

2-aminoethoxydiphenyl borate is a common activator of TRPV1, TRPV2, and TRPV3

The transient receptor potential (TRP) superfamily contains a large number of proteins encoding cation permeable channels that are further divided into TRPC (canonical), TRPM (melastatin), and TRPV (vanilloid) subfamilies. Among the six TRPV members, TRPV1, TRPV2, TRPV3, and TRPV4 form heat-activated cation channels, which serve diverse functions ranging from nociception to osmolality regulation. Although chemical activators for TRPV1 and TRPV4 are well documented, those for TRPV2 and TRPV3 are lacking. Here we show that in the absence of other stimuli, 2-aminoethoxydiphenyl borate (2APB) activates TRPV1, TRPV2, and TRPV3, but not TRPV4, TRPV5, and TRPV6 expressed in HEK293 cells. In contrast, 2APB inhibits the activity of TRPC6 and TRPM8 evoked by 1-oleolyl-2-acetyl-sn-glycerol and menthol, respectively. In addition, low levels of 2APB strongly potentiate the effect of capsaicin, protons, and heat on TRPV1 as well as that of heat on TRPV3 expressed in Xenopus oocytes. In dorsal root ganglia neurons, supra-additive stimulations were evoked by 2APB and capsaicin or 2APB and acid. Our data suggest the existence of a common activation mechanism for TRPV1, TRPV2, and TRPV3 that may serve as a therapeutic target for pain management and treatment for diseases caused by hypersensitivity and temperature misregulation.     

Regulation of Ca2+-dependent desensitization in the vanilloid receptor TRPV1 by calcineurin and cAMP-dependent protein kinase

The vanilloid receptor TRPV1 is a polymodal nonselective cation channel of nociceptive sensory neurons involved in the perception of inflammatory pain. TRPV1 exhibits desensitization in a Ca2+-dependent manner upon repeated activation by capsaicin or protons. The cAMP-dependent protein kinase (PKA) decreases desensitization of TRPV1 by directly phosphorylating the channel presumably at sites Ser116 and Thr370. In the present study we investigated the influence of protein phosphatase 2B (calcineurin) on Ca2+-dependent desensitization of capsaicin- and proton-activated currents. By using site-directed mutagenesis, we generated point mutations at PKA and protein kinase C consensus sites and studied wild type (WT) and mutant channels transiently expressed in HEK293t or HeLa cells under whole cell voltage clamp. We found that intracellular application of the cyclosporin A.cyclophilin A complex (CsA.CyP), a specific inhibitor of calcineurin, significantly decreased desensitization of capsaicin- or proton-activated TRPV1-WT currents. This effect was similar to that obtained by extracellular application of forskolin (FSK), an indirect activator of PKA. Simultaneous applications of CsA.CyP and FSK in varying concentrations suggested that these substances acted independently from each other. In mutation T370A, application of CsA.CyP did not reduce desensitization of capsaicin-activated currents as compared with WT and to mutant channels S116A and T144A. In a double mutation at candidate protein kinase C phosphorylation sites, application of CsA.CyP or FSK decreased desensitization of capsaicin-activated currents similar to WT channels. We conclude that Ca2+-dependent desensitization of TRPV1 might be in part regulated through channel dephosphorylation by calcineurin and channel phosphorylation by PKA possibly involving Thr370 as a key amino acid residue.