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PubMed2006

Pharmacological and electrophysiological characterization of store-operated currents and capacitative Ca(2+) entry in vascular smooth muscle cells.

Capacitative Ca(2+) entry (CCE) in vascular smooth muscle cells contributes to vasoconstrictor and mitogenic effects of vasoactive hormones. In A7r5 rat aortic smooth muscle cells, measurements of cytosolic free Ca(2+) concentration ([Ca(2+)](i)) have demonstrated that depletion of intracellular Ca(2+) stores activates CCE. However, there is disagreement in published studies regarding the regulation of this mechanism by the vasoconstrictor hormone [Arg(8)]-vasopressin (AVP). We have employed electrophysiological methods to characterize the membrane currents activated by store depletion [store-operated current (I(SOC))]. Because of different recording conditions, it has not been previously determined whether I(SOC) corresponds to CCE measured using fura-2; nor has the channel protein responsible for CCE been identified. In the present study, the pharmacological characteristics of I(SOC), including its sensitivity to blockade by 2-aminoethoxydiphenylborane, diethylstilbestrol, or micromolar Gd(3+), were found to parallel the effects of these drugs on thapsigargin- or AVP-activated CCE measured under identical external ionic conditions using fura-2. Thapsigargin-stimulated I(SOC) was also measured in freshly isolated rat mesenteric artery smooth muscle cells (MASMC). Members of the transient receptor potential (TRP) family of nonselective cation channels, TRPC1, TRPC4, and TRPC6, were detected by reverse transcription-polymerase chain reaction and Western blot in both A7r5 cells and MASMC. TRPC1 expression was reduced in a stable A7r5 cell line expressing a small interfering RNA (siRNA) or by infection of A7r5 cells with an adenovirus expressing a TRPC1 antisense nucleotide sequence. Thapsigargin-stimulated I(SOC) was reduced in both the TRPC1 siRNA- and TRPC1 antisense-expressing cells, suggesting that the TRPC1 channel contributes to the I(SOC)/CCE pathway.

Animals↗
PubMed1992

Identification of a Drosophila gene encoding a calmodulin-binding protein with homology to the trp phototransduction gene.

We have isolated a number of Drosophila cDNAs on the basis of their encoding calmodulin-binding proteins. A full-length cDNA clone corresponding to one of these genes has been cloned and sequenced. Conservation of amino acid sequence and tissue-specific expression are observed between this gene and the transient receptor potential (trp) gene. We propose the name transient receptor potential-like (trpl) to describe this newly isolated gene. The trpl protein contains two possible calmodulin-binding sites, six transmembrane regions, and a sequence homologous to an ankyrin-like repeat. Structurally, the trpl and trp proteins resemble cation channel proteins, particularly the brain isoform of the voltage-sensitive Ca2+ channel. The identification of a protein similar to the trp gene product, yet also able to bind Ca2+/calmodulin, allows for a reinterpretation of the phenotype of the trp mutations and suggests that both genes may encode light-sensitive ion channels.

Amino Acid Sequence↗
PubMed2004

Acid-induced pain and its modulation in humans.

Despite the discovery of ion channels that are activated by protons, we still know relatively little about the signaling of acid pain. We used a novel technique, iontophoresis of protons, to investigate acid-induced pain in human volunteers. We found that transdermal iontophoresis of protons consistently caused moderate pain that was dose-dependent. A marked desensitization occurred with persistent stimulation, with a time constant of approximately 3 min. Recovery from desensitization occurred slowly, over many hours. Acid-induced pain was significantly augmented in skin sensitized by acute topical application of capsaicin. However, skin desensitized by repeated capsaicin application showed no significant reduction in acid-induced pain, suggesting that both capsaicin-sensitive and insensitive sensory neurons contribute to acid pain. Furthermore, topical application of non-steroidal anti-inflammatory drugs (NSAIDs) significantly attenuated acid-evoked pain but did not affect the heat pain threshold, suggesting a specific interaction between NSAIDs and peripheral acid sensors. Subcutaneous injection of amiloride (1 mm) also significantly inhibited the pain induced by iontophoresis of acid, suggesting an involvement of acid-sensing ion channel (ASIC) receptors. Conversely, iontophoresis of acid over a wide range of skin temperatures from 4 to 40 degrees C produced only minor changes in the induced pain. Together these data suggest a prominent role for ASIC channels and only a minor role for transient receptor potential vanilloid receptor-1 as mediators of cutaneous acid-induced pain.

Acid Sensing Ion Channels↗
PubMed2002

Mechanosensory ion channels in charophyte cells: the response to touch and salinity stress.

Mechanosensitive (MS) ion channels are activated by mechanical stress and then transduce this information into electrical signals. These channels are involved in the growth, development and response to environmental stress in higher plants. Detailed analyses of the electrophysiology in higher plants are difficult because such plants are composed of complex tissues. The large cells of the charophytes facilitate electrophysiological measurements and allow us to study MS ion channels at the level of single cells. We draw parallels between the process of touch-perception in freshwater Chara, and the turgor-regulating response to osmotic shock in salt-tolerant Lamprothamnium. In terms of electrophysiology, these responses can be considered in three stages: (1) stimulus perception, (2) signal transmission and (3) induction of response. In Chara the first stage is due to the receptor potential (RPD), a transient depolarization with a critical threshold that triggers action potentials, which are responsible for stages (2) and (3). Receptor potentials are generated by MS ion channels. Action potentials involve a transient influx of Ca(2+) to the cytoplasm, effluxes of K(+) and Cl(-) and a temporary decrease of turgor pressure. Reducing cell turgor increases sensitivity to mechanical stimulation. In Lamprothamnium, a hypotonic shock produces an extended depolarization that resembles an extended RPD and is responsive to osmotic rather than ionic changes. Like the action potential, a critical threshold depolarization triggers Ca(2+) influx, opening of Ca(2+)-sensitive Cl(-) channels and K(+) channels; effluxes that last over an hour and result in turgor regulation. These processes show us, in primal form and at the level of single cells, how mechanoperception occurs in higher plants. Recent progress in research into the role of MS ion channels in the freshwater and salt-tolerant Characeae is reviewed and the relevance of these findings to plants in general is considered.

Cell Membrane↗
PubMed2005

1,25-Dihydroxyvitamin D and 25-hydroxyvitamin D--mediated regulation of TRPV6 (a putative epithelial calcium channel) mRNA expression in Caco-2 cells.

BACKGROUND: TRPV6 is a member of the vanilloid subfamily of transient receptor potential (TRP) proteins and likely functions as an epithelial calcium channel in calcium-transporting organs, such as the intestine, kidney, and placenta. TRPV6 mRNA expression is strongly regulated by 1,25-dihydroxyvitamin D (1,25VD), the active hormonal form of vitamin D, in intestine and in Caco-2 cells, a human colon cancer cell line. AIM OF THE STUDY: The aim of the present study was to characterise the mode of regulation of the 1,25VD-mediated TRPV6 mRNA expression and to test the effect of the precursor of 1,25VD namely 25 hydroxyvitamin D (25 VD) on TRPV6 mRNA expression in Caco-2 cells. METHODS: Caco-2 cells were treated in a 2 x 2 format with 1,25VD and the transcriptional inhibitor actinomycin D (AD, 4 microg/ml), and also with translational inhibitor cycloheximide (CHX, 10 microg/ml) after 14 days in culture and TRPV6 mRNA levels were determined using reverse transcription-real time PCR.TRPV6 mRNA half life studies were performed by inhibiting transcription followed by sampling at various time points for TRPV6 mRNA. Varying concentrations of 25 VD were used to test their effect on TRPV6 mRNA in the presence of 5% FBS and also in the absence of serum (but containing insulin-transferrin-selenium mixture) for 24 h. RESULTS: Treatment with 10(-7) M 1,25VD for 8 h resulted in a 60-fold increase in TRPV6 mRNA and this increase could be completely blocked with AD. Treatment with CHX to inhibit de novo protein synthesis did not prevent the initiation of 1,25VD-induced TRPV6 expression, although it did reduce the extent of TRPV6 mRNA accumulation. We found that TRPV6 mRNA half-life was 8 h in Caco-2 cells and was not altered by 1,25VD treatment. Finally, we observed that treatment with 10(-6) M of the pro-hormone 25 VD for 24 h resulted in a significant increase in TRPV6 expression in Caco-2 cells, which is consistent with the presence of 1alpha-hydroxylase (CYP27B1) expression in Caco-2 cells and a possible autocrine vitamin D signaling pathway in colon cells. CONCLUSIONS: 1,25 dihydroxyvitamin D regulates TRPV6 expression by a process that requires new mRNA and protein synthesis and the point of regulation lies likely at the transcriptional level especially since vitamin D did not increase the half life of TRPV6 mRNA. In addition, the prohormone form of 1,25 dihydroxyvitamin D, i. e. the 25 hydroxyvitamin D, induced TRPV6 mRNA expression in Caco-2 cells.

Caco-2 Cells↗
PubMed2003

Capacitative calcium entry and TRPC channel proteins are expressed in rat distal pulmonary arterial smooth muscle.

Mammalian homologs of transient receptor potential (TRP) genes in Drosophila encode TRPC proteins, which make up cation channels that play several putative roles, including Ca2+ entry triggered by depletion of Ca2+ stores in endoplasmic reticulum (ER). This capacitative calcium entry (CCE) is thought to replenish Ca2+ stores and contribute to signaling in many tissues, including smooth muscle cells from main pulmonary artery (PASMCs); however, the roles of CCE and TRPC proteins in PASMCs from distal pulmonary arteries, which are thought to be the major site of pulmonary vasoreactivity, remain uncertain. As an initial test of the possibility that TRPC channels contribute to CCE and Ca2+ signaling in distal PASMCs, we measured [Ca2+]i by fura-2 fluorescence in primary cultures of myocytes isolated from rat intrapulmonary arteries (>4th generation). In cells perfused with Ca2+-free media containing cyclopiazonic acid (10 microM) and nifedipine (5 microM) to deplete ER Ca2+ stores and block voltage-dependent Ca2+ channels, restoration of extracellular Ca2+ (2.5 mM) caused marked increases in [Ca2+]i whereas MnCl2 (200 microM) quenched fura-2 fluorescence, indicating CCE. SKF-96365, LaCl3, and NiCl2, blocked CCE at concentrations that did not alter Ca2+ responses to 60 mM KCl (IC50 6.3, 40.4, and 191 microM, respectively). RT-PCR and Western blotting performed on RNA and protein isolated from distal intrapulmonary arteries and PASMCs revealed mRNA and protein expression for TRPC1, -4, and -6, but not TRPC2, -3, -5, or -7. Our results suggest that CCE through TRPC-encoded Ca2+ channels could contribute to Ca2+ signaling in myocytes from distal intrapulmonary arteries.

Animals↗
PubMed2005

Identification of an N-terminal TRPC2 splice variant which inhibits calcium influx.

TRPC2 is a member of the transient receptor potential (TRP) superfamily of Ca2+-permeable channels expressed in nonexcitable cells. TRPC2 is involved in a number of physiological processes including sensory activation of the vomeronasal organ, sustained Ca2+ entry in sperm, and regulation of calcium influx by erythropoietin. Here, a new splice variant of TRPC2, called "Similar to mouse TRPC2" (smTRPC2), was identified consisting of 213 amino acids, largely coincident with the N-terminus of TRPC2 clone 17. This splice variant lacks all six TRPC2 transmembrane domains and the calcium pore. Expression of smTRPC2 was found in all tissues examined by RT-PCR and in primary erythroid cells by RT-PCR and Western blotting. Confocal microscopy of CHO-S cells transfected with TRPC2 clone 14 and smTRPC2 demonstrated that TRPC2 clone 14 and smTRPC2 both localize at or near the plasma membrane and in the perinuclear region. Cell surface localization of TRPC2 was confirmed with biotinylation, and was not substantially affected by smTRPC2 expression. Coassociation of TRPC2 c14 and alpha with smTRPC2 was confirmed by immunoprecipitation. To examine the functional significance of smTRPC2 expression, a CHO-S model was used to study its effect on calcium influx stimulated by Epo through TRPC2. Single CHO-S cells which express transfected Epo-R were identified by detection of green fluorescent protein (GFP). Cells that express transfected TRPC2 c14 or alpha were identified by detection of blue fluorescent protein (BFP). [Ca]i was quantitiated with Fura Red fluorescence using digital video imaging. Epo stimulated calcium influx through TRPC2 isoforms c14 and alpha, which was inhibited by coexpression of smTRPC2. These data demonstrate that a short splice variant of TRPC2 exists in many cell types, which associates with and modifies the activity of functional TRPC2 splice variants.

Alternative Splicing↗
PubMed2006

Critical evaluation of the colocalization between calcitonin gene-related peptide, substance P, transient receptor potential vanilloid subfamily type 1 immunoreactivities, and isolectin B4 binding in primary afferent neurons of the rat and mouse.

UNLABELLED: Calcitonin gene-related peptide (CGRP) and/or substance P (SP) immunoreactivity as well as isolectin B(4) (IB(4)) binding are commonly used to define peptidergic and non-peptidergic nociceptor populations, respectively. Although this demarcation is well supported in the mouse, there is accumulating evidence to suggest it is not so in the rat. Hence, this investigation was undertaken to evaluate and quantify the colocalization of the neuropeptides CGRP and SP with IB(4) binding sites and the transient receptor potential vanilloid subfamily type 1 (TRPV1) channel and to compare this colocalization between trigeminal (TG) and dorsal root ganglia (DRG) in adult rats. These findings illustrate that there is a substantial overlap ( approximately 45% in the DRG and approximately 30% in the TG) between peptidergic neurons (ie, CGRP- and SP-expressing) and neurons that bind IB(4) in rat sensory ganglia. However, there were also significant differences in the colocalization of these markers between the DRG and TG. For instance, in the DRG, significantly more CGRP-immunoreactive neurons also expressed IB(4) binding sites (44.5%) compared with the TG (27.5%). In contrast, significantly fewer CGRP-immunoreactive neurons in the DRG colocalized TRPV1 immunoreactivity (49.2%) compared with the TG (70%). Moreover, we directly assessed the colocalization of CGRP and IB(4) in the TG of rats and mice using a CGRP antibody that recognizes this peptide in both species. Thus, whereas only an approximately 10% overlap was observed in TG neurons of mouse, significantly greater overlap (approximately 35%) was observed in those of rat. PERSPECTIVE: These data indicate that in adult rat sensory ganglia, there is not a clear distinction between the peptidergic and non-peptidergic nociceptor subclasses as a function of IB(4) binding. Furthermore, there are significant differences between the TG and DRG in the degree to which commonly utilized nociceptive neuronal markers are co-expressed. Taken together, the present findings dictate prudence when extrapolating experimental conclusions about the neurochemical classification of neurons between sensory ganglia or between species, including humans.

Animals↗
PubMed2006

High-throughput random mutagenesis screen reveals TRPM8 residues specifically required for activation by menthol.

Menthol is a cooling compound derived from mint leaves and is extensively used as a flavoring chemical. Menthol activates transient receptor potential melastatin 8 (TRPM8), an ion channel also activated by cold, voltage and phosphatidylinositol-4,5-bisphosphate (PIP2). Here we investigated the mechanism by which menthol activates mouse TRPM8. Using a new high-throughput approach, we screened a random mutant library consisting of approximately 14,000 individual TRPM8 mutants for clones that are affected in their response to menthol while retaining channel function. We identified determinants of menthol sensitivity in two regions: putative transmembrane segment 2 (S2) and the C-terminal TRP domain. Analysis of these mutants indicated that activation by menthol involves a gating mechanism distinct and separable from gating by cold, voltage or PIP2. Notably, TRP domain mutations mainly attenuated menthol efficacy, suggesting that this domain influences events downstream of initial binding. In contrast, S2 mutations strongly shifted the concentration dependence of menthol activation, raising the possibility that S2 influences menthol binding.

Amino Acid Sequence↗
PubMed2004

Clues to understanding cold sensation: thermodynamics and electrophysiological analysis of the cold receptor TRPM8.

The cold and menthol receptor, TRPM8, also designated CMR1, is a member of the transient receptor potential (TRP) family of excitatory ion channels. TRPM8 is a channel activated by cold temperatures, voltage, and menthol. In this study, we characterize the cold- and voltage-induced activation of TRPM8 channel in an attempt to identify the temperature- and voltage-dependent components involved in channel activation. Under equilibrium conditions, decreasing temperature has two effects. (i) It shifts the normalized conductance vs. voltage curves toward the left, along the voltage axis. This effect indicates that the degree of order is higher when the channel is in the open configuration. (ii) It increases the maximum channel open probability, suggesting that temperature affects both voltage-dependent and -independent pathways. In the temperature range between 18 degrees C and 25 degrees C, large changes in enthalpy (DeltaH=-112 kcal/mol) and entropy (DeltaS=-384 cal/mol K) accompany the activation process. The Q10 calculated in the same temperature range is 24. This thermodynamic analysis strongly suggests that the process of opening involves large conformational changes of the channel-forming protein. Therefore, the highly temperature-dependent transition between open and closed configurations is possible because enthalpy and entropy are both large and compensate each other. Our data also demonstrate that temperature and voltage interact allosterically to enhance channel opening.

Cell Line↗
PubMed2003

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.

Animals↗
PubMed2003

Store-operated Ca2+ entry in first trimester and term human placenta.

We have examined whether store-operated Ca2+ entry, a common pathway for Ca2+ entry in non-excitable tissue, is apparent in the syncytiotrophoblast of both first trimester and term human placenta. Expression of transient receptor potential (TRPC) homologues, a family of channels thought to be involved in store-operated Ca2+ entry, was also studied at the mRNA and protein levels. [Ca2+]i in syncytiotrophoblast of first trimester and term placental villous fragments was measured by microfluorimetry using the Ca2+-sensitive dye fura-2. Store-operated Ca2+ entry was stimulated using 1 microM thapsigargin in Ca(2+)-free Tyrode buffer (no added Ca2+ + 1 mM EGTA) followed by superfusion with control (Ca2+-containing) buffer. In term fragments, this protocol resulted in a rapid increase in [Ca2+]i, which was inhibited in the presence of 150 microM GdCl3, 200 microM NiCl2, 200 microM CoCl2 or 30 microM SKF96365 but was unaffected by addition of 10 microM nifedipine. It was not possible to stimulate such a rise in [Ca2+]i in first trimester fragments. Messenger RNA encoding TRPC1, TRPC3, TRPC4, TRPC5 and TRPC6 was identified in both first trimester and term placentas. From Western blotting, TRPC3 and TRPC6 proteins were detected in term, but not in first trimester, placentas, while TRPC1 protein was not detected. By immunocytochemistry, TRPC3 and TRPC4 were localised to cytotrophoblast cells in first trimester placentas and to the syncytiotrophoblast in term placentas. TRPC6 staining was present in the syncytiotrophoblast of both first trimester and term placenta, but the intensity was much greater in the latter. We propose that store-operated Ca2+ entry may be an important route for Ca2+ entry into the syncytiotrophoblast of term, but not first trimester placentas, and that in human placenta TRPC channels may underlie this entry mechanism.

Adult↗
PubMed2002

Inhibition of endogenous TRP1 decreases capacitative Ca2+ entry and attenuates pulmonary artery smooth muscle cell proliferation.

Pulmonary vascular medial hypertrophy due to proliferation of pulmonary artery smooth muscle cells (PASMC) greatly contributes to the increased pulmonary vascular resistance in pulmonary hypertension patients. A rise in cytosolic free Ca2+ concentration ([Ca2+]cyt) is an important stimulus for cell growth in PASMC. Resting [Ca2+]cyt, intracellularly stored [Ca2+], capacitative Ca2+ entry (CCE), and store-operated Ca2+ currents (I(SOC)) are greater in proliferating human PASMC than in growth-arrested cells. Expression of TRP1, a transient receptor potential gene proposed to encode the channels responsible for CCE and I(SOC), was also upregulated in proliferating PASMC. Our aim was to determine if inhibition of endogenous TRP1 gene expression affects I(SOC) and CCE and regulates cell proliferation in human PASMC. Cells were treated with an antisense oligonucleotide (AS, for 24 h) specifically designed to cleave TRP1 mRNA and then returned to normal growth medium for 40 h before the experiments. Then, mRNA and protein expression of TRP1 was downregulated, and amplitudes of I(SOC) and CCE elicited by passive depletion of Ca2+ from the sarcoplasmic reticulum using cyclopiazonic acid were significantly reduced in the AS-treated PASMC compared with control. Furthermore, the rate of cell growth was decreased by 50% in AS-treated PASMC. These results indicate that TRP1 may encode a store-operated Ca2+ channel that plays a critical role in PASMC proliferation by regulating CCE and intracellular [Ca2+](cyt).

Blood Proteins↗
PubMed2006

Role of transient receptor potential C3 in TNF-alpha-enhanced calcium influx in human airway myocytes.

Previous studies have suggested that the proinflammatory cytokine, TNF-alpha, contributes to airway hyperresponsivness by altering airway smooth muscle (ASM) Ca(2+) responses to agonist stimulation. The present study examined the effects of TNF-alpha on Ca(2+) influx pathways in cultured human ASM cells (HASMCs). Proteins encoded by the transient receptor potential (TRP) gene family function as channels through which receptor-operated and store-operated Ca(2+) entry (SOCE) occur. In the present study, the presence of TRPC1, TRPC3, TRPC4, TRPC5, and TRPC6 mRNA and protein expression was confirmed in cultured HASMCs using RT-PCR and Western blot analysis. TNF-alpha treatment significantly increased TRPC3 mRNA and protein levels in HASMCs as well as SOCE. TNF-alpha treatment also increased both the peak and plateau intracellular Ca(2+) concentration responses in HASMCs elicited by acetylcholine and bradykinin. The effects of TNF-alpha treatment on SOCE and agonist-induced intracellular Ca(2+) concentration responses were attenuated using small interfering RNA transfection, which knocked down TRPC3 expression. Thus, in inflammatory airway diseases, TNF-alpha treatment may result in increased myocyte activation due to altered Ca(2+) influx pathways. These results suggest that TRPC3 may be an important therapeutic target in inflammatory airway diseases such as asthma and chronic obstructive pulmonary disease.

Bronchi↗
PubMed2006

Transient receptor potential vanilloid 1 is required for intrinsic osmoreception in organum vasculosum lamina terminalis neurons and for normal thirst responses to systemic hyperosmolality.

Recent studies have indicated that members of the transient receptor potential vanilloid (TRPV) family of cation channels are required for the generation of normal osmoregulatory responses, yet the mechanism of osmosensory transduction in primary osmoreceptor neurons of the CNS remains to be defined. Indeed, despite ample evidence suggesting that the organum vasculosum lamina terminalis (OVLT) serves as the primary locus of the brain for the detection of osmotic stimuli, evidence that neurons in the OVLT are intrinsically osmosensitive has remained elusive. Here we show that murine OVLT neurons are intrinsically sensitive to increases in the osmolality of the extracellular fluid. Hypertonic conditions provoked increases in membrane cation conductance that resulted in the generation of an inward current, depolarizing osmoreceptor potentials, and enhanced action potential discharge. Moreover, we found that this osmosensory signal transduction cascade was absent in OVLT neurons from TRPV1 knock-out (TRPV1-/-) mice and that responses of wild type (WT) OVLT neurons could be blocked by ruthenium red, an inhibitor of TRPV channels. Finally, TRPV1-/- mice showed significantly attenuated water intake in response to systemic hypertonicity compared with WT controls. These findings indicate that OVLT neurons act as primary osmoreceptors and that a product of the trpv1 gene is required for osmosensory transduction.

Animals↗
PubMed2005

Downregulation of transient receptor potential melastatin 8 by protein kinase C-mediated dephosphorylation.

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 Ca2+ 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.

Animals↗
PubMed2004

Protease-activated receptor 2 sensitizes the capsaicin receptor transient receptor potential vanilloid receptor 1 to induce hyperalgesia.

Inflammatory proteases (mast cell tryptase and trypsins) cleave protease-activated receptor 2 (PAR2) on spinal afferent neurons and cause persistent inflammation and hyperalgesia by unknown mechanisms. We determined whether transient receptor potential vanilloid receptor 1 (TRPV1), a cation channel activated by capsaicin, protons, and noxious heat, mediates PAR2-induced hyperalgesia. PAR2 was coexpressed with TRPV1 in small- to medium-diameter neurons of the dorsal root ganglia (DRG), as determined by immunofluorescence. PAR2 agonists increased intracellular [Ca2+] ([Ca2+]i) in these neurons in culture, and PAR2-responsive neurons also responded to the TRPV1 agonist capsaicin, confirming coexpression of PAR2 and TRPV1. PAR2 agonists potentiated capsaicin-induced increases in [Ca2+]i in TRPV1-transfected human embryonic kidney (HEK) cells and DRG neurons and potentiated capsaicin-induced currents in DRG neurons. Inhibitors of phospholipase C and protein kinase C (PKC) suppressed PAR2-induced sensitization of TRPV1-mediated changes in [Ca2+]i and TRPV1 currents. Activation of PAR2 or PKC induced phosphorylation of TRPV1 in HEK cells, suggesting a direct regulation of the channel. Intraplantar injection of a PAR2 agonist caused persistent thermal hyperalgesia that was prevented by antagonism or deletion of TRPV1. Coinjection of nonhyperalgesic doses of PAR2 agonist and capsaicin induced hyperalgesia that was inhibited by deletion of TRPV1 or antagonism of PKC. PAR2 activation also potentiated capsaicin-induced release of substance P and calcitonin gene-related peptide from superfused segments of the dorsal horn of the spinal cord, where they mediate hyperalgesia. We have identified a novel mechanism by which proteases that activate PAR2 sensitize TRPV1 through PKC. Antagonism of PAR2, TRPV1, or PKC may abrogate protease-induced thermal hyperalgesia.

Animals↗
PubMed2006

Twenty odd years of stretch-sensitive channels.

After formation of the giga-seal, the membrane patch can be stimulated by hydrostatic or osmotic pressure gradients applied across the patch. This feature led to the discovery of stretch-sensitive or mechanosensitive (MS) channels, which are now known to be ubiquitously expressed in cells representative of all the living kingdoms. In addition to mechanosensation, MS channels have been implicated in many basic cell functions, including regulation of cell volume, shape, and motility. The successful cloning, overexpression, and crystallization of bacterial MS channel proteins combined with patch clamp and modeling studies have provided atomic insight into the working of these nanomachines. In particular, studies of MS channels have revealed new understanding of how the lipid bilayer modulates membrane protein function. Three major membrane protein families, transient receptor potential, 2 pore domain K(+), and the epithelial Na(+) channels, have been shown to form MS channels in animal cells, and their polymodal activation embrace fields far beyond mechanosensitivity. The discovery of new drugs highly selective for MS channels ("mechanopharmaceutics") and the demonstration of MS channel involvement in several major human diseases ("mechanochannelopathies") provide added motivation for devising new techniques and approaches for studying MS channels.

Animals↗