Nanosecond pulsed electric field generators for the study of subcellular effects

Modeling and experimental studies have shown that pulsed electric fields of nanosecond duration and megavolt per meter amplitude affect subcellular structures but do not lead to the formation of large pores in the outer membrane. This "intracellular electromanipulation" requires the use of pulse generators which provide extremely high power but low energy pulses. In this study, we describe the concept of the required pulsed power sources, their design, operation, and the necessary diagnostics. Two types of pulse generators based on the Blumlein line principle have been developed and are described here. One system is designed to treat a large number of cells in cuvettes holding volumes from 0.1 to 0.8 ml. Pulses of up to 40 kV amplitude, with a duration of 10 ns and a rise time close to 1 ns can be applied to the cuvette. For an electrode gap of 1 mm this voltage corresponds to an average electric field of 40 MV/m. The second system allows for real time observation of individual cells under a microscope. It generates pulses of 10-300 ns duration with a rise time of 3.5 ns and voltage amplitudes up to 1 kV. Connected to a microreactor with an electrode gap of 100 microm, electric fields up to 10 MV/m are applied.     

Recruitment of the intracellular Ca by ultrashort electric stimuli: The impact of pulse duration

Nanosecond-duration electric stimuli are distinguished by the ability to permeabilize intracellular membranes and recruit Ca²⁺ from intracellular stores. We quantified this effect in non-excitable cells (CHO) using ratiometric Ca²⁺ imaging with Fura-2. In a Ca²⁺-free medium, 10-, 60-, and 300-ns stimuli evoked Ca²⁺ transients by mobilization of Ca²⁺ from the endoplasmic reticulum. With 2 mM external Ca²⁺, the transients included both extra- and intracellular components. The recruitment of intracellular Ca²⁺ increased as the stimulus duration decreased. At the threshold of 200–300 nM, the transients were amplified by calcium-induced calcium release. We conclude that nanosecond stimuli mimic Ca²⁺ signaling while bypassing the usual receptor- and channels-mediated cascades. The recruitment of the intracellular Ca²⁺ can be controlled by the duration of the stimulus.     

The platelet receptor for type III collagen (TIIICBP) is present in platelet membrane lipid microdomains (rafts)

Platelet interactions with collagen are orchestrated by the presence or the migration of platelet receptor(s) for collagen into lipid rafts, which are specialized lipid microdomains from the platelet plasma membrane enriched in signalling proteins. Electron microscopy shows that in resting platelets, TIIICBP, a receptor specific for type III collagen, is present on the platelet membrane and associated with the open canalicular system, and redistributes to the platelet membrane upon platelet activation. After platelet lysis by 1% Triton X-100 and the separation of lipid rafts on a discontinuous sucrose gradient, TIIICBP is recovered in lipid raft-containing fractions and Triton X-100 insoluble fractions enriched in cytoskeleton proteins. Platelet aggregation, induced by type III collagen, was inhibited after disruption of the lipid rafts by cholesterol depletion, whereas platelet adhesion under static conditions did not require lipid raft integrity. These results indicate that TIIICBP, a platelet receptor involved in platelet interaction with type III collagen, is localized within platelet lipid rafts where it could interact with other platelet receptors for collagen (GP VI and α2β1 integrin) for efficient platelet activation. Pascal Maurice and Ludovic Waeckel have contributed equally to this work.     

Functional role of the calmodulin- and inositol 1,4,5-trisphosphate receptor-binding (CIRB) site of TRPC6 in human platelet activation

Background

All identified mammalian TRPC channels show a C-terminal calmodulin (CaM)- and inositol 1,4,5-trisphosphate receptors (IP₃Rs)-binding (CIRB) site involved in the regulation of TRPC channel function.

Objectives

To assess the basis of CaM/IP₃Rs binding to the CIRB site of TRPC6 and its role in platelet physiology.

Methods

Protein association was detected by co-immunoprecipitation and Western blotting, Ca²⁺ mobilization was measured by fluorimetric techniques and platelet function was analyzed by aggregometry.

Results

Co-immunoprecipitation of TRPC6 with CaM or the IP₃Rs at different cytosolic free Ca²⁺ concentrations ([Ca²⁺]_c) indicates that the association between these proteins is finely regulated by cytosolic Ca²⁺ via association of CaM and displacement of the IP₃Rs at high [Ca²⁺]_c. Thrombin-stimulated association of TRPC6 with CaM or the IP₃Rs was sensitive to 2-APB and partially inhibited by dimethyl BAPTA loading, thus suggesting that the association between these proteins occurs through both Ca²⁺-dependent and -independent mechanisms. Incorporation of an anti-TRPC6 C-terminal antibody, whose epitope overlaps the CIRB region, impaired the dynamics of the association of TRPC6 with CaM and the IP₃Rs, which lead to both inhibition and enhancement of thrombin- and thapsigargin-evoked Ca²⁺ entry in the presence of low or high, respectively, extracellular Ca²⁺ concentrations, as well as altered thrombin-evoked platelet aggregation.

Conclusions

Our results indicate that the CIRB site of TRPC6 plays an important functional role in platelets both modulating Ca²⁺ entry and aggregation through its interaction with CaM and IP₃Rs.     

Involvement of SNARE proteins in thrombin-induced platelet aggregation: evidence for the relevance of Ca2+ entry

Thrombin induces platelet activation through a variety of intracellular mechanisms, including Ca(2+) mobilization. The protein of the exocytotic machinery SNAP-25, but not VAMPs, is required for store-operated Ca(2+) entry, the main mechanism for Ca(2+) influx in platelets. Hence, we have investigated the role of the SNAP-25 and VAMPs in thrombin-induced platelet aggregation. Platelet stimulation with thrombin or selective activation of thrombin receptors PAR-1, PAR-4 or GPIb-IX-V results in platelet aggregation that, except for GPIb-IX-V receptor, requires Ca(2+) entry for full activation. Depletion of the intracellular Ca(2+) stores using pharmacological tools was unable to induce aggregation except when cytosolic Ca(2+) concentration reached a critical level (around 1.5 microM). Electrotransjection of cells with anti-SNAP-25 antibody reduced thrombin-evoked platelet aggregation, while electrotransjection of anti-VAMP-1, -2 and -3 antibody had no effect. These findings support a role for SNAP-25 but not VAMP-1, -2 and -3 in platelet aggregation, which is likely mediated by the regulation of Ca(2+) mobilization in human platelets.     

Physical properties of membrane fractions isolated from human platelets: implications for chilling induced platelet activation

In previous studies, it has been suggested that chilling induced activation of human platelets is related to a lipid phase transition seen in membrane lipids. Those studies showed a single, surprisingly cooperative transition in human platelets, as determined by Fourier transform infrared (FTIR) spectroscopy, findings that are confirmed here with calorimetric measurements. Such transitions have now been studied in membrane fractions obtained from the platelets and it is reported that all fractions and purified phospholipids show similar transitions. In order to obtain these data it was necessary to develop means for separating these fractions. Therefore, a novel method for isolation and separation of dense tubular system (DTS) and plasma membranes in human platelets is described here. Lipid analysis showed that phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were the dominant phospholipids in both fractions, whereas cholesterol and sphingomyelin (SM) were predominantly located in the plasma membranes. Thermotropic phase transitions in the two membrane fractions, determined by differential scanning calorimetry (DSC) and FTIR spectroscopy were found to occur at about 15 degrees C, similar to the Tm of intact human platelets. These data are discussed in relation to the role of the DTS and plasma membranes in the cold-induced activation of human platelets.     

Mating aggregation of a stink bug,Megacopta punctissimum (Montandon) (Heteroptera: Plataspidae)

Summary Adults of a stink bug,Megacopta punctissimum, form aggregations on stems of the bush-clover,Lespedeza crytobotria, in spring. They do not oviposit there, but feed and mate within aggregations. Oviposition is made on other leguminous plants such as the kudzu-vine,Pueraria lobata. Mean size of aggregations (groups of two or more individuals which sit on a stem at distance less than 3 cm from their nearest neighbors) consisted of 4.4 individuals. The sex ratios within aggregations were similar to overall sex ratio of the population including solitary individuals (0.58). More than 50% of females found in aggregations were copulating. Percentage of femalesin copula in larger aggregations was higher than that in smaller aggregations, and this difference was considered to be caused by the higher chance of sexual enconters in larger aggregations. It was suggested that the aggregation of this species on the bush-clover is not a ‘harem’ (a male monopolizes a group of females reported in some other stink bugs) but is mating aggregation to raise the chance of mating. This work was partially supported by Grant-in-Aid (No. 56480039) from the Ministry of Education, Science and Culture.     

Discovery of a novel potent GABA(B) receptor agonist

Structure-activity studies have led to a discovery of 3-(4-pyridyl)methyl ether derivative 9d that has 25- to 50-fold greater functional potency than R-baclofen at human and rodent GABA(B) receptors in vitro. Mouse hypothermia studies confirm that this compound crosses the blood-brain barrier and is approximately 50-fold more potent after systemic administration.     

Synthesis, characterization and catalytic oxidation of cyclohexane using a novel host (zeolite-Y)/guest (binuclear transition metal complexes) nanocomposite materials

Transition metal (M = Mn(II), Co(II), Ni(II) and Cu(II)) complexes with octahydro-Schiff base (H₄-N₄O₄) = 2,7,13,18-tetramethyl-3,6,14,17-tetraazatricyclo-[17.3.1.1]-tetracosa-1(23),2,6,8(24),9,11,13,17,19,21-decaene-9,11,20,22-tetraol; H₄([H]₈-N₄O₄) = 2,7,13,,18-tetramethyl-3,6,14,17-tetraazatricyclo-[17.13.1.1.]-tetracosa-1(23),8(24),9,11,19,21-hexane-9,11,20,22-tetraol) have been encapsulated in nanopores of zeolite-Y; [M([H]₈-N₄O₄)]@NaY; with Flexible Ligand Method (FLM) for the first time. The new Host-Guest Nanocomposite Materials (HGNM) was characterized by several techniques: chemical analysis, spectroscopic methods (DRS, FT-IR and UV/Vis), BET technique, conductometric and magnetic measurements. The catalytic activities for oxidation of cyclohexane with HGNM complexes are reported. Zeolite encapsulated octahydro-Schiff base copper(II) complex; [Cu([H]₈-N₄O₄)]@NaY; was found to be more active than the corresponding cobalt(II), manganese(II) and nickel(II) complexes for cyclohexane oxidation. The catalytic properties of the complexes are influenced by their geometry and by the steric environment of the active sites. HGNM are stable enough to be reused and are suitable to be utilized as partial oxidation catalysts. The encapsulated catalysts systems; [M([H]₈-N₄O₄)]@NaY; were more active than the corresponding neat complexes; [M([H]₈-N₄O₄))].     

Phosphorylation of a tropomyosin-like (30 KD) protein during platelet activation

In this study, we have used the tumor promoter 12-o-tetradecanoylphorbol-13-acetate (TPA), as well as its biologically inactive analogue 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), to investigate platelet protein phosphorylation and its possible correlation with platelet activation. Our data show that TPA, but not 4 alpha-PDD, induces a preferential phosphorylation of a 30,000 dalton (30 KD) protein. This phosphoprotein is found to be physically associated with an actomyosin-containing platelet cytoskeleton complex. Further analysis using both standard two-dimensional gel electrophoresis and one-dimensional urea-SDS gel electrophoresis reveals that this 30 KD protein has several tropomyosin-like properties. Most importantly, the degree of TPA-induced phosphorylation of the 30 KD protein is directly proportional to the extent of platelet granule release and the shape change of the platelet, as well as to the degree of aggregation. We speculate that this phosphorylated tropomyosinlike protein may play a pivotal role in the regulation of actomyosin-mediated platelet contractility, which has been previously implicated in a variety of platelet functions.     

Neutrophil elastase acts as a biased agonist for proteinase-activated receptor-2 (PAR2)

Human neutrophil proteinases (elastase, proteinase-3, and cathepsin-G) are released at sites of acute inflammation. We hypothesized that these inflammation-associated proteinases can affect cell signaling by targeting proteinase-activated receptor-2 (PAR(2)). The PAR family of G protein-coupled receptors is triggered by a unique mechanism involving the proteolytic unmasking of an N-terminal self-activating tethered ligand (TL). Proteinases can either activate PAR signaling by unmasking the TL sequence or disarm the receptor for subsequent enzyme activation by cleaving downstream from the TL sequence. We found that none of neutrophil elastase, cathepsin-G, and proteinase-3 can activate G(q)-coupled PAR(2) calcium signaling; but all of these proteinases can disarm PAR(2), releasing the N-terminal TL sequence, thereby preventing G(q)-coupled PAR(2) signaling by trypsin. Interestingly, elastase (but neither cathepsin-G nor proteinase-3) causes a TL-independent PAR(2)-mediated activation of MAPK that, unlike the canonical trypsin activation, does not involve either receptor internalization or recruitment of β-arrestin. Cleavage of synthetic peptides derived from the extracellular N terminus of PAR(2), downstream of the TL sequence, demonstrated distinct proteolytic sites for all three neutrophil-derived enzymes. We conclude that in inflammation, neutrophil proteinases can modulate PAR(2) signaling by preventing/disarming the G(q)/calcium signal pathway and, via elastase, can selectively activate the p44/42 MAPK pathway. Our data illustrate a new mode of PAR regulation that involves biased PAR(2) signaling by neutrophil elastase and a disarming/silencing effect of cathepsin-G and proteinase-3.     

High Definition Oscillometry: a novel technique for non-invasive blood pressure monitoring in the cynomolgus monkey (Macaca fascicularis)

Background  Current approaches for accurate blood pressure determination rely predominantly on invasive techniques. High Definition Oscillometry (HDO) was evaluated as a potential non-invasive approach for accurate blood pressure recordings in cynomolgus monkeys.
Methods  In conscious animals, systolic, diastolic, mean arterial blood pressure (MAP) and pulse/minute were determined 15 times within approx. 9 minutes per individual. This session was performed during 3 consecutive days. Anaesthesia induced hypotension was controlled simultaneously with HDO and telemetry as reference.
Results  Repeated measurements were highly reproducible. After procedural habituation, mean MAP was 96.2 ± 13.7 mmHg in males and 86.9 ± 4.3 mmHg in females. Mean intraindividual coefficients of variation ranged between 10.8% and 2.4% depending on the session and parameter. Values determined by HDO corresponded to those reported for invasive techniques.
Conclusion  Our results demonstrate, using telemetry as reference, the accuracy of HDO-based non-invasive blood pressure measurements in macaques to detect drug-related cardiovascular changes.     

The hydraulic conductivity as a criterion for the membrane integrity of protoplasts fused by an electric field pulse

The hydraulic conductivity of the membrane, Lp, of fused plant protoplasts was measured and compared to that for unfused cells, in order to identify possible changes in membrane properties resulting from the fusion process. Fusion was achieved by an electric field pulse which induced breakdown in the membranes of protoplasts in close contact. Close membrane contact was established by dielectrophoresis. In some experiments pronase was added during field application; pronase stabilizes protoplasts against high field pulses and long exposure times to the field. The Lp-values were obtained from the shrinking and swelling kinetics in response to osmotic stress. The Lp-values of fused mesophyll cell protoplasts of Avena sativa L. and of mesophyll and guard cell protoplasts of Vicia faba L. were found to be 1.9±0.9·10^-6, 3.2±2.2·10^-6, and 0.8±0.7·10^-6 cm·bar^-1·s^-1, respectively. Within the limits of error, no changes in the Lp-values of fused protoplasts could be detected in comparison to unfused protoplasts. The Lp-values are in the range of those reported for walled cells of higher plants, as revealed by the pressure probe.Abbreviations GCP guard cell protoplast - Lp hydraulic conductivity - MCP mesophyll cell protoplast     

Pulsed electric field treatment as a potential method for microbial inactivation in scaffold materials for tissue engineering: the inactivation of bacteria in collagen gel

Aims: To investigate the effectiveness of pulsed electric field (PEF) treatment as a new method for inactivation of micro-organisms in complex biomatrices and to assess this by quantifying the inactivation of Escherichia coli seeded in collagen gels. Methods and Results: PEF was applied to E. coli seeded collagen gels in static (nonflowing) chambers. The influence of electric field strength, pulse number and seeded cell densities were investigated. The highest level of inactivation was obtained at the maximum field strength of 45 kV cm⁻¹. For low levels of E. coli contamination (10³ CFU ml⁻¹), PEF treatment resulted in no viable E. coli being recovered from the gels. However, PEF treatment of gels containing higher cell densities (≥10⁴ CFU ml⁻¹) did not achieve complete inactivation of E. coli. Conclusions: PEF treatment successfully inactivated E. coli seeded in collagen gels by 3 log₁₀ CFU ml⁻¹. Complete inactivation was hindered at high cell densities by the tailing effect observed. Significance and Impact of the Study: PEF shows potential as a novel, nondestructive method for decontamination of collagen-based matrices. Further investigation is required to ensure its compatibility with other proteins and therapeutic drugs for tissue engineering and drug delivery applications.     

Calpastatin domain L is a partial agonist of the calmodulin-binding site for channel activation in Cav1.2 Ca2+ channels

Cav1.2 Ca(2+) channel activity diminishes in inside-out patches (run-down). Previously, we have found that with ATP, calpastatin domain L (CSL) and calmodulin (CaM) recover channel activity from the run-down in guinea pig cardiac myocytes. Because the potency of the CSL repriming effect was smaller than that of CaM, we hypothesized that CSL might act as a partial agonist of CaM in the channel-repriming effect. To examine this hypothesis, we investigated the effect of the competitions between CSL and CaM on channel activity and on binding in the channel. We found that CSL suppressed the channel-activating effect of CaM in a reversible and concentration-dependent manner. The channel-inactivating effect of CaM seen at high concentrations of CaM, however, did not seem to be affected by CSL. In the GST pull-down assay, CSL suppressed binding of CaM to GST fusion peptides derived from C-terminal regions in a competitive manner. The inhibition of CaM binding by CSL was observed with the IQ peptide but not the PreIQ peptide, which is the CaM-binding domain in the C terminus. The results are consistent with the hypothesis that CSL competes with CaM as a partial agonist for the site in the IQ domain in the C-terminal region of the Cav1.2 channel, which may be involved in activation of the channel.     

Discovery and characterization of a positive allosteric modulator of transient receptor potential canonical 6 (TRPC6) channels

The non-selective second messenger-gated cation channel TRPC6 (transient receptor potential canonical 6) is activated by diacylglycerols (DAG) in a PKC-independent manner and plays important roles in a variety of physiological processes and diseases. In order to facilitate novel therapies, the development of potent inhibitors as well as channel-activating agents is of great interest. The screening of a chemical library, comprising about 17,000 small molecule compounds, revealed an agent, which induced increases in intracellular Ca²⁺ concentrations ([Ca²⁺]_i) in a concentration-dependent manner (EC₅₀ = 2.37 ± 0.25 μM) in stably TRPC6-expressing HEK293 cells. This new compound (C20) selectively acts on TRPC6, unlike OAG (1-oleoyl-1-acetyl-sn-glycerol), which also activates PKC and does not discriminate between TRPC6 and the closely related channels TRPC3 and TRPC7. Further evaluation by Ca²⁺ assays and electrophysiological studies revealed that C20 rather operated as an enhancer of channel activation than as an activator by itself and led to the assumption that the compound C20 is an allosteric modulator of TRPC6, enabling low basal concentrations of DAG to induce activation of the ion channel. Furthermore, C20 was tested in human platelets that express TRPC6. A combined activation of TRPC6 with C20 and OAG elicited a robust increase in [Ca²⁺]_i in human platelets. This potentiated channel activation was sensitive to TRPC6 channel blockers. To achieve sufficient amounts of C20 for biological studies, we applied a one-pot synthesis strategy. With regard to studies in native systems, the sensitizing ability of C20 can be a valuable pharmacological tool to selectively exaggerate TRPC6-dependent signals.     

Regulation of intracellular levels of NAD: a novel role for CD38

Nicotinamide adenine dinucleotide (NAD) plays key roles in many cellular functions. In addition to its well-known role in energy metabolism, NAD also plays a role in signal transduction, ageing, and cellular injury. NAD is also involved in many signal transduction pathways. Therefore, it is imperative to understand the mechanisms that control intracellular NAD levels. However, to date, the mechanisms that regulate intracellular levels of NAD have not been completely elucidated. CD38 is a multifunctional enzyme ubiquitously distributed in mammalian tissues. CD38 has been implicated as the enzyme responsible for the synthesis of the second messengers. However, its major enzymatic activity is the hydrolysis of NAD, in fact, CD38 will generate one molecule of cADPR for every 100 molecules of NAD hydrolyzed. To date, the role of CD38 as a modulator of levels of NAD has not been explored. We postulated that CD38 is the major NADase in mammalian cells and that it regulates intracellular NAD levels. In the current studies we examined the NADase activities and NAD levels in a variety of tissues from both wild-type and CD38 deficient mice. In accordance with our hypothesis, we found that tissue levels of NAD in CD38 deficient mice are 10- to 20-fold higher than in wild-type animals. In addition, NADase activity in the plasma membrane, mitochondria, sarcoplasmic reticulum, and nuclei is essentially absent in most tissues from CD38 deficient mice. These data support the novel concept that CD38 is a major regulator of cellular NAD levels. These findings have implications for understanding the mechanisms that regulate intracellular NAD levels and its role in energy homeostasis, signal transduction, and ageing.     

Cutting edge: T cells trigger CD40-dependent platelet activation and granular RANTES release: a novel pathway for immune response amplification

Platelets, in addition to exerting hemostatic activity, contribute to immunity and inflammation. The recent report that platelets express CD40 led us to hypothesize that CD40 ligand (CD40L)-positive T cells could bind to platelets, cause their activation, and trigger granular RANTES release, creating a T cell recruitment feedback loop. Platelets were cocultured with resting or activated autologous T cells and their activation was assessed by P-selectin expression. RANTES binding to endothelial cells was assessed by confocal microscopy, and its biological activity was demonstrated by a T cell adhesion assay. CD40L-positive T cells induced platelet activation through a contact-mediated, CD40-dependent pathway resulting in RANTES release, which bound to endothelial cells and mediated T cell recruitment. Soluble CD40L induced the same events via p38, but not extracellular signal-regulated kinase, phosphorylation. These results show the existence of a novel platelet-dependent pathway of immune response amplification which brings these nonimmune cells close to the level of pathogenic relevance traditionally attributed to classical immune cells.     

Bioefficacy of a novel calcium-potassium salt of (-)-hydroxycitric acid

Obesity is associated with cardiovascular disease, diabetes and certain forms of cancer. Popular strategies on weight loss often fail to address many key factors such as fat mass, muscle density, bone density, water mass, their inter-relationships and impact on energy production, body composition, and overall health and well-being. (−)-Hydroxycitric acid (HCA), a natural plant extract from the dried fruit rind of Garcinia cambogia, has been reported to promote body fat loss in humans without stimulating the central nervous system. The level of effectiveness of G. cambogia extract is typically attributed solely to HCA. However, other components by their presence or absence may significantly contribute to its therapeutic effectiveness. Typically, HCA used in dietary weight loss supplement is bound to calcium, which results in a poorly soluble (<50%) and less bioavailable form. Conversely, the structural characteristics of a novel Ca²⁺/K⁺ bound (−)-HCA salt (HCA-SX or Super CitriMax) make it completely water soluble as well as bioavailable. An efficacious dosage of HCA-SX (4500 mg/day t.i.d.) provides a good source of Ca²⁺ (495 mg, 49.5% of RDI) and K⁺ (720 mg, 15% of RDI). Ca²⁺ ions are involved in weight management by increasing lipid metabolism, enhancing thermogenesis, and increasing bone density. K⁺, on the other hand, increases energy, reduces hypertension, increases muscle strength and regulates arrhythmias. Both Ca and K act as buffers in pH homeostasis. HCA-SX has been shown to increase serotonin availability, reduce appetite, increase fat oxidation, improve blood lipid levels, reduce body weight, and modulate a number of obesity regulatory genes without affecting the mitochondrial and nuclear proteins required for normal biochemical and physiological functions.     

Furanocoumarins Are a Novel Class of Modulators for the Transient Receptor Potential Vanilloid Type 1 (TRPV1) Channel

Furanocoumarin imperatorin is the major active component of Angelica dahurica root extracts, widely used in traditional medicine to treat headache, toothache, and orbital eye pain. In this study, we investigated the mechanisms that may underlie the pain-relieving effects of the compound. We found that imperatorin significantly inhibited formalin- and capsaicin-induced nocifensive responses but did not alter baseline thermal withdrawal thresholds in the rat. We established that imperatorin is a weak agonist of TRPV1, a channel implicated in detecting several noxious stimuli, exhibiting a 50% effective concentration (EC₅₀) of 12.6 ± 3.2 μm. A specific TRPV1 antagonist, JNJ-17203212 (0.5 μm), potently inhibited imperatorin-induced TRPV1 activation. Site-directed mutagenesis studies revealed that imperatorin most likely acted via a site adjacent to or overlapping with the TRPV1 capsaicin-binding site. TRPV1 recovery from desensitization was delayed in the presence of imperatorin. Conversely, imperatorin sensitized TRPV1 to acid activation but did not affect the current amplitude and/or the activation-inactivation properties of Na_v1.7, a channel important for transmission of nociceptive information. Thus, our data indicate that furanocoumarins represent a novel group of TRPV1 modulators that may become important lead compounds in the drug discovery process aimed at developing new treatments for pain management.