Proton Activation Research Articles

Identification of Extracellular Domain Residues Required for Epithelial Na+ Channel Activation by Acidic pH

A growing body of evidence suggests that the extracellular domain of the epithelial Na(+) channel (ENaC) functions as a sensor that fine tunes channel activity in response to changes in the extracellular environment. We previously found that acidic pH increases the activity of human ENaC, which results from a decrease in Na(+) self-inhibition. In the current work, we identified extracellular domain residues responsible for this regulation. We found that rat ENaC is less sensitive to pH than human ENaC, an effect mediated in part by the γ subunit. We identified a group of seven residues in the extracellular domain of γENaC (Asp-164, Gln-165, Asp-166, Glu-292, Asp-335, His-439, and Glu-455) that, when individually mutated to Ala, decreased proton activation of ENaC. γ(E455) is conserved in βENaC (Glu-446); mutation of this residue to neutral amino acids (Ala, Cys) reduced ENaC stimulation by acidic pH, whereas reintroduction of a negative charge (by MTSES modification of Cys) restored pH regulation. Combination of the seven γENaC mutations with β(E446A) generated a channel that was not activated by acidic pH, but inhibition by alkaline pH was intact. Moreover, these mutations reduced the effect of pH on Na(+) self-inhibition. Together, the data identify eight extracellular domain residues in human β- and γENaC that are required for regulation by acidic pH.

Contribution of metal‐ion complex formation with organic substances to the acidity of water extracts of polluted soils

The formation of complexes of copper ions with organic substances in soil water extracts and the development of acid–base properties of the extracts were examined. Potentiometric titration of water extracts from several soils with copper nitrate solution with a consecutive determination of the activity of protons and copper ions in the extracts was performed. This demonstrated the existence of a functional relationship between the complexing of copper ions with organic substances and the acid–base properties of the water extracts. The parameters of the interrelated processes of formation and dissociation of metal–organic complexes and the protonation‐deprotonation of organic acids, the ligands of which participate in the complexing reactions, were determined. It was found that the formation of the complexes is accompanied by the acidification of the solution from the acid hydrolysis of the copper ions and the displacement of protons from the functional groups of organic acids. The protons released in the course of complexing reactions affected these reactions. Under their impact, the stability of the copper–organic complexes decreased; finally, the formation of the complexes and the release of protons stopped, and the pH of the extracts reached an equilibrium level.

The acyl-CoA binding protein is required for normal epidermal barrier function in mice

The acyl-CoA binding protein (ACBP) is a 10 kDa intracellular protein expressed in all eukaryotic species. Mice with targeted disruption of Acbp (ACBP(-/-) mice) are viable and fertile but present a visible skin and fur phenotype characterized by greasy fur and development of alopecia and scaling with age. Morphology and development of skin and appendages are normal in ACBP(-/-) mice; however, the stratum corneum display altered biophysical properties with reduced proton activity and decreased water content. Mass spectrometry analyses of lipids from epidermis and stratum corneum of ACBP(+/+) and ACBP(-/-) mice showed very similar composition, except for a significant and specific decrease in the very long chain free fatty acids (VLC-FFA) in stratum corneum of ACBP(-/-) mice. This finding indicates that ACBP is critically involved in the processes that lead to production of stratum corneum VLC-FFAs via complex phospholipids in the lamellar bodies. Importantly, we show that ACBP(-/-) mice display a ∼50% increased transepidermal water loss compared with ACBP(+/+) mice. Furthermore, skin and fur sebum monoalkyl diacylglycerol (MADAG) levels are significantly increased, suggesting that ACBP limits MADAG synthesis in sebaceous glands. In summary, our study shows that ACBP is required for production of VLC-FFA for stratum corneum and for maintaining normal epidermal barrier function.

Proton activity of Nafion 117 membrane measured from potential difference of hydrogen electrodes

The proton activity of the Nafion membrane was estimated from the potential difference between a normal hydrogen electrode (NHE) in 0.5 mol dm−3 H2SO4 and a dynamic hydrogen electrode (DHE) constructed on a Nafion 117 membrane. The potential difference between the DHE and the NHE was directly measured at the same temperature in a box chamber filled with N2 gas. As a result, the potential difference of ENHE − EDHE changes from 0.006 to −0.024 V with the increasing temperature from 289 to 313 K; however, the potential difference remains at around −0.024 V when the temperature is between 313 and 343 K. Based on these data in combination with the Nernst equation, the proton activity of the Nafion 117 membrane at 289 K is estimated to be 0.8, which increases up to ca. 2.4 with the increasing temperature.

Charge transfer dye-based PHEMA hydrogel sensor: its fluorescence responses to pH, metal ion, and humidity

Poly(2-hydroxyethylmethacrylate) (PHEMA) is one of the most well known synthetic hydrogels. In this study, PHEMA gel containing a charge transfer dye was synthesized using a copolymerization method. This polymer gel swelled appropriately in water with a swelling ratio (Q) of approximately 1.0, and swelled significantly in aprotic polar solvents, such as DMF and DMSO, with Q values of >8.0, whereas it never swelled in nonpolar solvents. The fluorescence (FL) emission band of the hydrogel polymer showed a red shift exclusively in water, whereas it showed a blue shift in the other solvents tested. The emission band also shifted to shorter wavelengths with increasing proton activity (with decrease in pH) or iron cation concentration, and the FL intensity increased gradually. The FL intensity also decreased significantly with increasing absolute humidity. A charge transfer dye-based PHEMA gel was synthesized using a copolymerization method. The FL emission band of the hydrogel polymer showed a red shift exclusively in water. The emission band shifted to shorter wavelengths with increasing in proton activity or iron cation concentration and the FL intensity gradually increased. The FL intensity deceased significantly with increasing absolute humidity.

Intracellular Proton-mediated Activation of TRPV3 Channels Accounts for the Exfoliation Effect of α-Hydroxyl Acids on Keratinocytes

α-Hydroxyl acids (AHAs) from natural sources act as proton donors and topical compounds that penetrate skin and are well known in the cosmetic industry for their use in chemical peels and improvement of the skin. However, little is known about how AHAs cause exfoliation to expose fresh skin cells. Here we report that the transient receptor potential vanilloid 3 (TRPV3) channel in keratinocytes is potently activated by intracellular acidification induced by glycolic acid. Patch clamp recordings and cell death assay of both human keratinocyte HaCaT cells and TRPV3-expressing HEK-293 cells confirmed that intracellular acidification led to direct activation of TRPV3 and promoted cell death. Site-directed mutagenesis revealed that an N-terminal histidine residue, His-426, known to be involved in 2-aminoethyl diphenylborinate-mediated TRPV3 activation, is critical for sensing intracellular proton levels. Taken together, our findings suggest that intracellular protons can strongly activate TRPV3, and TRPV3-mediated proton sensing and cell death in keratinocytes may serve as a molecular basis for the cosmetic use of AHAs and their therapeutic potential in acidic pH-related skin disorders.

Steady-state dissolution kinetics of mineral ferric phosphate in the presence of desferrioxamine-B and oxalate ligands at pH = 4–6 and T = 24 ± 0.6 °C

Ferric phosphate (FePO4·2H2O) is one of the most common secondary phosphate minerals in the environment. Nevertheless, few studies address the biological dissolution mechanism(s) of FePO4·2H2O. This paper reports steady-state dissolution rates of synthetic FePO4·2H2O at 4≤pH0≤6 by desferrioxamine-B (DFO-B) and oxalate (Ox) ligands. The composition of the influent solution was 10mM NaClO4, 5mM MES buffer. The influent solution was adjusted to 4≤pH0≤6 by adding aliquots of HNO3 or NaOH stock solution. The initial concentrations of DFO-B and Ox, [DFO-B]0 and [Ox]0, ranged from 0 to 135μM, and 0 to 345μM. Geochemical thermodynamic equilibrium modeling was conducted using MINEQL+ (Schecher and McAvoy, 1998). Speciation calculations were based on thermodynamic formation constants at 298.17K, K298 (infinite dilution reference state). Ligand-promoted dissolution rates were determined after steady-state values. Iron concentrations in the effluent solution were quantified (t>500h). Typical effluent-flow rate was maintained at 0.10±0.01mLmin−1. The measured dissolution rate of FePO4·2H2O by DFO-B and Ox, RDFO–OxObs, was compared to the sum of dissolution rates by DFO-B (RDFO-B) or Ox (ROx), RDFO–OxSum (RDFO–OxSum=RDFO‐B+ROx). Results were analyzed using the t student test. Obtained data values with p≤0.05 (⁎) and ≤0.01 (⁎⁎) were considered to differ statistically from control experiments. Dissolution rates by DFO-B (RDFO-B) increased with [DFOB]0, and no evidence of surface masking became apparent. By contrast, dissolution rates by Ox (ROx) varied with [Ox]0 and pH0. The kinetics of dissolution by Ox was not explained by a first-order mineral dissolution behavior. Dissolution rates by DFO-B and Ox (RDFO–OxObs) surpassed RDFO-B or ROx, and increased with proton activity. Reacting FePO4·2H2O with DFO-B and high amounts of Ox resulted in higher values for RDFO–OxObs relative to RDFO-B. Observed (RDFO–OxObs) to calculated (RDFO–OxSum=RDFO‐B+ROx) ratio was found to be highest at [DFOB]0=50μM and [Ox]0=49μM. Increases in the proton activity favors the dissolution of FePO4·2H2O by DFO-B and Ox, explained because the sequestration of Fe(III) at the surface vicinity in the form of adsorbed Fe(III)-oxalate complexes. A direct comparison between the dissolution behavior of FePO4·2H2O by DFO-B and Ox against those for goethite (α-FeOOH) and Al goethite (AlFeOOH) was conducted. The dissolution behavior was found to be a function of the mineral structure. RDFO-B values for FePO4·2H2O by 22.5μM DFO-B surpassed those for α-FeOOH or α-AlFeOOH by 20μM DFO-B, namely, 37, and 11.6 and 3–5μmolkg−1h−1, respectively. ROx values for FePO4·2H2O by 49mM Ox surpassed that for α-FeOOH by 70μM Ox or α-AlFeOOH by 50μM Ox, namely, i.e., 12, and 0.7 and 0.1μmolkg−1h−1. The latter results agree with the idea of the inhibition of Fe release in goethite because its sequestration in the form of adsorbed Fe(III) oxalate complexes. In contrast, a different scenario holds true for dissolution by 50μM DFO-B and 49μM Ox. The dissolution rates for FePO4·2H2O, α-FeOOH, and α-AlFeOOH correspond to 50, and 39–42 and 71–129μmolkg−1h−1, respectively. The high extent of iron release from Al goethite is best explained because high-energy surface sites formed after Al substitution in goethite.

Synthesis and optimization of proton exchange membranes by a pulsed plasma enhanced chemical vapor deposition technique

A pulsed plasma enhanced chemical vapor deposition technique, which involves alternative plasma “on” and plasma “off”, has been used to prepare sulfonated proton exchange membranes. Using such a plasma discharge technique, the degradation of monomer molecules can be well controlled, and the obtained membranes are found to be highly sulfonated. Compared with the commercial Nafion® 117, these plasma polymerized membranes contain higher contents of sulfonic acid groups and exhibit higher water uptake and IECs, comparable proton conductivity and activation energies for proton conduction, and low methanol permeability, which provide them with great potentials as PEMs for uses in direct alcohol fuel cells. In addition, the properties of the fabricated membranes are strongly dependent upon the conditions used for the membrane fabrication, such as the duty cycle and the partial pressure of monomers. For synthesis of membranes with desirable properties, the conditions used for the membrane fabrication should be well selected.

Backbone Modified Small Bite-Angle Diphosphines: Synthesis, Structure and Regioselective Thermal Rearrangements of Os3(CO)10{μ-Ph2PCH(Me)PPh2}

Addition of 1,1-bis(diphenylphosphino)ethane, Ph2PCH(Me)PPh2, to Os3(CO)12 in benzene at 60 °C in the presence of Me3NO affords Os3(CO)10{μ-Ph2PCH(Me)PPh2} (1) in almost quantitative yield. Thermolysis of 1 in boiling toluene gives the expected electronically-unsaturated 46-electron triosmium cluster Os3(CO)8{μ3-Ph2PCH(Me)P(Ph)C6H4}(μ-H) (2) as the major product formed from the regioselective activation of a phenyl ring lying anti to the methyl group in 1. A second minor product is Os3(CO)8{μ3-Ph2PC(Me)P(Ph)C6H4}(μ-H)2 (3) resulting from the activation of methine and phenyl protons. This complex contains an unusual semi-bridging tertiary phosphine ligand characterized by short [2.398(1) A] and long [2.872(1) A] osmium–phosphorus interactions which structurally resembles an intermediate during phosphine migration across a metal–metal edge. The extent of the bonding in this potential three-center, two-electron interaction was examined by electronic structure calculations. The Wiberg bond index for the short Os(1)–P(1) bond is as expected but the long Os(3)–P(1) interaction is nearly an order of magnitude smaller, which suggests that there is no significant interaction between the Os(3) and P(1) centers. Prolonged heating of 2 in boiling toluene leads to the formation of Os3(CO)7(η6-C6H5CH3){μ3-PhPCH(Me)P(Ph)C6H4} (4), while in octane Os3(CO)9{μ3-PhPCH(Me)P(Ph)C6H4} (5) is generated. Both contain the 6-electron μ3-PhPCH(Me)P(Ph)C6H4 ligand formed as a result of carbon–phosphorus bond cleavage, while in 4 toluene has displaced three carbonyls. The molecular structures of 1–4 have been determined by single crystal X-ray diffraction analysis.

Cyclic voltammetry of a nickel hydroxide electrode with a homogeneous two-phase structure

A mathematical model is suggested for charge-discharge processes taking place in an NiOOH/Ni(OH)2 electrode in conditions of cyclic voltammetry with a linearly scanned potential. The object of the analysis is a flat active material crystal of thickness h that models a particle 2h in size. The physicochemical properties of the NiOOH/Ni(OH)2 system are evaluated on the basis of notions of a homogeneous two-phase structure of the material with a variable phase concentration ratio. An equation is suggested for the relationship between the local proton activity coefficient in the crystal lattice and the state of the charge. The proton activity coefficient appears in the equilibrium potential, the charge-discharge kinetics, and the proton diffusion coefficient equations. The concept of a barrier layer governing the equilibrium potential’s hysteresis is introduced and consideration is given to the influence of the ohmic resistance of the solid phase and the effect of photon migration under the action of an electric field.

Does Activation of the Anti Proton, Rather than Concertedness, Determine the Stereochemistry of Base-Catalyzed 1,2-Elimination Reactions? Anti Stereospecificity in E1cB Eliminations of β-3-Trifluoromethylphenoxy Esters, Thioesters, and Ketones

As part of a comprehensive investigation on the stereochemical aspects of base-catalyzed 1,2-elimination reactions, we have studied a set of acyclic carbonyl substrates that react by an irreversible E1cB mechanism with largely anti stereospecificity. (2)H NMR data show that these reactions using KOH in EtOH/H(2)O under non-ion-pairing conditions produce a minimum of 85-89% anti elimination on stereospecifically labeled tert-butyl (2R*,3R*)- and (2R*,3S*)-3-(3-trifluoromethylphenoxy)-2,3-(2)H(2)-butanoate, S-tert-butyl (2R*,3R*)- and (2R*,3S*)-3-(3-trifluoromethylphenoxy)-2,3-(2)H(2)-butanethioate, and the related ketones, (4R*,5R*)- and (4R*,5S*)-5-(3-trifluoromethylphenoxy)-4,5-(2)H(2)-3-hexanone. With both diastereomers of each substrate available, the KIEs can be calculated and the innate stereoselectivities determined. The elimination reactions of the β-3-trifluoromethylphenoxy substrates occur by E1cB mechanisms with diffusionally equilibrated enolate-anion intermediates. Thus, it is clear that anti elimination does not depend solely upon concerted E2 mechanisms. Negative hyperconjugation provides a satisfactory explanation for the anti stereospecificity exhibited by our carbonyl substrates, where the leaving group activates the anti proton, leading to the enolate intermediate. The activation of the anti proton by negative hyperconjugation may also play a role in the concerted pathways of E2 mechanisms. We have also measured the rates of the hydroxide-catalyzed elimination reactions of butanoate, thiobutanoate, and ketone substrates in EtOH/H(2)O, with β-tosyloxy, acetoxy, and 3-trifluoromethylphenoxy nucleofuges.

Intramembrane Proton Binding Site Linked to Activation of Bacterial Pentameric Ion Channel

Prokaryotic orthologs of eukaryotic Cys-loop receptor channels recently emerged as structural and mechanistic surrogates to investigate this superfamily of intercellular signaling proteins. Here, we examine proton activation of the prokaryotic ortholog GLIC using patch clamp electrophysiology, mutagenesis, and molecular dynamics (MD) simulations. Whole-cell current recordings from human embryonic kidney (HEK) 293 cells expressing GLIC show half-maximal activation at pH 6, close to the pK(a) of histidine, implicating the three native His residues in proton sensing linked to activation. The mutation H235F abolishes proton activation, H277Y is without effect, and all nine mutations of His-127 prevent expression on the cell surface. In the GLIC crystal structure, His-235 on transmembrane (TM) α-helix 2, hydrogen bonds to the main chain carbonyl oxygen of Ile-259 on TM α-helix 3. MD simulations show that when His-235 is protonated, the hydrogen bond persists, and the channel remains in the open conformation, whereas when His-235 is deprotonated, the hydrogen bond dissociates, and the channel closes. Mutations of the proximal Tyr-263, which also links TM α-helices 2 and 3 via a hydrogen bond, alter proton sensitivity over a 1.5 pH unit range. MD simulations show that mutations of Tyr-263 alter the hydrogen bonding capacity of His-235. The overall findings show that His-235 in the TM region of GLIC is a novel proton binding site linked to channel activation.

Biogenic hydroxyapatite (Apatite II™) dissolution kinetics and metal removal from acid mine drainage

Apatite II™ is a biogenic hydroxyapatite (expressed as Ca5(PO4)OH) derived from fish bone. Using grains of Apatite II™ with a fraction size between 250 and 500μm, batch and flow-through experiments were carried out to (1) determine the solubility constant for the dissolution reactionCa5(PO4)3(OH)⇔5Ca2++3PO43−+OH−,(2) obtain steady-state dissolution rates over the pH range between 2.22 and 7.14, and (3) study the Apatite II™’s mechanisms to remove Pb2+, Zn2+, Mn2+, and Cu2+ from metal polluted water as it dissolves.The logKS value obtained was −50.8±0.82 at 25°C. Far-from-equilibrium fish-bone hydroxyapatite dissolution rates decrease by increasing pH. Assuming that the dissolution reaction is controlled by fast adsorption of a proton on a specific surface site that dominates through the pH range studied, probably PO−, followed by a slow hydrolysis step, the dissolution rate dependence is expressed in molm−2s−1 asRate25 °C=−8.9×10−10×9.96×105×aH+1+9.96×105×aH+where aH+ is the proton activity in solution.Removal of Pb2+, Zn2+, Mn2+ and Cu2+ was by formation of phosphate-metal compounds on the Apatite II™ substrate, whereas removal of Cd2+ was by surface adsorption. Increase in pH enhanced the removal of aqueous heavy metals. Using the kinetic parameters obtained (e.g., dissolution rate and pH-rate dependence law), reactive transport simulations reproduced the experimental variation of pH and concentrations of Ca, P and toxic divalent metal in a column experiment filled with Apatite II™ that was designed to simulate the Apatite II™–metal polluted water interaction.

Direct participation of counter anion in acid hydrolysis of glycoside

The mechanism of acid hydrolysis of glycoside has been investigated since the end of the 19th century accompanied by lots of literatures published on the mechanism, although little attention has surprisingly been paid to the action of counter anion of acid. In this paper, it was investigated whether or not counter anion of acid directly participates in acid hydrolysis of glycosides, methyl α- and β-D-glucopyranosides (MGP) in water, aqueous 74%, and 82% 1,4-dioxane systems. Because proton activity of a reaction system is the important rate-determining parameter in the universally acknowledged mechanism, it was carefully estimated in this study. The results suggested that bromide anion directly participates in the acid hydrolysis reaction of MGP in a water solvent system and the participation of bromide anion is further pronounced in aqueous 74% and 82% 1,4-dioxane solvent systems. It was also suggested that chloride anion directly participates in these dioxane solvent systems.

Predicting effects of cations on copper toxicity to lettuce (Lactuca sativa) by the biotic ligand model.

A biotic ligand model (BLM) was developed to estimate Cu toxicity to lettuce (Lactuca sativa) in terms of root elongation after 4 d of exposure. Effects of Na(+), K(+), Ca(2+), and Mg(2+) on Cu toxicity were examined. The addition of these cations resulted in a 50-fold difference in the copper median effective activity (EC50 cu2+). However, these variations could not be interpreted entirely as a function of the concentrations of these cations alone. In particular, only the relationship between EC50 cu2+ and the activity of protons was found to be significant in the whole range of pH examined from 5.0 to 7.0. The addition of K(+), Na(+), Ca(2+), and Mg(2+) at concentrations up to 20 mM resulted in a 16-fold difference in EC50 cu2+ values. This difference was significant, as indicated by non-overlapping standard deviations of the negative logarithm of EC50 cu2+ pEC50 cu2+) obtained with (7.37 ± 0.22) and without (6.76 ± 0.22) additions of K(+), Na(+), Ca(2+), and Mg(2+). The variations were not statistically significantly related to concentrations of these cations; therefore, only protons can be integrated in the BLM predicting Cu toxicity to lettuce L. sativa with the important parameters: log K(HBL) =6.27, log K(CuBL) =7.40, and [formula in text]. The lack of significant relationships between EC50 cu2+ and concentrations of the cations was not in line with the main assumption of the BLM about the competition between cations for binding sites.

Investigations of 89Y(p,x) 86,88,89gZr, 86m+g,87g,87m,88gY, 85gSr, and 84gRb nuclear processes up to 42 MeV

Production cross-sections of the 89Y(p,x) 86,88,89gZr, 86m+g,87g,87m,88gY, 85gSr, and 84gRb nuclear processes were investigated up to 42-MeV proton energy by using a stacked-foil activation technique at the MC-50 cyclotron of the Korea Institute of Radiological and Medical Sciences. Cumulative cross-sections of the 88gY radionuclide have been reported here for the first time from proton activation on natural yttrium target. The present data were compared with the available experimental data as well as theoretical calculations based on the TALYS and the ALICE-IPPE codes, and found in general good agreement among them. Due to the mono-isotopic characteristics of the natural yttrium, the investigated 89Y(p,x) 86,88,89gZr, 86m+g,87g,87m,88gY, 85gSr, and 84gRb processes are suitable for testing of nuclear reaction theories and/or mechanism. A quantitative analysis has also done to understand more accurately the predictive power of the model codes by performing calculations of deviation factors between the measured data and respective model calculations. The thick target integral yields, i.e., induced radioactivity per unit fluence of 42-MeV protons were also deduced from the measured cross-sections of the investigated radionuclides. Optimal production pathway for the 89Zr-PET radionuclide using a cyclotron is discussed elaborately. The measured cross-sections of 87gY, 88gY, 88Zr, and 89gZr radionuclides find importance due to their suitable decay characteristics leading to medical and thin layer activation applications. In addition, the present experimental results will play an important role in enrichment of the literature data base for proton-induced reactions on natural yttrium leading to various applications.

Transient receptor potential vanilloid-1 participates in the inhibitory effect of ginsenoside Rg1 on capsaicin-induced interleukin-8 and prostaglandin E2 production in HaCaT cells

Ginsenoside Rg1 (GRg1), one of the major active constituents of Panax notoginseng, has shown anti-inflammatory and antinocioceptic activity, but its role in keratinocytes needs further study. We have examined the inhibitory effect of GRg1 on transient receptor potential vanilloid-1 (TRPV1) activation in keratinocyte HaCaT cells and explored its involved mechanism. HEK 293T cells over-expressing exogenous TRPV1 were constructed and named HEK 293T-TRPV1 cells. The effects of GRg1 on production of interleukin-8 (IL-8) and prostaglandin E(2) (PGE(2) ), calcium influx, the expression of cyclooxygenase-2 (COX-2) and nuclear factor-κB (NF-κB) transcriptional activity in HEK 293T-TRPV1 and HaCaT cells were examined by ELISA, Fluo 3-AM fluorescence probe, Western blot and Dual-Luciferase Reporter Assay, respectively. The results showed that GRg1 blocked intracellular calcium by both capsaicin and proton activation in a TRPV1-dependent manner. Furthermore, GRg1 inhibited the expression of COX-2 and NF-κB transcriptional activity induced by capsaicin in keratinocytes. The inhibitory effect of GRg1 was similar to capsazepine, an antagonist of TRPV1. More importantly, GRg1 dose-dependently inhibited capsaicin-induced PGE(2) and IL-8 secretion in HaCaT cells and HEK 293T-TRPV1 cells. These data showed that GRg1 could inhibit TRPV1 mediated responses in HaCaT cells, indicating that GRg1 acted as a TRPV1 antagonist.

The Chimeric Approach Reveals That Differences in the TRPV1 Pore Domain Determine Species-specific Sensitivity to Block of Heat Activation

The capsaicin-, heat-, and proton-activated ion channel TRPV1, a member of the transient receptor potential cation channel family is a polymodal nociceptor. For almost a decade, TRPV1 has been explored by the pharmaceutical industry as a potential target for example for pain conditions. Antagonists which block TRPV1 activation by capsaicin, heat, and protons were developed by a number of pharmaceutical companies. The unexpected finding of hyperthermia as an on-target side effect in clinical studies using polymodal TRPV1 antagonists has prompted companies to search for ways to circumvent hyperthermia, for example by the development of modality-selective antagonists. The significant lack of consistency of the pharmacology of many TRPV1 antagonists across different species has been a further obstacle. JYL-1421 for example was shown to block capsaicin and heat responses in human and monkey TRPV1 while it was largely ineffective in blocking heat responses in rat TRPV1. These findings suggested structural dissimilarities between different TRPV1 species relevant for small compound antagonism for example of heat activation. Using a chimeric approach (human and rat TRPV1) in combination with a novel FLIPR-based heat activation assay and patch-clamp electrophysiology we have identified the pore region as being strongly linked to the observed species differences. We demonstrate that by exchanging the pore domains JYL-1421, which is modality-selective in rat can be made modality-selective in human TRPV1 and vice-versa.

Multiparametric Dissolution of Thorium–Cerium Dioxide Solid Solutions

The dissolution of Th(1-x)Ce(x)O(2) solid solutions samples prepared by thermal conversion of oxalate precursors was studied by varying independently several parameters (such as chemical composition, leachate acidity, leaching temperature, firing temperature, and crystallization state). The relative effects of these parameters on the normalized dissolution rate were examined. Either the obtained partial order related to the proton activity (n = 0.50 ± 0.01) or the activation energy (E(A) = 57 ± 6 kJ.mol(-1)) suggested that the dissolution was mainly driven by surface reactions occurring at the solid/liquid interface. Conversely to that observed for Th(1-x)U(x)O(2) and Ce(1-x)Nd(x)O(2-x/2) solid solutions, the chemical composition did not induce strong modifications of the chemical durability of the leached samples. While the initial normalized dissolution rate slightly depended on the elimination of crystal defects for firing temperatures below 800 °C, it was mainly independent of the crystallite size (T ≥ 900 °C). The role of crystal defects (then that of preparation conditions) appeared thus important to consider since they contributed to modifications of the normalized dissolution rates of the same order of magnitude than that of the leachate acidity.

Small Molecule Positive Allosteric Modulation of TRPV1 Activation by Vanilloids and Acidic pH

Transient receptor potential cation channel subfamily V member 1 (TRPV1) is a high-conductance, nonselective cation channel strongly expressed in nociceptive primary afferent neurons of the peripheral nervous system and functions as a multimodal nociceptor gated by temperatures greater than 43°C, protons, and small-molecule vanilloid ligands such as capsaicin. The ability to respond to heat, low pH, vanilloids, and endovanilloids and altered sensitivity and expression in experimental inflammatory and neuropathic pain models made TRPV1 a major target for the development of novel, nonopioid analgesics and resulted in the discovery of potent antagonists. In human clinical trials, observations of hyperthermia and the potential for thermal damage by suppressing the ability to sense noxious heat suggested that full-scale blockade of TRPV1 function can be counterproductive and subtler pharmacological approaches are necessary. Here we show that the dihydropyridine derivative 4,5-diethyl-3-(2-methoxyethylthio)-2-methyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS1477) behaves as a positive allosteric modulator of both proton and vanilloid activation of TRPV1. Under inflammatory-mimetic conditions of low pH (6.0) and protein kinase C phosphorylation, addition of MRS1477 further increased sensitivity of already sensitized TPRV1 toward capsaicin. MRS1477 does not affect inhibition by capsazepine or ruthenium red and remains effective in potentiating activation by pH in the presence of an orthosteric vanilloid antagonist. These results indicate a distinct site on TRPV1 for positive allosteric modulation that may bind endogenous compounds or novel pharmacological agents. Positive modulation of TRPV1 sensitivity suggests that it may be possible to produce a selective analgesia through calcium overload restricted to highly active nociceptive nerve endings at sites of tissue damage and inflammation.