Calmodulin Supports TRPA1 Channel Association with Opioid Receptors and Glutamate NMDA Receptors in the Nervous Tissue

Transient receptor potential ankyrin member 1 (TRPA1) belongs to the family of thermo TRP cation channels that detect harmful temperatures, acids and numerous chemical pollutants. TRPA1 is expressed in nervous tissue, where it participates in the genesis of nociceptive signals in response to noxious stimuli and mediates mechanical hyperalgesia and allodynia associated with different neuropathies. The glutamate N-methyl-d-aspartate receptor (NMDAR), which plays a relevant role in allodynia to mechanical stimuli, is connected via histidine triad nucleotide-binding protein 1 (HINT1) and type 1 sigma receptor (σ1R) to mu-opioid receptors (MORs), which mediate the most potent pain relief. Notably, neuropathic pain causes a reduction in MOR antinociceptive efficacy, which can be reversed by blocking spinal NMDARs and TRPA1 channels. Thus, we studied whether TRPA1 channels form complexes with MORs and NMDARs that may be implicated in the aforementioned nociceptive signals. Our data suggest that TRPA1 channels functionally associate with MORs, delta opioid receptors and NMDARs in the dorsal root ganglia, the spinal cord and brain areas. These associations were altered in response to pharmacological interventions and the induction of inflammatory and also neuropathic pain. The MOR-TRPA1 and NMDAR-TRPA1 associations do not require HINT1 or σ1R but appear to be mediated by calcium-activated calmodulin. Thus, TRPA1 channels may associate with NMDARs to promote ascending acute and chronic pain signals and to control MOR antinociception.     

Analysis of heregulin symmetry by weighted evolutionary tracing

Heregulins are members of the protein family of EGF-like growthand differentiation factors. The primary cell-surface targetsof heregulins are heterodimers of the EGF-receptor homolog HER2with either HER3 or HER4. We used a weighted evolutionary traceanalysis to identify structural features that distinguish theEGF-like domain (hrg) of heregulins from other members of theEGF family. In this analysis, each amino acid sequence is weightedaccording to its uniqueness and the variability in each positionis assigned by an amino acid substitution matrix. Conservedresidues in heregulin that are variable in other EGF-like domainsare considered possible specificity-conferring residues. Thisanalysis identifies two clusters of residues at the foot ofthe boot-shaped hrg domain. The residues in one cluster arerecruited from the N-terminus; those in the other are from the-loop region and show a weak sequence similarity to the N-terminalresidues at the opposite side of the boot. The remaining residueswith high conservation scores distribute themselves into thesetwo distinct surfaces on hrg. This pseudo-twofold symmetry andthe presence of two distinct interfaces may reflect the preferenceof hrg for heterodimeric versus homodimeric HER complexes.     

A mixed helix--beta-sheet model of the transmembrane region of the nicotinic acetylcholine receptor

We have modelled the transmembrane region of the 7 nicotinicacetylcholine receptor as a mixed -helical/ß-sheetstructure. The model was mainly based on the crystal structureof a pore-forming toxin, heat-labile enterotoxin. This is apentameric protein having a central pore or channel composedof five -helices, one from each of the 5 B subunits that formthis pentamer. The remainder of this structure is ß-sheet,loops and a short -helix, not included in the model. The modeluses this channel as a template to build the transmembrane region,from M1 to the middle of M3. The remainder of M3 and M4 werebuilt de novo as -helices. Great consideration was given tolabelling data available for the transmembrane region. In generalterms, the shape of the model agrees very well with that obtainedindependently by electron microscopic analysis and the secondarystructure predicted by the model is in accord with that estimatedindependently by Fourier transform infrared spectroscopy. TheM2 helical region of the model is only slightly kinked, contraryto what is inferred from electron microscopic analysis, buthas the same overall shape and form. On the membrane face ofthe model, the presence of deep pockets may provide the structuralbasis for the distinction between annular and non-annular lipidbinding sites. Also, the transmembrane region is clearly asymmetricin the direction perpendicular to the membrane, and this mayhave strong influence on the surrounding lipid composition ofeach leaflet of the cytoplasmic membrane.     

A mutational analysis of receptor binding sites of interleukin-1{beta}:differences in binding of human interleukin-1{beta} muteins to human and mouse receptors

The 3-D crystal structure of interleukin-1ß(IL-1ß)has been used to define its receptor binding surface by mutationalanalysis. The surface of IL-1ß was probed by site-directedmutagenesis. A total of 27 different IL-1ß muteinswere constructed, purified and analyzed. Receptor binding measurementson mouse and human cell lines were performed to identify receptoraffinities. IL-1ß muteins with modified receptor affinitywere evaluated for structural integrity by CD spectroscopy orX-ray crystallography. Changes in six surface loops, as wellas in the C- and N-termini, yielded muteins with lower bindingaffinities. Two muteins with intact binding affinities showed10- to 100-fold reduced biological activity. The surface regioninvolved in receptor binding constitutes a discontinuous areaof 1000 Å2 formed by discontinuous polypeptide chain stretches.Based on these results, a subdivision into two distinct localareas is proposed. Differences in receptor binding affinitiesfor human and mouse receptors have been observed for some muteins,but not for wild-type IL-1ß. This is the first timea difference in binding affinity of IL-1ß muteinsto human and mouse receptors has been demonstrated     

Enantiomeric Composition of Monoterpene Hydrocarbons in Some Conifers and Receptor Neuron Discrimination of α-Pinene and Limonene Enantiomers in the Pine Weevil, Hylobius abietis

The enantiomeric composition of seven monoterpene hydrocarbons in headspace volatiles of spruce sawdust and seedlings (Picea abies), pine seedlings (Pinus sylvestris), and branches of juniper (Juniperus communis) was determined by gas chromatographic separation on a -Cyclodextrin column. For the six monoterpenes, -pinene, camphene, -pinene, sabinene, limonene, and -phellandrene, both enantiomers were present, whereas for 3-carene only the (+)-configuration was found. The amount of each enantiomer varied considerably both in relation to total amount of all of them, and for the six pairs also in relation to the opposite enantiomer. One olfactory receptor neuron in the pine weevil (Hylobius abietis) showed a strong response to -pinene when stimulated with all four headspace materials via a GC equipped with a DB-WAX column. The same neuron was subsequently tested with repeated stimulations via the GC effluent containing the (+)- or (–)-enantiomer. A marked better response to (+)- than to (–)--pinene was elicited. Another olfactory receptor neuron that responded strongly to limonene when stimulated with the spruce volatiles was tested for enantiomers of limonene. This neuron responded more strongly to (–)- than to (+)-limonene, when stimulated alternately with each of the limonene enantiomers. Discrimination between enantiomers by plant olfactory receptor neurons suggests that the enantiomeric ratios of volatile compounds may be important in host location by the pine weevil.     

Highly ion-conductive solid polymer electrolytes based on polyethylene non-woven matrix

Highly ion-conductive solid polymer electrolyte (SPE) based on polyethylene (PE) non-woven matrix is prepared by filling poly(ethylene glycol) (PEG)-based crosslinked electrolyte inside the pores of the non-woven matrix. The PE non-woven matrix not only shows good mechanical strength for SPE to be a free-standing film, but also has very porous structure for high ion conductivity. The ion conductivity of SPE based on PE non-woven matrix can be enhanced by adding sufficient non-volatile plasticizer such as poly(ethylene glycol) dimethyl ether (PEGDME) into ion conduction phase without sacrificing mechanical strength. SPE with 20 wt.% crosslinking agent and 80 wt.% non-volatile plasticizer shows 3.1 × 10⁻⁴ S cm⁻¹ at room temperature (20 °C), to our knowledge, which is the highest level for SPEs. It is also electrochemically stable up to 5.2 V and has high transference number about 0.52 due to the introduction of anion receptor as an additive. The interfacial resistance between Li electrode and SPE is low enough to perform charge/discharge test of unit cell consisting of LiCoO₂/SPE/Li at room temperature. The discharge capacity of the unit cell shows 87% of theoretical value with 86% Coulombic efficiency.     

Behavioral and electrophysiological study of antifeedant mechanisms associated with polyhydroxy alkaloids

Eleven polyhydroxy alkaloids of plant origin were tested for anti-feedant effects against larvae of the lepidopteransSpodoptera littoralis, Spodoptera frugiperda, Heliothis virescens, andHelicoverpa armigera. Data from behavioral and electrophysiological investigations were correlated to reveal information on the mode of action of the antifeedants. The pyrrolidine DMDP was an effective antifeedant for all four species, whereas the piperidines fagomine and XZ-1 and the pyrrolizidine alexine were all ineffective as antifeedants. The activity of the pyrrolidines CYB-3 and DAB-1, the piperidines DNJ, DMJ, and BR1, and the bicyclic octahydroindolizine castanospermine varied among species. The investigation focuses on the structural similarities between some of the alkaloids and some common phagostimulatory sugars and illustrates a neural interaction involving the neurons that are differentially responsive to alkaloids and sugars. InS. littoralis, the neurons responding specifically to the alkaloids DMDP, DAB-1, and castanospermine and to the sugars fructose, sucrose, and glucose are more active when the compounds are applied singly than when an alkaloid and a sugar are applied together. The implications for the occurrence and functioning of different sugar receptor sites are discussed.     

Quantitative mass spectrometry to investigate epidermal growth factor receptor phosphorylation dynamics

Identifying proteins of signaling networks has received much attention, because an array of biological processes are entirely dependent on protein cross-talk and protein-protein interactions. Protein posttranslational modifications (PTM) add an additional layer of complexity, resulting in complex signaling networks. Of particular interest to our working group are the signaling networks of epidermal growth factor (EGF) receptor, a transmembrane receptor tyrosine kinase involved in various cellular processes, including cell proliferation, differentiation, and survival. Ligand binding to the N-terminal residue of the extracellular domain of EGF receptor induces conformational changes, dimerization, and (auto)-phosphorylation of intracellular tyrosine residues. In addition, activated EGF receptor may positively affect survival pathways, and thus determines the pathways for tumor growth and progression. Notably, in many human malignancies exaggerated EGF receptor activities are commonly observed. An understanding of the mechanism that results in aberrant phosphorylation of EGF receptor tyrosine residues and derived signaling cascades is crucial for an understanding of molecular mechanisms in cancer development. Here, we summarize recent labeling methods and discuss the difficulties in quantitative MS-based phosphorylation assays to probe for receptor tyrosine kinase (RTK) activity. We also review recent advances in sample preparation to investigate membrane-bound RTKs, MS-based detection of phosphopeptides, and the diligent use of different quantitative methods for protein labeling.     

Computer simulations of signal transduction mechanism in {alpha}1B-adrenergic and m3-muscarinic receptors

Molecular dynamices simulations of the hamster _1Badrenergicand the rat m3-muscarinic seven-helix bundle receptor modelshave been carried out. The free, agonist-bound and antagonist-boundforms have been considered. Moreover, three mutant forms ofthe m3-muscarinic recep-tor (N507A, N507D and N507S) have alsobeen simulated; among these, the N507S mutant shows a constitutiveactivity. A comparative structural/dynamics analysis has beenperformed to elucidate (i) the perturbations induced by thefunctionally different ligands upon binding to their targetreceptor, (ii) the features of the three single-point mutantswith respect to the receptor wild type and (iii) the propertiesshared by the agonist-boundforms of the _1B-adrenergic receptorand the m3-muscarinic receptor and by the constitutively activemutant N507S. The consistency obtained between the structuralrearrangement of the transmembrane seven-helix bundle modelsconsidered, and the experimental pharmacological efficaciesof the ligands and of the mutants, constitute an important validationof the 3-D models obtained and allow the inference of the mechanismof ligand- or mutation-induced receptor activation at the molecularlevel.