Expression of a novel versican variant in dorsal root ganglia from spared nerve injury rats.

The size and modular structure of versican and its gene suggest the existence of multiple splice variants. We have identified, cloned, and sequenced a previously unknown exon located within the noncoding gene sequence downstream of exon 8. This exon, which we have named exon 8β, specifies two stop-codons.

Neuronally produced versican V2 renders C-fiber nociceptors IB4 -positive.

A subpopulation of nociceptors, the glial cell line-derived neurotrophic factor (GDNF)-dependent, non-peptidergic C-fibers, expresses a cell-surface glycoconjugate that can be selectively labeled with isolectin B4 (IB4 ), a homotetrameric plant lectin from Griffonia simplicifolia. We show that versican is an IB4 -binding molecule in rat dorsal root ganglion neurons. Using reverse transcriptase polymerase chain reaction (RT-PCR), in situ hybridization and immunofluorescence experiments on rat lumbar dorsal root ganglion, we provide the first demonstration that versican is produced by neurons.

Light-triggered conversion of non-ionic into ionic surfactants: towards chameleon detergents for 2-D gel electrophoresis.

Caged non-ionic detergents, comprised of polar oligo(ethylene glycol) and non-polar alkyl chains joined by a photocleavable ortho-nitrobenzyl sulfonate linker have been synthesized and characterized. The light-triggered transformation of such chameleon surfactant from a charge-neutral into a charged form offers great potential to improve 2-D gel electrophoretic separation of complex protein mixtures.

Loop 3 of short neurotoxin II is an additional interaction site with membrane-bound nicotinic acetylcholine receptor as detected by solid-state NMR spectroscopy.

The contact area of neurotoxin II from Naja naja oxiana when interacting with the membrane-bound nicotinic acetylcholine receptor from Torpedo californica was determined by solid-state, magic-angle spinning NMR spectroscopy. For this purpose, the carbon signals for more than 90% of the residues of the bound neurotoxin were assigned. Differences between the solution and solid-state chemical shifts of the free and bound form of the toxin are confined to distinct surface regions.

Nicotinic acetylcholine receptors at atomic resolution.

While the structures of several G protein coupled receptors (GPCRs) have been solved by X-ray crystallography, the high-resolution structure of first ligand-gated ion channels (LGICs) has been available only recently. The ultrastructure of the prototypic LGIC nAChR is deduced from electron microscopy in combination with crystal structures of model proteins. From this analysis, agonist and antagonist binding sites and hints at the mechanism of channel gating become visible.

Identification and characterisation of novel tubulin-binding motifs located within the C-terminus of TRPV1.

Previously, we reported that TRPV1, the vanilloid receptor, interacts with soluble alphabeta-tubulin dimers as well as microtubules via its C-terminal cytoplasmic domain. The interacting region of TRPV1, however, has not been defined. We found that the TRPV1 C-terminus preferably interacts with beta-tubulin and less with alpha-tubulin.

TRPV1 at nerve endings regulates growth cone morphology and movement through cytoskeleton reorganization.

While the importance of Ca(2+) channel activity in axonal path finding is established, the underlying mechanisms are not clear. Here, we show that transient receptor potential vanilloid receptor 1 (TRPV1), a member of the TRP superfamily of nonspecific ion channels, is physically and functionally present at dynamic neuronal extensions, including growth cones. These nonselective cation channels sense exogenous ligands, such as resenifera toxin, and endogenous ligands, such as N-arachidonoyl-dopamine (NADA), and affect the integrity of microtubule cytoskeleton.

Intracellular domain of nicotinic acetylcholine receptor: the importance of being unfolded.

Bioinformatics methods with subsequent verification by experimental data were applied to the structural investigation of the intracellular loop of the delta-subunit of the nicotinic acetylcholine receptor (nAChR). Three complementary methods were used: prediction of secondary structure elements, prediction of ordered/disordered protein regions and prediction of short functional binding motifs. The output of five different algorithms was used for the secondary structure construction.

Rapid disassembly of dynamic microtubules upon activation of the capsaicin receptor TRPV1.

The transmission of pain signalling involves the cytoskeleton, but mechanistically this is poorly understood. We recently demonstrated that the capsaicin receptor TRPV1, a non-selective cation channel expressed by nociceptors that is capable of detecting multiple pain-producing stimuli, directly interacts with the tubulin cytoskeleton. We hypothesized that the tubulin cytoskeleton is a downstream effector of TRPV1 activation.

Characterization of rat transient receptor potential vanilloid 1 receptors lacking the N-glycosylation site N604.

Whole-cell patch-clamp recordings were performed on HEK293 cells transiently transfected with the rat (r) wild-type transient receptor potential vanilloid 1 (TRPV1) (rTRPV1) receptor or with a mutant that lacks the potential N-glycosylation site at position N604 (rTRPV1-N604T). Replacement of Asn by Thr (N604T) depressed the maximum of the concentration-response curve for capsaicin and decreased the EC50 value of this agonist. Further, such a manipulation modified the sensitivity to the TRPV1 receptor-antagonist capsazepine and altered the dependence of the capsaicin effect on extracellular pH.

Analysis of the dorsal spinal cord synaptic architecture by combined proteome analysis and in situ hybridization.

The proteomic analysis of tissue samples is an analytical challenge, because identified gene products not only have to be assigned to subcellular structures, but also to cell subpopulations. We here report a strategy of combined subcellular proteomic profiling and in situ hybridization to assign proteins to subcellular sites in subsets of cells within the dorsal region of rat spinal cord. With a focus on synaptic membranes, which represent a complex membrane protein structure composed of multiple integral membrane proteins and networks of accessory structural proteins, we also compared different two-dimensional gel electrophoresis systems for the separation of the proteins.

Identification of versican as an isolectin B4-binding glycoprotein from mammalian spinal cord tissue.

Nociceptors are specialized nerve fibers that transmit noxious pain stimuli to the dorsal horn of the spinal cord. A subset of nociceptors, the nonpeptidergic C-fibers, is characterized by its reactivity for the plant isolectin B4 (IB4) from Griffonia simplicifolia. The molecular nature of the IB4-reactive glycoconjugate, although used as a neuroanatomical marker for more than a decade, has remained unknown.

Identification and characterization of a Ca2+ -sensitive interaction of the vanilloid receptor TRPV1 with tubulin.

The vanilloid receptor TRPV1 plays a well-established functional role in the detection of a range of chemical and thermal noxious stimuli, such as those associated with tissue inflammation and the resulting pain. TRPV1 activation results in membrane depolarization, but may also trigger intracellular Ca2+ -signalling events. In a proteomic screen for proteins associated with the C-terminal sequence of TRPV1, we identified beta-tubulin as a specific TRPV1-interacting protein.

Intracellular domains of the delta-subunits of Torpedo and rat acetylcholine receptors--expression, purification, and characterization.

There are quite detailed structural data on the extracellular ligand-binding domain and the intramembrane channel-forming domain of the nicotinic acetylcholine receptors (nAChR). However, the structure of the intracellular domain, which has variable amino acid sequences in different nAChR subunits, remains unknown. We expressed in Escherichia coli the intracellular loops (between transmembrane fragments TM3 and TM4) of the delta-subunits from the Torpedo californica and Rattus norvegicus muscle nAChRs.

Towards structure determination of neurotoxin II bound to nicotinic acetylcholine receptor: a solid-state NMR approach.

Solid-state magic-angle spinning nuclear magnetic resonance (NMR) has sufficient resolving power for full assignment of resonances and structure determination of immobilised biological samples as was recently shown for a small microcrystalline protein. In this work, we show that highly resolved spectra may be obtained from a system composed of a receptor-toxin complex. The NMR sample used for our studies consists of a membrane preparation of the nicotinic acetylcholine receptor from the electric organ of Torpedo californica which was incubated with uniformly 13C-,15N-labelled neurotoxin II.

Snake and snail toxins acting on nicotinic acetylcholine receptors: fundamental aspects and medical applications.

This review covers recent data on interactions of nicotinic acetylcholine receptors (AChR) with snake venom proteins (alpha- and kappa-neurotoxins, 'weak' toxins recently shown to act on AChRs), as well as with peptide alpha-conotoxins from Conus snails. Mutations of AChRs and toxins, X-ray/nuclear magnetic resonance structures of alpha-neurotoxin bound to AChR fragments, and the X-ray structure of the acetylcholine-binding protein were used by several groups to build models for the alpha-neurotoxin-AChR complexes. Application of snake toxins and alpha-conotoxins for pharmacological distinction of muscle, neuronal and neuronal-like AChR subtypes and for other medical purposes is briefly discussed.

Dual expression of mouse and rat VRL-1 in the dorsal root ganglion derived cell line F-11 and biochemical analysis of VRL-1 after heterologous expression.

The vanilloid-like TRP-channel VRL-1 (TRPV2) is a nonselective cation channel expressed by primary sensory neurons and non-neuronal tissues [Caterina, M.J., Rosen, T.

Identification of PSF as a protein kinase Calpha-binding protein in the cell nucleus.

Protein kinase C (PKC) isoforms are present in the cell nucleus in diverse cell lines and tissues. Since little is known about proteins interacting with PKC inside the cell nucleus, we used Neuro-2a neuroblastoma cells, in which PKCalpha is present in the nucleus, to screen for nuclear binding partners for PKC. Applying overlay assays, we detected several nuclear proteins which bind to PKCalpha.

Structure-activity relationships of methoctramine-related polyamines as muscular nicotinic receptor noncompetitive antagonists. 3. Effect of inserting the tetraamine backbone into a macrocyclic structure.

The present article expands on the study of another aspect of structure-activity relationships of the polymethylene tetraamines, namely, the effect of inserting the tetraamine backbone into a macrocyclic structure. To this end, compounds 8-12 were designed by linking the two terminal nitrogen atoms of prototype methoctramine 2 to an aryl moiety. Alternatively, 2 was first modified to achieve compounds 6 and 7, which in turn were cyclized by linking the two terminal primary amine functions to a polyphenyl spacer, affording 13-20.

Structure-activity relationships of methoctramine-related polyamines as muscular nicotinic receptor noncompetitive antagonists. 2. Role of polymethylene chain lengths separating amine functions and of substituents on the terminal nitrogen atoms.

Polymethylene tetraamine methoctramine (1) is a prototypical antimuscarinic ligand endowed with a significant affinity for muscular nAChRs. Thus, according to the universal template approach, structural modifications were performed on 1 in order to improve affinity and selectivity for the muscle-type nAChR. The polyamine derivatives synthesized were tested at both frog rectus and Torpedo nAChRs and at guinea pig left atria (M(2)) and ileum longitudinal muscle (M(3)) mAChRs.

Activation by acidic pH of CLC-7 expressed in oocytes from Xenopus laevis.

ClC chloride channels are important in diverse physiological functions such as transepithelial transport, cell volume regulation, excitability, and acidification of intracellular organelles. We have investigated the expression of CLC-7 in oocytes from Xenopus laevis with the two electrode voltage clamp technique and Western blot analysis. Using a specific antibody against CLC-7, we found an approximately 80 kDa protein in oocytes, previously injected with CLC-7-cRNA.

Identification of orcokinins in single neurons in the stomatogastric nervous system of the crayfish, Cherax destructor.

The orcokinins are a highly conserved family of crustacean peptides that enhance hindgut contractions in the crayfish Orconectes limosus (Stangier et al. [1992] Peptides 13:859-864). By combining immunocytochemical and mass spectrometrical analysis of the stomatogastric nervous system (STNS) in the crayfish Cherax destructor, we show that multiple orcokinins are synthesized in single neurons.

Biochemical characterization of the vanilloid receptor 1 expressed in a dorsal root ganglia derived cell line.

The vanilloid receptor VR1 is an ion channel predominantly expressed by primary sensory neurons involved in nociception. Here we describe its biochemical properties and assess the subcellular localization, the glycosylation state and the quaternary structure of VR1 expressed in HEK293 cells and in the DRG-derived cell line F-11 (N18TG2 mouse neuroblastoma x rat dorsal root ganglia, hybridoma). VR1 was found to be glycosylated in both cell types.