99 research outputs found
Binding of Ala-scanning analogs of ω-conotoxin MVIIC to N- and P/Q-type calcium channels
Abstractω-Conotoxin MVIIC binds to P/Q-type calcium channels with high affinity and N-type channels with low affinity. To reveal the residues essential for subtype selectivity, we synthesized Ala-scanning analogs of MVIIC. Binding assays using rat cerebellar P2 membranes suggested that Thr11, Tyr13 and Lys2 are essential for binding to both N- and P/Q-type channels, whereas Lys4 and Arg22 are important for binding to P/Q-type channels. These results suggest that MVIIC interacts with P/Q-type channels via a large surface, in good agreement with previous observations using chimeric analogs
Interaction of a synaptobrevin (VAMP)-syntaxin complex with presynaptic calcium channels
AbstractNerve terminal protein complexes implicated in exocytosis were examined by immuno-isolation from rat brain synaptosomes. Immunoprecipitation with anti-syntaxin or anti-VAMP antibodies revealed a syntaxin-SNAP25-VAMP-synaptotagmin complex. Anti-VAMP antibodies also trapped a distinct VAMP-synaptophysin complex. A similar fraction (about 70%) of N-type calcium channels ([125I]ω conotoxin GVIA receptors), was immunoprecipitated by either anti-syntaxin or anti-VAMP antibodies, but not by anti-synaptophysin antibodies (< 4%). The majority of N- but not L-type calcium channels ([3H]PN200-110 receptors), appear to be associated with a synaptic vesicle prefusion complex
The Tetrodotoxin Receptor of Voltage-Gated Sodium Channels—Perspectives from Interactions with μ-Conotoxins
Neurotoxin receptor site 1, in the outer vestibule of the conducting pore of voltage-gated sodium channels (VGSCs), was first functionally defined by its ability to bind the guanidinium-containing agents, tetrodotoxin (TTX) and saxitoxin (STX). Subsequent studies showed that peptide μ-conotoxins competed for binding at site 1. All of these natural inhibitors block single sodium channels in an all-or-none manner on binding. With the discovery of an increasing variety of μ-conotoxins, and the synthesis of numerous derivatives, observed interactions between the channel and these different ligands have become more complex. Certain μ-conotoxin derivatives block single-channel currents partially, rather than completely, thus enabling the demonstration of interactions between the bound toxin and the channel’s voltage sensor. Most recently, the relatively small μ-conotoxin KIIIA (16 amino acids) and its variants have been shown to bind simultaneously with TTX and exhibit both synergistic and antagonistic interactions with TTX. These interactions raise new pharmacological possibilities and place new constraints on the possible structures of the bound complexes of VGSCs with these toxins
Phoneutria nigriventer toxin 1: a novel statedependent inhibitor of neuronal sodium channels that interacts with mu-conotoxin binding sites
ABSTRACT A toxin was purified to homogeneity from the venom of the South American armed spider Phoneutria nigriventer and found to have a molecular mass of 8600 Da and a C-terminally amidated glycine residue. It appears to be identical to Toxin 1 (Tx1) isolated previously from this venom. Tx1 reversibly inhibited sodium currents in Chinese hamster ovary cells expressing recombinant sodium (Na v 1.2) channels without affecting their fast biophysical properties. The kinetics of inhibition of peak sodium current varied with membrane potential, with on-rates increasing and off-rates decreasing with more depolarized holding potentials in the Ϫ100 to Ϫ50 mV range. Thus, the apparent affinity of Tx1 for the channel increases as the membrane is depolarized. A mono[ 125 I]iodo-Tx1 derivative displayed high-affinity binding to a single class of sites (K D ϭ 80 pM, B max ϭ 0.43 pmol/mg protein) in rat brain membranes. Solubilized binding sites were immunoprecipitated by antibodies directed against a conserved motif in sodium channel ␣ subunits. 125 I-Tx1 binding was competitively displaced by conotoxin GIIIB (IC 50 ϭ 0.5 M) but not by 1 M tetrodotoxin. However, the inhibition of 125 I-Tx1 binding by conotoxin GIIIB was abrogated in the presence of tetrodotoxin (1 M). Patch-clamp and binding data indicate that P. nigriventer Tx1 is a novel, state-dependent sodium-channel blocker that binds to a site in proximity to pharmacological site 1, overlapping conotoxin but not tetrodotoxin binding sites
Retinal glycoprotein enrichment by concanavalin a enabled identification of novel membrane autoantigen synaptotagmin-1 in equine recurrent uveitis.
Complete knowledge of autoantigen spectra is crucial for understanding pathomechanisms of autoimmune diseases like equine recurrent uveitis (ERU), a spontaneous model for human autoimmune uveitis. While several ERU autoantigens were identified previously, no membrane protein was found so far. As there is a great overlap between glycoproteins and membrane proteins, the aim of this study was to test whether pre-enrichment of retinal glycoproteins by ConA affinity is an effective tool to detect autoantigen candidates among membrane proteins. In 1D Western blots, the glycoprotein preparation allowed detection of IgG reactions to low abundant proteins in sera of ERU patients. Synaptotagmin-1, a Ca2+-sensing protein in synaptic vesicles, was identified as autoantigen candidate from the pre-enriched glycoprotein fraction by mass spectrometry and was validated as a highly prevalent autoantigen by enzyme-linked immunosorbent assay. Analysis of Syt1 expression in retinas of ERU cases showed a downregulation in the majority of ERU affected retinas to 24%. Results pointed to a dysregulation of retinal neurotransmitter release in ERU. Identification of synaptotagmin-1, the first cell membrane associated autoantigen in this spontaneous autoimmune disease, demonstrated that examination of tissue fractions can lead to the discovery of previously undetected novel autoantigens. Further experiments will address its role in ERU pathology
Molecular interactions of the plasma membrane calcium ATPase 2 at pre- and post-synaptic sites in rat cerebellum.
The plasma membrane calcium extrusion mechanism, PMCA (plasma membrane calcium ATPase) isoform 2 is richly expressed in the brain and particularly the cerebellum. Whilst PMCA2 is known to interact with a variety of proteins to participate in important signalling events [Strehler EE, Filoteo AG, Penniston JT, Caride AJ (2007) Plasma-membrane Ca(2+) pumps: structural diversity as the basis for functional versatility. Biochem Soc Trans 35 (Pt 5):919-922], its molecular interactions in brain synapse tissue are not well understood. An initial proteomics screen and a biochemical fractionation approach identified PMCA2 and potential partners at both pre- and post-synaptic sites in synapse-enriched brain tissue from rat. Reciprocal immunoprecipitation and GST pull-down approaches confirmed that PMCA2 interacts with the post-synaptic proteins PSD95 and the NMDA glutamate receptor subunits NR1 and NR2a, via its C-terminal PDZ (PSD95/Dlg/ZO-1) binding domain. Since PSD95 is a well-known partner for the NMDA receptor this raises the exciting possibility that all three interactions occur within the same post-synaptic signalling complex. At the pre-synapse, where PMCA2 was present in the pre-synapse web, reciprocal immunoprecipitation and GST pull-down approaches identified the pre-synaptic membrane protein syntaxin-1A, a member of the SNARE complex, as a potential partner for PMCA2. Both PSD95-PMCA2 and syntaxin-1A-PMCA2 interactions were also detected in the molecular and granule cell layers of rat cerebellar sagittal slices by immunohistochemistry. These specific molecular interactions at cerebellar synapses may allow PMCA2 to closely control local calcium dynamics as part of pre- and post-synaptic signalling complexes
Dosage de l'activité endoprotéolytique de neurotoxines clostridiales
AIX-MARSEILLE2-BU Méd/Odontol. (130552103) / SudocSudocFranceF
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