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Development of a fluorescence assay for the characterization of brevenal binding to rat brain synaptosomes.

ABSTRACT: The marine dinoflagellate Karenia brevis produces a family of neurotoxins known as brevetoxins. Brevetoxins elicit their effects by binding to and activating voltage-sensitive sodium channels (VSSCs) in cell membranes. K. brevis also produces brevenal, a brevetoxin antagonist, which is able to inhibit and/or negate many of the detrimental effects of brevetoxins. Brevenal binding to VSSCs has yet to be fully characterized, in part due to the difficulty and expense of current techniques. In this study, we have developed a novel fluorescence binding assay for the brevenal binding site. Several fluorescent compounds were conjugated to brevenal to assess their effects on brevenal binding. The assay was validated against the radioligand assay for the brevenal binding site and yielded comparable equilibrium inhibition constants. The fluorescence-based assay was shown to be quicker and far less expensive and did not generate radioactive waste or need facilities for handling radioactive materials. In-depth studies using the brevenal conjugates showed that, while brevenal conjugates do bind to a binding site in the VSSC protein complex, they are not displaced by known VSSC site specific ligands. As such, brevenal elicits its action through a novel mechanism and/or currently unknown receptor site on VSSCs.

SUBMITTER: McCall JR

PROVIDER: S-EPMC4176390 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Development of a fluorescence assay for the characterization of brevenal binding to rat brain synaptosomes.

McCall Jennifer R JR Goodman Allan J AJ Jacocks Henry M HM Thompson Alysha M AM Baden Daniel G DG Bourdelais Andrea J AJ

Journal of natural products 20140917 9

The marine dinoflagellate Karenia brevis produces a family of neurotoxins known as brevetoxins. Brevetoxins elicit their effects by binding to and activating voltage-sensitive sodium channels (VSSCs) in cell membranes. K. brevis also produces brevenal, a brevetoxin antagonist, which is able to inhibit and/or negate many of the detrimental effects of brevetoxins. Brevenal binding to VSSCs has yet to be fully characterized, in part due to the difficulty and expense of current techniques. In this s ...[more]

PMID: 25226846

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Project description:Brevetoxins are a family of ladder-frame polyether toxins produced during blooms of the marine dinoflagellate Karenia brevis. Inhalation of brevetoxins aerosolized by wind and wave action can lead to asthma-like symptoms in beach goers. Consumption of either shellfish or finfish exposed to K. brevis blooms can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are due to activation of voltage-sensitive sodium channels (VSSCs) in cell membranes. Binding of brevetoxin analogs and competitors to site 5 on these channels has historically been measured using a radioligand competition assay that is fraught with difficulty, including slow analysis time, production of radioactive waste, and cumbersome and expensive methods associated with the generation of radioactive labeled ligands. In this study, we describe the development of a novel fluorescent synaptosome binding assay for the brevetoxin receptor. BODIPY(®)-conjugated to PbTx-2 was used as the labeled ligand. The BODIPY(®)-PbTx-2 conjugate was found to displace [(3)H]-PbTx-3 from its binding site on VSSCs on rat brain synaptosomes with an equilibrium inhibition constant of 0.11 nM. We have shown that brevetoxin A and B analogs are all able to compete for binding with the fluorescent ligand. Most importantly, this assay was validated against the current site 5 receptor binding assay standard, the radioligand receptor assay for the brevetoxin receptor using [(3)H]-PbTx-3 as the labeled ligand. The fluorescence based assay yielded equilibrium inhibition constants comparable to the radioligand assay for all brevetoxin analogs. The fluorescence based assay was quicker, far less expensive, and did not generate radioactive waste or need radioactive facilities. As such, this fluorescence-based assay can be used to replace the current radioligand assay for site 5 on voltage-sensitive sodium channels and will be a vital tool for future experiments examining the binding affinity of various ligands for site 5 on sodium channels.

Project description:Previous findings from our laboratory and others indicate that two-dimensional gel electrophoresis (2-DE) can be used to study protein expression in defined brain regions, but mainly the proteins which are present in high abundance in glia are readily detected. The current study was undertaken to determine the protein profile in a synaptosomal subcellular fraction isolated from the cerebral cortex of the rat. Both 2-DE and liquid chromatography - tandem mass spectrometry (LC-MS/MS) procedures were used to isolate and identify proteins in the synaptosomal fraction and accordingly >900 proteins were detected using 2-DE; the 167 most intense gel spots were isolated and identified with matrix-assisted laser desorption/ionization - time of flight peptide mass fingerprinting or LC-MS/MS. In addition, over 200 proteins were separated and identified with the LC-MS/MS "shotgun proteomics" technique, some in post-translationally modified form. The following classes of proteins associated with synaptic function were detected: (a) proteins involved in synaptic vesicle trafficking-docking (e.g., SNAP-25, synapsin I and II, synaptotagmin I, II, and V, VAMP-2, syntaxin 1A and 1B, etc.); (b) proteins that function as transporters or receptors (e.g., excitatory amino acid transporters 1 and 2, GABA transporter 1); (c) proteins that are associated with the synaptic plasma membrane (e.g., post-synaptic density-95/synapse-associated protein-90 complex, neuromodulin (GAP-43), voltage-dependent anion-selective channel protein (VDACs), sodium-potassium ATPase subunits, alpha 2 spectrin, septin 7, etc.); and (d) proteins that mediate intracellular signaling cascades that modulate synaptic function (e.g., calmodulin, calcium-calmodulin-dependent protein kinase subunits, etc.). Other identified proteins are associated with mitochondrial or general cytosolic function. Of the two proteins identified as endoplasmic reticular, both interact with the synaptic SNARE complex to regulate vesicle trafficking. Taken together, these results suggest that the integrity of the synaptosomes was maintained during the isolation procedure and that this subcellular fractionation technique enables the enrichment of proteins associated with synaptic function. The results also suggest that this experimental approach can be used to study the differential expression of multiple proteins involved in alterations of synaptic function.

Project description:RationaleNovel synthetic "bath salt" cathinones continue to appear on the street as abused and addictive drugs. The range of subjective experiences produced by different cathinones suggests that some compounds have primarily dopaminergic activity (possible stimulants) while others have primarily serotonergic activity (possible empathogenics). An understanding of the structure activity relationships (SARs) of these compounds will help in assessing the likely behavioral effects of future novel structures, and to define potential therapeutic strategies to reverse any reinforcing effects.ObjectivesA series of methcathinone analogs was systematically studied for their activity at the dopamine and serotonin transporters. Compound structures varied at the aromatic group, either by substituent or by replacement of the phenyl ring with a naphthalene or indole ring.MethodsA novel, high-yielding synthesis of methcathinone hydrochlorides was developed which avoids isolation of the unstable free bases. Neurotransmitter transporter release activity was determined in rat brain synaptosomes as previously reported. Compounds were also screened for activity at the norepinephrine transporter.ResultsTwenty-eight methcathinone analogs were analyzed and fully characterized in dopamine and serotonin transporter release assays. Compounds substituted at the 2-position (ortho) were primarily dopaminergic. Compounds substituted at the 3-position (meta) were found to be much less dopaminergic, with some substituents favoring serotonergic activity. Compounds substituted at the 4-position (para) were found to be far more serotonergic, as were disubstituted compounds and other large aromatic groups. One exception was the fluoro-substituted analogs which seem to favor the dopamine transporter.ConclusionsThe dopaminergic to serotonergic ratio can be manipulated by choice of substituent and location on the aromatic ring. It is therefore likely possible to tweak the subjective and reinforcing effects of these compounds by adjusting their structure. Certain substituents like a fluoro group tend to favor the dopamine transporter, while others like a trifluoromethyl group favor the serotonin transporter.

Dataset Information

Development of a fluorescence assay for the characterization of brevenal binding to rat brain synaptosomes.

Publications

Development of a fluorescence assay for the characterization of brevenal binding to rat brain synaptosomes.

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