Affinity purification of rat cortical and chicken forebrain synaptosomes using a biotinylated derivative of omega-CgTx GVIA.
We describe a magnetophoretic method for the affinity purification of synaptosomes expressing omega-CgTx GVIA-sensitive, N-type voltage-sensitive calcium channels (VSCCs). The method utilizes a biotinylated derivative of omega-CgTx GVIA which retains its ability to displace [125I] omega-CgTx GVIA from its binding sites on rat synaptic membranes. When coupled to streptavidin coated magnetizable beads, the hexanoyl spacer between omega-CgTx GVIA and the biotin:streptavidin bead complex is sufficiently long to allow flexibility of the toxin to bind to its receptor on synaptosomes. We have used this ligand successfully to isolate deaggregated synaptosomes from the parent fractions of chicken forebrain and rat cortex. In the chicken synaptosome parent fraction, omega-CgTx GVIA (1 nM-1 microM) produced a concentration-dependent block of the KCl-induced intracellular free Ca2+, [Ca2+]i, elevation with an IC50 of 28 nM. After affinity magnetophoresis no increase in [Ca2+]i elevation was observed in either the bound or unbound fractions. In the rat synaptosome parent fraction, the KCl-induced increase in free intracellular Ca2+ ([Ca2+]i) elevation was partially blocked by omega-CgTx GVIA (17 +/- 2% / 1 microM) and to a greater extent by omega-Aga IVA (55 +/- / 1 microM): a combination of the two toxins was additive (72 +/- 4% / 1 microM). The block obtained by omega-CgTx GVIA (1 microM) in the unbound fraction was reduced to 3 +/- 2%, whereas that by omega-Aga IVA (1 microM) increased to 82 +/- 3%. The block obtained by a combination of both toxins (83 +/- 2%) was the same as that with omega-Aga IVA alone (82 +/- 3%). No increase in free [Ca2+]i elevation was observed in the bound fraction although single synaptosome-like structures, displaying synaptophysin immunoreactivity, were detected on the beads. We conclude that omega-CgTx GVIA-sensitive N-type calcium channels are present on all chicken forebrain synaptosomes but only a subset of rat cortical synaptosomes.
Lilly Research Centre Ltd, Windlesham, Surrey, U.K.
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Bowman
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Smith
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McCormack
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D002645:Chickens; D004305:Dose-Response Relationship, Drug; D007150:Immunohistochemistry; D010455:Peptides / Q000494:pharmacology*; D011189:Potassium Chloride / Q000494:pharmacology; D016548:Prosencephalon / Q000378:metabolism*; D011869:Radioligand Assay; D051381:Rats; D011919:Rats, Inbred Strains; D013574:Synaptosomes / Q000378:metabolism*; D013997:Time Factors; D020866:omega-Conotoxin GVIA
D002121:Calcium Channel Blockers; D010455:Peptides; D011189:Potassium Chloride; D020866:omega-Conotoxin GVIA; D002118:Calcium