Affinity purification of rat cortical and chicken forebrain synaptosomes using a biotinylated derivative of co-CgTx GVIA

Abstract

We describe a magnetophoretic method for the affinity purification of synaptosomes expressing ω-CgTx GVIA-sensitive, N-type voltage-sensitive calcium channels (VSCCs). The method utilizes a biotinylated derivative of ω-CgTx GVIA which retains its ability to displace [ 125I] ω-CgTx GVIA from its binding sites on rat synaptic membranes. When coupled to streptavidin coated magnetizable beads, the hexanoyl spacer between ω-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, ω-CgTx GVIA (1 nM-1 μM) produced a concentration-dependent block of the KCl-induced intracellular free Ca 2+, [Ca 2+] i, elevation with an IC 50 of 28 nM. After affinity magnetophoresis no increase in [Ca 2+] i elevation was observed in either the bound or unbound fractions. In the rat synaptosome parent fraction, the KCl-induced increase in free intracellular Ca 2+ ([Ca 2+] i) elevation was partially blocked by ω-CgTx GVIA (17 ± 2% @ 1 μM) and to a greater extent by ω-Aga IVA (55 ± 5% @ 1 μM): a combination of the two toxins was additive (72 ± 4% @ 1 μM). The block obtained by co-CgTx GVIA (1 μM) in the unbound fraction was reduced to 3 ± 2%, whereas that by ω-Aga IVA (1 μM) increased to 82 ± 3%. The block obtained by a combination of both toxins (83 ± 2%) was the same as that with μ-Aga IVA alone (82 ± 3%). No increase in free [Ca 2+] i elevation was observed in the bound fraction although single synaptosome-like structures, displaying synaptophysin immunoreactivity, were detected on the beads. We conclude that ω-CgTx GVIA-sensitive N-type calcium channels are present on all chicken forebrain synaptosomes but only a subset of rat cortical synaptosomes.