ω-Agatoxin-TK is a useful tool to study P-type Ca 2+ channel-mediated changes in internal Ca 2+ and glutamate release in depolarised brain nerve terminals

Abstract

The present study shows that ω-agatoxin-TK, a toxin of the venom of Agelenopsis aperta, which is 10 times more concentrated than the P/Q type Ca 2+ channel blocker, ω-agatoxin-IVA in the venom, inhibits the high K + depolarisation-induced rise in internal Ca 2+ (Ca i, as determined with fura-2) dose dependently in cerebral (striatal and hippocampal) isolated nerve endings, with calculated IC 50's of about 60 nM. The maximal inhibition exerted by ω-agatoxin-TK in striatal synaptosomes (61 ± 11%) is 10% larger than in hippocampal synaptosomes, suggesting a larger population of ω-agatoxin-TK-sensitive Ca 2+ channels in striatal than in hippocampal nerve endings. The N-type Ca 2+ channel blocker, ω-conotoxin-GVIA (1 μM), inhibits part of the ω-agatoxin-TK-insensitive rise in Ca i induced by high K +. In contrast to the inhibition exerted by ω-agatoxin-TK on the Ca i response to high K +, ω-agatoxin-TK failed to inhibit the tetrodotoxin-sensitive elevations in Ca i and in internal Na + (Na i, as determined with SBFI) induced by veratridine, indicating that the Ca 2+ influx activated by veratridine does not involve ω-agatoxin-TK-sensitive channels. High K + does not increase Na i. In [ 3H]Glu preloaded hippocampal synaptosomes super-fused with low Na + Krebs Ringer HEPES (a condition that guarantees the elimination of neurotransmitter transporters-mediated release), the release of [ 3H]Glu induced by high K + is absolutely dependent on the entrance of external Ca 2+. This exocytotic release of [ 3H]Glu attained in the absence of a chemical Na + gradient is inhibited with the same potency and efficacy by ω-agatoxin-TK and by ω-agatoxin-IVA, which is known to differ from ω-agatoxin-TK in its amino terminal moiety. These results indicate that ω-agatoxin-TK represents a good pharmacological tool to study P/Q type Ca 2+ channel-mediated responses in cerebral nerve endings.