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Omega-agatoxin-TK is a useful tool to study P-type Ca2+ channel-mediated changes in internal Ca2+ and glutamate
María Sitges1, Carlos Alberto Galindo
1Departmento de Biología Celular y Fisiología, Instituto de Investigaciones Biomédicas, Apartado Postal 70228,Ciudad Universitaria 04510, México, DF. sitgas@biomedicas.unam.mx
Abstract:
The present study shows that omega-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, omega-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 60nM. The maximal inhibition exerted by omega-agatoxin-TK in striatal synaptosomes (61 +/- 11%) is 10% larger than in hippocampal synaptosomes, suggesting a larger population of omega-agatoxin-TK-sensitive Ca(2+) channels in striatal than in hippocampal nerve endings. The N-type Ca(2+) channel blocker, omega-conotoxin-GVIA (1muM), inhibits part of the omega-agatoxin-TK-insensitive rise in Ca(i) induced by high K(+). In contrast to the inhibition exerted by omega-agatoxin-TK on the Ca(i) response to high K(+), omega-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 omega-agatoxin-TK-sensitive channels. High K(+) does not increase Na(i). In [(3)H]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 [(3)H]Glu induced by high K(+) is absolutely dependent on the entrance of external Ca(2+). This exocytotic release of [(3)H]Glu attained in the absence of a chemical Na(+) gradient is inhibited with the same potency and efficacy by omega-agatoxin-TK and by omega-agatoxin-IVA, which is known to differ from omega-agatoxin-TK in its amino terminal moiety. These results indicate that omega-agatoxin-TK represents a good pharmacological tool to study P/Q type Ca(2+) channel-mediated responses in cerebral nerve endings.
Insights
Omega-agatoxin-TK, a potent toxin from Agelenopsis aperta venom, effectively blocks P/Q type calcium channels in brain nerve endings. This toxin is a valuable tool for studying calcium channel-mediated responses in the brain.
Area of Science:
- Neuroscience
- Pharmacology
- Molecular Biology
Background:
- Calcium channels play crucial roles in neuronal function, including neurotransmitter release.
- Different subtypes of voltage-gated calcium channels (VGCCs) mediate distinct physiological processes.
- Omega-agatoxin-TK is a toxin found in Agelenopsis aperta venom, known to interact with calcium channels.
Purpose of the Study:
- To investigate the inhibitory effects of omega-agatoxin-TK on depolarization-induced calcium influx in cerebral nerve endings.
- To determine the selectivity of omega-agatoxin-TK for specific calcium channel subtypes.
- To evaluate omega-agatoxin-TK as a pharmacological tool for studying P/Q type calcium channel-mediated neurotransmitter release.
Main Methods:
- Measurement of intracellular calcium ([Ca(i)]) using fura-2 in isolated striatal and hippocampal nerve endings.
- Application of high potassium (K(+)) or veratridine to induce depolarization and calcium influx.
- Use of specific calcium channel blockers, including omega-agatoxin-TK, omega-agatoxin-IVA, and omega-conotoxin-GVIA.
- Measurement of intracellular sodium ([Na(i)]) using SBFI.
- Assessment of [(3)H]glutamate release from synaptosomes under conditions minimizing transporter-mediated release.
Main Results:
- Omega-agatoxin-TK dose-dependently inhibited high K(+)-induced Ca(i) rise in cerebral nerve endings with an IC(50) of approximately 60 nM.
- Striatal synaptosomes showed a greater maximal inhibition (61%) by omega-agatoxin-TK compared to hippocampal synaptosomes (10% difference).
- Omega-agatoxin-TK did not inhibit veratridine-induced Ca(i) and Na(i) elevations, indicating it does not target tetrodotoxin-sensitive channels.
- High K(+)-induced exocytotic release of [(3)H]glutamate was inhibited by omega-agatoxin-TK and omega-agatoxin-IVA with similar potency and efficacy.
Conclusions:
- Omega-agatoxin-TK is a potent inhibitor of P/Q type calcium channels in cerebral nerve endings.
- The toxin exhibits differential sensitivity in striatal versus hippocampal nerve endings.
- Omega-agatoxin-TK is a valuable pharmacological tool for investigating P/Q type calcium channel function and associated neurotransmitter release.
