Calcium/calmodulin inhibits the binding of specific [125I]omega-conotoxin GVIA to chick brain membranes.

The effect of Ca2+/calmodulin (CaM) on the specific binding of [125I]omega-conotoxin GVIA (125I-omega-CTX) to crude membranes from chick brain was investigated. When we examined the effects of the activation of various endogenous protein kinases on specific [125I]omega-CTX binding to crude membranes, we observed that Ca2+/CaM had an inhibitory effect regardless of whether or not the standard medium contained ATP (0.5 mM). Ca2+/CaM also had an inhibitory effect in a simple binding-assay medium containing HEPES-HCl buffer, BSA, Ca2+ and CaM, and this effect was dependent on the concentration of Ca2+. The effect of Ca2+/CaM was attenuated by the CaM antagonists W-7 and CaM-kinase II fragment (290-309). An experiment with modified ELISA using purified anti omega-CTX antibody indicated that Ca2+/CaM did not affect the direct binding of [125I]omega-CTX and CaM. These results suggest that Ca2+/CaM either directly or indirectly affects specific [125I]omega-CTX binding sites, probably N-type Ca2+ channels in crude membranes from chick whole brain.

Proportions of Ca2+ channel subtypes in chick or rat P2 fraction and NG108-15 cells using various Ca2+ blockers.

The proportions of calcium (Ca2+) channel subtypes in chick or rat P2 fraction and NG 108-15 cells were investigated using selective L-, N-, P- and P/Q- type Ca2+ channel blockers. KCl-stimulated 45Ca2+ uptake by chick P2 fraction was blocked by 40-50% using N-type Ca2+ channel blockers [omega-conotoxin GVIA, aminoglycoside antibiotics and dynorphin A(1-13)], but was not inhibited by P- or P/Q-type blockers (omega-agatoxin IVA or omega-conotoxin MVIIC). On the other hand, KCl-stimulated 45Ca2+ uptake by rat P2 fraction was blocked by 30 approximately 40% using P- or P/Q-type Ca2+ channel blockers, but was not inhibited by N-type Ca2+ channel blockers. The L-type Ca2+ channel blockers 1,4-dihydropyridines, diltiazem and verapamil, but not calciseptine (CaS), inhibited both KCl-stimulated 45Ca2+ uptake and veratridine-induced 22Na+ uptake by chick or rat P2 fraction with similar IC50 values. CaS did not have any effect on 45Ca2+ uptake by either chick or rat P2 fraction. In NG108-15 cells, CaS, omega-agatoxin IVA and omega-conotoxin MVIIC, but not omega-conotoxin GVIA, inhibited KCl-stimulated 45Ca2+ uptake by 30-40%. Various combinations of these Ca2+ channel blockers had no significant additional effects in chick or rat P2 fraction or NG 108-15 cells. These findings suggest that KCl-stimulated 45Ca2+ uptake by chick or rat P2 fraction and NG 108-15 cells is a convenient and useful model for screening whether or not natural or synthetic substances have selective effects as L-, N-, P-, or P/Q- type Ca2+ channel antagonists or agonists.