Study on the possible involvement of protein kinases in the modulation of brain presynaptic sodium channels; comparison with calcium channels

Abstract

A possible modulatory role of kinases on voltage sensitive Na+ channels of presynaptic brain nerve endings was investigated by testing the effect of several kinase activators and inhibitors on the elevation of [Nai] induced by veratridine in mouse brain synaptosomes loaded with a selective Na+ indicator dye. Veratridine (20 mM) increases the basal [Nai] level (20 mM) more than twofold. This increase is independent of external Ca2, but abolished by tetrodotoxin (1 mM). Activation of cAMP dependent protein kinase with forskolin or cAMP analogs, or of protein kinase C with diacylglycerol did not affect the veratridine-induced elevation in [Nai]. Drugs reported to inhibit calmodulin-dependent events, as well as the regulatory domain of protein kinase C, were potent and effective inhibitors of the increase in [Nai] induced by veratridine, as well as other veratridine induced responses, namely elevation of [Cai] (monitored with the Ca2 indicator dye fura-2) and neurotransmitter (GABA) release. Drugs that inhibit kinases by binding to the catalytic site were ineffective, however, as was the phosphatase inhibitor, okadaic acid. A selective inhibitor of Ca2 and calmodulin dependent protein kinase II also did not affect the elevation of [Nai] induced by veratridine, but markedly diminished the elevation of [Cai] induced by depolarization either with veratridine or with high K+ (15 and 30 mM). On the basis of these results it is concluded that, the dramatic inhibition exerted by some of the drugs tested on the elevation of [Nai] induced by veratridine is not due to their effects on kinases, but to a possible interaction of these compounds with an intracellular site of the Na+ channel. On the other hand, while Ca2 and calmodulin dependent protein kinase II is unable to modulate brain presynaptic voltage sensitive Na+ channels, it facilitates the activation of brain presynaptic voltage sensitive Ca2 channels.