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

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 [Na-i] induced by veratridine in mouse brain synaptosomes loaded with a selective Na+ indicator dye. Veratridine (20 mu M) increases the basal [Na-i] level (20 mM) more than twofold. This increase is independent of external Ca2+, but abolished by tetrodotoxin (1 mu M). 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 [Na-i]. 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 [Na-i] induced by veratridine, as well as other veratridine induced responses, namely elevation of [Ca-i] (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 [Na-i] induced by veratridine, but markedly diminished the elevation of [Ca-i] 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 [Na-i] 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 Naf 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. (C) 1998 Elsevier Science Ltd. All rights reserved.