Studies from this laboratory have demonstrated that phenytoin, crbamazepine, and the benzodiazepines (BZ) inhibit Ca++ calmodulin regulated protein phosphorylation and neurotransmitter release in several synaptic preparations. An hypothesis was proposed from these results stating that the Ca++regulated phosphorylation of specific brain proteins, especially at the synapse, may play an important role in regulating neuronal excitability and the action of specific anticonvulsant drugs. This investigation will systematically examine this hypothesis. Initial studies will be directed at isolating the Ca++ calmodulin kinase system and characterizing the effects of Ca++, calmodulin, and anticonvulsant compounds on protein phosphorylation, neurotransmitter release, and Ca++regulated functions in various synaptic preparations. We will correlate the potency of several benzodiazepine derivatives to inhibit these Ca++ regulated events with their potency for inhibiting maximal electric shock induced convulsions. Results from this laboratory have identified a novel anticonvulsant binding site in brain membrane. The potency of the benzodiazepines to bind to this site correlate very significantly with their ability to inhibit maximal electric shock induced seizures and Ca++ calmodulin kinases activity. These results provide evidence for the existence of an anticonvulsant binding site in brain membrane that may regulate the activity of the Ca++ calmodulin kinase system and modulate the excitability of synaptic membrane. Employing photoaffinity labelling experiments with highly purified Ca++ calmodulin kinase and membrane fractions, we will be able to determine if a subunit or modulator of the kinase system is part of the membrane anticonvulsant binding site. Studies will also be initiated to characterize a compound recently isolated from brain that is a potent inhibitor of the Ca++ calmodulin kinase system. Our preliminary studies indicate that this new compound is also a potent anticonvulsant in inhibiting maximal electric shock induced seizures. The proposed experiments will allow us to characterize this new endogenous anticonvulsant substance and determine if it is a """"""""natural"""""""" anticonvulsant released from brain during seizure activity with the resultant suppression of seizure activity.
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