Low threshold calcium (Ca2+) spikes mediated by T-type Ca2+ channels play a key role in neuronal excitability. These channels open after small fluctuations in the neuronal membrane potential, leading to a further depolarization and the opening of other channels, such as voltage-gated sodium (Na +) channels and high voltage-activated Ca 2+ channels, often leading to bursts of neuronal activity. Over-active burst firing of thalamic neurons is thought to trigger not only absence epileptic seizures, but have also been implicated in a wide range of mental disorders characterized by the presence of thalamocortical dysrhythmias. The discovery of three genes encoding T-type channels has led to many breakthroughs in our understanding of their physiology, and the renewal this grant will extend these studies further into their biophysics, pharmacology, and structure-function of T-type channels. One hypothesis to be tested is that mutations in a T channel gene triggers childhood absence epilepsy (CAE). This channelopathy hypothesis will be tested by introducing these mutations into the channel, and measuring how this affects functional activity. Half of the CAE mutations are clustered in a particular region of the channel, so studies will explore its role in channel function. One goal of these studies is to provide the proof of concept that developing a novel T-type channel blocker will produce an effective antiepileptic drug. Such proof might also come from studies on the mechanism of action of new generation antiepileptic drugs that can treat many types of epilepsy and neuropathic pain. Studies will explore the selectivity of these drugs using patch clamp electrophysiology of cells engineered to express human Na + and Ca2+ channels. Novel compounds have been synthesized that block both channels at lower doses than the parent drug, the antiepileptic phenytoin. Therefore a final goal of this grant will be to use computer modeling to design novel compounds. A fluorescence-based assay has been developed that allows for medium throughput screening for active compounds. Lead compounds will be sent to the NIH Anticonvulsant Drug Development Program for in vivo testing. These studies will test the hypothesis that more potent channel blockers are better antiepileptics and the importance of selectivity.
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