Molecular Analysis of Neuronal T Type CA++ Channels

描述(由申请人提供)：由T-型钙通道介导的低阈值钙(Ca~(2+))峰在神经元兴奋性中起关键作用。这些通道在神经细胞膜电位微小波动后开放，导致进一步的去极化和其他通道的开放，如电压门控钠(Na+)通道和高电压激活的钙通道，通常导致神经元活动的爆发。丘脑神经元过度活跃的爆发式放电被认为不仅引发失神癫痫发作，而且还与以丘脑皮质节律失常为特征的一系列精神障碍有关。三个编码T型通道的基因的发现使我们对它们的生理认识取得了许多突破，而这一拨款的更新将使这些研究进一步扩展到它们的生物物理学、药理学和T型通道的结构与功能。一个有待检验的假设是，T通道基因的突变会引发儿童失神癫痫(CAE)。通过将这些突变引入通道，并测量这如何影响功能活动，将检验这一通道病假说。一半的CAE突变聚集在通道的特定区域，因此研究将探索它在通道功能中的作用。这些研究的目的之一是提供概念上的证据，即开发一种新型的T型通道阻滞剂将产生一种有效的抗癫痫药物。这一证据也可能来自对新一代抗癫痫药物作用机制的研究，这些药物可以治疗多种类型的癫痫和神经性疼痛。研究将利用表达人钠和钙通道的工程细胞的膜片钳电生理学来探索这些药物的选择性。已经合成了新的化合物，它们以比母药抗癫痫药苯妥英更低的剂量阻断这两个通道。因此，这笔赠款的最终目标将是使用计算机建模来设计新的化合物。开发了一种基于荧光的分析方法，允许对活性化合物进行中等吞吐量的筛选。先导化合物将被送往NIH抗惊厥药物开发计划进行体内试验。这些研究将检验这样一种假设，即更有效的通道阻滞剂是更好的抗癫痫药，以及选择性的重要性。

DESCRIPTION (provided by applicant): 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.