Molecular Analysis of Neuronal T Type CA++ Channels

描述（由申请人提供）：由t型Ca2+通道介导的低阈值钙（Ca2+）峰值在神经元兴奋性中起关键作用。这些通道在神经元膜电位的微小波动后打开，导致进一步的去极化和其他通道的打开，如电压门控钠（Na +）通道和高压激活的ca2 +通道，通常导致神经元活动的爆发。丘脑神经元过度活跃的突发放电被认为不仅会引发癫痫发作，而且还与以丘脑皮质节律异常为特征的多种精神障碍有关。编码t型通道的三个基因的发现使我们对其生理学的理解取得了许多突破，这笔拨款的续期将进一步扩展这些研究到t型通道的生物物理学、药理学和结构功能。一个有待验证的假设是，T通道基因的突变会引发儿童期缺失性癫痫（CAE）。通过将这些突变引入通道，并测量其如何影响功能活动，将对这种通道病假说进行检验。一半的CAE突变聚集在通道的特定区域，因此研究将探讨其在通道功能中的作用。这些研究的一个目标是为开发一种新型t型通道阻滞剂将产生一种有效的抗癫痫药物的概念提供证据。新一代抗癫痫药物可以治疗多种类型的癫痫和神经性疼痛，其作用机制的研究也可能证明这一点。研究将探索这些药物的选择性使用膜片钳电生理细胞工程表达人类Na +和Ca2+通道。新的化合物已经被合成，以较低的剂量阻断这两个通道，比母体药物，抗癫痫的苯妥英。因此，这项拨款的最终目标将是使用计算机建模来设计新的化合物。已经开发了一种基于荧光的测定方法，允许对活性化合物进行中等通量筛选。先导化合物将被送到美国国立卫生研究院抗惊厥药物开发项目进行体内测试。这些研究将检验更有效的通道阻滞剂是更好的抗癫痫药的假设和选择性的重要性。

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.