Molecular Mechanisms of Na Channel Slow Inactivation

描述（由申请人提供）：电压门控钠通道的失活是可兴奋膜传递动作电位能力的重要决定因素。钠离子通道的快速失活发生在毫秒的时间过程中，这在一定程度上限制了动作电位的持续时间。一种更持久的失活形式，即所谓的慢失活，发生在几百毫秒以上，可能通过限制参与动作电位上升的Na通道的数量来调节膜的兴奋性。最近的研究表明，人类疾病如高钾血症性周期性麻痹中存在缓慢失活的紊乱。虽然参与快速失活的结构已经被很好地描述，但慢失活门控机制的性质和位置仍然知之甚少。更好地了解这些结构可以为更合理地治疗膜兴奋性障碍提供基础。在我们最初的提案中，我们概述了我们的计划，通过检查可能充当孔隙闭塞“门”的区域，以及可能赋予电压敏感性的结构，来定义参与缓慢失活的Na通道区域。自我们提交论文以来，已经出现了几条证据线，并导致了一个尚未经过验证的建议，即在离子传导孔的外表面附近的塌陷阻碍了Na电流的缓慢失活。我们已经测试了这一假设，并产生了证据表明，当通道缓慢失活时，钠渗透途径的外口保持开放。[这次重新提交为期两年的延期有三个具体目的：1)测试部分或全部S4电压传感器在缓慢失活期间固定在外向位置的假设。(2)为了验证缓慢失活门控涉及孔隙细胞质部分构象变化的假设，而不是之前假设的外部孔隙区域。3)表征野生型和疾病相关突变通道在短暂重复去极化过程中的慢失活门控，这将为慢失活的生理相关性提供更深入的了解。申请人的长期职业规划是将正在进行的工作中的电生理学方法与他以前在细胞和分子生物学方面的培训结合起来，研究神经系统疾病中的兴奋性障碍。额外的两年培训需要从仅仅熟悉电生理技术过渡到有经验建立和指导一个研究项目，其中电生理技术是一个组成部分。

DESCRIPTION (provided by applicant): Inactivation of voltage-gated Na channels is a critically important determinant of the ability of an excitable membrane to transmit an action potential. Fast-inactivation of Na channels occurs over a time course of milliseconds and in part limits the duration of the action potential. A more prolonged form of inactivation, so-called slow inactivation, takes place over several hundred milliseconds and may serve to regulate the excitability of the membrane by limiting the number of Na channels available to participate in the upstroke of an action potential. Recent studies have implicated derangements of slow inactivation in human diseases such as Hyperkalemic Periodic Paralysis. Whereas the structures that participate in fast inactivation are well described, the nature and location of the slow inactivation gating mechanism remains poorly understood. Better understanding of these structures may provide a basis for more rational therapies for disorders of membrane excitability. In our initial proposal we outlined our plan to define those regions of the Na channel that participate in slow inactivation, by examining both regions that may act as the pore-occluding "gate," as well as those structures which may confer voltage sensitivity to slow inactivation. Since our submission, several lines of evidence have emerged and led to the as-yet untested proposal that a collapse near the external face of the ion-conducting pore impedes Na current during slow inactivation. We have tested this hypothesis and have generated evidence that the outer mouth of the Na permeation pathway remains open while the channel is slow inactivated. [This resubmission for a two-year extension has three Specific Aims: 1) To test the hypothesis that some or all of the S4 voltage sensors are immobilized in the outward position during slow inactivation. (2) To test the hypothesis that slow inactivation gating involves conformational shifts in the cytoplasmic portion of the pore, rather than the external pore region as previously hypothesized. 3) To characterize slow inactivation gating of wild-type and disease-associated mutant channels during trains of brief repetitive depolarizations, which will provide greater insight on the physiological relevance of slow inactivation. The long-range career plan of the applicant is to combine the electrophysiological approaches of the on-going work with his former training in cellular and molecular biology to investigate disorders of excitability in neurological disease. The additional two years of training are required to make the transition from being merely acquainted with the techniques of electrophysiology to having the experience to set-up and direct a research program for which electrophysiological techniques are an integral component.]